Interactions of phosphatidylinositol kinase, GTPase-activating protein (GAP), and GAP-associated proteins with the colony-stimulating factor 1 receptor.

PubMed Central

Reedijk, M; Liu, X Q; Pawson, T

1990-01-01

The interactions of the macrophage colony-stimulating factor 1 (CSF-1) receptor with potential targets were investigated after ligand stimulation either of mouse macrophages or of fibroblasts that ectopically express mouse CSF-1 receptors. In Rat-2 cells expressing the mouse CSF-1 receptor, full activation of the receptor and cellular transformation require exogenous CSF-1, whereas NIH 3T3 cells expressing mouse c-fms are transformed by autocrine stimulation. Activated CSF-1 receptors physically associate with a phosphatidylinositol (PI) 3'-kinase. A mutant CSF-1 receptor with a deletion of the kinase insert region was deficient in its ability to bind functional PI 3'-kinase and to induce PI 3'-kinase activity precipitable with antiphosphotyrosine antibodies. In fibroblasts, CSF-1 stimulation also induced the phosphorylation of the GTPase-activating protein (GAP)-associated protein p62 on tyrosine, although GAP itself was a relatively poor substrate. In contrast to PI 3'-kinase association, phosphorylation of p62 and GAP was not markedly affected by deletion of the kinase insert region. These results indicate that the kinase insert region selectively enhances the CSF-1-dependent association of the CSF-1 receptor with active PI 3'-kinase. The insert deletion mutant retains considerable transforming activity in NIH 3T3 cells (G. Taylor, M. Reedijk, V. Rothwell, L. Rohrschneider, and T. Pawson, EMBO J. 8:2029-2037, 1989). This mutant was more seriously impaired in Rat-2 cell transformation, although mutant-expressing Rat-2 cells still formed small colonies in soft agar in the presence of CSF-1. Therefore, phosphorylation of GAP and p62 through activation of the CSF-1 receptor does not result in full fibroblast transformation. The interaction between the CSF-1 receptor and PI 3'-kinase may contribute to c-fms fibroblast transformation and play a role in CSF-1-stimulated macrophages. Images PMID:2172781

Possible involvement of MSX-2 homeoprotein in v-ras-induced transformation.

PubMed

Takahashi, C; Akiyama, N; Kitayama, H; Takai, S; Noda, M

1997-04-01

A truncated MSX-2 homeoprotein was found to induce flat reversion when expressed in v-Ki-ras-transformed NIH3T3 cells. Although the expression of endogenous MSX-2 gene is low in most of the normal adult tissues examined, it is frequently activated in carcinoma-derived cell lines. Likewise, the gene is inactive in untransformed cells but is transcriptionally activated after transformation by v-Ki-ras oncogene, suggesting that the intact MSX-2 may play a positive, rather than suppressive, role in cell transformation. To test this possibility, we isolated a full-length human MSX-2 cDNA and tested its activities in two cell systems: fibroblast and myoblast. In NIH3T3 fibroblasts, although the gene by itself failed to confer a transformed phenotype, antisense MSX-2 cDNA as well as truncated MSX-2 cDNA interfered with the transforming activities of both v-Ki-ras and v-raf oncogene. In C2C12 myoblasts, MSX-2 was found to suppress MyoD gene expression, as do activated ras oncogenes, under certain culture conditions, and truncated MSX-2 cDNA was found to inhibit the activities of both MSX-2 and ras in this system as well. Our findings not only suggest that the truncated version MSX-2 may act as a dominant suppressor of intact MSX-2 but also raise the possibility that MSX-2 gene may be an important downstream target for the Ras signaling pathways.

Integrin-mediated signal transduction linked to Ras pathway by GRB2 binding to focal adhesion kinase.

PubMed

Schlaepfer, D D; Hanks, S K; Hunter, T; van der Geer, P

The cytoplasmic focal adhesion protein-tyrosine kinase (FAK) localizes with surface integrin receptors at sites where cells attach to the extracellular matrix. Increased FAK tyrosine phosphorylation occurs upon integrin engagement with fibronectin. Here we show that adhesion of murine NIH3T3 fibroblasts to fibronectin promotes SH2-domain-mediated association of the GRB2 adaptor protein and the c-Src protein-tyrosine kinase (PTK) with FAK in vivo, and also results in activation of mitogen-activated protein kinase (MAPK). In v-Src-transformed NIH3T3, the association of v-Src, GRB2 and Sos with FAK is independent of cell adhesion to fibronectin. The GRB2 SH2 domain binds directly to tyrosine-phosphorylated FAK. Mutation of tyrosine residue 925 of FAK (YENV motif) to phenylalanine blocks GRB2 SH2-domain binding to FAK in vitro. Our results show that fibronectin binding to integrins on NIH3T3 fibroblasts promotes c-Src and FAK association and formation of an integrin-activated signalling complex. Phosphorylation of FAK at Tyr 925 upon fibronectin stimulation creates an SH2-binding site for GRB2 which may link integrin engagement to the activation of the Ras/MAPK signal transduction pathway.

Cell-free chromatin from dying cancer cells integrate into genomes of bystander healthy cells to induce DNA damage and inflammation

PubMed Central

Mittra, Indraneel; Samant, Urmila; Sharma, Suvarna; Raghuram, Gorantla V; Saha, Tannistha; Tidke, Pritishkumar; Pancholi, Namrata; Gupta, Deepika; Prasannan, Preeti; Gaikwad, Ashwini; Gardi, Nilesh; Chaubal, Rohan; Upadhyay, Pawan; Pal, Kavita; Rane, Bhagyeshri; Shaikh, Alfina; Salunkhe, Sameer; Dutt, Shilpee; Mishra, Pradyumna K; Khare, Naveen K; Nair, Naveen K; Dutt, Amit

2017-01-01

Bystander cells of the tumor microenvironment show evidence of DNA damage and inflammation that can lead to their oncogenic transformation. Mediator(s) of cell–cell communication that brings about these pro-oncogenic pathologies has not been identified. We show here that cell-free chromatin (cfCh) released from dying cancer cells are the key mediators that trigger both DNA damage and inflammation in the surrounding healthy cells. When dying human cancer cells were cultured along with NIH3T3 mouse fibroblast cells, numerous cfCh emerged from them and rapidly entered into nuclei of bystander NIH3T3 cells to integrate into their genomes. This led to activation of H2AX and inflammatory cytokines NFκB, IL-6, TNFα and IFNγ. Genomic integration of cfCh triggered global deregulation of transcription and upregulation of pathways related to phagocytosis, DNA damage and inflammation. None of these activities were observed when living cancer cells were co-cultivated with NIH3T3 cells. However, upon intravenous injection into mice, both dead and live cells were found to be active. Living cancer cells are known to undergo extensive cell death when injected intravenously, and we observed that cfCh emerging from both types of cells integrated into genomes of cells of distant organs and induced DNA damage and inflammation. γH2AX and NFκB were frequently co-expressed in the same cells suggesting that DNA damage and inflammation are closely linked pathologies. As concurrent DNA damage and inflammation is a potent stimulus for oncogenic transformation, our results suggest that cfCh from dying cancer cells can transform cells of the microenvironment both locally and in distant organs providing a novel mechanism of tumor invasion and metastasis. The afore-described pro-oncogenic pathologies could be abrogated by concurrent treatment with chromatin neutralizing/degrading agents suggesting therapeutic possibilities. PMID:28580170

Continuous release of interleukin 12 from microencapsulated engineered cells for colon cancer therapy

PubMed Central

Zheng, Shu; Xiao, Zuo-Xiang; Pan, Yue-Long; Han, Ming-Yong; Dong, Qi

2003-01-01

AIM: To explore the anti-tumor immunity against CT26 colon tumor of the microencapsulated cells modified with murine interleukine-12 (mIL-12) gene. METHODS: Mouse fibroblasts (NIH3T3) were stably transfected to express mIL-12 using expression plasmids carrying mIL-12 gene (p35 and p40), and NIH3T3-mIL-12 cells were encapsulated in alginate microcapsules for long-term delivery of mIL-12. mIL-12 released from the microencapsulated NIH3T3-mIL-12 cells was confirmed using ELISA assay. Transplantation of the microencapsulated NIH3T3-mIL-12 cells was performed in the tumor-bearing mice with CT26 cells. The anti-tumor responses and the anti-tumor activities of the microencapsulated NIH3T3-mIL-12 cells were evaluated. RESULTS: Microencapsulated NIH3T3-mIL-12 cells could release mIL-12 continuously and stably for a long time. After the microencapsulated NIH3T3-mIL-12 cells were transplanted subcutaneously into the tumor-bearing mice for 21 d, the serum concentrations of mIL-12, mIL-2 and mIFN-γ, the cytotoxicity of the CTL from the splenocytes and the NK activity in the treatment group were significantly higher than those in the controls. Moreover, mIL-12 released from the microencapsulated NIH3T3-mIL-12 cells resulted in a significant inhibition of tumor proliferation and a prolonged survival of tumor-bearing mice. CONCLUSION: The microencapsulated NIH3T3-mIL-12 cells have a significant therapeutic effect on the experimental colon tumor by activating anti-tumor immune responses in vivo. Microencapsulated and genetically engineered cells may be an extremely versatile tool for tumor gene therapy. PMID:12717836

Mutations in the Polybasic Juxtamembrane Sequence of Both Plasma Membrane- and Endoplasmic Reticulum-localized Epidermal Growth Factor Receptors Confer Ligand-independent Cell Transformation*

PubMed Central

Bryant, Kirsten L.; Antonyak, Marc A.; Cerione, Richard A.; Baird, Barbara; Holowka, David

2013-01-01

Deregulation of ErbB receptor-tyrosine kinases is a hallmark of many human cancers. Conserved in the ErbB family is a cluster of basic amino acid residues in the cytoplasmic juxtamembrane region. We found that charge-silencing mutagenesis within this juxtamembrane region of the epidermal growth factor receptor (EGFR) results in the generation of a mutant receptor (EGFR Mut R1-6) that spontaneously transforms NIH 3T3 cells in a ligand-independent manner. A similar mutant with one additional basic residue, EGFR Mut R1-5, fails to exhibit ligand-independent transformation. The capacity of EGFR Mut R1-6 to mediate this transformation is maintained when this mutant is retained in the endoplasmic reticulum via a single point mutation, L393H, which we describe. We show that EGFR Mut R1-6 with or without L393H exhibits enhanced basal tyrosine phosphorylation when ectopically expressed, and the ligand-independent transforming activity of EGFR Mut R1-6 is sensitive to inhibition of EGFR kinase activity and is particularly dependent on PI3K and mTOR activity. Similar to EGFR Mut R1-6/L393H in NIH 3T3 cells, EGFR variant type III, a highly oncogenic mutant form of EGFR linked to human brain cancers, confers transforming activity while it is wholly endoplasmic reticulum-retained in U87 cells. Our findings highlight the importance of the polybasic juxtamembrane sequence in regulating the oncogenic potential of EGFR signaling. PMID:24142702

LABORATORY MEASUREMENTS OF NiH BY FOURIER TRANSFORM DISPERSED FLUORESCENCE

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vallon, Raphael; Richard, Cyril; Crozet, Patrick

2009-05-01

Red and orange bands of laser-induced fluorescence in NiH have been recorded on a Fourier transform interferometer at Doppler resolution. The spectra show strong transitions to low-lying vibronic states which are not thermally populated in a laboratory source, and therefore do not appear in laser excitation spectra, but which would be expected to contribute significantly to any stellar spectrum. The strongest bands belong to the G[{omega}' 5/2]-X {sub 2} {sup 2}{delta}{sub 3/2}, I[{omega}' 3/2]-X {sub 2}, and {sup 2}{delta}{sub 3/2} I[{omega}' 3/2]-W {sub 1} {sup 2}{pi}{sub 3/2} systems. Measurements are reported for {sup 58}NiH, {sup 60}NiH, and {sup 62}NiH.

Treatment with LPS plus INF-γ induces the expression and function of muscarinic acetylcholine receptors, modulating NIH3T3 cell proliferation: participation of NOS and COX.

PubMed

Español, A J; Maddaleno, M O; Lombardi, M G; Cella, M; Martínez Pulido, P; Sales, M E

2014-11-01

LPS and IFN-γ are potent stimuli of inflammation, a process in which fibroblasts are frequently involved. We analysed the effect of treatment with LPS plus IFN-γ on the expression and function of muscarinic acetylcholine receptors in NIH3T3 fibroblasts with regards to proliferation of these cells. We also investigated the participation of NOS and COX, and the role of NF-κB in this process. NIH3T3 cells were treated with LPS (10 ng·mL(-1)) plus IFN-γ (0.5 ng·mL(-1)) for 72 h (iNIH3T3 cells). Cell proliferation was evaluated with MTT and protein expression by Western blot analysis. NOS and COX activities were measured by the Griess method and radioimmunoassay respectively. The cholinoceptor agonist carbachol was more effective at stimulating proliferation in iNIH3T3 than in NIH3T3 cells, probably due to the de novo induction of M3 and M5 muscarinic receptors independently of NF-κB activation. iNIH3T3 cells produced higher amounts of NO and PGE2 than NIH3T3 cells, concomitantly with an up-regulation of NOS1 and COX-2, and with the de novo induction of NOS2/3 in inflamed cells. We also found a positive feedback between NOS and COX that could potentiate inflammation. Inflammation induced the expression of muscarinic receptors and, therefore,stimulated carbachol-induced proliferation of fibroblasts. Inflammation also up-regulated the expression of NOS and COX-2, thus potentiating the effect of carbachol on NO and PGE2 production. A positive crosstalk between NOS and COX triggered by carbachol in inflamed cells points to muscarinic receptors as potential therapeutic targets in inflammation. © 2014 The British Pharmacological Society.

Treatment with LPS plus INF-γ induces the expression and function of muscarinic acetylcholine receptors, modulating NIH3T3 cell proliferation: participation of NOS and COX

PubMed Central

Español, A J; Maddaleno, M O; Lombardi, M G; Cella, M; Martínez Pulido, P; Sales, M E

2014-01-01

Background and Purpose LPS and IFN-γ are potent stimuli of inflammation, a process in which fibroblasts are frequently involved. We analysed the effect of treatment with LPS plus IFN-γ on the expression and function of muscarinic acetylcholine receptors in NIH3T3 fibroblasts with regards to proliferation of these cells. We also investigated the participation of NOS and COX, and the role of NF-κB in this process. Experimental Approach NIH3T3 cells were treated with LPS (10 ng·mL−1) plus IFN-γ (0.5 ng·mL−1) for 72 h (iNIH3T3 cells). Cell proliferation was evaluated with MTT and protein expression by Western blot analysis. NOS and COX activities were measured by the Griess method and radioimmunoassay respectively. Key Results The cholinoceptor agonist carbachol was more effective at stimulating proliferation in iNIH3T3 than in NIH3T3 cells, probably due to the de novo induction of M3 and M5 muscarinic receptors independently of NF-κB activation. iNIH3T3 cells produced higher amounts of NO and PGE2 than NIH3T3 cells, concomitantly with an up-regulation of NOS1 and COX-2, and with the de novo induction of NOS2/3 in inflamed cells. We also found a positive feedback between NOS and COX that could potentiate inflammation. Conclusions and Implications Inflammation induced the expression of muscarinic receptors and, therefore,stimulated carbachol-induced proliferation of fibroblasts. Inflammation also up-regulated the expression of NOS and COX-2, thus potentiating the effect of carbachol on NO and PGE2 production. A positive crosstalk between NOS and COX triggered by carbachol in inflamed cells points to muscarinic receptors as potential therapeutic targets in inflammation. PMID:24990429

[Effects of fasudil on bleomycin-induced pulmonary fibrosis in mice and on the biological behaviors in NIH3T3 mouse fibroblast cell line].

PubMed

Jiang, Chunguo; Huang, Hui; Liu, Jia; Wang, Yanxun; Zhao, Yuyue; Xu, Zuojun

2014-09-01

To determine the beneficial effects and mechanisms of fasudil, a selective ROCK inhibitor, on bleomycin-induced pulmonary fibrosis in mice and to determine the effects and mechanisms of fasudil on the biological behaviors in NIH3T3 mouse fibroblast cell line. The BPF model was induced by a single dosage of 2.5 mg/kg bleomycin intratracheal injection in mice and fasudil intraperitoneal injection was given to the mice. The fibrosis degree was determined pathologically by using the Ashcroft scoring method and biochemically by hydroxyproline assay in lung tissue. NIH3T3 mouse fibroblast cell line was cultured in vitro and fasudil was given to the cell. The proliferation activity in NIH3T3 cells were detected by MTT assay and flat colony forming experiment. The migration activity in NIH3T3 cells were detected by scratch test and transwell chamber experiment. The expression of CyclinD1, MMP2 and TIMP1 mRNA in NIH3T3 cells was detected by RT-PCR. The expression of CyclinD1, MMP2 and TIMP1 protein and the level of MYPT1 phosphorylation in NIH3T3 cells was detected by Western blot. Compare to the mice administrated by bleomycin, the Ashcroft score and hydroxyproline content were significantly decreased in the mice administered fasudil. Administration of fasudil can reduce the ability of proliferation and migration in a dose-dependent manner in NIH3T3 cells. The effect of fasudil was possibly related to increase the production of TIMP1 and decrease the production of CyclinD1 and MMP2. Administration of fasudil can attenuate pulmonary fibrosis both in vivo and in vitro. These findings suggest that fasudil may be a potential therapeutic candidate for the treatment of pulmonary fibrosis.

Lunasin-aspirin combination against NIH/3T3 cells transformation induced by chemical carcinogens.

PubMed

Hsieh, Chia-Chien; Hernández-Ledesma, Blanca; de Lumen, Ben O

2011-06-01

Carcinogenesis is a multistage process involving a number of molecular pathways sensitive to intervention. Chemoprevention is defined as the use of natural and/or synthetic substances to block, reverse, or retard the process of carcinogenesis. To achieve greater inhibitory effects on cancer cells, combination of two or more chemopreventive agents is commonly considered as a better preventive and/or therapeutic strategy. Lunasin is a promising cancer preventive peptide identified in soybean and other seeds. Its efficacy has been demonstrated by both in vitro and in vivo models. This peptide has been found to inhibit human breast cancer MDA-MB-231 cells proliferation, suppressing cell cycle progress and inducing cell apoptosis. Moreover, lunasin potentiates the effects on these cells of different synthetic and natural compounds, such as aspirin and anacardic acid. This study explored the role of lunasin, alone and in combination with aspirin and anacardic acid on cell proliferation and foci formation of transformed NIH/3T3 cells induced by chemical carcinogens 7,12-dimethylbenz[a]anthracene or 3-methylcholanthrene. The results revealed that lunasin, acting as a single agent, inhibits cell proliferation and foci formation. When combined with aspirin, these effects were significantly increased, indicating that this combination might be a promising strategy to prevent/treat cancer induced by chemical carcinogens.

Genotoxicity of a Low-Dose Nitrosamine Mixture as Drinking Water Disinfection Byproducts in NIH3T3 Cells.

PubMed

Wang, Hai-Yan; Qin, Ming; Dong, Lei; Lv, Jia-Ying; Wang, Xia

2017-01-01

N - nitrosamines (NAms), which can arise as byproducts of disinfection agents, are reportedly found in drinking water, and their potential carcinogenicity is a concern; however, little research exists regarding the genotoxicity or carcinogenicity of NAms exposure as a low-dose mixture. The three most common NAms components in China's drinking water are N -nitrosodimethylamine (NDMA), N -nitrosodiethylamine (NDEA) and N -nitrosomethylethylamine (NMEA). Thus, we measured the genotoxic and carcinogenic potential of these compounds and measured the cell cycle and gene expression. The data show that exposure to the NAms-mixture doubled the revertants in the TA98 and TA100 S. typhimurium strains and increased the DNA double-strand breaks and the micronuclear frequency in the NIH3T3 cells compared to a single exposure. After long-term NAms mixture exposure, a malignant transformation of NIH3T3 and a significantly increased G2/M distribution were observed. Furthermore, P53, CDK1, P38, CDC25A and CyclinB expressions were down-regulated in the NAms-mixture exposure group; however, P21 and GADD45A genes were up-regulated. Interestingly, the CHK1/CHK2 and CDC25A genes had two responses, depending on the NAms concentrations. Thus, we observed mutagenic, genotoxic and carcinogenic effects after a low-dose NAms-mixture exposure in drinking water, and DNA repair and apoptosis pathways may contribute to these adverse effects.

Genotoxicity of a Low-Dose Nitrosamine Mixture as Drinking Water Disinfection Byproducts in NIH3T3 Cells

PubMed Central

Wang, Hai-yan; Qin, Ming; Dong, Lei; Lv, Jia-ying; Wang, Xia

2017-01-01

N-nitrosamines (NAms), which can arise as byproducts of disinfection agents, are reportedly found in drinking water, and their potential carcinogenicity is a concern; however, little research exists regarding the genotoxicity or carcinogenicity of NAms exposure as a low-dose mixture. The three most common NAms components in China's drinking water are N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA) and N-nitrosomethylethylamine (NMEA). Thus, we measured the genotoxic and carcinogenic potential of these compounds and measured the cell cycle and gene expression. The data show that exposure to the NAms-mixture doubled the revertants in the TA98 and TA100 S. typhimurium strains and increased the DNA double-strand breaks and the micronuclear frequency in the NIH3T3 cells compared to a single exposure. After long-term NAms mixture exposure, a malignant transformation of NIH3T3 and a significantly increased G2/M distribution were observed. Furthermore, P53, CDK1, P38, CDC25A and CyclinB expressions were down-regulated in the NAms-mixture exposure group; however, P21 and GADD45A genes were up-regulated. Interestingly, the CHK1/CHK2 and CDC25A genes had two responses, depending on the NAms concentrations. Thus, we observed mutagenic, genotoxic and carcinogenic effects after a low-dose NAms-mixture exposure in drinking water, and DNA repair and apoptosis pathways may contribute to these adverse effects. PMID:28924367

SUMOylated IRF-1 shows oncogenic potential by mimicking IRF-2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Park, Sun-Mi; School of Biological Sciences and Biotechnology, Chonnam National University, Gwangju 500-757; Chae, Myounghee

2010-01-01

Interferon regulatory factor-1 (IRF-1) is an interferon-induced transcriptional activator that suppresses tumors by impeding cell proliferation. Recently, we demonstrated that the level of SUMOylated IRF-1 is elevated in tumor cells, and that SUMOylation of IRF-1 attenuates its tumor-suppressive function. Here we report that SUMOylated IRF-1 mimics IRF-2, an antagonistic repressor, and shows oncogenic potential. To demonstrate the role of SUMOylated IRF-1 in tumorigenesis, we used SUMO-IRF-1 recombinant protein. Stable expression of SUMO-IRF-1 in NIH3T3 cells resulted in focus formation and anchorage-independent growth in soft agar. Inoculation of SUMO-IRF-1-transfected cells into athymic nude mice resulted in tumor formation and infiltration ofmore » adipose tissues. Finally, we demonstrated that SUMO-IRF-1 transforms NIH3T3 cells in a dose-dependent manner suggesting that SUMOylated IRF-1 may act as an oncogenic protein in tumor cells.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jenei, Veronika; Andersson, Tommy; Jakus, Judit

E3B1, a human homologue of the mouse gene product Abi-1, has been implicated in growth-factor-mediated regulation of the small GTPases p21{sup Ras} and Rac. E3b1 is a regulator of Rac because it can form a complex with Sos-1 and eps8, and such a Sos-1-e3B1-eps8 complex serves as a guanine nucleotide exchange factor for Rac. In the present study, we found that overexpression of e3B1 in NIH3T3/EGFR cells sensitized EGF-induced activation of Rac1, whereas it had no impact on EGF-induced activation of p21{sup Ras}. Remarkably, we found that EGF-induced activation of the p21{sup Ras}-related GTPase Rap1 was also sensitized in NIH3T3/EGFR-e3B1more » cells. Thus, in NIH3T3/EGFR-e3B1 cells, maximal EGF-induced activation of Rap1 occurs with a dose of EGF much lower than in NIH3T3/EGFR cells. We also report that overexpression of e3B1 in NIH3T3/EGFR cells renders EGF-induced activation of Rap1 completely dependent on Src tyrosine kinases but not on c-Abl. However, EGF-induced tyrosine phosphorylation of the Rap GEF C3G occurred regardless of whether e3B1 was overexpressed or not, and this did not involve Src tyrosine kinases. Accordingly, we propose that overexpression of e3B1 in NIH3T3/EGFR cells leads to mobilization of Src tyrosine kinases that participate in EGF-induced activation of Rap1 and inhibition of cell proliferation.« less

Density-dependent induction of apoptosis by transforming growth factor-beta 1 in a human ovarian carcinoma cell line.

PubMed

Mathieu, C; Jozan, S; Mazars, P; Côme, M G; Moisand, A; Valette, A

1995-01-01

Transforming growth factor-beta 1 inhibited proliferation of a human ovarian carcinoma cell line (NIH-OVCAR-3). The inhibition of NIH-OVCAR-3 cell proliferation was accompanied by a decrease in clonogenic potential, evidenced by the reduced ability of TGF-beta 1-treated NIH-OVCAR-3 cells to form colonies on a plastic substratum. This rapid decrease of clonogenic potential, which was detected 6 h after addition of TGF-beta 1 was dose-dependent (IC50 = 4 pM). Fluorescence microscopy of DAPI-stained cells supported by electron-microscopic examination showed that TGF-beta 1 induced chromatin condensation and nuclear fragmentation. In addition, oligonucleosomal-sized fragments were detected in the TGF-beta 1-treated cells. These features indicated that TGF-beta 1 induced NIH-OVCAR-3 cell death by an apoptosis-like mechanism. This TGF-beta 1 apoptotic effect was subject to modulation by cell density. It was observed that an increase in cell density (up to 20 x 10(3) cells/cm2) protected NIH-OVCAR-3 cells against apoptosis induced by TGF-beta 1. Conditioned medium from high-density cultures of NIH-OVCAR-3 cells did not inhibit apoptosis induced by TGF-beta 1 on NIH-OVCAR-3 cells cultured at low density, suggesting that the protective effect of cell density was not related to the cell secretion of a soluble survival factor.

Identification and characterization of an alternative splice variant of Mpl with a high affinity for TPO and its activation of ERK1/2 signaling.

PubMed

Wang, Qiong; Sun, Rui; Wu, Leyan; Huang, Junfeng; Wang, Ping; Yuan, Hailong; Qiu, Feifei; Xu, Xiaohong; Wu, Di; Yu, Ying; Liu, Xin; Zhang, Qing

2013-12-01

The thrombopoietin receptor is a crucial element in thrombopoietin-initiated signaling pathways, which stimulates the differentiation of normal hematopoietic progenitor cells, the maturation of megakaryocytes, and the generation of platelets. In this study, we identified a novel activating variant of thrombopoietin receptor, termed Mpl-D, in human megakaryoblastic leukemia Dami cells and demonstrated that the binding affinity of the Mpl-D receptor for thrombopoietin is enhanced. Cell cycle analysis revealed that in the presence of thrombopoietin, most Mpl-D expressing NIH3T3 (NIH3T3/Mpl-D) cells were prevalent in G1 phase while the S and G2/M populations were less frequently observed. Unexpectedly, thrombopoietin induced strong and prolonged ERK1/2 signaling in NIH3T3/Mpl-D cells compared with its receptor wild-type expressing NIH3T3 (NIH3T3/Mpl-F) cells. Further analysis of the mRNA levels of cyclin D1/D2 in NIH3T3/Mpl-D cells demonstrated markedly down-regulated expression compared to NIH3T3/Mpl-F cells in the presence of thrombopoietin. Thus, the prolonged activation of ERK1/2 by Mpl-D might lead to G1 cell cycle arrest through a profound reduction of cyclin D1/D2 in order to support cell survival without proliferation. We also provided tertiary structural basis for the Mpl-D and thrombopoietin interaction, which might provide insights into how Mpl-D effectively increases binding to thrombopoietin and significantly contributes to its specific signaling pathway. These results suggest a new paradigm for the regulation of cytokine receptor expression and function through the alternative splicing variant of Mpl in Dami cells, which may play a role in the pathogenesis of megakaryoblastic leukemia. Copyright © 2013 Elsevier Ltd. All rights reserved.

Reactive oxygen species-dependent HSP90 protein cleavage participates in arsenical As(+3)- and MMA(+3)-induced apoptosis through inhibition of telomerase activity via JNK activation.

PubMed

Shen, Shing-Chuan; Yang, Liang-Yo; Lin, Hui-Yi; Wu, Chin-Yen; Su, Tsung-Hsien; Chen, Yen-Chou

2008-06-01

The effects of six arsenic compounds including As(+3), MMA(+3), DMA(+3), As(+5), MMA(+5), and DMA(+5) on the viability of NIH3T3 cells were examined. As(+3) and MMA(+3), but not the others, exhibited significant cytotoxic effects in NIH3T3 cells through apoptosis induction. The apoptotic events such as DNA fragmentation and chromosome condensation induced by As(+3) and MMA(+3) were prevented by the addition of NAC and CAT, and induction of HO-1 gene expression in accordance with cleavage of the HSP90 protein, and suppression of telomerase activity were observed in NIH3T3 cells under As(+3) and MMA(+3) treatments. An increase in the intracellular peroxide level was examined in As(+3)- and MMA(+3)-treated NIH3T3 cells, and As(+3)- and MMA(+3)-induced apoptotic events were blocked by NAC, CAT, and DPI addition. HSP90 inhibitors, GA and RD, significantly attenuated the telomerase activity in NIH3T3 cells with an enhancement of As(+3)- and MMA(+3)-induced cytotoxicity. Suppression of JNKs significantly inhibited As(+3)- and MMA(+3)-induced apoptosis by blocking HSP90 protein cleavage and telomerase reduction in NIH3T3 cells. Furthermore, Hb, SnPP, and dexferosamine showed no effect against As(+3)- and MMA(+3)-induced apoptosis, and overexpression of HO-1 protein or inhibition of HO-1 protein expression did not affect the apoptosis induced by As(+3) or MMA(+3). These data provide the first evidence to indicate that apoptosis induced by As(+3) and MMA(+3) is mediated by an ROS-dependent degradation of HSP90 protein and reduction of telomerase via JNK activation, and HO-1 induction might not be involved.

New anti-cancer chemicals Ertredin and its derivatives, regulate oxidative phosphorylation and glycolysis and suppress sphere formation in vitro and tumor growth in EGFRvIII-transformed cells.

PubMed

Atsumi, Sonoko; Nosaka, Chisato; Adachi, Hayamitsu; Kimura, Tomoyuki; Kobayashi, Yoshihiko; Takada, Hisashi; Watanabe, Takumi; Ohba, Shun-Ichi; Inoue, Hiroyuki; Kawada, Manabu; Shibasaki, Masakatsu; Shibuya, Masabumi

2016-07-19

EGFRvIII is a mutant form of the epidermal growth factor receptor gene (EGFR) that lacks exons 2-7. The resulting protein does not bind to ligands and is constitutively activated. The expression of EGFRvIII is likely confined to various types of cancer, particularly glioblastomas. Although an anti-EGFRvIII vaccine is of great interest, low-molecular-weight substances are needed to obtain better therapeutic efficacy. Thus, the purpose of this study is to identify low molecular weight substances that can suppress EGFRvIII-dependent transformation. We constructed a new throughput screening system and searched for substances that decreased cell survival of NIH3T3/EGFRvIII spheres under 3-dimensional (3D)-culture conditions, but retained normal NIH3T3 cell growth under 2D-culture conditions. In vivo activity was examined using a mouse transplantation model, and derivatives were chemically synthesized. Functional characterization of the candidate molecules was investigated using an EGFR kinase assay, immunoprecipitation, western blotting, microarray analysis, quantitative polymerase chain reaction analysis, and measurement of lactate and ATP synthesis. In the course of screening 30,000 substances, a reagent, "Ertredin" was found to inhibit anchorage-independent 3D growth of sphere-forming cells transfected with EGFRvIII cDNA. Ertredin also inhibited sphere formation in cells expressing wild-type EGFR in the presence of EGF. However, it did not affect anchorage-dependent 2D growth of parental NIH3T3 cells. The 3D-growth-inhibitory activity of some derivatives, including those with new structures, was similar to Ertredin. Furthermore, we demonstrated that Ertredin suppressed tumor growth in an allograft transplantation mouse model injected with EGFRvIII- or wild-type EGFR-expressing cells; a clear toxicity to host animals was not observed. Functional characterization of Ertredin in cells expressing EGFRvIII indicated that it stimulated EGFRvIII ubiquitination, suppressed both oxidative phosphorylation and glycolysis under 3D conditions, and promoted cell apoptosis. We developed a high throughput screening method based on anchorage-independent sphere formation induced by EGFRvIII-dependent transformation. In the course of screening, we identified Ertredin, which inhibited anchorage-independent 3D growth and tumor formation in nude mice. Functional analysis suggests that Ertredin suppresses both mitochondrial oxidative phosphorylation and cytosolic glycolysis in addition to promoting EGFRvIII degradation, and stimulates apoptosis in sphere-forming, EGFRvIII-overexpressing cells.

A comparison of the in vitro cytotoxicity of conventional and resin modified glass ionomer cements

PubMed Central

Selimović-Dragaš, Mediha; Huseinbegović, Amina; Kobašlija, Sedin; Hatibović-Kofman, Šahza

2012-01-01

To evaluate cytotoxicity of experimental conventional and resin modified glass-ionomer cements on UMR-106 osteoblast cell cultures and cell cultures of NIH3T3 mouse fibroblasts specimens were prepared for every experimental material and divided into: group 1. Conventional glass-ionomer cements: GC Fuji IX GP Fast, GC Fuji Triage and Ketac Silver; group 2. Resin modified glass-ionomer cements: GC Fuji II LC, GC Fuji Plus and Vitrebond; group 3. Positive control was presented by specimens of composite Vit-l-ecence® and negative control-group 4. was presented by α-minimum essential medium for UMR-106 – osteoblast-like cells and Dulbecco’s Modified Eagle’s Medium for NIH3T3 mouse fibroblast cells. Both cell cultures were exposed to 10% of eluate of each single specimen of each experimental material. Experimental dishes were incubated for 24 h. Cell metabolism was evaluated using methyltetrazolium assay. Kruskal-Wallis test and Tukey-Kramer post hoc test for the materials evaluated on NIH3T3 mouse fibroblast cells, as well as UMR-106 osteoblast-like cells showed significantly more cytotoxicity of RMGICs, predominantly Vitrebond to both GICs and composite-Vit-l-ecence®. The lowest influence on cell’s metabolism on UMR-106 osteoblas-like cells was shown by Ketac Silver and the lowest influence on cell’s metabolism on NIH3T3 mouse fibroblast cells was shown by Fuji IX GP Fast. Statistical evaluation of sensitivity of cell lines UMR-106 osteoblast-like cells and NIH3T3 mouse fibroblast cells, using Mann-Whitney test, showed that NIH3T3 mouse fibroblast cells were more sensitive for the evaluation of cytotoxicity of dental materials. PMID:23198945

Preparation of Proper Immunogen by Cloning and Stable Expression of cDNA coding for Human Hematopoietic Stem Cell Marker CD34 in NIH-3T3 Mouse Fibroblast Cell Line

PubMed Central

Shafaghat, Farzaneh; Abbasi-Kenarsari, Hajar; Majidi, Jafar; Movassaghpour, Ali Akbar; Shanehbandi, Dariush; Kazemi, Tohid

2015-01-01

Purpose: Transmembrane CD34 glycoprotein is the most important marker for identification, isolation and enumeration of hematopoietic stem cells (HSCs). We aimed in this study to clone the cDNA coding for human CD34 from KG1a cell line and stably express in mouse fibroblast cell line NIH-3T3. Such artificial cell line could be useful as proper immunogen for production of mouse monoclonal antibodies. Methods: CD34 cDNA was cloned from KG1a cell line after total RNA extraction and cDNA synthesis. Pfu DNA polymerase-amplified specific band was ligated to pGEMT-easy TA-cloning vector and sub-cloned in pCMV6-Neo expression vector. After transfection of NIH-3T3 cells using 3 μg of recombinant construct and 6 μl of JetPEI transfection reagent, stable expression was obtained by selection of cells by G418 antibiotic and confirmed by surface flow cytometry. Results: 1158 bp specific band was aligned completely to reference sequence in NCBI database corresponding to long isoform of human CD34. Transient and stable expression of human CD34 on transfected NIH-3T3 mouse fibroblast cells was achieved (25% and 95%, respectively) as shown by flow cytometry. Conclusion: Cloning and stable expression of human CD34 cDNA was successfully performed and validated by standard flow cytometric analysis. Due to murine origin of NIH-3T3 cell line, CD34-expressing NIH-3T3 cells could be useful as immunogen in production of diagnostic monoclonal antibodies against human CD34. This approach could bypass the need for purification of recombinant proteins produced in eukaryotic expression systems. PMID:25789221

Treatment of Ras-induced cancers by the F-actin cappers tensin and chaetoglobosin K, in combination with the caspase-1 inhibitor N1445.

PubMed

Tikoo, A; Cutler, H; Lo, S H; Chen, L B; Maruta, H

1999-01-01

For transforming normal fibroblasts to malignant cells, oncogenic Ras mutants such as v-Ha-ras require Rho family GTPases (Rho, Rac, and CDC42) that are responsible for controlling actin-cytoskeleton organization. Ras activates Rac through a PI-3 kinase-mediated pathway. Rac causes uncapping of actin filaments (F-actin) at the plus-ends, through phosphatidylinositol 4,5 bisphosphate (PIP2), and eventually induces membrane ruffling. Several distinct F-actin/PIP2-binding proteins, such as gelsolin, which severs and caps the plus-ends of actin filaments, or HS1, which cross-links actin filaments, have been shown to suppress v-Ha-Ras-induced malignant transformation when they are overexpressed. Interestingly, an F-actin cross-linking drug (photosensitizer) called MKT-077 suppresses Ras transformation. Thus, an F-actin capping/severing drug might also have an anticancer potential. This study was conducted to determine first whether Ras-induced malignant phenotype (anchorage-independent growth) is suppressed by overexpression of the gene encoding a large plus-end F-actin capping protein called tensin and second to test the anti-Ras potential of a unique fungal antibiotic (small compound) called chaetoglobosin K (CK) that also caps the plus-ends of actin filaments. DNA transfection with a retroviral vector carrying the tensin cDNA was used to overexpress tensin in v-Ha-Ras-transformed NIH 3T3 cells. All stable tensin transfectants rarely formed colonies in soft agar, indicating that tensin suppresses the anchorage-independent growth. The anti-Ras action of CK was determined by incubating the Ras-transformants in the presence of CK in soft agar. Two microM CK almost completely inhibited their colony formation, indicating that CK also suppresses the malignant phenotype. However, unlike tensin, CK causes an apoptosis of Ras-transformed NIH 3T3 cells and, less effectively, of normal NIH 3T3 cells, indicating that CK has an F-actin capping-independent side effect(s). CK-induced apoptosis is at least in part caused by CK-induced inhibition of the kinase PKB/AKT. However, a specific ICE/caspase-1 inhibitor called N1445 completely abolished the CK-induced apoptosis by reactivating PKB, but without affecting the CK-induced suppression of Ras transformation. Like the F-actin cross-linking drug MKT-077, the F-actin capping drug CK may be useful for the treatment of Ras-associated cancers if it is combined with the ICE inhibitor N1445, which abolishes the side effect of CK. Our observations that two distinct F-actin capping molecules (i.e., tensin and CK) suppress Ras-induced malignant phenotype strongly suggest, if not prove, that capping of actin filaments at the plus-ends alone is sufficient to block one of the Ras signaling pathways essential for its oncogenicity. This notion is compatible with the fact that Ras induces the uncapping of actin filaments at the plus-ends through the Rac/PIP2 pathway.

Expression of the invertebrate sea urchin P16 protein into mammalian MC3T3 osteoblasts transforms and reprograms them into "osteocyte-like" cells.

PubMed

Alvares, Keith; Ren, Yinshi; Feng, Jian Q; Veis, Arthur

2016-01-01

P16 is an acidic phosphoprotein important in both sea urchin embryonic spicule development and transient mineralization during embryogenesis, syncytium formation, and mineralization in mature urchin tooth. Anti-P16 has been used to localize P16 to the syncytial membranes and the calcite mineral. Specific amino acid sequence motifs in P16 are similar to sequences in DSPP, a protein common to all vertebrate teeth, and crucial for their mineralization. Here, we examine the effect of P16 on vertebrate fibroblastic NIH3T3 cells and osteoblastic MC3T3 cells. Transfection of NIH3T3 cells with P16 cDNA resulted in profound changes in the morphology of the cells. In culture, the transfected cells sent out long processes that contacted processes from neighboring cells forming networks or syncytia. There was a similar change in morphology in cultured osteoblastic MC3T3 cells. In addition, the MC3T3 developed numerous dendrites as found in osteocytes. Importantly, there was also a change in the expression of the osteoblast and osteocyte specific genes. MC3T3 cells transfected with P16 showed an 18-fold increase in expression of the osteocyte specific Dentin matrix protein (DMP1) gene, accompanied by decreased expression of osteoblast specific genes: Bone sialoprotein (BSP), osteocalcin (OCN), and β-catenin decreased by 70%, 64%, and 68 %, respectively. Thus, invertebrate urchin P16 with no previously known analog in vertebrates was able to induce changes in both cell morphology and gene expression, converting vertebrate-derived osteoblast-like precursor cells to an "osteocyte-like" phenotype, an important process in bone biology. The mechanisms involved are presently under study. © 2015 Wiley Periodicals, Inc.

Expression of the invertebrate sea urchin P16 protein into mammalian MC3T3 osteoblasts transforms and reprograms them into “osteocyte-like” cells

PubMed Central

Alvares, Keith; Ren, Yinshi; Feng, Jian Q.; Veis, Arthur

2015-01-01

P16 is an acidic phosphoprotein important in both sea urchin embryonic spicule development and transient mineralization during embryogenesis, and syncytium formation and mineralization in mature urchin tooth. Anti-P16 has been used to localize P16 to the syncytial membranes and the calcite mineral. Specific amino acid sequence motifs in P16 are similar to sequences in DSPP a protein common to all vertebrate teeth, and crucial for their mineralization. Here we examine the effect of P16 on vertebrate fibroblastic NIH3T3 cells and osteoblastic MC3T3 cells. Transfection of NIH3T3 cells with P16 cDNA resulted in profound changes in the morphology of the cells. In culture the transfected cells sent out long processes that contacted processes from neighboring cells forming networks or syncytia. There was a similar change in morphology in cultured osteoblastic MC3T3 cells. In addition, the MC3T3 developed numerous dendrites as found in osteocytes. Importantly, there was also a change in the expression of the osteoblast and osteocyte specific genes. MC3T3 cells transfected with P16 showed an 18 fold increase in expression of the osteocyte specific Dentin matrix protein (DMP1) gene, accompanied by decreased expression of osteoblast specific genes: Bone sialoprotein (BSP), osteocalcin (OCN) and β-catenin decreased by 70%, 64% and 68 %, respectively. Thus, invertebrate urchin P16 with no previously known analog in vertebrates was able to induce changes in both cell morphology and gene expression, converting vertebrate-derived osteoblast-like precursor cells to an “osteocyte-like” phenotype, an important process in bone biology. The mechanisms involved are presently under study. PMID:26581835

Estimation of Viscoelastic Properties of Cells Using Acoustic Tweezing Cytometry.

PubMed

Yang, Chunmei; Chen, Di; Hong, Xiaowei

2016-12-01

Recently developed acoustic tweezing cytometry uses ultrasound-responsive targeted microbubbles for biomechanical stimulation of live cells at the subcellular level. The purpose of this research was to estimate the viscoelastic characteristics of cells from the displacements of cell-bound microbubbles in response to ultrasound pulses on acoustic tweezing cytometry. Microbubbles were bound to NIH/3T3 fibroblasts and ATDC5 cells through an integrin-cytoskeleton linkage. The evolution of microbubble behaviors under irradiation by ultrasound pulses was captured by a high-speed camera and tracked by a customized algorithm. The total damping constant, stiffness, and rigidity of the cells were estimated by fitting the measured temporal displacement profiles to a Kelvin-Voigt-based model. The mean maximum displacement of the microbubbles attached to NIH/3T3 fibroblasts was much greater than that for ATDC5 cells. The mean fitted damping constant and stiffness ± SD for ATDC5 cells were 28.16 ± 7.08 mg/s and 0.5041 ± 0.1381 mN/m, respectively, and the values for NIH/3T3 fibroblasts were 13.12 ± 4.23 mg/s and 0.2591 ± 0.0715 mN/m. The rigidity for ATDC5 cells was 331.46 ± 106.50 MPa, whereas that for NIH/3T3 fibroblasts was 117.92 ± 34.83 MPa. The Arg-Gly-Asp-integrin-cytoskeleton system of NIH/3T3 fibroblasts appears to be softer than that of ATDC5 cells. The rigidity of ATDC5 cells was significantly greater than that of NIH/3T3 fibroblasts at the 95% confidence level. This strategy provides a novel way to determine the viscoelastic properties of the live cells. © 2016 by the American Institute of Ultrasound in Medicine.

Nickel-Refining Fumes Induced DNA Damage and Apoptosis of NIH/3T3 Cells via Oxidative Stress

PubMed Central

Wang, Yue; Wang, Sheng-Yuan; Jia, Li; Zhang, Lin; Ba, Jing-Chong; Han, Dan; Yu, Cui-Ping; Wu, Yong-Hui

2016-01-01

Although there have been numerous studies examining the toxicity and carcinogenicity of nickel compounds in humans and animals, its molecular mechanisms of action are not fully elucidated. In our research, NIH/3T3 cells were exposed to nickel-refining fumes at the concentrations of 0, 6.25, 12.50, 25, 50 and 100 μg/mL for 24 h. Cell viability, cell apoptosis, reactive oxygen species (ROS) level, lactate dehydrogenase (LDH) assay, the level of glutathione (GSH), activities of superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA) level were detected. The exposure of NIH/3T3 cells to nickel-refining fumes significantly reduced cell viability and induced cell apoptotic death in a dose-dependent manner. Nickel-refining fumes significantly increased ROS levels and induced DNA damage. Nickel-refining fumes may induce the changes in the state of ROS, which may eventually initiate oxidative stress, DNA damage and apoptosis of NIH/3T3 cells. PMID:27347984

ETS target genes: Identification of Egr1 as a target by RNA differential display and whole genome PCR techniques

PubMed Central

Robinson, Lois; Panayiotakis, Alexandra; Papas, Takis S.; Kola, Ismail; Seth, Arun

1997-01-01

ETS transcription factors play important roles in hematopoiesis, angiogenesis, and organogenesis during murine development. The ETS genes also have a role in neoplasia, for example in Ewing’s sarcomas and retrovirally induced cancers. The ETS genes encode transcription factors that bind to specific DNA sequences and activate transcription of various cellular and viral genes. To isolate novel ETS target genes, we used two approaches. In the first approach, we isolated genes by the RNA differential display technique. Previously, we have shown that the overexpression of ETS1 and ETS2 genes effects transformation of NIH 3T3 cells and specific transformants produce high levels of the ETS proteins. To isolate ETS1 and ETS2 responsive genes in these transformed cells, we prepared RNA from ETS1, ETS2 transformants, and normal NIH 3T3 cell lines and converted it into cDNA. This cDNA was amplified by PCR and displayed on sequencing gels. The differentially displayed bands were subcloned into plasmid vectors. By Northern blot analysis, several clones showed differential patterns of mRNA expression in the NIH 3T3-, ETS1-, and ETS2-expressing cell lines. Sixteen clones were analyzed by DNA sequence analysis, and 13 of them appeared to be unique because their DNA sequences did not match with any of the known genes present in the gene bank. Three known genes were found to be identical to the CArG box binding factor, phospholipase A2-activating protein, and early growth response 1 (Egr1) genes. In the second approach, to isolate ETS target promoters directly, we performed ETS1 binding with MboI-cleaved genomic DNA in the presence of a specific mAb followed by whole genome PCR. The immune complex-bound ETS binding sites containing DNA fragments were amplified and subcloned into pBluescript and subjected to DNA sequence and computer analysis. We found that, of a large number of clones isolated, 43 represented unique sequences not previously identified. Three clones turned out to contain regulatory sequences derived from human serglycin, preproapolipoprotein C II, and Egr1 genes. The ETS binding sites derived from these three regulatory sequences showed specific binding with recombinant ETS proteins. Of interest, Egr1 was identified by both of these techniques, suggesting strongly that it is indeed an ETS target gene. PMID:9207063

A Protocol for Using Förster Resonance Energy Transfer (FRET)-force Biosensors to Measure Mechanical Forces across the Nuclear LINC Complex.

PubMed

Arsenovic, Paul T; Bathula, Kranthidhar; Conway, Daniel E

2017-04-11

The LINC complex has been hypothesized to be the critical structure that mediates the transfer of mechanical forces from the cytoskeleton to the nucleus. Nesprin-2G is a key component of the LINC complex that connects the actin cytoskeleton to membrane proteins (SUN domain proteins) in the perinuclear space. These membrane proteins connect to lamins inside the nucleus. Recently, a Förster Resonance Energy Transfer (FRET)-force probe was cloned into mini-Nesprin-2G (Nesprin-TS (tension sensor)) and used to measure tension across Nesprin-2G in live NIH3T3 fibroblasts. This paper describes the process of using Nesprin-TS to measure LINC complex forces in NIH3T3 fibroblasts. To extract FRET information from Nesprin-TS, an outline of how to spectrally unmix raw spectral images into acceptor and donor fluorescent channels is also presented. Using open-source software (ImageJ), images are pre-processed and transformed into ratiometric images. Finally, FRET data of Nesprin-TS is presented, along with strategies for how to compare data across different experimental groups.

Compendium of the FY1988 and FY1989 Research Reviews for the Research Methods Branch

DTIC Science & Technology

1993-02-01

14, contained a very aggressive cholangiocarcinoma . DNA from these tissues was examined by primary transfection assay. DNA isolated from the MAM-Ac...exposed animals appeared to correlate with the degree of malignancy observed in the liver. DNA isolated from the cholangiocarcinoma lesion in MD6-14 was...transfection. DNA isolated from the cholangiocarcinoma lesion of MD6-14 very rapidly transformed NIH 3T3 cells as determined by standard focus assay

Transforming capacity of two novel genes JS-1 and JS-2 located in chromosome 5p and their overexpression in human esophageal squamous cell carcinoma.

PubMed

Fatima, Sarwat; Chui, Chung H; Tang, Wing K; Hui, Kin S; Au, Ho W; Li, Wing Y; Wong, Mei M; Cheung, Filly; Tsao, S W; Lam, King Y; Beh, Philip S L; Wong, John; Law, Simon; Srivastava, Gopesh; Ho, Kwok P; Chan, Albert S C; Tang, Johnny C O

2006-01-01

Esophageal squamous cell carcinoma (ESCC) has a high mortality rate and geographic differences in incidence. Previous studies of comparative genomic hybridization (CGH) showed that chromosomal 5p is frequently amplified in cell lines and primary ESCC of Hong Kong Chinese origin. In this report, attempt was made to study two novel genes, named as JS-1 and JS-2, which are located in chromosome 5p15.2 and are 5' upstream to delta catenin for their roles in molecular pathogenesis of ESCC. Eleven cell lines, 27 primary ESCC cases and multiple human tissue cDNA panels (MTC) of digestive system were studied for the expression level of JS-1 and JS-2 by RT-PCR. The full-length cDNA sequences of JS-1 and JS-2 were determined from a non-tumor esophageal epithelial cell line by 3' and 5' rapid amplification of cDNA ends (RACE). The transforming capacity of JS-1 and JS-2 was also investigated by transfecting NIH 3T3 cells with the expression vector pcDNA3.1(-) cloned with the full coding sequences and it was followed by the study of foci formation of the transfected cells under confluence growth and the anchorage-independent growth in soft agar. Forty-five percent (5/11) and 18% (2/11) of the ESCC cell lines showed overexpression of JS-1 and JS-2 respectively, while 55% (15/27) and 14% (3/22) primary ESCC cases showed overexpression of JS-1 and JS-2 respectively. JS-1 overexpression was most common in patients with stage II ESCC (6/27; 22%) whereas JS-2 was only overexpressed in a dysplastic lesion (1/22; 4%) and stage III tumors (2/22; 9%). The expression levels of JS-1 and JS-2 are both low in normal esophageal tissues. Overexpression of JS-1 in NIH 3T3 cells caused foci formation in confluence growth and colony formation in soft agar but not for JS-2. A high grade sarcoma was formed in the athymic nude mice when NIH 3T3 cells overexpressing JS-1 were injected subcutaneously. Our results thus indicate that the frequent overexpression of JS-1 in ESCC and its transforming capacity in normal cells may play a critical role in the molecular pathogenesis of ESCC. The present study also forms the ground work for further identification of novel mechanisms of molecular carcinogenesis in ESCC and other cancers.

[Influence of extremely low-frequency magnetic field on circadian rhythm of cryptochrome in mouse embryonic fibroblasts].

PubMed

Sun, Z Y; Geng, D Y; Chen, C F; Wang, P P; Song, T

2017-06-20

Objective: To investigate the influence of extremely low-frequency magnetic field on periodical expression of cryptochrome ( Cry ) gene in mouse embryonic fibroblast NIH3T3 cells. Methods: The NIH3T3 cells were divided into magnetic field group and sham-exposure group. The NIH3T3 cells in the magnetic field group were stimulated by horse serum and then exposed to an extremely low-frequency magnetic field (50 Hz and 0.3 mT) for 48 hours, and those in the sham-exposure group were also stimulated by horse serum and then exposed to a coil for 48 hours. The NIH3T3 cells were collected, total RNA was extracted, and cDNA was obtained via reverse transcription. Real-time fluorescent quantitative RT-PCR was used to measure the changes in transcription cycles of Cry and Period genes in both groups. Results: There was no significant difference in the proliferation rate at 0, 12, 24, and 48 hours of exposure between the two groups ( P >0.05) . Both sham-exposure group and magnetic field group showed a rhythmic change in the expression of Cry gene, and compared with the sham-exposure group, the magnetic field group had a significantly shortened circadian rhythm of Cry gene in NIH3T3 cells ( t =2.57, P <0.05) . Both groups had rhythmic and periodical expression of Period gene and there was no significant difference between the two groups ( t =0.70, P >0.05) . Conclusion: Extremely low-frequency magnetic field can significantly shorten the circadian rhythm of Cry gene in mouse embryonic fibroblasts, while there is no significant change in the circadian rhythm of Period gene.

Limits of transforming competence of SV40 nuclear and cytoplasmic large T mutants with altered Rb binding sequences.

PubMed

Tedesco, D; Fischer-Fantuzzi, L; Vesco, C

1993-03-01

Multiple amino acid substitutions were introduced into the SV40 large T region that harbors the retinoblastoma protein (Rb) binding site and the nuclear transport signal, changing either one or both of these determinants. Mutant activities were examined in a set of assays allowing different levels of transforming potential to be distinguished; phenotypic changes in established and pre-crisis rat embryo fibroblasts (REFs) were detected under isogenic cell conditions, and comparisons made with other established rodent cells. The limit of the transforming ability of mutants with important substitutions in the Rb binding site fell between two transformation levels of the same established rat cells. Such cells could be induced to form dense foci but not agar colonies (their parental pre-crises REFs, as expected, were untransformed either way). Nonetheless, agar colony induction was possible in other cell lines, such as mouse NIH3T3 and (for one of the mutants) rat F2408. All these mutants efficiently immortalized pre-crisis REFs. The transforming ability of cytoplasmic mutants appeared to depend on the integrity of the Rb-binding sequence to approximately the same extent as that of the wild-type large T, although evidence of in vivo Rb-cytoplasmic large T complexes was not found. The presence or absence of small t was critical when the transforming task of mutants was near the limit of their abilities.

Use of electroporation for high-molecular-weight DNA-mediated gene transfer.

PubMed

Jastreboff, M M; Ito, E; Bertino, J R; Narayanan, R

1987-08-01

Electroporation was used to introduce high-molecular-weight DNA into murine hematopoietic cells and NIH3T3 cells. CCRF-CEM cells were stably transfected with SV2NEO plasmid and the genomic DNA from G-418-resistant clones (greater than 65 kb) was introduced into mouse bone marrow and NIH3T3 cells by electroporation. NEO sequences and expression were detected in the hematopoietic tissues of lethally irradiated mice, with 24% of individual spleen colonies expressing NEO. The frequency of genomic DNA transfer into NIH3T3 cells was 0.25 X 10(-3). Electroporation thus offers a powerful mode of gene transfer not only of cloned genes but also of high-molecular-weight DNA into cells.

Identification of the zinc finger 216 (ZNF216) in human carcinoma cells: a potential regulator of EGFR activity

PubMed Central

Mincione, Gabriella; Di Marcantonio, Maria Carmela; Tarantelli, Chiara; Savino, Luca; Ponti, Donatella; Marchisio, Marco; Lanuti, Paola; Sancilio, Silvia; Calogero, Antonella; Di Pietro, Roberta; Muraro, Raffaella

2016-01-01

Epidermal Growth Factor Receptor (EGFR), a member of the ErbB family of receptor tyrosine kinase (RTK) proteins, is aberrantly expressed or deregulated in tumors and plays pivotal roles in cancer onset and metastatic progression. ZNF216 gene has been identified as one of Immediate Early Genes (IEGs) induced by RTKs. Overexpression of ZNF216 protein sensitizes 293 cell line to TNF-α induced apoptosis. However, ZNF216 overexpression has been reported in medulloblastomas and metastatic nasopharyngeal carcinomas. Thus, the role of this protein is still not clearly understood. In this study, the inverse correlation between EGFR and ZNF216 expression was confirmed in various human cancer cell lines differently expressing EGFR. EGF treatment of NIH3T3 cells overexpressing both EGFR and ZNF216 (NIH3T3-EGFR/ZNF216), induced a long lasting activation of EGFR in the cytosolic fraction and an accumulation of phosphorylated EGFR (pEGFR) more in the nuclear than in the cytosolic fraction compared to NIH3T3-EGFR cells. Moreover, EGF was able to stimulate an increased expression of ZNF216 in the cytosolic compartment and its nuclear translocation in a time-dependent manner in NIH3T3-EGFR/ZNF216. A similar trend was observed in A431 cells endogenously expressing the EGFR and transfected with Znf216. The increased levels of pEGFR and ZNF216 in the nuclear fraction of NIH3T3-EGFR/ZNF216 cells were paralleled by increased levels of phospho-MAPK and phospho-Akt. Surprisingly, EGF treatment of NIH3T3-EGFR/ZNF216 cells induced a significant increase of apoptosis thus indicating that ZNF216 could sensitize cells to EGF-induced apoptosis and suggesting that it may be involved in the regulation and effects of EGFR signaling. PMID:27732953

Biocompatibility of bio based calcium carbonate nanocrystals aragonite polymorph on NIH 3T3 fibroblast cell line.

PubMed

Kamba, Abdullahi Shafiu; Ismail, Maznah; Ibrahim, Tengku Azmi Tengku; Zakaria, Zuki Abu Bakar

2014-01-01

Currently, there has been extensive research interest for inorganic nanocrystals such as calcium phosphate, iron oxide, silicone, carbon nanotube and layered double hydroxide as a drug delivery system especially in cancer therapy. However, toxicological screening of such particles is paramount importance before use as delivery carrier. In this study we examine the biocompatibility of CaCO3 nanocrystal on NIH 3T3 cell line. Transmission and field emission scanning electron microscopy (TEM and FESEM) were used for the characterisation of CaCO3 nanocrystals. Cytotoxicity and genotoxic effect of calcium carbonate nanocrystals in cultured mouse embryonic fibroblast NIH 3T3 cell line using various bioassays including MTT, and Neutral red/Trypan blue double-staining assays. LDH, BrdU and reactive oxygen species were used for toxicity analysis. Cellular morphology was examined by scanning electron microscopy (SEM) and confocal fluorescence microscope. The outcome of the analyses revealed a clear rod-shaped aragonite polymorph of calcium carbonate nanocrystal. The analysed cytotoxic and genotoxicity of CaCO3 nanocrystal on NIH 3T3 cells using different bioassays revealed no significance differences as compared to control. A slight decrease in cell viability was noticed when the cells were exposed to higher concentrations of 200 to 400 µg/ml, while increase in ROS generation and LDH released at 200 and 400 µg/ml was observed. The study has shown that CaCO3 nanocrystal is biocompatible and non toxic to NIH 3T3 fibroblast cells. The analysed results offer a promising potential of CaCO3 nanocrystal for the development of intracellular drugs, genes and other macromolecule delivery systems.

Characterization of mutagen-activated cellular oncogenes that confer anchorage independence to human fibroblasts and tumorigenicity to NIH 3T3 cells: Sequence analysis of an enzymatically amplified mutant HRAS allele

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stevens, C.W.; Manoharan, T.H.; Fahl, W.E.

1988-06-01

Treatment of diploid human fibroblasts with an alkylating mutagen has been shown to induce stable, anchorage-independent cell populations at frequencies consistent with an activating mutation. After treatment of human foreskin fibroblasts with the mutagen benzo({alpha})pyrene ({plus minus})anti-7,8-dihydrodiol 9,10-epoxide and selection in soft agar, 17 anchorage-independent clones were isolated and expanded, and their cellular DNA was used to cotransfect NIH 3T3 cells along with pSV2neo. DNA from 11 of the 17 clones induced multiple NIH 3T3 cell tumors in recipient nude mice. Southern blot analyses showed the presence of human Alu repetitive sequences in all of the NIH 3T3 tumor cellmore » DNAs. Intact, human HRAS sequences were observed in 2 of the 11 tumor groups, whereas no hybridization was detected when human KRAS or NRAS probes were used. Slow-migrating ras p21 proteins, consistent with codon 12 mutations, were observed in the same two NIH 3T3 tumor cell groups that contained the human HRAS bands. Genomic DNA from one of these two human anchorage-independent cell populations (clone 21A) was used to enzymatically amplify a portion of exon 1 of the HRAS gene. The results demonstrate that exposure of normal human cells to a common environmental mutagen yields HRAS GC {yields} TA codon 12 transversions that have been commonly observed in human tumors.« less

Protective properties of Huperzine A through activation Nrf2/ARE-mediated transcriptional response in X-rays radiation-induced NIH3T3 cells.

PubMed

Zhu, Huan-Feng; Yan, Peng-Wei; Wang, Li-Jun; Liu, Ya-Tian; Wen, Jing; Zhang, Qian; Fan, Yan-Xin; Luo, Yan-Hong

2018-06-22

Huperzine A (HupA), derived from Huperzia Serrata, has exhibited a variety of biological actions, in particular neuroprotective effect. However, the protective activities of HupA on murine embryonic fibroblast NIH3T3 cells after X-rays radiation have not been fully elucidated. Herein, HupA treatment dramatically promoted cell viability, abated a G0/G1 peak accumulation, and ameliorated increase of cell apoptosis in NIH3T3 cells after X-rays radiation. Simultaneously, HupA notably enhanced activities of anti-oxidant enzymes, inhibited activity of lipid peroxide, and efficiently eliminated production of reactive oxygen species in NIH3T3 cells after X-rays radiation. Dose-dependent increase of antioxidant genes by HupA were associated with up-regulated Nrf2 and down-regulated Keap-1 expression, which was confirmed by increasing nuclear accumulation, and inhibiting of degradation of Nrf2. Notably, augmented luciferase activity of ARE may explained Nrf2/ARE-mediated signaling pathways behind HupA protective properties. Moreover, expression of Nrf2 HupA-mediated was significant attenuated by AKT inhibitor (LY294002), p38 MAPK inhibitor (SB202190) and ERK inhibitor (PD98059). Besides, HupA-mediated cell viability, and ROS production were dramatically bated by LY294002, SB202190, and PD98059. Taken together, HupA effectively ameliorated X-rays radiation-induced damage Nrf2-ARE-mediated transcriptional response via activation AKT, p38, and ERK signaling in NIH3T3 cells. © 2018 Wiley Periodicals, Inc.

The anti-apoptotic activity associated with phosphatidylinositol transfer protein alpha activates the MAPK and Akt/PKB pathway.

PubMed

Schenning, Martijn; Goedhart, Joachim; Gadella, Theodorus W J; Avram, Diana; Wirtz, Karel W A; Snoek, Gerry T

2008-10-01

The conditioned medium (CM) from mouse NIH3T3 fibroblast cells overexpressing phosphatidylinositol transfer protein alpha (PI-TPalpha; SPIalpha cells) demonstrates an increased anti-apoptotic activity compared with CM from wild type NIH3T3 (wtNIH3T3) cells. As previously shown, the anti-apoptotic activity acts by activating a G protein-coupled receptor, most probably a cannabinoid 1 (CB1)-like receptor as the activity was blocked by both pertussis toxin and rimonabant [M. Schenning, C.M. van Tiel, D. Van Manen, J.C. Stam, B.M. Gadella, K.W. Wirtz and G.T. Snoek, Phosphatidylinositol transfer protein alpha regulates growth and apoptosis of NIH3T3 cells: involvement of a cannabinoid 1-like receptor, J. Lipid Res. 45 (2004) 1555-1564]. The CB1 receptor appears to be expressed in mouse fibroblast cells, at levels in the order SPIalpha>wtNIH3T3>SPIbeta cells (i.e. wild type cells overexpressing PI-TPbeta). Upon incubation of SPIbeta cells with the PI-TPalpha-dependent anti-apoptotic factors, both the ERK/MAP kinase and the Akt/PKB pathway are activated in a CB1 receptor dependent manner as shown by Western blotting. In addition, activation of ERK2 was also shown by EYFP-ERK2 translocation to the nucleus, as visualized by confocal laser scanning microscopy. The subsequent activation of the anti-apoptotic transcription factor NF-kappaB is in line with the increased resistance towards UV-induced apoptosis. On the other hand, receptor activation by CM from SPIalpha cells was not linked to phospholipase C activation as the YFP-labelled C2-domain of protein kinase C was not translocated to the plasma membrane of SPIbeta cells as visualized by confocal laser scanning microscopy.

Feline leukemia virus infection requires a post-receptor binding envelope-dependent cellular component.

PubMed

Hussain, Naveen; Thickett, Kelly R; Na, Hong; Leung, Cherry; Tailor, Chetankumar S

2011-12-01

Gammaretrovirus receptors have been suggested to contain the necessary determinants to mediate virus binding and entry. Here, we show that murine NIH 3T3 and baby hamster kidney (BHK) cells overexpressing receptors for subgroup A, B, and C feline leukemia viruses (FeLVs) are weakly susceptible (10(1) to 10(2) CFU/ml) to FeLV pseudotype viruses containing murine leukemia virus (MLV) core (Gag-Pol) proteins, whereas FeLV receptor-expressing murine Mus dunni tail fibroblast (MDTF) cells are highly susceptible (10(4) to 10(6) CFU/ml). However, NIH 3T3 cells expressing the FeLV subgroup B receptor PiT1 are highly susceptible to gibbon ape leukemia virus pseudotype virus, which differs from the FeLV pseudotype viruses only in the envelope protein. FeLV resistance is not caused by a defect in envelope binding, low receptor expression levels, or N-linked glycosylation. Resistance is not alleviated by substitution of the MLV core in the FeLV pseudotype virus with FeLV core proteins. Interestingly, FeLV resistance is alleviated by fusion of receptor-expressing NIH 3T3 and BHK cells with MDTF or human TE671 cells, suggesting the absence of an additional cellular component in NIH 3T3 and BHK cells that is required for FeLV infection. The putative FeLV-specific cellular component is not a secreted factor, as MDTF conditioned medium does not alleviate the block to FeLV infection. Together, our findings suggest that FeLV infection requires an additional envelope-dependent cellular component that is absent in NIH 3T3 and BHK cells but that is present in MDTF and TE671 cells.

Challenges of T3 and T4 Translational Research

ERIC Educational Resources Information Center

Vukotich, Charles J., Jr.

2016-01-01

Translational research is a new and important way of thinking about research. It is a major priority of the National Institutes of Health (NIH) in the United States. NIH has created the Clinical and Translational Science Awards to promote this priority. NIH has defined T1 and T2 phases of translational research in the medical field, in order to…

Involvement of up-regulated Necl-5/Tage4/PVR/CD155 in the loss of contact inhibition in transformed NIH3T3 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Minami, Yukiko; Department of Surgery and Clinical Oncology, Osaka University Graduate School of Medicine/Faculty of Medicine, Suita 565-0871, Osaka; Ikeda, Wataru

2007-01-26

Normal cells show contact inhibition of cell movement and proliferation, but this is lost following transformation. We found that Necl-5, originally identified as a poliovirus receptor and up-regulated in many cancer cells, enhances growth factor-induced cell movement and proliferation. We showed that when cells contact other cells, Necl-5 interacts in trans with nectin-3 and is removed by endocytosis from the cell surface, resulting in a reduction of cell movement and proliferation. We show here that up-regulation of the gene encoding Necl-5 by the oncogene V12-Ki-Ras causes enhanced cell movement and proliferation. Upon cell-cell contact, de novo synthesis of Necl-5 exceedsmore » the rate of Necl-5 endocytosis, eventually resulting in a net increase in the amount of Necl-5 at the cell surface. In addition, expression of the gene encoding nectin-3 is markedly reduced in transformed cells. Thus, up-regulation of Necl-5 following transformation contributes to the loss of contact inhibition in transformed cells.« less

Targeted Gene Therapy for Breast Cancer

DTIC Science & Technology

1999-08-01

recognizing conformational and polysaccharide epitopes, cell ELISA using NIH3T3 and NIH/189 cells was employed to detect such clones. Cells (lxl04/well...77T 541-542, 1997. 26. Zhou, Y. and Lee, A. S. Mechanism for the suppression of the mammalian stress response by genistein, an anticancer

Interactions of 1D- and 2D-layered inorganic nanoparticles with fibroblasts and human mesenchymal stem cells

PubMed Central

Rashkow, Jason Thomas; Talukdar, Yahfi; Lalwani, Gaurav; Sitharaman, Balaji

2015-01-01

Aim This study investigates the effects of tungsten disulfide nanotubes (WSNTs) and molybdenum disulfide nanoplatelets (MSNPs) on fibroblasts (NIH-3T3) and mesenchymal stem cells (MSCs) to determine safe dosages for potential biomedical applications. Materials & methods Cytotoxicity of MSNPs and WSNTs (5–300 µg/ml) on NIH-3T3 and MSCs was assessed at 6, 12 or 24 h. MSC differentiation to adipocytes and osteoblasts was assessed following treatment for 24 h. Results Only NIH-3T3 cells treated with MSNPs showed dose or time dependent increase in cytotoxicity. Differentiation markers of MSCs in treated groups were unaffected compared with untreated controls. Conclusion MSNPs and WSNTs at concentrations less than 50 µg/ml are potentially safe for treatment of fibroblasts or MSCs for up to 24 h. PMID:26080694

A novel mechanism by which tissue transglutaminase activates signaling events that promote cell survival.

PubMed

Boroughs, Lindsey K; Antonyak, Marc A; Cerione, Richard A

2014-04-04

Tissue transglutaminase (tTG) functions as a GTPase and an acyl transferase that catalyzes the formation of protein cross-links. tTG expression is frequently up-regulated in human cancer, where it has been implicated in various aspects of cancer progression, including cell survival and chemo-resistance. However, the extent to which tTG cooperates with other proteins within the context of a cancer cell, versus its intrinsic ability to confer transformed characteristics to cells, is poorly understood. To address this question, we asked what effect the ectopic expression of tTG in a non-transformed cellular background would have on the behavior of the cells. Using NIH3T3 fibroblasts stably expressing a Myc-tagged form of tTG, we found that tTG strongly protected these cells from serum starvation-induced apoptosis and triggered the activation of the PI3-kinase/mTOR Complex 1 (mTORC1)/p70 S6-kinase pathway. We determined that tTG forms a complex with the non-receptor tyrosine kinase c-Src and PI3-kinase, and that treating cells with inhibitors to block tTG function (monodansylcadaverine; MDC) or c-Src kinase activity (PP2) disrupted the formation of this complex, and prevented tTG from activating the PI3-kinase pathway. Moreover, treatment of fibroblasts over-expressing tTG with PP2, or with inhibitors that inactivate components of the PI3-kinase pathway, including PI3-kinase (LY294002) and mTORC1 (rapamycin), ablated the tTG-promoted survival of the cells. These findings demonstrate that tTG has an intrinsic capability to stimulate cell survival through a novel mechanism that activates PI3-kinase signaling events, thus highlighting tTG as a potential target for the treatment of human cancer.

The shed ectodomain of Nr-CAM stimulates cell proliferation and motility, and confers cell transformation.

PubMed

Conacci-Sorrell, Maralice; Kaplan, Anna; Raveh, Shani; Gavert, Nancy; Sakurai, Takeshi; Ben-Ze'ev, Avri

2005-12-15

Nr-CAM, a cell-cell adhesion molecule of the immunoglobulin-like cell adhesion molecule family, known for its function in neuronal outgrowth and guidance, was recently identified as a target gene of beta-catenin signaling in human melanoma and colon carcinoma cells and tissue. Retrovirally mediated transduction of Nr-CAM into fibroblasts induces cell motility and tumorigenesis. We investigated the mechanisms by which Nr-CAM can confer properties related to tumor cell behavior and found that Nr-CAM expression in NIH3T3 cells protects cells from apoptosis in the absence of serum by constitutively activating the extracellular signal-regulated kinase and AKT signaling pathways. We detected a metalloprotease-mediated shedding of Nr-CAM into the culture medium of cells transfected with Nr-CAM, and of endogenous Nr-CAM in B16 melanoma cells. Conditioned medium and purified Nr-CAM-Fc fusion protein both enhanced cell motility, proliferation, and extracellular signal-regulated kinase and AKT activation. Moreover, Nr-CAM was found in complex with alpha4beta1 integrins in melanoma cells, indicating that it can mediate, in addition to homophilic cell-cell adhesion, heterophilic adhesion with extracellular matrix receptors. Suppression of Nr-CAM levels by small interfering RNA in B16 melanoma inhibited the adhesive and tumorigenic capacities of these cells. Stable expression of the Nr-CAM ectodomain in NIH3T3 cells conferred cell transformation and tumorigenesis in mice, suggesting that the metalloprotease-mediated shedding of Nr-CAM is a principal route for promoting oncogenesis by Nr-CAM.

Interactions of 1D- and 2D-layered inorganic nanoparticles with fibroblasts and human mesenchymal stem cells

DOE PAGES

Rashkow, Jason Thomas; Talukdar, Yahfi; Lalwani, Gaurav; ...

2015-06-01

Here, this study investigates the effects of tungsten disulfide nanotubes (WSNTs) and molybdenum disulfide nanoplatelets (MSNPs) on fibroblasts (NIH-3T3) and mesenchymal stem cells (MSCs) to determine safe dosages for potential biomedical applications. Cytotoxicity of MSNPs and WSNTs (5–300 μg/ml) on NIH-3T3 and MSCs was assessed at 6, 12 or 24 h. MSC differentiation to adipocytes and osteoblasts was assessed following treatment for 24 h. Only NIH-3T3 cells treated with MSNPs showed dose or time dependent increase in cytotoxicity. Differentiation markers of MSCs in treated groups were unaffected compared with untreated controls. In conclusion, MSNPs and WSNTs at concentrations less thanmore » 50 μg/ml are potentially safe for treatment of fibroblasts or MSCs for up to 24 h.« less

A functional simian virus 40 origin of replication is required for the generation of a super T antigen with a molecular weight of 100,000 in transformed mouse cells.

PubMed Central

Chen, S; Grass, D S; Blanck, G; Hoganson, N; Manley, J L; Pollack, R E

1983-01-01

We used two recombinant plasmids, one containing wild-type simian virus 40 DNA (pSVR1) and the other containing a simian virus 40 genome with a defective origin of replication (pSVR1-origin-minus) to transfect NIH3T3 cells. Quantitation of T-antigen synthesis by indirect immunofluorescence at 48 h after transfection with either DNA revealed the same percentage of T-positive nuclei. The transformation frequencies observed were also similar with both plasmids. Immunoprecipitation of [35S]methionine-labeled cell extracts showed the expected 94,000-dalton (94K) T and 17K t antigens in all clones examined. In pSVR1-generated transformants, a 100K super T antigen was also detected. Transformants isolated from pSVR1-origin-minus transfection, however, never expressed this 100K super T antigen, and some of these clones originally also showed greatly reduced levels of 94K T antigen. However, after growth in culture for several generations, the levels of 94K T antigen synthesis in these underproducer clones were dramatically increased. A direct correlation between the amounts of T antigen synthesized and the ability to grow independently of anchorage was observed. The mechanism which brings about increasing levels of T-antigen synthesis in some of the clones is not clear, but it appears not to be due to changes in either the copy number or the methylation pattern of the integrated simian virus 40 DNA. Images PMID:6312105

Mitogenic signals and transforming potential of Nyk, a newly identified neural cell adhesion molecule-related receptor tyrosine kinase.

PubMed Central

Ling, L; Kung, H J

1995-01-01

Nyk/Mer is a recently identified receptor tyrosine kinase with neural cell adhesion molecule-like structure (two immunoglobulin G-like domains and two fibronectin III-like domains) in its extracellular region and belongs to the Ufo/Axl family of receptors. The ligand for Nyk/Mer is presently unknown, as are the signal transduction pathways mediated by this receptor. We constructed and expressed a chimeric receptor (Fms-Nyk) composed of the extracellular domain of the human colony-stimulating factor 1 receptor (Fms) and the transmembrane and cytoplasmic domains of human Nyk/Mer in NIH 3T3 fibroblasts in order to investigate the mitogenic signaling and biochemical properties of Nyk/Mer. Colony-stimulating factor 1 stimulation of the Fms-Nyk chimeric receptor in transfected NIH 3T3 fibroblasts leads to a transformed phenotype and generates a proliferative response in the absence of other growth factors. We show that phospholipase C gamma, phosphatidylinositol 3-kinase/p70 S6 kinase, Shc, Grb2, Raf-1, and mitogen-activated protein kinase are downstream components of the Nyk/Mer signal transduction pathways. In addition, Nyk/Mer weakly activates p90rsk, while stress-activated protein kinase, Ras GTPase-activating protein (GAP), and GAP-associated p62 and p190 proteins are not activated or tyrosine phosphorylated by Nyk/Mer. An analysis comparing the Nyk/Mer signal cascade with that of the epidermal growth factor receptor indicates substrate preferences by these two receptors. Our results provide a detailed description of the Nyk/Mer signaling pathways. Given the structural similarity between the Ufo/Axl family receptors, some of the information may also be applied to other members of this receptor tyrosine kinase family. PMID:8524223

Oncogenic transformation induced by cell-free nucleic acids circulating in plasma (genometastasis) remains after the surgical resection of the primary tumor: a pilot study.

PubMed

García-Olmo, Damián; García-Olmo, Dolores C; Domínguez-Berzosa, Carolina; Guadalajara, Hector; Vega, Luz; García-Arranz, Mariano

2012-06-01

The oncogenic transformation by cell-free nucleic acids circulating in plasma has been named as genometastasis. The feasibility of this phenomenon has been demonstrated and now it is necessary to value the impact of this phenomenon and to determine what conditions could promote or inhibit it. The goal of this study was to examine the transforming ability of plasma from colorectal cancer patients in a long-term follow-up after the surgical excision of the primary tumor, and to try correlate it with the clinical picture of patients. Blood samples were taken from eight patients with K-ras-mutated colorectal tumors, who were under surgical primary tumor resection at least 2 years before. Plasma was isolated by two centrifugations and added to cultures of NIH-3T3 cells and human adipose-derived stem cells (hASCs). In two cases, plasma was separated from cells by a membrane with 0.4-μm pores. The presence of mutated and non-mutated human K-ras sequences was tested by real-time PCR in cultured cells. After 30 days, cells were subcutaneously injected into athymic nude mice in order to test their ability to generate tumors. In four of the eight patients analyzed after surgery, tumor DNA was detected in plasma. Plasmas from three of them were able to oncogenically transform NIH-3T3 cells in culture and, when those cells were injected in mice, carcinomas were generated. After a 2-year follow-up, metastases were found in two of the three patients whose plasmas were able to transform cells, and in two of the four in whom plasma tumor DNA was not detected. Thus, after a mean follow-up of 29.5 months, only four of 13 patients (30.8%) were alive and disease-free. Primary tumor resection does not assure a complete clean of blood of circulating oncogenes, in spite of a disease-free clinical picture. Moreover, in some cases plasma kept their oncogenic capabilities. The value of these findings as prognosis factor remains unclear and needs further investigations.

Growth-inhibitory effects of the red alga Gelidium amansii on cultured cells.

PubMed

Chen, Yue-Hwa; Tu, Ching-Jung; Wu, Hsiao-Ting

2004-02-01

The objective of this study was to investigate the effects of Gelidium amansii, an edible red agar cultivated off the northeast coast of Taiwan, on the growth of two lines of cancer cells, murine hepatoma (Hepa-1) and human leukemia (HL-60) cells, as well as a normal cell line, murine embryo fibroblast cells (NIH-3T3). The potential role of G. amansii on the induction of apoptosis was also examined. The results indicated that all extracts from G. amansii, including phosphate-buffered saline (PBS) and methanol extracts from dried algae as well as the dimethyl sulfoxide (DMSO) extract from freeze-dried G. amansii agar, inhibited the growth of Hepa-1 and NIH-3T3 cells, but not the growth of HL-60 cells. Annexin V-positive cells were observed in methanol and DMSO extract-treated, but not PBS extract-treated Hepa-1 and NIH-3T3 cells, suggesting that the lipid-soluble extracts of G. amansii induced apoptosis. In summary, extracts of G. amansii from various preparations exhibited antiproliferative effects on Hepa-1 and NIH-3T3 cells, and apoptosis may play a role in the methanol and DMSO extract-induced inhibitory effects. However, the antiproliferative effects of PBS extracts was not through apoptosis. Moreover, the growth-inhibitory effects of G. amansii were not specific to cancer cells.

Mutation of the Salt Bridge-forming Residues in the ETV6-SAM Domain Interface Blocks ETV6-NTRK3-induced Cellular Transformation*

PubMed Central

Cetinbas, Naniye; Huang-Hobbs, Helen; Tognon, Cristina; Leprivier, Gabriel; An, Jianghong; McKinney, Steven; Bowden, Mary; Chow, Connie; Gleave, Martin; McIntosh, Lawrence P.; Sorensen, Poul H.

2013-01-01

The ETV6-NTRK3 (EN) chimeric oncogene is expressed in diverse tumor types. EN is generated by a t(12;15) translocation, which fuses the N-terminal SAM (sterile α-motif) domain of the ETV6 (or TEL) transcription factor to the C-terminal PTK (protein-tyrosine kinase) domain of the neurotrophin-3 receptor NTRK3. SAM domain-mediated polymerization of EN leads to constitutive activation of the PTK domain and constitutive signaling of the Ras-MAPK and PI3K-Akt pathways, which are essential for EN oncogenesis. Here we show through complementary biophysical and cellular biological techniques that mutation of Lys-99, which participates in a salt bridge at the SAM polymer interface, reduces self-association of the isolated SAM domain as well as high molecular mass complex formation of EN and abrogates the transformation activity of EN. We also show that mutation of Asp-101, the intermolecular salt bridge partner of Lys-99, similarly blocks transformation of NIH3T3 cells by EN, reduces EN tyrosine phosphorylation, inhibits Akt and Mek1/2 signaling downstream of EN, and abolishes tumor formation in nude mice. In contrast, mutations of Glu-100 and Arg-103, residues in the vicinity of the interdomain Lys-99–Asp-101 salt bridge, have little or no effect on these oncogenic characteristics of EN. Our results underscore the importance of specific electrostatic interactions for SAM polymerization and EN transformation. PMID:23798677

Mutation of the salt bridge-forming residues in the ETV6-SAM domain interface blocks ETV6-NTRK3-induced cellular transformation.

PubMed

Cetinbas, Naniye; Huang-Hobbs, Helen; Tognon, Cristina; Leprivier, Gabriel; An, Jianghong; McKinney, Steven; Bowden, Mary; Chow, Connie; Gleave, Martin; McIntosh, Lawrence P; Sorensen, Poul H

2013-09-27

The ETV6-NTRK3 (EN) chimeric oncogene is expressed in diverse tumor types. EN is generated by a t(12;15) translocation, which fuses the N-terminal SAM (sterile α-motif) domain of the ETV6 (or TEL) transcription factor to the C-terminal PTK (protein-tyrosine kinase) domain of the neurotrophin-3 receptor NTRK3. SAM domain-mediated polymerization of EN leads to constitutive activation of the PTK domain and constitutive signaling of the Ras-MAPK and PI3K-Akt pathways, which are essential for EN oncogenesis. Here we show through complementary biophysical and cellular biological techniques that mutation of Lys-99, which participates in a salt bridge at the SAM polymer interface, reduces self-association of the isolated SAM domain as well as high molecular mass complex formation of EN and abrogates the transformation activity of EN. We also show that mutation of Asp-101, the intermolecular salt bridge partner of Lys-99, similarly blocks transformation of NIH3T3 cells by EN, reduces EN tyrosine phosphorylation, inhibits Akt and Mek1/2 signaling downstream of EN, and abolishes tumor formation in nude mice. In contrast, mutations of Glu-100 and Arg-103, residues in the vicinity of the interdomain Lys-99-Asp-101 salt bridge, have little or no effect on these oncogenic characteristics of EN. Our results underscore the importance of specific electrostatic interactions for SAM polymerization and EN transformation.

Evidence for in vivo phosphorylation of the Grb2 SH2-domain binding site on focal adhesion kinase by Src-family protein-tyrosine kinases.

PubMed

Schlaepfer, D D; Hunter, T

1996-10-01

Focal adhesion kinase (FAK) is a nonreceptor protein-tyrosine kinase (PTK) that associates with integrin receptors and participates in extracellular matrix-mediated signal transduction events. We showed previously that the c-Src nonreceptor PTK and the Grb2 SH2/SH3 adaptor protein bound directly to FAK after fibronectin stimulation (D. D. Schlaepfer, S.K. Hanks, T. Hunter, and P. van der Geer, Nature [London] 372:786-791, 1994). Here, we present evidence that c-Src association with FAK is required for Grb2 binding to FAK. Using a tryptic phosphopeptide mapping approach, the in vivo phosphorylation of the Grb2 binding site on FAK (Tyr-925) was detected after fibronectin stimulation of NIH 3T3 cells and was constitutively phosphorylated in v-Src-transformed NIH 3T3 cells. In vitro, c-Src phosphorylated FAK Tyr-925 in a glutathione S-transferase-FAK C-terminal domain fusion protein, whereas FAK did not. Using epitope-tagged FAK constructs, transiently expressed in human 293 cells, we determined the effect of site-directed mutations on c-Src and Grb2 binding to FAK. Mutation of FAK Tyr-925 disrupted Grb2 binding, whereas mutation of the c-Src binding site on FAK (Tyr-397) disrupted both c-Src and Grb2 binding to FAK in vivo. These results support a model whereby Src-family PTKs are recruited to FAK and focal adhesions following integrin-induced autophosphorylation and exposure of FAK Tyr-397. Src-family binding and phosphorylation of FAK at Tyr-925 creates a Grb2 SH2-domain binding site and provides a link to the activation of the Ras signal transduction pathway. In Src-transformed cells, this pathway may be constitutively activated as a result of FAK Tyr-925 phosphorylation in the absence of integrin stimulation.

Molecular Determinants of Antiestrogen and Drug Sensitivity in Breast Carcinoma Cells

DTIC Science & Technology

1997-08-01

W.S. human HT1080 fibrosarcoma cells (a gift of Pear, personal communication). G.R. Stark), susceptible to ecotropic retrovirus In several...transfection with equal amounts of plasmid pOPRSVIluc (generated by replacing CAT of pORRSVICAT with luc) which expresses luc from R02 promoter, and control...retroviral transduction into (Fig. 1). During transient co-transfection of NIH 3T3 human HT1080 fibrosarcoma or mouse NIH 3T3 cell cells with a laac expressing

[The proliferative characteristics of cells in culture during perfusion of the medium].

PubMed

Akatov, V S; Lavrovskaia, V P; Lezhnev, E I

1991-01-01

The proliferation of Chinese hamster fibroblasts (CHF) and NIH 3T3 cells was studied at different flow rates immediately above the cells. It is shown that there is a limiting density of saturation of the perfused culture that accounts for 1.7 x 10(6) - 2.0 x 10(6) cells/cm2 for NIH 3T3 cells, and for 6 x 10(6) - 7 x 10(6) cells/cm2 for CHF. The growth curves and the distribution of cells with respect to the phases of the cell cycle during cultivation with and without perfusion are presented. Based on the results obtained it is assumed that the limit of saturation density of perfused CHF culture is due to the mass transfer of the growth-inhibiting metabolites inside the multilayer structures. The assumption seems to hold true for NIH 3T3 cells, too, even though the multilayer character of growth of this culture is less pronounced than in CHF.

MedlinePlus FAQ: Can you tell me how to cite MedlinePlus pages?

MedlinePlus

... article . Conuel T. Finding answers in a beauty shop. NIH MedlinePlus: the magazine [Internet]. 2012 Fall [cited ... identified Conuel T. Finding answers in a beauty shop. NIH MedlinePlus: the magazine. 2012 Fall; 7(3): ...

Ultrasound-mediated structural changes in cells revealed by FTIR spectroscopy: A contribution to the optimization of gene and drug delivery

NASA Astrophysics Data System (ADS)

Grimaldi, Paola; Di Giambattista, Lucia; Giordani, Serena; Udroiu, Ion; Pozzi, Deleana; Gaudenzi, Silvia; Bedini, Angelico; Giliberti, Claudia; Palomba, Raffaele; Congiu Castellano, Agostina

2011-12-01

Ultrasound effects on biological samples are gaining a growing interest concerning in particular, the intracellular delivery of drugs and genes in a safe and in a efficient way. Future progress in this field will require a better understanding of how ultrasound and acoustic cavitation affect the biological system properties. The morphological changes of cells due to ultrasound (US) exposure have been extensively studied, while little attention has been given to the cells structural changes. We have exposed two different cell lines to 1 MHz frequency ultrasound currently used in therapy, Jurkat T-lymphocytes and NIH-3T3 fibroblasts, both employed as models respectively in the apoptosis and in the gene therapy studies. The Fourier Transform Infrared (FTIR) Spectroscopy was used as probe to reveal the structural changes in particular molecular groups belonging to the main biological systems. The genotoxic damage of cells exposed to ultrasound was ascertained by the Cytokinesis-Block Micronucleus (CBMN) assay. The FTIR spectroscopy results, combined with multivariate statistical analysis, regarding all cellular components (lipids, proteins, nucleic acids) of the two cell lines, show that Jurkat cells are more sensitive to therapeutic ultrasound in the lipid and protein regions, whereas the NIH-3T3 cells are more sensitive in the nucleic acids region; a meaningful genotoxic effect is present in both cell lines only for long sonication times while in the Jurkat cells also a significant cytotoxic effect is revealed for long times of exposure to ultrasound.

Antiproliferative activity of flower hexane extract obtained from Mentha spicata associated with Mentha rotundifolia against the MCF7, KB, and NIH/3T3 cell lines.

PubMed

Nedel, Fernanda; Begnini, Karine; Carvalho, Pedro Henrique de Azambuja; Lund, Rafael Guerra; Beira, Fátima T A; Del Pino, Francisco Augusto B

2012-11-01

This study assessed the antiproliferative effect in vitro of the flower hexane extract obtained from Mentha spicata associated with Mentha rotundifolia against the human breast adenocarcinoma (MCF-7), human mouth epidermal carcinoma (KB), and mouse embryonic fibroblast (NIH 3T3) cell lines, using sulforhodamine B (SRB) assay. A cell density of 2×10(4)/well was seeded in 96-well plates, and samples at different concentrations ranging from 10 to 500 mg/mL were tested. The optical density was determined in an ELISA multiplate reader (Thermo Plate TP-Reader). Results demonstrated that the hexane extract presented antiproliferative activity against both the tumor cell lines KB and MCF-7, presenting a GI(50) (MCF-7=13.09 mg/mL), TGI (KB=37.76 mg/mL), and IL(50) (KB=291.07 mg/mL). Also, the hexane extract presented antiproliferative activity toward NIH 3T3 cells GI(50) (183.65 mg/mL), TGI (280.54 mg/mL), and IL(50) (384.59 mg/mL). The results indicate that the flower hexane extract obtained from M. spicata associated with M. rotundifolia presents an antineoplastic activity against KB and MCF-7, although an antiproliferative effect at a high concentration of the extract was observed toward NIH 3T3.

[Induction of robust senescence-associated secretory phenotype in mouse NIH-3T3 cells by mitomycin C].

PubMed

Huang, Wei-Xing; Guo, Xiao-Xuan; Peng, Zhong-Zhi; Weng, Chun-Liang; Huang, Chun-Yan; Shi, Ben-Yan; Yang, Jie; Liao, Xiao-Xin; Li, Xiao-Yi; Zheng, Hui-Ling; Liu, Xin-Guang; Sun, Xue-Rong

2017-02-25

Senescence-associated secretory phenotype (SASP) is often a concomitant result of cell senescence, embodied by the enhanced function of secretion. The SASP factors secreted by senescent cells include cytokines, proteases and chemokines, etc, which can exert great influence on local as well as systemic environment and participate in the process of cell senescence, immunoregulation, angiogenesis, cell proliferation and tumor invasion, etc. Relative to the abundance of SASP models in human cells, the in vitro SASP model derived from mouse cells is scarce at present. Therefore, the study aimed to establish a mouse SASP model to facilitate the research in the field. With this objective, we treated the INK4a-deficient mouse NIH-3T3 cells and the wildtype mouse embryonic fibroblasts (MEF) respectively with mitomycin C (MMC), an anticarcinoma drug which could induce DNA damage. The occurring of cell senescence was evaluated by cell morphology, β-gal staining, integration ratio of EdU and Western blot. Quantitative RT-PCR and ELISA were used to detect the expression and secretion of SASP factors, respectively. The results showed that, 8 days after the treatment of NIH-3T3 cells with MMC (1 μg/mL) for 12 h or 24 h, the cells became enlarged and the ratios of β-gal-positive (blue-stained) cells significantly increased, up to 77.4% and 90.4%, respectively. Meanwhile, the expression of P21 protein increased and the integration ratios of EdU significantly decreased (P < 0.01). Quantitative RT-PCR detection showed that the mRNA levels of several SASP genes, including IL-6, TNF-α, IL-1α and IL-1β increased evidently. ELISA detection further observed an enhanced secretion of IL-6 (P < 0.01). On the contrary, although wildtype MEF could also be induced into senescence by MMC treatment for 12 h or 24 h, embodied by the enlarged cell volume, increased ratios of β-gal-positive cells (up to 71.7% and 80.2%, respectively) and enhanced expression of P21 protein, the secretion of IL-6 displayed no significant change. Our study indicated that, although MMC could induce senescence in both mouse NIH-3T3 cells and wildtype MEF, only senescent NIH-3T3 cells displayed the canonical SASP phenomena. Current study suggested that senescent NIH-3T3 cells might be an appropriate in vitro SASP model of mouse cells.

The expression of a truncated HMGI-C gene induces gigantism associated with lipomatosis.

PubMed

Battista, S; Fidanza, V; Fedele, M; Klein-Szanto, A J; Outwater, E; Brunner, H; Santoro, M; Croce, C M; Fusco, A

1999-10-01

Rearrangements of the HMGI-C gene have frequently been detected in human benign tumors of mesenchymal origin, including lipomas. The HMGI-C protein has three AT-hook domains and an acidic COOH-terminal tail. The HMGI-C modifications consist in the loss of the C-tail and the fusion with ectopic sequences. Recent results show that the loss of the COOH-terminal region, rather than the acquisition of new sequences, is sufficient to confer to HMGI-C the ability to transform NIH3T3 cells. Therefore, transgenic mice carrying a HMGI-C construct (HMGI-C/T), containing only the three AT-hook domains, were generated. The HMGI-C/T mice showed a giant phenotype, together with a predominantly abdominal/pelvic lipomatosis, suggesting a pivotal role of the HMGI-C truncation in the generation of human lipomas.

Riboflavin Depletion Promotes Tumorigenesis in HEK293T and NIH3T3 Cells by Sustaining Cell Proliferation and Regulating Cell Cycle-Related Gene Transcription.

PubMed

Long, Lin; He, Jian-Zhong; Chen, Ye; Xu, Xiu-E; Liao, Lian-Di; Xie, Yang-Min; Li, En-Min; Xu, Li-Yan

2018-05-07

Riboflavin is an essential component of the human diet and its derivative cofactors play an established role in oxidative metabolism. Riboflavin deficiency has been linked with various human diseases. The objective of this study was to identify whether riboflavin depletion promotes tumorigenesis. HEK293T and NIH3T3 cells were cultured in riboflavin-deficient or riboflavin-sufficient medium and passaged every 48 h. Cells were collected every 5 generations and plate colony formation assays were performed to observe cell proliferation. Subcutaneous tumorigenicity assays in NU/NU mice were used to observe tumorigenicity of riboflavin-depleted HEK293T cells. Mechanistically, gene expression profiling and gene ontology analysis were used to identify abnormally expressed genes induced by riboflavin depletion. Western blot analyses, cell cycle analyses, and chromatin immunoprecipitation were used to validate the expression of cell cycle-related genes. Plate colony formation of NIH3T3 and HEK293T cell lines was enhanced >2-fold when cultured in riboflavin-deficient medium for 10-20 generations. Moreover, we observed enhanced subcutaneous tumorigenicity in NU/NU mice following injection of riboflavin-depleted compared with normal HEK293T cells (55.6% compared with 0.0% tumor formation, respectively). Gene expression profiling and gene ontology analysis revealed that riboflavin depletion induced the expression of cell cycle-related genes. Validation experiments also found that riboflavin depletion decreased p21 and p27 protein levels by ∼20%, and increased cell cycle-related and expression-elevated protein in tumor (CREPT) protein expression >2-fold, resulting in cyclin D1 and CDK4 levels being increased ∼1.5-fold, and cell cycle acceleration. We also observed that riboflavin depletion decreased intracellular riboflavin levels by 20% and upregulated expression of riboflavin transporter genes, particularly SLC52A3, and that the changes in CREPT and SLC52A3 correlated with specific epigenetic changes in their promoters in riboflavin-depleted HEK293T cells. Riboflavin depletion contributes to HEK293T and NIH3T3 cell tumorigenesis and may be a risk factor for tumor development.

Substrate flexibility regulates growth and apoptosis of normal but not transformed cells

NASA Technical Reports Server (NTRS)

Wang, H. B.; Dembo, M.; Wang, Y. L.

2000-01-01

One of the hallmarks of oncogenic transformation is anchorage-independent growth (27). Here we demonstrate that responses to substrate rigidity play a major role in distinguishing the growth behavior of normal cells from that of transformed cells. We cultured normal or H-ras-transformed NIH 3T3 cells on flexible collagen-coated polyacrylamide substrates with similar chemical properties but different rigidity. Compared with cells cultured on stiff substrates, nontransformed cells on flexible substrates showed a decrease in the rate of DNA synthesis and an increase in the rate of apoptosis. These responses on flexible substrates are coupled to decreases in cell spreading area and traction forces. In contrast, transformed cells maintained their growth and apoptotic characteristics regardless of substrate flexibility. The responses in cell spreading area and traction forces to substrate flexibility were similarly diminished. Our results suggest that normal cells are capable of probing substrate rigidity and that proper mechanical feedback is required for regulating cell shape, cell growth, and survival. The loss of this response can explain the unregulated growth of transformed cells.

Enhancement of cell growth on honeycomb-structured polylactide surface using atmospheric-pressure plasma jet modification

NASA Astrophysics Data System (ADS)

Cheng, Kuang-Yao; Chang, Chia-Hsing; Yang, Yi-Wei; Liao, Guo-Chun; Liu, Chih-Tung; Wu, Jong-Shinn

2017-02-01

In this paper, we compare the cell growth results of NIH-3T3 and Neuro-2A cells over 72 h on flat and honeycomb structured PLA films without and with a two-step atmospheric-pressure nitrogen-based plasma jet treatment. We developed a fabrication system used for forming of a uniform honeycomb structure on PLA surface, which can produce two different pore sizes, 3-4 μm and 7-8 μm, of honeycomb pattern. We applied a previously developed nitrogen-based atmospheric-pressure dielectric barrier discharge (DBD) jet system to treat the PLA film without and with honeycomb structure. NIH-3T3 and a much smaller Neuro-2A cells were cultivated on the films under various surface conditions. The results show that the two-step plasma treatment in combination with a honeycomb structure can enhance cell growth on PLA film, should the cell size be not too smaller than the pore size of honeycomb structure, e.g., NIH-3T3. Otherwise, cell growth would be better on flat PLA film, e.g., Neuro-2A.

Anti-proliferative and mutagenic activities of aqueous and methanol extracts of leaves from Pereskia bleo (Kunth) DC (Cactaceae).

PubMed

Er, Hui Meng; Cheng, En-Hsiang; Radhakrishnan, Ammu Kutty

2007-09-25

The anti-proliferative effects of the aqueous and methanol extracts of leaves of Pereskia bleo (Kunth) DC (Cactaceae) against a mouse mammary cancer cell line (4T1) and a normal mouse fibroblast cell line (NIH/3T3) were evaluated under an optimal (in culture medium containing 10% foetal bovine serum (FBS)) and a sub-optimal (in culture medium containing 0.5% FBS) conditions. Under the optimal condition, the aqueous extract showed a significant (p<0.05) anti-proliferative effect at 200 microg/mL and 300 microg/mL in 4T1 cells and 300 microg/mL in NIH/3T3 cells, whereas the methanol extract did not show any notable anti-proliferative effect in these cell lines, at any of the concentrations tested. Under the sub-optimal condition, the aqueous extract showed a significant (p<0.05) anti-proliferative effect at 200 microg/mL and 300 microg/mL in NIH/3T3 cells, whilst the methanol extract showed a significant (p<0.05) anti-proliferative effect at 200 microg/mL and 300 microg/mL in both cell lines. An upward trend of apoptosis was observed in both 4T1 and NIH/3T3 cells treated with increasing concentrations of the aqueous extract. The level of apoptosis observed at all the concentrations of the aqueous extract tested was consistently higher than necrosis. There was a significant (p<0.05) increase in the level of necrosis observed in the 4T1 cells treated with 300 microg/mL of the methanol extract. Generally, the level of necrosis was noted to be higher than that of apoptosis in the methanol extract-treated cells. The mutagenicity assay performed showed that in the absence of S-9 liver metabolic activation, the extract was not mutagenic up to the concentration of 165 microg/mL . However, in the presence of S-9 liver metabolic activation, the aqueous extract was mutagenic at all the concentrations tested. This study shows that both the aqueous and methanol extracts of the leaves from Pereskia bleo (Kunth) DC (Cactaceae) do not have appreciable anti-proliferative effect on the 4T1 and NIH/3T3 cells as the EC(50) values obtained are greater than 50 microg/mL when tested under optimal culture condition. Moreover, the aqueous extract may form mutagenic compound(s) upon the metabolisation by liver enzymes.

Enhanced preferential cytotoxicity through surface modification: synthesis, characterization and comparative in vitro evaluation of TritonX-100 modified and unmodified zinc oxide nanoparticles in human breast cancer cell (MDA-MB-231).

PubMed

Kc, Biplab; Paudel, Siddhi Nath; Rayamajhi, Sagar; Karna, Deepak; Adhikari, Sandeep; Shrestha, Bhupal G; Bisht, Gunjan

2016-01-01

Nanoparticles (NPs) are receiving increasing interest in biomedical research owing to their comparable size with biomolecules, novel properties and easy surface engineering for targeted therapy, drug delivery and selective treatment making them a better substituent against traditional therapeutic agents. ZnO NPs, despite other applications, also show selective anticancer property which makes it good option over other metal oxide NPs. ZnO NPs were synthesized by chemical precipitation technique, and then surface modified using Triton X-100. Comparative study of cytotoxicity of these modified and unmodified NPs on breast cancer cell line (MDA-MB-231) and normal cell line (NIH 3T3) were carried out. ZnO NPsof average size 18.67 ± 2.2 nm and Triton-X modified ZnO NPs of size 13.45 ± 1.42 nm were synthesized and successful characterization of synthesized NPs was done by Fourier transform infrared spectroscopy (FT-IR), X-Ray diffraction (XRD), transmission electron microscopy (TEM) analysis. Surface modification of NPs was proved by FT-IR analysis whereas structure and size by XRD analysis. Morphological analysis was done by TEM. Cell viability assay showed concentration dependent cytotoxicity of ZnO NPs in breast cancer cell line (MDA-MB-231) whereas no positive correlation was found between cytotoxicity and increasing concentration of stress in normal cell line (NIH 3T3) within given concentration range. Half maximum effective concentration (EC50) value for ZnO NPs was found to be 38.44 µg/ml and that of modified ZnO NPs to be 55.24 µg/ml for MDA-MB-231. Crystal violet (CV) staining image showed reduction in number of viable cells in NPs treated cell lines further supporting this result. DNA fragmentation assay showed fragmented bands indicating that the mechanism of cytotoxicity is through apoptosis. Although use of surfactant decreases particle size, toxicity of modified ZnO NPs were still less than unmodified NPs on MDA-MB-231 contributed by biocompatible surface coating. Both samples show significantly less toxicity towards NIH 3T3 in concentration independent manner. But use of Triton-X, a biocompatible polymer, enhances this preferentiality effect. Since therapeutic significance should be analyzed through its comparative effect on both normal and cancer cells, possible application of biocompatible polymer modified nanoparticles as therapeutic agent holds better promise.Graphical abstractSurface coating, characterization and comparative in vitro cytotoxicity study on MDA-MB 231 and NIH 3T3 of ZnO NPs showing enhanced preferentiality by biocompatible surface modification.

In vitro antibacterial activity of poly (amidoamine)-G7 dendrimer.

PubMed

Gholami, Mitra; Mohammadi, Rashin; Arzanlou, Mohsen; Akbari Dourbash, Fakhraddin; Kouhsari, Ebrahim; Majidi, Gharib; Mohseni, Seyed Mohsen; Nazari, Shahram

2017-06-05

Nano-scale dendrimers are synthetic macromolecules that frequently used in medical and health field. Traditional anibiotics are induce bacterial resistence so there is an urgent need for novel antibacterial drug invention. In the present study seventh generation poly (amidoamine) (PAMAM-G7) dendrimer was synthesized and its antibacterial activities were evaluated against representative Gram- negative and Gram-positive bacteria. PAMAM-G7 was synthesized with divergent growth method. The structural and surface of PAMAM-G7 were investigated by transmission electron microscopy, scanning electron microscope and fourier transform infrared. Pseudomonas. aeruginosa (n = 15), E. coli (n = 15), Acinetobacter baumanni (n = 15), Shigella dysenteriae (n = 15), Klebsiella pneumoniae (n = 10), Proteus mirabilis (n = 15), Staphylococcus aureus (n = 15) and Bacillus subtilis (n = 10) have been used for antibacterial activity assay. Additionally, representative standard strains for each bacterium were included. Minimum Inhibitory Concentration (MIC) was determined using microdilution method. Subsequently, Minimum Bactericidal Concentration (MBC) was determined by sub-culturing each of the no growth wells onto Mueller Hinton agar medium. The cytotoxicity of PAMAM-G7 dendrimer were evaluated in HCT116 and NIH 3 T3 cells by MTT assay. The average size of each particle was approximately 20 nm. PAMAM-G7 was potentially to inhibit both Gram positive and gram negative growth. The MIC50 and MIC90 values were determined to be 2-4 μg/ml and 4-8 μg/ml, respectively. The MBC50 and MBC90 values were found to be 64-256 μg/ml and 128-256 μg/ml, respectively. The cytotoxity effect of dendrimer on HCT116 and NIH 3 T3 cells is dependent upon exposure time to and concentration of dendrimers. The most reduction (44.63 and 43%) in cell viability for HCT116 and NIH 3 T3 cells was observed at the highest concentration, 0.85 μM after 72 h treatmentm, respectively. This study we conclude that PAMAM-G7 dendrimer could be a potential candidate as a novel antibacterial agent.

Gene expression profiling defined pathways correlated with fibroblast cell proliferation induced by Opisthorchis viverrini excretory/secretory product.

PubMed

Thuwajit, Chanitra; Thuwajit, Peti; Uchida, Kazuhiko; Daorueang, Daoyot; Kaewkes, Sasithorn; Wongkham, Sopit; Miwa, Masanao

2006-06-14

To investigate the mechanism of fibroblast cell proliferation stimulated by the Opisthorchis viverrini excretory/secretory (ES) product. NIH-3T3, mouse fibroblast cells were treated with O. viverrini ES product by non-contact co-cultured with the adult parasites. Total RNA from NIH-3T3 treated and untreated with O. viverrini was extracted, reverse transcribed and hybridized with the mouse 15K complementary DNA (cDNA) array. The result was analyzed by ArrayVision version 5 and GeneSpring version 5 softwares. After normalization, the ratios of gene expression of parasite treated to untreated NIH-3T3 cells of 2-and more-fold upregulated was defined as the differentially expressed genes. The expression levels of the signal transduction genes were validated by semi-quantitative SYBR-based real-time RT-PCR. Among a total of 15,000 genes/ESTs, 239 genes with established cell proliferation-related function were 2 fold-and more-up-regulated by O. viverrini ES product compared to those in cells without exposure to the parasitic product. These genes were classified into groups including energy and metabolism, signal transduction, protein synthesis and translation, matrix and structural protein, transcription control, cell cycle and DNA replication. Moreover, the expressions of serine-threonine kinase receptor, receptor tyrosine kinase and collagen production-related genes were up-regulated by O. viverrini ES product. The expression level of signal transduction genes; pkC, pdgfr alpha, jak 1, eps 8, tgf beta 1i4, strap and h ras measured by real-time RT-PCR confirmed their expression levels to those obtained from cDNA array. However, only the up-regulated expression of pkC, eps 8 and tgfbeta 1i4 which are the downstream signaling molecules of either epidermal growth factor (EGF) or transforming growth factor-beta (TGF-beta) showed statistical significance (P < 0.05). O. viverrini ES product stimulates the significant changes of gene expression in several functional categories and these mainly include transcripts related to cell proliferation. The TGF-beta and EGF signal transduction pathways are indicated as the possible pathways of O. viverrini-driven cell proliferation.

Reconstituted human gingival epithelium: nonsubmerged in vitro model.

PubMed

Delcourt-Huard, A; Corlu, A; Joffre, A; Magloire, H; Bonnaure-Mallet, M

1997-01-01

Many studies have shown that human gingival keratinocytes grown in submerged culture fail to attain optimal differentiation. This study reports an in vitro culture system for oral gingival epithelial cells, in which they are grown at the air-liquid interface, on polycarbonate inserts, in the presence of an NIH-3T3 feeder layer. This model was compared with two submerged culture methods for gingival keratinocytes, on type 1 collagen gel and on an NIH-3T3 feeder layer. Transmission electron microscopy showed an advanced level of stratification (over six layers of cells) for cultures grown at the air-liquid interface. Immunofluorescence and electrophoretic patterns showed the presence of cytokeratins 10 and 11 in cytoskeletal protein extracts of these cultured keratinocytes. In this air-liquid interface culture model, in the presence of NIH-3T3 feeder cells, keratinocytes can achieve an advanced level of stratification and differentiation and a resemblance to in vivo gingiva. The obtention of a highly differentiated epithelium will permit in vitro pharmacological studies and studies on the biocompatability of certain alloys with the superficial periodontium; it will also provide grafts for patients undergoing periodontal surgery.

[Effects of methyl tertiary butyl ether on cell cycle and cell apoptosis].

PubMed

Zhou, W; Huang, G; Zhang, H; Ye, S

2000-07-01

To explore the effects of the new gasoline additive, methyl tertiary butyl ether (MTBE) on cell cycle and cell apoptosis. Flow cytometry was used to evaluate the effect of MTBE (1, 2, 4 microl/ml, 24 h) on NIH/3T3 cell cycles; and the effect of MTBE on Hela cell apoptosis was evaluated by detecting cell survival using crystal violet staining. Flow cytometry showed that MTBE could change NIH/3T3 cell cycles, decrease the number of cells in S stage, and arrest cells at G(2) + M stage. The results suggested that MTBE could affect NIH/3T3 cell cycles and induce cell proliferation. This situation existed 48 hours after the treatment, and cell cycles came back normal 96 hours after the treatment. By detecting cell survival using crystal violet staining, we found that MTBE could inhibit the apoptosis of Hela cells which was induced by tumor necrosis factor (TNF)alpha and cycloheximide. MTBE's carcinogenicity to animals may relate to induction of cell proliferation and inhibition of cell apoptosis.

[The relationship of the saturation density of multilayer cell cultures to their mass exchange with the medium].

PubMed

Akatov, V S; Lavrovskaia, V P

1991-01-01

Chinese hamster fibroblasts (CHF) and NIH 3T3 cells were cultured on a glass substrate at different distances from the porous membrane separating the cells from the perfusing medium. It is shown that with perfusion of medium above the membrane there is no movement of the medium near the cells. In both the types of culture, the cells grow in multilayers, however the multilayer character of growth in CHF is more pronounced than in NIH 3T3 cells. The saturation density of the cultures depends on the cell-membrane separation, and at separations of no more than 0.2 mm exceeds the saturation density in the monolayer by 8-10 fold. The dependences of the saturation density on separation are different for CHE and NIH 3T3 cells, indicating qualitative differences in the inhibition of cell growth in monolayers between these cultures. The results obtained indicate that the inhibition of cell growth in monolayer is due to mass exchange limitations, rather than to intercellular contact interactions.

Modafinil inhibits K(Ca)3.1 currents and muscle contraction via a cAMP-dependent mechanism.

PubMed

Choi, Shinkyu; Kim, Moon Young; Joo, Ka Young; Park, Seonghee; Kim, Ji Aee; Jung, Jae-Chul; Oh, Seikwan; Suh, Suk Hyo

2012-07-01

Modafinil has been used as a psychostimulant for the treatment of narcolepsy. However, its primary mechanism of action remains elusive. Therefore, we examined the effects of modafinil on K(Ca)3.1 channels and vascular smooth muscle contraction. K(Ca)3.1 currents and channel activity were measured using a voltage-clamp technique and inside-out patches in mouse embryonic fibroblast cell line, NIH-3T3 fibroblasts. Intracellular adenosine 3',5'-cyclic monophosphate (cAMP) concentration was measured, and the phosphorylation of K(Ca)3.1 channel protein was examined using western blotting in NIH-3T3 fibroblasts and/or primary cultured mouse aortic smooth muscle cells (SMCs). Muscle contractions were recorded from mouse aorta and rat pulmonary artery by using a myograph developed in-house. Modafinil was found to inhibit K(Ca)3.1 currents in a concentration-dependent manner, and the half-maximal inhibition (IC(50)) of modafinil for the current inhibition was 6.8 ± 0.7 nM. The protein kinase A (PKA) activator forskolin also inhibited K(Ca)3.1 currents. The inhibitory effects of modafinil and forskolin on K(Ca)3.1 currents were blocked by the PKA inhibitors PKI(14-22) or H-89. In addition, modafinil relaxed blood vessels (mouse aorta and rat pulmonary artery) in a concentration-dependent manner. Modafinil increased cAMP concentrations in NIH-3T3 fibroblasts or primary cultured mouse aortic SMCs and phosphorylated K(Ca)3.1 channel protein in NIH-3T3 fibroblasts. However, open probability and single-channel current amplitudes of K(Ca)3.1 channels were not changed by modafinil. From these results, we conclude that modafinil inhibits K(Ca)3.1 channels and vascular smooth muscle contraction by cAMP-dependent phosphorylation, suggesting that modafinil can be used as a cAMP-dependent K(Ca)3.1 channel blocker and vasodilator. Copyright © 2012 Elsevier Ltd. All rights reserved.

Activation of K-ras by codon 13 mutations in C57BL/6 X C3H F1 mouse tumors induced by exposure to 1,3-butadiene.

PubMed

Goodrow, T; Reynolds, S; Maronpot, R; Anderson, M

1990-08-01

1,3-Butadiene has been detected in urban air, gasoline vapors, and cigarette smoke. It has been estimated that 65,000 workers are exposed to this chemical in occupational settings in the United States. Lymphomas, lung, and liver tumors were induced in female and male C57BL/6 X C3H F1 (hereafter called B6C3F1) mice by inhalation of 6.25 to 625 ppm 1,3-butadiene for 1 to 2 years. The objective of this study was to examine these tumors for the presence of activated protooncogenes by the NIH 3T3 transfection and nude mouse tumorigenicity assays. Transfection of DNA isolated from 7 of 9 lung tumors and 7 of 12 liver tumors induced morphological transformation of NIH 3T3 cells. Southern blot analysis indicated that the transformation induced by 6 lung and 3 liver tumor DNA samples was due to transfer of a K-ras oncogene. Four of the 7 liver tumors that were positive upon transfection contained an activated H-ras gene. The identity of the transforming gene in one of the lung tumors has not been determined but was not a member of the ras family or a met or raf gene. Eleven 1,3-butadiene-induced lymphomas were examined for transforming genes using the nude mouse tumorigenicity assay. Activated K-ras genes were detected in 2 of the 11 lymphomas assayed. DNA sequencing of polymerase chain reaction-amplified ras gene exons revealed that 9 of 11 of the activating K-ras mutations were G to C transversions in codon 13. One liver tumor contained an activated K-ras gene with mutations in both codons 60 and 61. The activating mutation in one of the K-ras genes from a lymphoma was not identified but DNA sequence analysis of amplified regions in proximity to codons 12, 13, and 61 demonstrated that the mutation was not located in or near these codons. Activation of K-ras genes by codon 13 mutations has not been found in any lung or liver tumors or lymphomas from untreated B6C3F1 mice. Thus, the K-ras activation found in 1,3-butadiene-induced B6C3F1 mouse tumors probably occurred as a result of genotoxic effects of this chemical. The oncogenes most frequently detected in human pulmonary adenocarcinomas are K-ras genes. Activated K-ras genes have also been found in some human lymphomas. This suggest that activation of K-ras may be important in the induction of human pulmonary adenocarcinomas and lymphomas.(ABSTRACT TRUNCATED AT 400 WORDS)

Ultrasound-mediated structural changes in cells revealed by FTIR spectroscopy: a contribution to the optimization of gene and drug delivery.

PubMed

Grimaldi, Paola; Di Giambattista, Lucia; Giordani, Serena; Udroiu, Ion; Pozzi, Deleana; Gaudenzi, Silvia; Bedini, Angelico; Giliberti, Claudia; Palomba, Raffaele; Castellano, Agostina Congiu

2011-12-15

Ultrasound effects on biological samples are gaining a growing interest concerning in particular, the intracellular delivery of drugs and genes in a safe and in a efficient way. Future progress in this field will require a better understanding of how ultrasound and acoustic cavitation affect the biological system properties. The morphological changes of cells due to ultrasound (US) exposure have been extensively studied, while little attention has been given to the cells structural changes. We have exposed two different cell lines to 1 MHz frequency ultrasound currently used in therapy, Jurkat T-lymphocytes and NIH-3T3 fibroblasts, both employed as models respectively in the apoptosis and in the gene therapy studies. The Fourier Transform Infrared (FTIR) Spectroscopy was used as probe to reveal the structural changes in particular molecular groups belonging to the main biological systems. The genotoxic damage of cells exposed to ultrasound was ascertained by the Cytokinesis-Block Micronucleus (CBMN) assay. The FTIR spectroscopy results, combined with multivariate statistical analysis, regarding all cellular components (lipids, proteins, nucleic acids) of the two cell lines, show that Jurkat cells are more sensitive to therapeutic ultrasound in the lipid and protein regions, whereas the NIH-3T3 cells are more sensitive in the nucleic acids region; a meaningful genotoxic effect is present in both cell lines only for long sonication times while in the Jurkat cells also a significant cytotoxic effect is revealed for long times of exposure to ultrasound. Copyright © 2011 Elsevier B.V. All rights reserved.

The proto-oncogene product c-Crk associates with insulin receptor substrate-1 and 4PS. Modulation by insulin growth factor-I (IGF) and enhanced IGF-I signaling.

PubMed

Beitner-Johnson, D; Blakesley, V A; Shen-Orr, Z; Jimenez, M; Stannard, B; Wang, L M; Pierce, J; LeRoith, D

1996-04-19

The Crk proto-oncogene product is an SH2 and SH3 domain-containing adaptor protein which we have previously shown to become rapidly tyrosine phosphorylated in response to stimulation with insulin-like growth factor I (IGF-I) in NIH-3T3 cells. In order to further characterize the role of Crk in the IGF-I signaling pathway, NIH-3T3 and 293 cells were stably transfected with an expression vector containing the Crk cDNA. The various resultant 3T3-Crk clones expressed Crk at approximately 2-15-fold higher levels than parental 3T3 cells. In 3T3-Crk cells, Crk immunoreactivity was detected in insulin receptor substrate-1 (IRS-1) immunoprecipitates. Stimulation with IGF-I resulted in a dissociation of Crk protein from IRS-1. In contrast, the association of the related adaptor protein Grb2 with IRS-1 was enhanced by IGF-I stimulation. Similar results were obtained in stably transfected 293-Crk cells, which express both IRS-1 and the IRS-1-related signaling protein 4PS. In these cells, IRS-1 and 4PS both associated with Crk, and this association was also decreased by IGF-I treatment, whereas the association of Grb2 with IRS-1 and 4PS was enhanced by IGF-I. Overexpression of Crk also enhanced IGF-I-induced mitogenesis of NIH-3T3 cells, as measured by [3H]thymidine incorporation. The levels of IGF-I-induced mitogenesis were proportional to the level of Crk expression. These results suggest that Crk is a positive effector of IGF-I signaling, and may mediate its effects via interaction with IRS-1 and/or 4PS.

Cytoskeletal stiffness, friction, and fluidity of cancer cell lines with different metastatic potential.

PubMed

Coughlin, Mark F; Bielenberg, Diane R; Lenormand, Guillaume; Marinkovic, Marina; Waghorne, Carol G; Zetter, Bruce R; Fredberg, Jeffrey J

2013-03-01

We quantified mechanical properties of cancer cells differing in metastatic potential. These cells included normal and H-ras-transformed NIH3T3 fibroblast cells, normal and oncoprotein-overexpressing MCF10A breast cancer cells, and weakly and strongly metastatic cancer cell line pairs originating from human cancers of the skin (A375P and A375SM cells), kidney (SN12C and SN12PM6 cells), prostate (PC3M and PC3MLN4 cells), and bladder (253J and 253JB5 cells). Using magnetic twisting cytometry, cytoskeletal stiffness (g') and internal friction (g″) were measured over a wide frequency range. The dependencies of g' and g″ upon frequency were used to determine the power law exponent x which is a direct measure of cytoskeletal fluidity and quantifies where the cytoskeleton resides along the spectrum of solid-like (x = 1) to fluid-like (x = 2) states. Cytoskeletal fluidity x increased following transformation by H-ras oncogene expression in NIH3T3 cells, overexpression of ErbB2 and 14-3-3-ζ in MCF10A cells, and implantation and growth of PC3M and 253J cells in the prostate and bladder, respectively. Each of these perturbations that had previously been shown to enhance cancer cell motility and invasion are shown here to shift the cytoskeleton towards a more fluid-like state. In contrast, strongly metastatic A375SM and SN12PM6 cells that disseminate by lodging in the microcirculation of peripheral organs had smaller x than did their weakly metastatic cell line pairs A375P and SN12C, respectively. Thus, enhanced hematological dissemination was associated with decreased x and a shift towards a more solid-like cytoskeleton. Taken together, these results are consistent with the notion that adaptations known to enhance metastatic ability in cancer cell lines define a spectrum of fluid-like versus solid-like states, and the position of the cancer cell within this spectrum may be a determinant of cancer progression.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Soeda, Shuhei; Nakayama, Yuji, E-mail: nakayama@mb.kyoto-phu.ac.jp; Department of Biochemistry and Molecular Biology, Kyoto Pharmaceutical University, 5 Nakauchi-cho, Misasagi, Yamashina-ku, Kyoto 607-8414

Src-family tyrosine kinases are aberrantly activated in cancers, and this activation is associated with malignant tumor progression. v-Src, encoded by the v-src transforming gene of the Rous sarcoma virus, is a mutant variant of the cellular proto-oncogene c-Src. Although investigations with temperature sensitive mutants of v-Src have shown that v-Src induces many oncogenic processes, the effects on cell division are unknown. Here, we show that v-Src inhibits cellular proliferation of HCT116, HeLa S3 and NIH3T3 cells. Flow cytometry analysis indicated that inducible expression of v-Src results in an accumulation of 4N cells. Time-lapse analysis revealed that binucleation is induced throughmore » the inhibition of cytokinesis, a final step of cell division. The localization of Mklp1, which is essential for cytokinesis, to the spindle midzone is inhibited in v-Src-expressing cells. Intriguingly, Aurora B, which regulates Mklp1 localization at the midzone, is delocalized from the spindle midzone and the midbody but not from the metaphase chromosomes upon v-Src expression. Mklp2, which is responsible for the relocation of Aurora B from the metaphase chromosomes to the spindle midzone, is also lost from the spindle midzone. These results suggest that v-Src inhibits cytokinesis through the delocalization of Mklp1 and Aurora B from the spindle midzone, resulting in binucleation. -- Highlights: • v-Src inhibits cell proliferation of HCT116, HeLa S3 and NIH3T3 cells. • v-Src induces binucleation together with cytokinesis failure. • v-Src causes delocalization of Mklp1, Aurora B and INCENP from the spindle midzone.« less

Accumulation and Toxicity of Superparamagnetic Iron Oxide Nanoparticles in Cells and Experimental Animals

PubMed Central

Jarockyte, Greta; Daugelaite, Egle; Stasys, Marius; Statkute, Urte; Poderys, Vilius; Tseng, Ting-Chen; Hsu, Shan-Hui; Karabanovas, Vitalijus; Rotomskis, Ricardas

2016-01-01

The uptake and distribution of negatively charged superparamagnetic iron oxide (Fe3O4) nanoparticles (SPIONs) in mouse embryonic fibroblasts NIH3T3, and magnetic resonance imaging (MRI) signal influenced by SPIONs injected into experimental animals, were visualized and investigated. Cellular uptake and distribution of the SPIONs in NIH3T3 after staining with Prussian Blue were investigated by a bright-field microscope equipped with digital color camera. SPIONs were localized in vesicles, mostly placed near the nucleus. Toxicity of SPION nanoparticles tested with cell viability assay (XTT) was estimated. The viability of NIH3T3 cells remains approximately 95% within 3–24 h of incubation, and only a slight decrease of viability was observed after 48 h of incubation. MRI studies on Wistar rats using a clinical 1.5 T MRI scanner were showing that SPIONs give a negative contrast in the MRI. The dynamic MRI measurements of the SPION clearance from the injection site shows that SPIONs slowly disappear from injection sites and only a low concentration of nanoparticles was completely eliminated within three weeks. No functionalized SPIONs accumulate in cells by endocytic mechanism, none accumulate in the nucleus, and none are toxic at a desirable concentration. Therefore, they could be used as a dual imaging agent: as contrast agents for MRI and for traditional optical biopsy by using Prussian Blue staining. PMID:27548152

Single-particle tracking of endocytosis and exocytosis of single-walled carbon nanotubes in NIH-3T3 cells.

PubMed

Jin, Hong; Heller, Daniel A; Strano, Michael S

2008-06-01

Over 10000 individual trajectories of nonphotobleaching single-walled carbon nanotubes (SWNT) were tracked as they are incorporated into and expelled from NIH-3T3 cells in real time on a perfusion microscope stage. An analysis of mean square displacement allows the complete construction of the mechanistic steps involved from single duration experiments. We observe the first conclusive evidence of SWNT exocytosis and show that the rate closely matches the endocytosis rate with negligible temporal offset. We identify and study the endocytosis and exocytosis pathway that leads to the previously observed aggregation and accumulation of SWNT within the cells.

Accumulation and Toxicity of Superparamagnetic Iron Oxide Nanoparticles in Cells and Experimental Animals.

PubMed

Jarockyte, Greta; Daugelaite, Egle; Stasys, Marius; Statkute, Urte; Poderys, Vilius; Tseng, Ting-Chen; Hsu, Shan-Hui; Karabanovas, Vitalijus; Rotomskis, Ricardas

2016-08-19

The uptake and distribution of negatively charged superparamagnetic iron oxide (Fe₃O₄) nanoparticles (SPIONs) in mouse embryonic fibroblasts NIH3T3, and magnetic resonance imaging (MRI) signal influenced by SPIONs injected into experimental animals, were visualized and investigated. Cellular uptake and distribution of the SPIONs in NIH3T3 after staining with Prussian Blue were investigated by a bright-field microscope equipped with digital color camera. SPIONs were localized in vesicles, mostly placed near the nucleus. Toxicity of SPION nanoparticles tested with cell viability assay (XTT) was estimated. The viability of NIH3T3 cells remains approximately 95% within 3-24 h of incubation, and only a slight decrease of viability was observed after 48 h of incubation. MRI studies on Wistar rats using a clinical 1.5 T MRI scanner were showing that SPIONs give a negative contrast in the MRI. The dynamic MRI measurements of the SPION clearance from the injection site shows that SPIONs slowly disappear from injection sites and only a low concentration of nanoparticles was completely eliminated within three weeks. No functionalized SPIONs accumulate in cells by endocytic mechanism, none accumulate in the nucleus, and none are toxic at a desirable concentration. Therefore, they could be used as a dual imaging agent: as contrast agents for MRI and for traditional optical biopsy by using Prussian Blue staining.

[Regulation of [12Asp]K-ras4B on transcriptional activity of estrogen receptor in endometrial carcinoma HEC-1A cell lines].

PubMed

Gui, Li-ming; Wei, Li-hui; Xu, Ming-xu; Wang, Jian-liu; Zhong, Ying-cheng; Li, Xiao-ping; Tu, Zheng; Sun, Peng-ming; Ma, Da-long

2004-01-01

To investigate the effect of mutant-type [(12)Asp]K-ras4B gene on the expression of estrogen receptor (ER) alpha and beta and their transcriptional activity as a transcription factor in endometrial carcinoma HEC-1A cell line. (1) Effect of [(12)Asp]K-ras4B on the expression of ER alpha and beta were determined using Western blot assay. (2) Eukaryotic expression plasmid pGL3-luciferase-ERE containing luciferase report gene and estrogen receptor element (ERE) was constructed, and co-transfected into NIH3T3 and HEC-1A cell lines with pEGFP-N1 to examine the effect of [(12)Asp]K-ras4B on ER transcription that is regulated by estradiol. In addition, they were transfected into pSV5-HER0 (containing full length wide type ERalpha cDNA) and pCMV-rafS621A (inhibiting raf kinase) plasmids to test the effect of [(12)Asp]K-ras4B/raf signal pathway on transcriptional activity of ER proteins. (1) Protein level of ERs expressed in pcDI transfected control cells was low while it was increased for 3.6-fold (97 +/- 25, 349 +/- 67, P < 0.01) and 1.9-fold (128 +/- 37, 349 +/- 30, P < 0.05) in ERalpha and ERbeta, respectively, in pcDI-[(12)Asp]K-ras4B NIH3T3 cells after transfection. (2) In pcDI-[(12)Asp]K-ras4B NIH3T3 cells, the ratios for ERalpha and and ERbeta levels before transfection of rafS621A plasmids to that after the transfection, were 2.4:1 (724 +/- 45, 310 +/- 46, P < 0.05) and 1.8:1 (493 +/- 20, 284 +/- 20, P < 0.01), respectively; In HEC-1A cells, these ratios were 2.1:1 (566 +/- 22, 279 +/- 30, P < 0.01) and 2.4:1 (405 +/- 33, 165 +/- 15, P < 0.01), respectively. (3) In low serum (2%) culture condition, estradiol (E(2)) stimulated luciferase activity with an increase of 13-fold (130 +/- 42, 1681 +/- 242, P < 0.01) in pcDI-[(12)Asp] K-ras4B NIH3T3 cells, 19-fold (141 +/- 39, 2644 +/- 331, P < 0.001) in HEC-1A cells, respectively, when compared with those in the absence of E(2). (4) In pSV5-HER0 transfected pcDI-[(12)Asp] K-ras4B NIH3T3 cells and HEC-1A cells, compared to the untransfected cells, the ER transcriptional activity in the transfected cells increased markedly. The luciferase activity was increased for 8-fold (1048 +/- 91, 8099 +/- 452, P < 0.01) and 6-fold (2148 +/- 259, 12,705 +/- 2670, P < 0.001), respectively. rafS621A mutant had suppressive effects on luciferase activities in HEC-1A cells and pcDI-[(12)Asp]K-ras4B NIH3T3 cells. The ratio of luciferase activities in pcDI-[(12)Asp]K-ras4B NIH3T3 and HEC-1A cells, before and after transfection was 7.8:1 (1184 +/- 168, 152 +/- 27, P < 0.05) and 6.4:1 (1949 +/- 212, 304 +/- 60, P < 0.01), respectively. (1) [(12)Asp]K-ras4B can enhance the expression of ERalpha and beta proteins. This may be correlated with [(12)Asp]K-ras4B/raf signaling pathway. (2) The effect of mutant-type [(12)Asp]K-ras4B gene on ERs transcriptional activity in HEC-1A cells appears to need E(2).

Effect of hyperbaric oxygen on BDNF-release and neuroprotection: Investigations with human mesenchymal stem cells and genetically modified NIH3T3 fibroblasts as putative cell therapeutics.

PubMed

Schulze, Jennifer; Kaiser, Odett; Paasche, Gerrit; Lamm, Hans; Pich, Andreas; Hoffmann, Andrea; Lenarz, Thomas; Warnecke, Athanasia

2017-01-01

Hyperbaric oxygen therapy (HBOT) is a noninvasive widely applied treatment that increases the oxygen pressure in tissues. In cochlear implant (CI) research, intracochlear application of neurotrophic factors (NTFs) is able to improve survival of spiral ganglion neurons (SGN) after deafness. Cell-based delivery of NTFs such as brain-derived neurotrophic factor (BDNF) may be realized by cell-coating of the surface of the CI electrode. Human mesenchymal stem cells (MSC) secrete a variety of different neurotrophic factors and may be used for the development of a biohybrid electrode in order to release endogenously-derived neuroprotective factors for the protection of residual SGN and for a guided outgrowth of dendrites in the direction of the CI electrode. HBOT could be used to influence cell behaviour after transplantation to the inner ear. The aim of this study was to investigate the effect of HBOT on the proliferation, BDNF-release and secretion of neuroprotective factors. Thus, model cells (an immortalized fibroblast cell line (NIH3T3)-native and genetically modified) and MSCs were repeatedly (3 x - 10 x) exposed to 100% oxygen at different pressures. The effects of HBO on cell proliferation were investigated in relation to normoxic and normobaric conditions (NOR). Moreover, the neuroprotective and neuroregenerative effects of HBO-treated cells were analysed by cultivation of SGN in conditioned medium. Both, the genetically modified NIH3T3/BDNF and native NIH3T3 fibroblasts, showed a highly significant increased proliferation after five days of HBOT in comparison to normoxic controls. By contrast, the number of MSCs was decreased in MSCs treated with 2.0 bar of HBO. Treating SGN cultures with supernatants of fibroblasts and MSCs significantly increased the survival rate of SGN. HBO treatment did not influence (increase / reduce) this effect. Secretome analysis showed that HBO treatment altered the protein expression pattern in MSCs.

High Prevalence of Polycystic Ovary Syndrome in Type 1 Diabetes Mellitus Adolescents: Is There a Difference Depending on the NIH and Rotterdam Criteria?

PubMed

Busiah, Kanetee; Colmenares, Ana; Bidet, Maud; Tubiana-Rufi, Nadia; Levy-Marchal, Claire; Delcroix, Christine; Jacquin, Paul; Martin, Delphine; Benadjaoud, Lila; Jacqz-Aigrain, Evelyne; Laborde, Kathleen; Robert, Jean-Jacques; Samara-Boustani, Dinane; Polak, Michel

2017-01-01

Polycystic ovary syndrome (PCOS) is more frequently observed in type 1 diabetes mellitus (T1DM) adult women than in nondiabetic women. No such prevalence has yet been studied in adolescent girls with T1DM. The aim of this study was to evaluate the prevalence of PCOS in adolescent girls with T1DM and to determine the clinical and hormonal features associated with the disorder. A cross-sectional study of 53 adolescent girls (gynecological age >2 years) referred for routine evaluation for T1DM was conducted. We diagnosed PCOS using the National Institutes of Health (NIH) and Rotterdam criteria. 26.4 and 47.9% of adolescents had PCOS according to NIH (NIH-PCOS) and Rotterdam (Rotterdam-PCOS) criteria. 66.7% of NIH-PCOS adolescents had a complete phenotype associated with hyperandrogenism, oligomenorrhea, and polycystic ovarian morphology, unlike only 33.3% of the Rotterdam-PCOS adolescents. A family history of type 2 diabetes mellitus (T2DM) was more frequent in PCOS than in non-PCOS girls, whichever criteria were used. Late pubertal development and a T1DM diagnosis close to puberty were factors associated with NIH-PCOS. Adolescents with T1DM had a high prevalence of PCOS. More differences between PCOS and non-PCOS patients were found using the NIH criteria, suggesting that clinical characteristics might be more accurate for diagnosing PCOS in girls with T1DM. A family history of T2DM is associated with a high risk of PCOS. © 2017 S. Karger AG, Basel.

Three dimensional spheroid cell culture for nanoparticle safety testing.

PubMed

Sambale, Franziska; Lavrentieva, Antonina; Stahl, Frank; Blume, Cornelia; Stiesch, Meike; Kasper, Cornelia; Bahnemann, Detlef; Scheper, Thomas

2015-07-10

Nanoparticles are widely employed for many applications and the number of consumer products, incorporating nanotechnology, is constantly increasing. A novel area of nanotechnology is the application in medical implants. The widespread use of nanoparticles leads to their higher prevalence in our environment. This, in turn, raises concerns regarding potential risks to humans. Previous studies have shown possible hazardous effects of some nanoparticles on mammalian cells grown in two-dimensional (2D) cultures. However, 2D in vitro cell cultures display several disadvantages such as changes in cell shape, cell function, cell responses and lack of cell-cell contacts. For this reason, the development of better models for mimicking in vivo conditions is essential. In the present work, we cultivated A549 cells and NIH-3T3 cells in three-dimensional (3D) spheroids and investigated the effects of zinc oxide (ZnO-NP) and titanium dioxide nanoparticles (TiO2-NP). The results were compared to cultivation in 2D monolayer culture. A549 cells in 3D cell culture formed loose aggregates which were more sensitive to the toxicity of ZnO-NP in comparison to cells grown in 2D monolayers. In contrast, NIH-3T3 cells showed a compact 3D spheroid structure and no differences in the sensitivity of the NIH-3T3 cells to ZnO-NP were observed between 2D and 3D cultures. TiO2-NP were non-toxic in 2D cultures but affected cell-cell interaction during 3D spheroid formation of A549 and NIH-3T3 cells. When TiO2-NP were directly added during spheroid formation in the cultures of the two cell lines tested, several smaller spheroids were formed instead of a single spheroid. This effect was not observed if the nanoparticles were added after spheroid formation. In this case, a slight decrease in cell viability was determined only for A549 3D spheroids. The obtained results demonstrate the importance of 3D cell culture studies for nanoparticle safety testing, since some effects cannot be revealed in 2D cell culture. Copyright © 2015 Elsevier B.V. All rights reserved.

A transfected cell model for the renal toxin transporter, rOCT2.

PubMed

Pan, B F; Sweet, D H; Pritchard, J B; Chen, R; Nelson, J A

1999-02-01

A cDNA for the organic cation transporter (rOCT2) of the rat kidney was inserted into the retroviral plasmid pLXSN. This plasmid was used to stably transfect NIH3T3 cells. The transfected cell line exhibited an enhanced rate of tetraethylammonium (TEA) uptake and efflux compared to wild-type NIH3T3 cells. Uptake of TEA by the transfected cells was markedly reduced upon incubation at 4 degrees C. When the extracellular pH was lowered from 8.1 to 5.9, uptake was also reduced, suggesting inhibition of rOCT2 by extracellular protons. The apparent K(m) for TEA in the transfected cells was 141 microM. The classical organic cation transport inhibitors, cyanine 863 and cimetidine, produced noncompetitive inhibition with apparent Ki values of 0.81 and 198 microM, respectively. Daunomycin, vinblastine, and the deoxyadenosine analogs, 2'-deoxytubercidin and 2-chlorodeoxyadenosine, did not appear to be substrates for rOCT2. However, the anticancer drug, cisplatin, competitively inhibited TEA uptake by rOCT2 with an apparent Ki value of 925 microM, suggesting that rOCT2 may play a role in its renal secretion. In summary, transfected NIH3T3 cells provide a facile system by which this and other organic ion transporters can be studied.

Nanofiber Alignment Regulates NIH3T3 Cell Orientation and Cytoskeletal Gene Expression on Electrospun PCL+Gelatin Nanofibers.

PubMed

Fee, Timothy; Surianarayanan, Swetha; Downs, Crawford; Zhou, Yong; Berry, Joel

2016-01-01

To examine the influence of substrate topology on the behavior of fibroblasts, tissue engineering scaffolds were electrospun from polycaprolactone (PCL) and a blend of PCL and gelatin (PCL+Gel) to produce matrices with both random and aligned nanofibrous orientations. The addition of gelatin to the scaffold was shown to increase the hydrophilicity of the PCL matrix and to increase the proliferation of NIH3T3 cells compared to scaffolds of PCL alone. The orientation of nanofibers within the matrix did not have an effect on the proliferation of adherent cells, but cells on aligned substrates were shown to elongate and align parallel to the direction of substrate fiber alignment. A microarray of cyotoskeleton regulators was probed to examine differences in gene expression between cells grown on an aligned and randomly oriented substrates. It was found that transcriptional expression of eight genes was statistically different between the two conditions, with all of them being upregulated in the aligned condition. The proteins encoded by these genes are linked to production and polymerization of actin microfilaments, as well as focal adhesion assembly. Taken together, the data indicates NIH3T3 fibroblasts on aligned substrates align themselves parallel with their substrate and increase production of actin and focal adhesion related genes.

[Screening and identification of anoikis-resistant gene UBCH7 in esophageal cancer cells].

PubMed

Yang, Yang; Wang, Bo-Shi; Wang, Xiao-Min; Zhang, Yu; Wang, Ming-Rong; Jia, Xue-Mei

2012-02-01

Anoikis is a kind of programmed cell death induced by loss of extracellular matrix (ECM) adhesion, which is one of key factors for homestasis. Resistance to anoikis is required for tumor cell metastasis. We have previously shown several anoikis-resistance genes in esophageal squamous cell carcinoma (ESCC). In order to find novel anoikis-resistant genes in ESCC, we constructed retroviral cDNA library using total RNA from ESCC cell lines. NIH 3T3 cells, which are sensitive to anoikis, were infected with the library constructed. The cells were cultured in soft agar, and the clones which can survive in detached states were selected. The cDNAs inserted into the anoikis-resistant NIH3T3 clones were amplified using retroviral specific primers. Sequencing analysis showed that a cDNA fragment inserted into the anoikis-resistant clone contains full coding sequence (ORF) of human UBCH7/UBE2L3 gene. By infection with retrovirus encoding UBCH7 ORF (pMSCV-UBCH7), forced expression of UBCH7 increased the anoikis-resistance of NIH3T3 cells. More importantly, knockdown of UBCH7 expression by siRNA transfection reduced the anoikis-resistant ability of esophageal cancer MLuC1 cells. The data suggest that UBCH7/UBE2L3 gene would be involved in anoikis-resistance in ESCC.

Mammalian knock out cells reveal prominent roles for atlastin GTPases in ER network morphology

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhao, Guohua; Zhu, Peng-Peng; Renvoisé, Benoît

Atlastins are large, membrane-bound GTPases that participate in the fusion of endoplasmic reticulum (ER) tubules to generate the polygonal ER network in eukaryotes. They also regulate lipid droplet size and inhibit bone morphogenetic protein (BMP) signaling, though mechanisms remain unclear. Humans have three atlastins (ATL1, ATL2, and ATL3), and ATL1 and ATL3 are mutated in autosomal dominant hereditary spastic paraplegia and hereditary sensory neuropathies. Cellular investigations of atlastin orthologs in most yeast, plants, flies and worms are facilitated by the presence of a single or predominant isoform, but loss-of-function studies in mammalian cells are complicated by multiple, broadly-expressed paralogs. Wemore » have generated mouse NIH-3T3 cells lacking all three mammalian atlastins (Atl1/2/3) using CRISPR/Cas9-mediated gene knockout (KO). ER morphology is markedly disrupted in these triple KO cells, with prominent impairment in formation of three-way ER tubule junctions. This phenotype can be rescued by expression of distant orthologs from Saccharomyces cerevisiae (Sey1p) and Arabidopsis (ROOT HAIR DEFECTIVE3) as well as any one of the three human atlastins. Minimal, if any, changes are observed in the morphology of mitochondria and the Golgi apparatus. Alterations in BMP signaling and increased sensitivity to ER stress are also noted, though effects appear more modest. Finally, atlastins appear required for the proper differentiation of NIH-3T3 cells into an adipocyte-like phenotype. These findings have important implications for the pathogenesis of hereditary spastic paraplegias and sensory neuropathies associated with atlastin mutations. - Highlights: • NIH-3T3 cells lacking all three atlastin paralogs were generated using CRISPR/Cas9. • Cells lacking all atlastin GTPases exhibit far fewer 3-way ER tubule junctions. • ER morphology defects in atlastin knockout cells are rescued by distant plant and yeast orthologs. • Atlastin knock out cells also exhibit differences in ER stress and BMP signaling. • Atlastins appear important for adipocyte-like differentiation of NIH-3T3 cells.« less

Bone marrow-derived cultured mast cells and peritoneal mast cells as targets of a growth activity secreted by BALB/3T3 fibroblasts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jozaki, K.; Kuriu, A.; Hirota, S.

1991-03-01

When fibroblast cell lines were cultured in contact with bone marrow-derived cultured mast cells (CMC), both NIH/3T3 and BALB/3T3 cell lines supported the proliferation of CMC. In contrast, when contact between fibroblasts and CMC was prohibited by Biopore membranes or soft agar, only BALB/3T3 fibroblasts supported CMC proliferation, suggesting that BALB/3T3 but not NIH/3T3 cells secreted a significant amount of a mast cell growth activity. Moreover, the BALB/3T3-derived growth activity induced the incorporation of (3H)thymidine by CMC and the clonal growth of peritoneal mast cells in methylcellulose. The mast cell growth activity appeared to be different from interleukin 3 (IL-3)more » and interleukin 4 (IL-4), because mRNAs for these interleukins were not detectable in BALB/3T3 fibroblasts. Although mast cells are genetically deficient in tissues of W/Wv mice, CMC did develop when bone marrow cells of W/Wv mice were cultured with pokeweed mitogen-stimulated spleen cell-conditioned medium. Because BALB/3T3 fibroblast-conditioned medium (BALB-FCM) did not induce the incorporation of (3H)thymidine by W/Wv CMC, the growth activity in BALB-FCM appeared to be a ligand for the receptor encoded by the W (c-kit) locus. Because CMC and peritoneal mast cells are obtained as homogeneous suspensions rather easily, these cells may be potentially useful as targets for the fibroblast-derived mast cell growth activity.« less

[Development of a mouse cell line containing stably integrated copies of pMCLacI/Neo plasmid: a model for studying mutations in vitro].

PubMed

Lu, Y; Li, H; Fu, J

2000-04-01

To establish a suitable model for studying the different mechanisms of mutation between expressed and non-expressed genes in mammalian cells. The NIH3T3 cells were transfected with the linearized pMCLacI/Neo DNAs by liposome-mediated transfection, and grew in the presence of G418. One drug resistant cell clone was selected to proliferate and to be analyzed with Southern blot and RT-PCR analyses on its genomic DNAs. (1) Multiple copies of pMCLacI/Neo plasmid DNA were intactly integrated in the genomic DNAs of the cell clone. (2) One of lac I target genes in the integrated plasmid could be transcribed in the NIH3T3 cells while the other could not. (3) The pMCLacI/Neo plasmid DNA could be efficiently rescued from the genomic DNAs of the cell clone with the average rescue efficiency of 410 cfu/microg DNA. The NIH3T3 cell line containing copies of a stably integrated pMCLacI/Neo has been established. The two lacI target genes in the cell line could imitate the functional states of expressed and non-expressed genes in mammalian cells respectively. The cell line will be a useful model for studying the different mechanisms of mutation between expressed and non-expressed genes in mammalian cells.

Fluorous Peptide Nucleic Acids: PNA Analogues with Fluorine in Backbone (γ-CF2-apg-PNA) Enhance Cellular Uptake.

PubMed

Ellipilli, Satheesh; Ganesh, Krishna N

2015-09-18

Fluorous PNA analogues possessing fluorine as inherent part of aminopropylglycine (apg) backbone (γ-CF2-apg PNA) have been synthesized and evaluated for biophysical and cell penetrating properties. These form duplexes of higher thermal stability with cRNA than cDNA, although destabilized compared to duplexes of standard aeg-PNA. Cellular uptake of the fluorinated γ-CF2-apg PNAs in NIH 3T3 and HeLa cells was 2-3-fold higher compared to that of nonfluorinated apg PNA, with NIH 3T3 cells showing better permeability compared to HeLa cells. The backbone fluorinated PNAs, which are first in this class, when combined with other chemical modifications may have potential for future PNA-based antisense agents.

Oxidative changes and apoptosis induced by 1800-MHz electromagnetic radiation in NIH/3T3 cells.

PubMed

Hou, Qingxia; Wang, Minglian; Wu, Shuicai; Ma, Xuemei; An, Guangzhou; Liu, Huan; Xie, Fei

2015-03-01

To investigate the potential adverse effects of mobile phone radiation, we studied reactive oxygen species (ROS), DNA damage and apoptosis in mouse embryonic fibroblasts (NIH/3T3) after intermittent exposure (5 min on/10 min off, for various durations from 0.5 to 8 h) to an 1800-MHz GSM-talk mode electromagnetic radiation (EMR) at an average specific absorption rate of 2 W/kg. A 2',7'-dichlorofluorescin diacetate fluorescence probe was used to detect intracellular ROS levels, immunofluorescence was used to detect γH2AX foci as a marker for DNA damage, and flow cytometry was used to measure apoptosis. Our results showed a significant increase in intracellular ROS levels after EMR exposure and it reached the highest level at an exposure time of 1 h (p < 0.05) followed by a slight decrease when the exposure continued for as long as 8 h. No significant effect on the number of γH2AX was detected after EMR exposure. The percentage of late-apoptotic cells in the EMR-exposed group was significantly higher than that in the sham-exposed groups (p < 0.05). These results indicate that an 1800-MHz EMR enhances ROS formation and promotes apoptosis in NIH/3T3 cells.

Revelation of Different Nanoparticle-Uptake Behavior in Two Standard Cell Lines NIH/3T3 and A549 by Flow Cytometry and Time-Lapse Imaging

PubMed Central

Jochums, André; Friehs, Elsa; Sambale, Franziska; Lavrentieva, Antonina; Bahnemann, Detlef; Scheper, Thomas

2017-01-01

The uptake of nanomaterials into different cell types is a central pharmacological issue for the determination of nanotoxicity as well as for the development of drug delivery strategies. Most responses of the cells depend on their intracellular interactions with nanoparticles (NPs). Uptake behavior can be precisely investigated in vitro, with sensitive high throughput methods such as flow cytometry. In this study, we investigated two different standard cell lines, human lung carcinoma (A549) and mouse fibroblast (NIH/3T3) cells, regarding their uptake behavior of titanium dioxide NPs. Cells were incubated with different concentrations of TiO2 NPs and samples were taken at certain time points to compare the uptake kinetics of both cell lines. Samples were analyzed with the help of flow cytometry by studying changes in the side and forward scattering signal. To additionally enable a detection via fluorescence, NPs were labeled with the fluorescent dye fluorescein isothiocyanate (FITC) and propidium iodide (PI). We found that NIH/3T3 cells take up the studied NPs more efficiently than A549 cells. These findings were supported by time-lapse microscopic imaging of the cells incubated with TiO2 NPs. Our results confirm that the uptake behavior of individual cell types has to be considered before interpreting any results of nanomaterial studies. PMID:29051447

Transfection of Fv-1 permissive and restrictive mouse cells with integrated DNA of murine leukemia viruses

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hsu, I.C.; Yang, W.K.; Tennant, R.W.

1978-03-01

Whole-cell DNA preparations isolated from SC-1 cells chronically infected with N- or B-tropic murine leukemia viruses (MuLV) were tested for infectious activity in an Fv-I/sup n/ (NIH-3T3) and two Fv-I/sup b/ (C57BL/6 and SV-A31) cell cultures. Efficiency of transfection for all DNAs was better in the NIH-3T3 cells than in C57BL/6 or SV-A31 cells; and an (N-tropic MuLV)SC-1 cell DNA preparation was slightly more infectious than a (B-tropic MuLV)SC-1 cell DNA preparation in all three cell cultures, regardless of their Fv-1 genotypes. Progeny viruses from the transfection showed N- or B-tropism corresponding to that of the parent viruses produced bymore » the infected SC-1 cells that were used for the DNA preparation. DNA dose-response studies in NIH-3T3 cells revealed a one-hit mechanism for both the (B-tropic MuLV)SC-1 cell DNA and the (N-tropic MuLV)SC-1 cell DNA preparation. These results demonstrate that, in contrast to virion infection, transfection of N- and B-tropic MuLV with DNA preparations from chronically infected cells is not affected by the Fv-1 gene.« less

Transfection of Fv-1 permissive and restrictive mouse cells with integrated DNA of murine leukemia viruses (host range restriction/Fv-1 gene/DNA transfection)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hsu, I.C.; Yang, W.K.; Tennant, R.W.

1978-03-01

Whole-cell DNA preparations isolated from SC-1 cells chronically infected with N- or B-tropic murine leukemia viruses (MuLV) were tested for infectious activity in an Fv-1/sup n/ (NIH-3T3) and two Fv-1/sup b/ (C57BL/6 and SV-A31) cell cultures. Efficiency of transfection for all DNAs was better in the NIH-3T3 cells than in C57BL/6 or SV-A31 cells; and an (N-tropic MuLV)SC-1 cell DNA preparation was slightly more infectious than a (B-tropic MuLV)SC-1 cell DNA preparation in all three cell cultures, regardless of their Fv-1 geonotypes. Progeny viruses from the transfection showed N- or B-tropism corresponding to that of the parent viruses produced bymore » the infected SC-I cells that were used for the DNA preparation. DNA dose-response studies in NIH-3T3 cells revealed a one-hit mechanism for both the (B-tropic MuLV)SC-1 cell DNA and the (N-tropic MuLV)SC-1 cell DNA preparation. These results demonstrate that, in contrast to virion infection, transfection of N- and B-tropic MuLV with DNA preparations from chronically infected cells is not affected by the Fv-1 gene.« less

Microfabricated Nanotopological Surfaces for Study of Adhesion-dependent Cell mechanosensitivity**

PubMed Central

Chen, Weiqiang; Sun, Yubing

2014-01-01

Cells display high sensitivity and exhibit diverse responses to the intrinsic nanotopography of the extracellular matrix through their nanoscale cellular sensing machinery. Here, we reported a simple microfabrication method for precise control and spatial patterning of the local nanoroughness on glass surfaces using photolithography and reactive ion etching (RIE). Using RIE-generated nanorough glass surfaces, we demonstrated that local nanoroughness could provide a potent biophysical signal to regulate a diverse array of NIH/3T3 fibroblast behaviors, including cell morphology, adhesion, proliferation and migration. We further showed that cellular responses to nanotopography might be regulated by cell adhesion signaling and actin cytoskeleton remodeling. To further investigate the role of cytoskeleton contractility in nanoroughness sensing, we applied the RIE method to generate nanoroughness on the tops of an array of elastomeric poly-dimethylsiloxane (PDMS) microposts. We utilized the PDMS microposts as force sensors and demonstrated that nanoroughness could indeed regulate the cytoskeleton contractility of NIH/3T3 fibroblasts. Our results suggested that a feedback regulation and mechano-chemical integration mechanism involving adhesion signaling, actin cytoskeleton, and intracellular mechanosensory components might play an important role in regulating mechanosensitive behaviors of NIH/3T3 fibroblasts. The capability to control and further predict cellular responses to nanoroughness might suggest novel methods for developing biomaterials mimicking nanotopographic structures in vivo and suitable local cellular microenvironments for functional tissue engineering. PMID:22887768

Sorting of tropomyosin isoforms in synchronised NIH 3T3 fibroblasts: evidence for distinct microfilament populations.

PubMed

Percival, J M; Thomas, G; Cock, T A; Gardiner, E M; Jeffrey, P L; Lin, J J; Weinberger, R P; Gunning, P

2000-11-01

The nonmuscle actin cytoskeleton consists of multiple networks of actin microfilaments. Many of these filament systems are bound by the actin-binding protein tropomyosin (Tm). We investigated whether Tm isoforms could be cell cycle regulated during G0 and G1 phases of the cell cycle in synchronised NIH 3T3 fibroblasts. Using Tm isoform-specific antibodies, we investigated protein expression levels of specific Tms in G0 and G1 phases and whether co-expressed isoforms could be sorted into different compartments. Protein levels of Tms 1, 2, 5a, 6, from the alpha Tm(fast) and beta-Tm genes increased approximately 2-fold during mid-late G1. Tm 3 levels did not change appreciably during G1 progression. In contrast, Tm 5NM gene isoform levels (Tm 5NM-1-11) increased 2-fold at 5 h into G1 and this increase was maintained for the following 3 h. However, Tm 5NM-1 and -2 levels decreased by a factor of three during this time. Comparison of the staining of the antibodies CG3 (detects all Tm 5NM gene products), WS5/9d (detects only two Tms from the Tm 5NM gene, Tm 5NM-1 and -2) and alpha(f)9d (detects specific Tms from the alpha Tm(fast) and beta-Tm genes) antibodies revealed 3 spatially distinct microfilament systems. Tm isoforms detected by alpha(f)9d were dramatically sorted from isoforms from the Tm 5NM gene detected by CG3. Tm 5NM-1 and Tm 5NM-2 were not incorporated into stress fibres, unlike other Tm 5NM isoforms, and marked a discrete, punctate, and highly polarised compartment in NIH 3T3 fibroblasts. All microfilament systems, excluding that detected by the WS5/9d antibody, were observed to coalign into parallel stress fibres at 8 h into G1. However, Tms detected by the CG3 and alpha(f)9d antibodies were incorporated into filaments at different times indicating distinct temporal control mechanisms. Microfilaments in NIH 3T3 cells containing Tm 5NM isoforms were more resistant to cytochalasin D-mediated actin depolymerisation than filaments containing isoforms from the alpha Tm(fast) and beta-Tm genes. This suggests that Tm 5NM isoforms may be in different microfilaments to alpha Tm(fast) and beta-Tm isoforms even when present in the same stress fibre. Staining of primary mouse fibroblasts showed identical Tm sorting patterns to those seen in cultured NIH 3T3 cells. Furthermore, we demonstrate that sorting of Tms is not restricted to cultured cells and can be observed in human columnar epithelial cells in vivo. We conclude that the expression and localisation of Tm isoforms are differentially regulated in G0 and G1 phase of the cell cycle. Tms mark multiple microfilament compartments with restricted tropomyosin composition. The creation of distinct microfilament compartments by differential sorting of Tm isoforms is observable in primary fibroblasts, cultured 3T3 cells and epithelial cells in vivo. Copyright 2000 Wiley-Liss, Inc.

Genomic response to Wnt signalling is highly context-dependent - Evidence from DNA microarray and chromatin immunoprecipitation screens of Wnt/TCF targets

DOE Office of Scientific and Technical Information (OSTI.GOV)

Railo, Antti; Pajunen, Antti; Itaeranta, Petri

2009-10-01

Wnt proteins are important regulators of embryonic development, and dysregulated Wnt signalling is involved in the oncogenesis of several human cancers. Our knowledge of the downstream target genes is limited, however. We used a chromatin immunoprecipitation-based assay to isolate and characterize the actual gene segments through which Wnt-activatable transcription factors, TCFs, regulate transcription and an Affymetrix microarray analysis to study the global transcriptional response to the Wnt3a ligand. The anti-{beta}-catenin immunoprecipitation of DNA-protein complexes from mouse NIH3T3 fibroblasts expressing a fusion protein of {beta}-catenin and TCF7 resulted in the identification of 92 genes as putative TCF targets. GeneChip assays ofmore » gene expression performed on NIH3T3 cells and the rat pheochromocytoma cell line PC12 revealed 355 genes in NIH3T3 and 129 genes in the PC12 cells with marked changes in expression after Wnt3a stimulus. Only 2 Wnt-regulated genes were shared by both cell lines. Surprisingly, Disabled-2 was the only gene identified by the chromatin immunoprecipitation approach that displayed a marked change in expression in the GeneChip assay. Taken together, our approaches give an insight into the complex context-dependent nature of Wnt pathway transcriptional responses and identify Disabled-2 as a potential new direct target for Wnt signalling.« less

Reversion of autocrine transformation by a dominant negative platelet-derived growth factor mutant.

PubMed Central

Vassbotn, F S; Andersson, M; Westermark, B; Heldin, C H; Ostman, A

1993-01-01

A non-receptor-binding mutant of the platelet-derived growth factor (PDGF) A chain, PDGF-0, was generated by exchanging 7 amino acids in the sequence. The mutant chains formed dimers that were similar to wild-type PDGF-AA with regard to stability and rate of processing to the mature 30-kDa secreted forms. Moreover, the mutant chains formed disulfide-bonded heterodimers with the PDGF B chain in NIH 3T3 cells heterodimer underwent the same processing and secretion as PDGF-AB. Transfection of c-sis-expressing 3T3 cells with PDGF-0 significantly inhibited the transformed phenotype of these cells, as determined by the following criteria. (i) Compared with PDGF-0-negative clones, PDGF-0-producing clones showed a reverted morphology. (ii) Clones producing PDGF-0 grew more slowly than PDGF-0-negative clones, with a fivefold difference in cell number after 14 days in culture. (iii) The expression of PDGF-0 completely inhibited the ability of the c-sis-expressing 3T3 cells to form colonies in soft agar; this inhibition was overcome by the addition of recombinant PDGF-BB to the culture medium, showing that the lack of colony formation of these cells was not due to a general unresponsiveness to PDGF. The specific expression of a PDGF-0/PDGF wild-type heterodimer in COS cells revealed that the affinity of the mutant heterodimer for the PDGF alpha receptor was decreased by approximately 50-fold compared with that of PDGF-AA. Thus, we show that a non-receptor-binding PDGF A-chain mutant neutralizes in a trans-dominant manner the autocrine transforming potential of the c-sis/PDGF B chain by forming low-affinity heterodimers with wild-type PDGF chains. This method of specifically antagonizing the effect of PDGF may be useful in investigations of the role of PDGF in normal and pathological conditions. Images PMID:8321214

Reversion of autocrine transformation by a dominant negative platelet-derived growth factor mutant.

PubMed

Vassbotn, F S; Andersson, M; Westermark, B; Heldin, C H; Ostman, A

1993-07-01

A non-receptor-binding mutant of the platelet-derived growth factor (PDGF) A chain, PDGF-0, was generated by exchanging 7 amino acids in the sequence. The mutant chains formed dimers that were similar to wild-type PDGF-AA with regard to stability and rate of processing to the mature 30-kDa secreted forms. Moreover, the mutant chains formed disulfide-bonded heterodimers with the PDGF B chain in NIH 3T3 cells heterodimer underwent the same processing and secretion as PDGF-AB. Transfection of c-sis-expressing 3T3 cells with PDGF-0 significantly inhibited the transformed phenotype of these cells, as determined by the following criteria. (i) Compared with PDGF-0-negative clones, PDGF-0-producing clones showed a reverted morphology. (ii) Clones producing PDGF-0 grew more slowly than PDGF-0-negative clones, with a fivefold difference in cell number after 14 days in culture. (iii) The expression of PDGF-0 completely inhibited the ability of the c-sis-expressing 3T3 cells to form colonies in soft agar; this inhibition was overcome by the addition of recombinant PDGF-BB to the culture medium, showing that the lack of colony formation of these cells was not due to a general unresponsiveness to PDGF. The specific expression of a PDGF-0/PDGF wild-type heterodimer in COS cells revealed that the affinity of the mutant heterodimer for the PDGF alpha receptor was decreased by approximately 50-fold compared with that of PDGF-AA. Thus, we show that a non-receptor-binding PDGF A-chain mutant neutralizes in a trans-dominant manner the autocrine transforming potential of the c-sis/PDGF B chain by forming low-affinity heterodimers with wild-type PDGF chains. This method of specifically antagonizing the effect of PDGF may be useful in investigations of the role of PDGF in normal and pathological conditions.

An Integrin-Targeting RGDK-Tagged Nanocarrier: Anticancer Efficacy of Loaded Curcumin.

PubMed

Das, Krishnendu; Nimushakavi, Sahithi; Chaudhuri, Arabinda; Das, Prasanta Kumar

2017-05-22

Herein we report the design and development of α 5 β 1 integrin-specific noncovalent RGDK-lipopeptide-functionalized single-walled carbon nanotubes (SWNTs) that selectively deliver the anticancer drug curcumin to tumor cells. RGDK tetrapeptide-tagged amphiphiles were synthesized that efficiently disperse SWNTs with a suspension stability index of >80 % in cell culture media. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)- and lactate dehydrogenase (LDH)-based cell viability assays in tumor (B16F10 melanoma) and noncancerous (NIH3T3 mouse fibroblast) cells revealed the non-cytotoxic nature of these RGDK-lipopeptide-SWNT conjugates. Cellular uptake experiments with monoclonal antibodies against α v β 3 , α v β 5 , and α 5 β 1 integrins showed that these SWNT nanovectors deliver their cargo (Cy3-labeled oligonucleotides, Cy3-oligo) to B16F10 cells selectively via α 5 β 1 integrin. Notably, the nanovectors failed to deliver the Cy3-oligo to NIH3T3 cells. The RGDK-SWNT is capable of delivering the anticancer drug curcumin to B16F10 cells more efficiently than NIH3T3 cells, leading to selective killing of B16F10 cells. Results of Annexin V binding based flow cytometry experiments are consistent with selective killing of tumor cells through the late apoptotic pathway. Biodistribution studies in melanoma (B16F10)-bearing C57BL/6J mice showed tumor-selective accumulation of curcumin intravenously administered via RGDK-lipopeptide-SWNT nanovectors. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Suppression of human fibrosarcoma cell growth by transcription factor, Egr-1, involves down-regulation of Bcl-2.

PubMed

Huang, R P; Fan, Y; Peng, A; Zeng, Z L; Reed, J C; Adamson, E D; Boynton, A L

1998-09-11

Previously, we showed that the transcription factor Egr-1 suppressed the proliferation of v-sis transformed NIH3T3 cells and also a number of human tumor cells. Here, we investigate the possible mechanisms responsible for this function. We show that transfected Egr-1 in human fibrosarcoma cells HT1080 leads to down-regulation of Bcl-2. Transient CAT transfection assays reveal that expression of Egr-1 suppresses Bcl-2 promoter activity in a dose-dependent manner. Furthermore, overexpression of Bcl-2 in Egr-1-expressing HT1080 cells enhanced cell proliferation in monolayer culture and increased anchorage-independent growth. Our results suggest that suppression of tumor cell proliferation by Egr-1 may be at least partially mediated through the down-regulation of Bcl-2.

[Morphological spectrum of USP6 rearranged lesions].

PubMed

Mechtersheimer, G; Werner, M

2018-03-01

USP6, also known as Tre-2 and TRE17, is an ubiquitase-specific proteinase that was identified more than two decades ago as a potential oncogene when it exhibited transforming properties upon overexpression in NIH 3T3 cells. Until recently, however, little was known about the function and the oncogenetic activation of USP6. The identification of rearrangements of the USP6 gene in aneurysmal bone cyst and in nodular fasciitis has not only led to a better understanding of the pathogenesis of these entities, but is also a useful tool in their diagnosis and differential diagnostic delineation from morphological mimics. In this review, the clinical, pathomorphological, and molecular genetic aspects of aneurysmal bone cyst and of nodular fasciitis, as well as from related lesions, are presented and discussed.

Inhibition by Chondroitin Sulfate E Can Specify Functional Wnt/β-Catenin Signaling Thresholds in NIH3T3 Fibroblasts*

PubMed Central

Willis, Catherine M.; Klüppel, Michael

2012-01-01

Aberrant activation of the Wnt/β-catenin signaling pathway is frequently associated with human disease, including cancer, and thus represents a key therapeutic target. However, Wnt/β-catenin signaling also plays critical roles in many aspects of normal adult tissue homeostasis. The identification of mechanisms and strategies to selectively inhibit the disease-related functions of Wnt signaling, while preserving normal physiological functions, is in its infancy. Here, we report the identification of exogenous chondroitin sulfate-E (CS-E) as an inhibitor of specific molecular and biological outcomes of Wnt3a signaling in NIH3T3 fibroblasts. We demonstrate that CS-E can decrease Wnt3a signaling through the negative regulation of LRP6 receptor activation. However, this inhibitory effect of CS-E only affected Wnt3a-mediated induction, but not repression, of target gene expression. We went on to identify a critical Wnt3a signaling threshold that differentially affects target gene induction versus repression. This signaling threshold also controlled the effects of Wnt3a on proliferation and serum starvation-induced apoptosis. Limiting Wnt3a signaling to this critical threshold, either by CS-E treatment or by ligand dilution, interfered with Wnt3a-mediated stimulation of proliferation but did not impair Wnt3a-mediated reduction of serum starvation-induced apoptosis. Treatment with pharmacological inhibitors demonstrated that both induction and repression of Wnt3a target genes in NIH3T3 cells require the canonical Wnt/β-catenin signaling cascade. Our data establish the feasibility of selective inhibition of Wnt/β-catenin transcriptional programs and biological outcomes through the exploitation of intrinsic signaling thresholds. PMID:22915582

The NESH/Abi-3-based WAVE2 complex is functionally distinct from the Abi-1-based WAVE2 complex.

PubMed

Sekino, Saki; Kashiwagi, Yuriko; Kanazawa, Hitoshi; Takada, Kazuki; Baba, Takashi; Sato, Seiichi; Inoue, Hiroki; Kojima, Masaki; Tani, Katsuko

2015-10-01

Abl interactor (Abi) family proteins play significant roles in actin cytoskeleton organization through participation in the WAVE complex. Mammals possess three Abi proteins: Abi-1, Abi-2, and NESH/Abi-3. Abi-1 and Abi-2 were originally identified as Abl tyrosine kinase-binding proteins. It has been disclosed that Abi-1 acts as a bridge between c-Abl and WAVE2, and c-Abl-mediated WAVE2 phosphorylation promotes actin remodeling. We showed previously that NESH/Abi-3 is present in the WAVE2 complex, but neither binds to c-Abl nor promotes c-Abl-mediated phosphorylation of WAVE2. In this study, we characterized NESH/Abi-3 in more detail, and compared its properties with those of Abi-1 and Abi-2. NESH/Abi-3 was ectopically expressed in NIH3T3 cells, in which Abi-1, but not NESH/Abi-3, is expressed. The expression of NESH/Abi-3 caused degradation of endogenous Abi-1, which led to the formation of a NESH/Abi-3-based WAVE2 complex. When these cells were plated on fibronectin-coated dishes, the translocation of WAVE2 to the plasma membrane was significantly reduced and the formation of peripheral lamellipodial structures was disturbed, suggesting that the NESH/Abi-3-based WAVE2 complex was unable to help produce lamellipodial protrusions. Next, Abi-1, Abi-2, or NESH/Abi-3 was expressed in v-src-transformed NIH3T3 cells. Only in NESH/Abi-3-expressed cells did treatment with an Abl kinase inhibitor, imatinib mesylate, or siRNA-mediated knockdown of c-Abl promote the formation of invadopodia, which are ventral membrane protrusions with extracellular matrix degradation activity. Structural studies showed that a linker region between the proline-rich regions and the Src homology 3 (SH3) domain of Abi-1 is crucial for its interaction with c-Abl and c-Abl-mediated phosphorylation of WAVE2. The NESH/Abi-3-based WAVE2 complex is functionally distinct from the Abi-1-based one, and NESH/Abi-3 may be involved in the formation of ventral protrusions under certain conditions.

Identification of the bombesin receptor on murine and human cells by cross-linking experiments.

PubMed

Kris, R M; Hazan, R; Villines, J; Moody, T W; Schlessinger, J

1987-08-15

The bombesin receptor present on the surface of murine and human cells was identified using 125I-labeled gastrin-releasing peptide as a probe, the cross-linking agent disuccinimidyl suberate, and sodium dodecyl sulfate gels. A clone of NIH-3T3 cells which possesses approximately 80,000 bombesin receptors/cell with a single binding constant of approximately 1.9 X 10(-9) M was used in these studies. In addition, we used Swiss 3T3 cells and a human glioma cell line which possesses approximately 100,000 and approximately 55,000 bombesin receptors/cell, respectively. Under conditions found optimal for binding, it is demonstrated that 125I-labeled gastrin-releasing peptide can be cross-linked specifically to a glycoprotein of apparent molecular mass of 65,000 daltons on the surface of the NIH-3T3 cells. Similar results were obtained when the cross-linked product was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing or non-reducing conditions. Moreover, the cross-linking reaction is specific and saturable and the 65,000-dalton polypeptide is not observed when the cross-linking experiments were performed with a NIH-3T3 cell line which is devoid of bombesin receptors. Interestingly, glycoproteins with apparent molecular weights of 75,000 were labeled specifically by 125I-labeled gastrin-releasing peptide when similar experiments were performed with Swiss 3T3 cells and with human glioma cell line GM-340. These different molecular weights may indicate differential glycosylation as treatment with the enzyme N-glycanase reduced the apparent molecular weight of the cross-linked polypeptide to 45,000. On the basis of these results it is concluded that the cross-linked polypeptides represent the bombesin receptor or the ligand-binding subunit of a putative larger bombesin receptor expressed on the surface of these cells.

Strong Inverse Correlation Between MicroRNA-125b and Human Papillomavirus DNA in Productive Infection

PubMed Central

Nuovo, Gerard J.; Wu, Xin; Volinia, Stefano; Yan, Fengting; di Leva, Gianpiero; Chin, Nena; Nicol, Alcina F.; Jiang, Jinmai; Otterson, Gregory; Schmittgen, Thomas D.; Croce, Carlo

2014-01-01

Infection by the human papillomavirus (HPV) is a cause of cervical intraepithelial neoplasia (CIN) and cancer. microRNA (miRNA) in situ analysis of the transformation zone epithelia, the site of initial cervical HPV infection, showed that miRNAs let-7c, — 99a, 26a, and 125b were the most abundantly expressed. In situ testing of CIN 1 showed a dramatic reduction in miR-125b expression in the koilocytes, the cytologic marker of productive HPV infection. A marked reduction in miR-125b was likewise observed in the HPV-infected cells of the condyloma acuminatum, verruca vulgaris, and epidermodysplasia verruciformis. Reverse transcriptase in situ polymerase chain reaction (PCR) showed that the pre-miRNA 125b was present in the koilocyte, suggesting direct inactivation of the mature miRNA. HEK cells transfected with only the antimiR-125b showed perinuclear halos equivalent to HPV-infected koilocytes. NIH 3T3 cells transfected with the HPV 16 full-length genome and mimetic miR-125b showed a marked reduction in viral DNA and protein synthesis by quantitative PCR and in situ-based analyses, respectively (P=0.002). Alternatively, cotransfection with anti-miR-125b and HPV 16 markedly increased HPV DNA (P=0.002). Sequence analyses showed strong homology between L2 of different HPV genotypes and miR-125b. Transfection with HPV 16 L2 resulted in a marked reduction in miR-125b levels in the NIH 3T3 cells. HPV L2-induced inactivation of miR-125b is associated with the classic cytologic changes of the koilocyte, and the exogenous application of mimetic miR-125b markedly inhibits HPV DNA synthesis. PMID:20736742

Strong inverse correlation between microRNA-125b and human papillomavirus DNA in productive infection.

PubMed

Nuovo, Gerard J; Wu, Xin; Volinia, Stefano; Yan, Fengting; di Leva, Gianpiero; Chin, Nena; Nicol, Alcina F; Jiang, Jinmai; Otterson, Gregory; Schmittgen, Thomas D; Croce, Carlo

2010-09-01

Infection by the human papillomavirus (HPV) is a cause of cervical intraepithelial neoplasia (CIN) and cancer. microRNA (miRNA) in situ analysis of the transformation zone epithelia, the site of initial cervical HPV infection, showed that miRNAs let-7c, -99a, 26a, and 125b were the most abundantly expressed. In situ testing of CIN 1 showed a dramatic reduction in miR-125b expression in the koilocytes, the cytologic marker of productive HPV infection. A marked reduction in miR-125b was likewise observed in the HPV-infected cells of the condyloma acuminatum, verruca vulgaris, and epidermodysplasia verruciformis. Reverse transcriptase in situ polymerase chain reaction (PCR) showed that the pre-miRNA 125b was present in the koilocyte, suggesting direct inactivation of the mature miRNA. HEK cells transfected with only the antimiR-125b showed perinuclear halos equivalent to HPV-infected koilocytes. NIH 3T3 cells transfected with the HPV 16 full-length genome and mimetic miR-125b showed a marked reduction in viral DNA and protein synthesis by quantitative PCR and in situ-based analyses, respectively (P=0.002). Alternatively, cotransfection with anti-miR-125b and HPV 16 markedly increased HPV DNA (P=0.002). Sequence analyses showed strong homology between L2 of different HPV genotypes and miR-125b. Transfection with HPV 16 L2 resulted in a marked reduction in miR-125b levels in the NIH 3T3 cells. HPV L2-induced inactivation of miR-125b is associated with the classic cytologic changes of the koilocyte, and the exogenous application of mimetic miR-125b markedly inhibits HPV DNA synthesis.

Glycopolymer micelles with reducible ionic cores for hepatocytes-targeting delivery of DOX.

PubMed

Wang, Yanxia; Zhang, Xinge; Yu, Peien; Li, Chaoxing

2013-01-30

A novel galactose-decorated cross-linked micelles (cl-micelles) with ionic cores using cystamine (Cys) as a biodegradable cross-linker was prepared by using block ionomer complexes of poly(ethylene glycol)-b-poly(2-acryloxyethyl-galactose)-b-poly(acrylic acid) (PEG-b-PAEG-b-PAA) and Ca(2+) (PEG-b-PAEG-b-PAA cl-micelles/Cys). Doxorubicin (DOX) was successfully incorporated into the ionic cores of such micelles via electrostatic interactions. Proton nuclear magnetic resonance spectrum and Fourier transform infrared spectrometer indicated galactose ligands were exposed at the micellar surface. The micelles were spherical in shape, with an average size of 100nm. The in vitro release studies confirmed that DOX-loaded PEG-b-PAEG-b-PAA cl-micelles/Cys accomplished rapid drug release under reducing condition. Remarkably, PEG-b-PAEG-b-PAA cl-micelles/Cys efficiently delivered and released DOX into the cell nucleus of HepG2 cells, and the intensity of fluorescence observed in HepG2 cells was stronger than that incubated with the micelles without galactose ligands. In contrast, little fluorescence was observed in NIH3T3 cells after incubation with PEG-b-PAEG-b-PAA cl-micelles/Cys. Interestingly, cytotoxicity assays showed that DOX-loaded PEG-b-PAEG-b-PAA cl-micelles/Cys retained higher cell inhibition efficiency in HepG2 cells as compared with NIH3T3 cells, and were more potent than the micelles without galactose ligands and the micelles with non degradable cross-links. These results indicate that PEG-b-PAEG-b-PAA cl-micelles/Cys have great potential in liver tumor-targeted chemotherapy. Copyright © 2012 Elsevier B.V. All rights reserved.

Effect of Handling, Storage and Cycling on Ni-H2 Cells: Second Plateau Phenomenon

NASA Technical Reports Server (NTRS)

Vaidyanathan, Hari; Rao, Gopalakrishna

2001-01-01

Proper handling of Ni-H2 cells/batteries in storage, during I&T, and at launch site is very important to preserve the useful energy and to extend the mission life. Cell reversal test is not a prudent test to verify or quantify the nickel pre-charge in Ni-H2 cells/batteries. The second plateau is due to the formation of Ni(+3) that is electrochemically inactive. Gas analysis of the cell, and chemical analysis of the positive plate are confirmatory tests to determine the nature of pre-charge in Ni-H2 cells.

Rhodamine B conjugates of triterpenoic acids are cytotoxic mitocans even at nanomolar concentrations.

PubMed

Sommerwerk, Sven; Heller, Lucie; Kerzig, Christoph; Kramell, Annemarie E; Csuk, René

2017-02-15

Triterpenoic acids 1-6 exhibited very low or no cytotoxicity at all, but their corresponding 2,3-di-O-acetyl-piperazinyl amides 13-18 showed low EC 50 values for several human tumor cell lines. Their cytotoxicity, however, was also high for the non-malignant mouse fibroblasts NIH 3T3. A significant improvement was achieved by preparing the rhodamine B derivatives 19-24. While rhodamine B is not cytotoxic (up to a concentration of 30μM - cut-off of the assay), the triterpenoid piperazine-spacered rhodamine B derivatives were cytotoxic in nano-molar concentration. Compound 24 (a diacetylated maslinic acid derivative) was most toxic for several human tumor cell lines but less toxic for mouse fibroblasts NIH 3T3. Staining and double-staining experiments revealed 24 to act as a mitocan. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Tetraspanin CD151 regulates alpha6beta1 integrin adhesion strengthening

NASA Technical Reports Server (NTRS)

Lammerding, Jan; Kazarov, Alexander R.; Huang, Hayden; Lee, Richard T.; Hemler, Martin E.

2003-01-01

The tetraspanin CD151 molecule associates specifically with laminin-binding integrins, including alpha6beta1. To probe strength of alpha6beta1-dependent adhesion to laminin-1, defined forces (0-1.5 nN) were applied to magnetic laminin-coated microbeads bound to NIH 3T3 cells. For NIH 3T3 cells bearing wild-type CD151, adhesion strengthening was observed, as bead detachment became more difficult over time. In contrast, mutant CD151 (with the C-terminal region replaced) showed impaired adhesion strengthening. Static cell adhesion to laminin-1, and detachment of beads coated with fibronectin or anti-alpha6 antibody were all unaffected by CD151 mutation. Hence, CD151 plays a key role in selectively strengthening alpha6beta1 integrin-mediated adhesion to laminin-1.

Role of NADP+-dependent isocitrate dehydrogenase (NADP+-ICDH) on cellular defence against oxidative injury by gamma-rays.

PubMed

Lee, S H; Jo, S H; Lee, S M; Koh, H J; Song, H; Park, J W; Lee, W H; Huh, T L

2004-09-01

To investigate the regulation of NADPH-producing isocitrate dehydrogenase (ICDH) in cytosol (IDPc) and mitochondria (IDPm) upon gamma-ray irradiation, and the roles of IDPc and IDPm in the protection against cellular damage induced by gamma-ray irradiation. Changes of IDPc and IDPm proteins upon gamma-ray irradiation to NIH3T3 cells were analysed by immunoblotting. To increase or decrease the expression of IDPc or IDPm, NIH3T3 cells were stably transfected with mouse IDPc or IDPm cDNA in either the sense or the antisense direction. The transfected cells with either increased or decreased IDPc or IDPm were exposed to gamma-rays, and the levels of reactive oxygen species generation, protein oxidation and lipid peroxidation were measured. Both IDPc and IDPm activities were induced by gamma-ray in NIH3T3 cells. Cells with decreased expression of IDPc or IDPm had elevated reactive oxygen species generation, lipid peroxidation and protein oxidation. Conversely, overproduction of IDPc or IDPm protein partially protected the cells from oxidative damage induced by gamma-ray irradiation. The protective role of IDPc and IDPm against gamma-ray-induced cellular damage can be attributed to elevated NADPH, reducing equivalents needed for recycling reduced glutathione in the cytosol and mitochondria. Thus, a primary biological function of the ICDHs may be production of NADPH, which is a prerequisite for some cellular defence systems against oxidative damage.

Low Copy Numbers of DC-SIGN in Cell Membrane Microdomains: Implications for Structure and Function

PubMed Central

Liu, Ping; Wang, Xiang; Itano, Michelle S.; Neumann, Aaron K.; de Silva, Aravinda M.; Jacobson, Ken; Thompson, Nancy L.

2014-01-01

Presently, there are few estimates of the number of molecules occupying membrane domains. Using a total internal reflection fluorescence microscopy (TIRFM) imaging approach, based on comparing the intensities of fluorescently labeled microdomains with those of single fluorophores, we measured the occupancy of DC-SIGN, a C-type lectin, in membrane microdomains. DC-SIGN or its mutants were labeled with primary monoclonal antibodies (mAbs) in either dendritic cells (DCs) or NIH3T3 cells, or expressed as GFP fusions in NIH3T3 cells. The number of DC-SIGN molecules per microdomain ranges from only a few to over 20, while microdomain dimensions range from the diffraction limit to > 1μm. The largest fraction of microdomains, appearing at the diffraction limit, in either immature DCs or 3T3 cells contains only 4-8 molecules of DC-SIGN, consistent with our preliminary super-resolution Blink microscopy estimates. We further show that these small assemblies are sufficient to bind and efficiently internalize a small (~50nm) pathogen, dengue virus, leading to infection of host cells. PMID:24313910

Graphene as a nanocarrier for tamoxifen induces apoptosis in transformed cancer cell lines of different origins.

PubMed

Misra, Santosh K; Kondaiah, Paturu; Bhattacharya, Santanu; Rao, C N R

2012-01-09

A cationic amphiphile, cholest-5en-3β-oxyethyl pyridinium bromide (PY(+) -Chol), is able to efficiently disperse exfoliated graphene (GR) in water by the physical adsorption of PY(+) -Chol on the surface of GR to form stable, dark aqueous suspensions at room temperature. The GR-PY(+) -Chol suspension can then be used to solubilize Tamoxifen Citrate (TmC), a breast cancer drug, in water. The resulting TmC-GR-PY(+) -Chol is stable for a long time without any precipitation. Fluorescence emission and UV absorption spectra indicate the existence of noncovalent interactions between TmC, GR, and PY(+) -Chol in these suspensions. Electron microscopy shows the existence of segregated GR sheets and TmC 'ribbons' in the composite suspensions. Atomic force microscopy indicates the presence of 'extended' structures of GR-PY(+) -Chol, which grows wider in the presence of TmC. The slow time-dependent release of TmC is noticed in a reconstituted cell culture medium, a property useful as a drug carrier. TmC-GR-PY(+) -Chol selectively enhanced the cell death (apoptosis) of the transformed cancer cells compared to normal cells. This potency is found to be true for a wide range of transformed cancer cells viz. HeLa, A549, ras oncogene-transformed NIH3T3, HepG2, MDA-MB231, MCF-7, and HEK293T compared to the normal cell HEK293 in vitro. Confocal microscopy confirmed the high efficiency of TmC-GR-PY(+) -Chol in delivering the drug to the cells, compared to the suspensions devoid of GR. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The state of research funding from the National Institutes of Health for criminal justice health research.

PubMed

Ahalt, Cyrus; Bolano, Marielle; Wang, Emily A; Williams, Brie

2015-03-03

Over 20 million Americans are currently or have been incarcerated. Most are from medically underserved populations; 1 in 3 African American men and 1 in 6 Latino men born in 2001 are projected to go to prison during their lifetime. The amount of funding from the National Institutes of Health (NIH) to understand and improve the health of persons involved with the criminal justice system is unknown. To describe NIH funding for research on the health and health care needs of criminal justice-involved persons. Review of NIH grants (2008-2012) in the RePORT (Research Portfolio Online Reporting Tools) database. U.S. criminal justice system. Criminal justice-involved persons participating in NIH-funded clinical research. NIH research and training grants awarded, by number, type, research area, institute or center, and dollar amount. Of more than 250 000 NIH-funded grants, 180 (<0.1%) focused on criminal justice health research. The 3 most common foci were substance use or HIV (64%), mental health (11%), and juvenile health (8%). The National Institute on Drug Abuse and the National Institute of Mental Health funded 78% of all grants. In 2012, the NIH invested $40.9 million in criminal justice health research, or 1.5% of the $2.7 billion health disparities budget for that year. NIH-supported research that did not explicitly include current or former prisoners but may have relevance to criminal justice health was not included. Federal funding for research focused on understanding and improving the health of criminal justice-involved persons is small, even compared with the NIH's overall investment in health disparities research. The NIH is well-positioned to transform the care of current and former prisoners by investing in this critical yet overlooked research area.

The Human Toxome Project

EPA Science Inventory

The Human Toxome project, funded as an NIH Transformative Research grant 2011--‐ 2016, is focused on developing the concepts and the means for deducing, validating, and sharing molecular Pathways of Toxicity (PoT). Using the test case of estrogenic endocrine disruption, the respo...

Second-site changes affect viability of amphotropic/ecotropic chimeric enveloped murine leukemia viruses.

PubMed

O'Reilly, L; Roth, M J

2000-01-01

Chimeras were previously generated between the ecotropic (Moloney-MuLV) and amphotropic (4070A) SU and TM proteins of murine leukemia virus (MuLV). After passage in D17 cells, three chimeras with junctions in the C terminus of SU (AE5, AE6, and AE7), showed improved kinetics of viral spreading, suggesting that they had adapted. Sequencing of the viruses derived from the D17 cell lines revealed second-site changes within the env gene. Changes were detected in the receptor binding domain, the proline-rich region, the C terminus of SU, and the ectodomain of TM. Second-site changes were subcloned into the parental DNA, singly and in combination, and tested for viability. All viruses had maintained their original cloned mutations and junctions. Reconstruction and passage of AE7 or AE6 virus with single point mutations recovered the additional second-site changes identified in the parental population. The AE5 isolate required changes in the VRA, the VRC, the VRB-hinge region, and the C terminus of SU for efficient infection. Passage of virus, including the parental 4070A, in D17 cells resulted in a predominant G100R mutation within the receptor binding domain. Viruses were subjected to titer determination in three cell types, NIH 3T3, canine D17, and 293T. AE6 viruses with changes in the proline-rich region initially adapted for growth on D17 cells could infect all cell types tested. AE6-based chimeras with additional mutations in the C terminus of SU could infect D17 and 293T cells. Infection of NIH 3T3 cells was dependent on the proline-rich mutation. AE7-based chimeras encoding L538Q and G100R were impaired in infecting NIH 3T3 and 293T cells.

Second-Site Changes Affect Viability of Amphotropic/Ecotropic Chimeric Enveloped Murine Leukemia Viruses

PubMed Central

O'Reilly, Lucille; Roth, Monica J.

2000-01-01

Chimeras were previously generated between the ecotropic (Moloney-MuLV) and amphotropic (4070A) SU and TM proteins of murine leukemia virus (MuLV). After passage in D17 cells, three chimeras with junctions in the C terminus of SU (AE5, AE6, and AE7), showed improved kinetics of viral spreading, suggesting that they had adapted. Sequencing of the viruses derived from the D17 cell lines revealed second-site changes within the env gene. Changes were detected in the receptor binding domain, the proline-rich region, the C terminus of SU, and the ectodomain of TM. Second-site changes were subcloned into the parental DNA, singly and in combination, and tested for viability. All viruses had maintained their original cloned mutations and junctions. Reconstruction and passage of AE7 or AE6 virus with single point mutations recovered the additional second-site changes identified in the parental population. The AE5 isolate required changes in the VRA, the VRC, the VRB-hinge region, and the C terminus of SU for efficient infection. Passage of virus, including the parental 4070A, in D17 cells resulted in a predominant G100R mutation within the receptor binding domain. Viruses were subjected to titer determination in three cell types, NIH 3T3, canine D17, and 293T. AE6 viruses with changes in the proline-rich region initially adapted for growth on D17 cells could infect all cell types tested. AE6-based chimeras with additional mutations in the C terminus of SU could infect D17 and 293T cells. Infection of NIH 3T3 cells was dependent on the proline-rich mutation. AE7-based chimeras encoding L538Q and G100R were impaired in infecting NIH 3T3 and 293T cells. PMID:10623753

The nucleolar protein SURF-6 is essential for viability in mouse NIH/3T3 cells.

PubMed

Polzikov, Mikhail; Magoulas, Charalambos; Zatsepina, Olga

2007-09-01

SURF-6 is a bona fide nucleolar protein comprising an evolutionary conserved family that extends from human to yeast. The expression of the mammalian SURF-6 has been recently found to be regulated during the cell cycle. In order to determine the importance of SURF-6 in mammalian cells, we applied the Tet-On system to regulate conditionally, in response to tetracycline, the expression of an antisense RNA (asRNA) that targets Surf-6 mRNA in mouse NIH/3T3 cells. Induced Surf-6 asRNA caused an effective depletion of SURF-6 protein resulted in cell death and in an apparent arrest in the G1 phase of the cell cycle. These results provide for the first time evidence that expression of SURF-6 is essential for mammalian cell viability, and suggest that SURF-6 might participate in the progression of cell cycle.

Human HST1 (HSTF1) gene maps to chromosome band 11q13 and coamplifies with the INT2 gene in human cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yoshida, Michihiro C.; Wada, Makio; Satoh, Hitoshi

1988-07-01

The human HST1 gene, previously designated the hst gene, and now assigned the name HSTF1 for heparin-binding secretory transforming factor in human gene nomenclature, was originally identified as a transforming gene in DNAs from human stomach cancers by transfection assay with mouse NIH 3T3 cells. The amino acid sequence of the product deduced from DNA sequences of the HST1 cDNA and genomic clones had approximately 40% homology to human basic and acidic fibroblast growth factors and mouse Int-2-encoded protein. The authors have mapped the human HST1 gene to chromosome 11 at band q13.3 by Southern blot hybridization analysis of amore » panel of human and mouse somatic cell hybrids and in situ hybridization with an HST1 cDNA probe. The HST1 gene was found to be amplified in DNAs obtained from a stomach cancer and a vulvar carcinoma cell line, A431. In all of these samples of DNA, the INT2 gene, previously mapped to human chromosome 11q13, was also amplified to the same degree as the HST1 gene.« less

Annexin A1, Annexin A2, and Dyrk 1B are upregulated during GAS1-induced cell cycle arrest.

PubMed

Pérez-Sánchez, Gilberto; Jiménez, Adriana; Quezada-Ramírez, Marco A; Estudillo, Enrique; Ayala-Sarmiento, Alberto E; Mendoza-Hernández, Guillermo; Hernández-Soto, Justino; Hernández-Hernández, Fidel C; Cázares-Raga, Febe E; Segovia, Jose

2018-05-01

GAS1 is a pleiotropic protein that has been investigated because of its ability to induce cell proliferation, cell arrest, and apoptosis, depending on the cellular or the physiological context in which it is expressed. At this point, we have information about the molecular mechanisms by which GAS1 induces proliferation and apoptosis; but very few studies have been focused on elucidating the mechanisms by which GAS1 induces cell arrest. With the aim of expanding our knowledge on this subject, we first focused our research on finding proteins that were preferentially expressed in cells arrested by serum deprivation. By using a proteomics approach and mass spectrometry analysis, we identified 17 proteins in the 2-DE protein profile of serum deprived NIH3T3 cells. Among them, Annexin A1 (Anxa1), Annexin A2 (Anxa2), dual specificity tyrosine-phosphorylation-regulated kinase 1B (Dyrk1B), and Eukaryotic translation initiation factor 3, F (eIf3f) were upregulated at transcriptional the level in proliferative NIH3T3 cells. Moreover, we demonstrated that Anxa1, Anxa2, and Dyrk1b are upregulated at both the transcriptional and translational levels by the overexpression of GAS1. Thus, our results suggest that the upregulation of Anxa1, Anxa2, and Dyrk1b could be related to the ability of GAS1 to induce cell arrest and maintain cell viability. Finally, we provided further evidence showing that GAS1 through Dyrk 1B leads not only to the arrest of NIH3T3 cells but also maintains cell viability. © 2017 Wiley Periodicals, Inc.

Titanium-hydroxyapatite composites sintered at low temperature for tissue engineering: in vitro cell support and biocompatibility.

PubMed

Comín, Romina; Cid, Mariana P; Grinschpun, Luciano; Oldani, Carlos; Salvatierra, Nancy A

2017-04-26

In clinical orthopedics, a critical problem is the bone tissue loss produced by a disease or injury. The use of composites from titanium and hydroxyapatite for biomedical applications has increased due to the resulting advantageous combination of hydroxyapatite bioactivity and favorable mechanical properties of titanium. Powder metallurgy is a simple and lower-cost method that uses powder from titanium and hydroxyapatite to obtain composites having hydroxyapatite phases in a metallic matrix. However, this method has certain limitations arising from thermal decomposition of hydroxyapatite in the titanium-hydroxyapatite system above 800°C. We obtained a composite from titanium and bovine hydroxyapatite powders sintered at 800°C and evaluated its bioactivity and cytocompatibility according to the ISO 10993 standard. Surface analysis and bioactivity of the composite was evaluated by X-ray diffraction and SEM. MTT assay was carried out to assess cytotoxicity on Vero and NIH3T3 cells. Cell morphology and cell adhesion on the composite surface were analyzed using fluorescence and SEM. We obtained a porous composite with hydroxyapatite particles well integrated in titanium matrix which presented excellent bioactivity. Our data did not reveal any toxicity of titanium-hydroxyapatite composite on Vero or NIH3T3 cells. Moreover, extracts from composite did not affect cell morphology or density. Finally, NIH3T3 cells were capable of adhering to and proliferating on the composite surface. The composite obtained displayed promising biomedical applications through the simple method of powder metallurgy. Additionally, these findings provide an in vitro proof for adequate biocompatibility of titanium-hydroxyapatite composite sintered at 800°C.

In vitro and in vivo tetracycline-controlled myogenic conversion of NIH-3T3 cells: evidence of programmed cell death after muscle cell transplantation.

PubMed

Del Bo, R; Torrente, Y; Corti, S; D'Angelo, M G; Comi, G P; Fagiolari, G; Salani, S; Cova, A; Pisati, F; Moggio, M; Ausenda, C; Scarlato, G; Bresolin, N

2001-01-01

Ex vivo gene therapy of Duchenne muscular dystrophy based on autologous transplantation of genetically modified myoblasts is limited by their premature senescence. MyoD-converted fibroblasts represent an alternative source of myogenic cells. In this study the forced MyoD-dependent conversion of murine NIH-3T3 fibroblasts into myoblasts under the control of an inducible promoter silent in the presence of tetracycline was evaluated. After tetracycline withdrawal this promoter drives the transcription of MyoD in the engineered fibroblasts, inducing their myogenesis and giving rise to beta-galactosidase-positive cells. MyoD-expressing fibroblasts withdrew from the cell cycle, but were unable to fuse in vitro into multinucleated myotubes. Five days following implantation of engineered fibroblasts in muscles of C57BL/10J mice we observed a sevenfold increase of beta-galactosidase-positive regenerating myofibers in animals not treated with antibiotic compared with treated animals. After 1 week the number of positive fibers decreased and several apoptotic myonuclei were detected. Three weeks following implantation of MyoD-converted fibroblasts in recipient mice, no positive "blue" fiber was observed. Our results suggest that transactivation by tetracycline of MyoD may drive an in vivo myogenic conversion of NIH-3T3 fibroblasts and that, in this experimental setting, apoptosis plays a relevant role in limiting the efficacy of engineered fibroblast transplantation. This work opens the question whether apoptotic phenomena also play a general role as limiting factors of cell-mediated gene therapy of inherited muscle disorders.

Mammalian transcription factor LSF is a target of ERK signaling

PubMed Central

Pagon, Zrinka; Volker, Janet; Cooper, Geoffrey M.; Hansen, Ulla

2012-01-01

LSF is a mammalian transcription factor that is rapidly and quantitatively phosphorylated upon growth induction of resting, peripheral human T cells, as assayed by a reduction in its electrophoretic mobility. The DNA-binding activity of LSF in primary T cells is greatly increased after this phosphorylation event [Volker et al., 1997]. We demonstrate here that LSF is also rapidly and quantitatively phosphorylated upon growth induction in NIH 3T3 cells, although its DNA-binding activity is not significantly altered. Three lines of experimentation established that ERK is responsible for phosphorylating LSF upon growth induction in both cell types. First, phosphorylation of LSF by ERK is sufficient to cause the reduced electrophoretic mobility of LSF. Second, the amount of ERK activity correlates with the extent of LSF phosphorylation in both primary human T cells and NIH 3T3 cells. Finally, specific inhibitors of the Ras/Raf/MEK/ERK pathway inhibit LSF modification in vivo. This phosphorylation by ERK is not sufficient for activation of LSF DNA-binding activity, as evidenced both in vitro and in mouse fibroblasts. Nonetheless, activation of ERK is a prerequisite for the substantial increase in LSF DNA-binding activity upon activation of resting T cells, indicating that ERK phosphorylation is necessary but not sufficient for activation of LSF in this cell type. PMID:12858339

Expression of human choline kinase in NIH 3T3 fibroblasts increases the mitogenic potential of insulin and insulin-like growth factor I.

PubMed

Chung, T; Huang, J S; Mukherjee, J J; Crilly, K S; Kiss, Z

2000-05-01

In mammalian cells, growth factors, oncogenes, and carcinogens stimulate phosphocholine (PCho) synthesis by choline kinase (CK), suggesting that PCho may regulate cell growth. To validate the role of PCho in mitogenesis, we determined the effects of insulin, insulin-like growth factor I (IGF-I), and other growth factors on DNA synthesis in NIH 3T3 fibroblast sublines highly expressing human choline kinase (CK) without increasing phosphatidylcholine synthesis. In serum-starved CK expressor cells, insulin and IGF-I stimulated DNA synthesis, p70 S6 kinase (p70 S6K) activity, phosphatidylinositol 3-kinase (PI3K) activity, and activating phosphorylation of p42/p44 mitogen-activated protein kinases (MAPK) to greater extents than in the corresponding vector control cells. Furthermore, the CK inhibitor hemicholinium-3 (HC-3) inhibited insulin- and IGF-I-induced DNA synthesis in the CK overexpressors, but not in the vector control cells. The results indicate that high cellular levels of PCho potentiate insulin- and IGF-I-induced DNA synthesis by MAPK- and p70 S6K-regulated mechanisms.

1800MHz Microwave Induces p53 and p53-Mediated Caspase-3 Activation Leading to Cell Apoptosis In Vitro

PubMed Central

Xing, Fuqiang; Zhan, Qiuqiang; He, Yiduo; Cui, Jiesheng; He, Sailing; Wang, Guanyu

2016-01-01

Recent studies have reported that exposure of mammalian cells to microwave radiation may have adverse effects such as induction of cell apoptosis. However, the molecular mechanisms underlying microwave induced mammalian cell apoptosis are not fully understood. Here, we report a novel mechanism: exposure to 1800MHz microwave radiation induces p53-dependent cell apoptosis through cytochrome c-mediated caspase-3 activation pathway. We first measured intensity of microwave radiation from several electronic devices with an irradiation detector. Mouse NIH/3T3 and human U-87 MG cells were then used as receivers of 1800MHz electromagnetic radiation (EMR) at a power density of 1209 mW/m2. Following EMR exposure, cells were analyzed for viability, intracellular reactive oxygen species (ROS) generation, DNA damage, p53 expression, and caspase-3 activity. Our analysis revealed that EMR exposure significantly decreased viability of NIH/3T3 and U-87 MG cells, and increased caspase-3 activity. ROS burst was observed at 6 h and 48 h in NIH/3T3 cells, while at 3 h in U-87 MG cells. Hoechst 33258 staining and in situ TUNEL assay detected that EMR exposure increased DNA damage, which was significantly restrained in the presence of N-acetyl-L-cysteine (NAC, an antioxidant). Moreover, EMR exposure increased the levels of p53 protein and p53 target gene expression, promoted cytochrome c release from mitochondrion, and increased caspase-3 activity. These events were inhibited by pretreatment with NAC, pifithrin-α (a p53 inhibitor) and caspase inhibitor. Collectively, our findings demonstrate, for the first time, that 1800MHz EMR induces apoptosis-related events such as ROS burst and more oxidative DNA damage, which in turn promote p53-dependent caspase-3 activation through release of cytochrome c from mitochondrion. These findings thus provide new insights into physiological mechanisms underlying microwave-induced cell apoptosis. PMID:27689798

Chemical composition of the essential oil from basil (Ocimum basilicum Linn.) and its in vitro cytotoxicity against HeLa and HEp-2 human cancer cell lines and NIH 3T3 mouse embryonic fibroblasts.

PubMed

Kathirvel, Poonkodi; Ravi, Subban

2012-01-01

This study examines the chemical composition and in vitro anticancer activity of the essential oil from Ocimum basilicum Linn. (Lamiaceae), cultivated in the Western Ghats of South India. The chemical compositions of basil fresh leaves were identified by GC-MS: 11 components were identified. The major constituents were found to be methyl cinnamate (70.1%), linalool (17.5%), β-elemene (2.6%) and camphor (1.52%). The results revealed that this plant may belong to the methyl cinnamate and linalool chemotype. A methyl thiazol tetrazolium assay was used for in vitro cytotoxicity screening against the human cervical cancer cell line (HeLa), human laryngeal epithelial carcinoma cell line (HEp-2) and NIH 3T3 mouse embryonic fibroblasts. The IC(50) values obtained were 90.5 and 96.3 µg mL(-1), respectively, and the results revealed that basil oil has potent cytotoxicity.

2-D or 3-D Mammography? The Future of Breast Cancer Detection

MedlinePlus

... D or 3-D Mammography?: The Future of Breast Cancer Detection NIH-supported clinical trial tests diagnostic imaging ... new trial may be the answer for finding breast cancer in women who don’t have symptoms. The ...

Synthesis, Characterization, and Study of In Vitro Cytotoxicity of ZnO-Fe3O4 Magnetic Composite Nanoparticles in Human Breast Cancer Cell Line (MDA-MB-231) and Mouse Fibroblast (NIH 3T3).

PubMed

Bisht, Gunjan; Rayamajhi, Sagar; Kc, Biplab; Paudel, Siddhi Nath; Karna, Deepak; Shrestha, Bhupal G

2016-12-01

Novel magnetic composite nanoparticles (MCPs) were successfully synthesized by ex situ conjugation of synthesized ZnO nanoparticles (ZnO NPs) and Fe 3 O 4 NPs using trisodium citrate as linker with an aim to retain key properties of both NPs viz. inherent selectivity towards cancerous cell and superparamagnetic nature, respectively, on a single system. Successful characterization of synthesized nanoparticles was done by XRD, TEM, FTIR, and VSM analyses. VSM analysis showed similar magnetic profile of thus obtained MCPs as that of naked Fe 3 O 4 NPs with reduction in saturation magnetization to 16.63 emu/g. Also, cell viability inferred from MTT assay showed that MCPs have no significant toxicity towards noncancerous NIH 3T3 cells but impart significant toxicity at similar concentration to breast cancer cell MDA-MB-231. The EC50 value of MCPs on MDA-MB-231 is less than that of naked ZnO NPs on MDA-MB-231, but its toxicity on NIH 3T3 was significantly reduced compared to ZnO NPs. Our hypothesis for this prominent difference in cytotoxicity imparted by MCPs is the synergy of selective cytotoxicity of ZnO nanoparticles via reactive oxygen species (ROS) and exhausting scavenging activity of cancerous cells, which further enhance the cytotoxicity of Fe 3 O 4 NPs on cancer cells. This dramatic difference in cytotoxicity shown by the conjugation of magnetic Fe 3 O 4 NPs with ZnO NPs should be further studied that might hold great promise for the development of selective and site-specific nanoparticles. Schematic representation of the conjugation, characterization and cytotoxicity analysis of Fe 3 O 4 -ZnO magnetic composite particles (MCPs).

The impact of National Institutes of Health funding on U.S. cardiovascular disease research.

PubMed

Lyubarova, Radmila; Itagaki, Brandon K; Itagaki, Michael W

2009-07-29

Intense interest surrounds the recent expansion of US National Institutes of Health (NIH) budgets as part of economic stimulus legislation. However, the relationship between NIH funding and cardiovascular disease research is poorly understood, making the likely impact of this policy change unclear. The National Library of Medicine's PubMed database was searched for articles published from 1996 to 2006, originating from U.S. institutions, and containing the phrases "cardiolog," "cardiovascular," or "cardiac," in the first author's department. Research methodology, journal of publication, journal impact factor, and receipt of NIH funding were recorded. Differences in means and trends were tested with t-tests and linear regression, respectively, with P < or = 0.05 for significance. Of 117,643 world cardiovascular articles, 36,684 (31.2%) originated from the U.S., of which 10,293 (28.1%) received NIH funding. The NIH funded 40.1% of U.S. basic science articles, 20.3% of overall clinical trials, 18.1% of randomized-controlled, and 12.2% of multicenter clinical trials. NIH-funded and total articles grew significantly (65 articles/year, P < 0.001 and 218 articles/year, P < 0.001, respectively). The proportion of articles receiving NIH funding was stable, but grew significantly for basic science and clinical trials (0.87%/year, P < 0.001 and 0.67%/year, P = 0.029, respectively). NIH-funded articles had greater journal impact factors than non NIH-funded articles (5.76 vs. 3.71, P < 0.001). NIH influence on U.S. cardiovascular research expanded in the past decade, during the period of NIH budget doubling. A substantial fraction of research is now directly funded and thus likely sensitive to budget fluctuations, particularly in basic science research. NIH funding predicts greater journal impact.

An in vitro bioassay for xenobiotics using the SXR-driven human CYP3A4/lacZ reporter gene.

PubMed

Lee, Mi R; Kim, Yeon J; Hwang, Dae Y; Kang, Tae S; Hwang, Jin H; Lim, Chae H; Kang, Hyung K; Goo, Jun S; Lim, Hwa J; Ahn, Kwang S; Cho, Jung S; Chae, Kap R; Kim, Yong K

2003-01-01

The dose and time effect of nine xenobiotics, including 17beta-estradiol, corticosterone, dexamethasone, progesterone, nifedipine, bisphenol A, rifampicin, methamphetamine, and nicotine were investigated, in vitro, using human steroid and xenobiotics receptor (SXR)-binding sites on the human CYP3A4 promoter, which can enhance the linked lacZ reporter gene transcription. To test this, liver-specific SAP (human serum amyloid P component)-SXR (SAP/SXR) and human CYP3A4 promoter-regulated lacZ (hCYP3A4/lacZ) constructs were transiently transfected into HepG2 and NIH3T3 cells to compare the xenobiotic responsiveness between human and nonhuman cell lines. In the HepG2 cells, rifampicin, followed by corticosterone, nicotine, methamphetamine, and dexamethasone, exhibited enhanced levels of the lacZ transcript, whereas those of bisphenol A and nifedipine were found to be reduced. No significant responses were observed with 17beta-estradiol or progesterone. In addition, 17beta-estradiol and progesterone did not change the levels of the lacZ transcripts in the HepG2 cells, but did induce significant increases in the transcripts of the NIH3T3 cells. Treatment with corticosterone and dexamethasone, which were highly expressed in the HepG2 cells, did not affect the levels of the lacZ transcript in NIH3T3 cells. These results show that lacZ transcripts can be measured, rapidly and reproducibly, using reverse transcriptase-polymerase chain reaction (RT-PCR) based on the expression of the hCYP3A4/lacZ reporter gene, and was mediated by the SXR. Thus, this in vitro reporter gene bioassay is useful for measuring xenobiotic activities, and is a means to a better relevant bioassay, using human cells, human genes and human promoters, in order to get a closer look at actual human exposure.

Immunotherapy in a human ovarian cancer xenograft model with two bispecific monoclonal antibodies: OV-TL 3/CD3 and OC/TR.

PubMed

van Ravenswaay Claasen, H H; Eggermont, A M; Nooyen, Y A; Warnaar, S O; Fieuren, G J

1994-02-01

The bispecific antibodies (bs-mAbs) OV-TL 3/CD3 and OC/TR (MOv18/CD3) efficiently mediate ovarian tumor cell lysis by cytotoxic T cells and activated peripheral blood lymphocytes (PBL) in vitro. OV-TL 3/CD3 and OC/TR are reactive with tumor-associated antigens on ovarian carcinoma cells (OA3 and CA-MOv18, respectively), and CD3 on activated PBL, bridging both cells and simultaneously inducing activation of the effector cells. In a comparative study we investigated the therapeutic efficacy of OV-TL 3/CD3 and OC/TR by targeting activated PBL with the bs-mAbs against intraperitoneally growing NIH:OVCAR-3 human ovarian carcinoma cells. As they have good tumor localization characteristics, HPLC-purified bispecific F(ab')2 fragments were used to target highly active PHA and IL-2-stimulated PBL effector cells. The efficacy of OV-TL 3/CD3 was compared to OC/TR with respect to tumor-associated antigen (TAA) binding on NIH:OVCAR-3 ascites cells and NIH:OVCAR-3 tumor cell lysis in vitro. In this report we show that ip ovarian cancer-bearing nude mice treated with IL-2 and activated PBL coated with bispecific F(ab')2 had a significantly longer survival than the untreated mice. No significant difference in survival was found between the OC/TR or OV-TL 3/CD3 bispecific antibody, although MOv18 expression was higher on NIH:OVCAR-3 ascites cells and PBL targeted with OC/TR induced slightly higher tumor cell lysis in vitro. Thus, the therapeutic efficacy of these bs-mAbs in vivo could not be predicted by TAA expression or bs-mAb-mediated tumor cell lysis in vitro.

Houttuynia cordata inhibits lipopolysaccharide-induced rapid pulmonary fibrosis by up-regulating IFN-γ and inhibiting the TGF-β1/Smad pathway.

PubMed

Du, Shaohui; Li, Hui; Cui, Yinghai; Yang, Lina; Wu, Jingjing; Huang, Haiyuan; Chen, Yangyan; Huang, Wei; Zhang, Rong; Yang, Jun; Chen, Dongfeng; Li, Yiwei; Zhang, Saixia; Zhou, Jianhong; Wei, Zhijun; Chow, Ngai Tan

2012-07-01

This study aimed to explore the effect and mechanism of H. cordata vapor extract on acute lung injury (ALI) and rapid pulmonary fibrosis (RPF). We applied the volatile extract of HC to an RPF rat model and analyzed the effect on ALI and RPF using hematoxylin-eosin (H&E) staining, routine blood tests, a cell count of bronchoalveolar lavage fluid (BALF), lactate dehydrogenase (LDH) content, van Gieson (VG) staining, hydroxyproline (Hyp) content and the dry/wet weight ratio. The expression of IFN-γ/STAT(1), IL-4/STAT(6) and TGF-β(1)/Smads was analyzed using ELISA, immunohistochemistry and western blotting methods. The active ingredients of the HC vapor extract were analyzed using a gas chromatograph-mass spectrometer (GC-MS), and the effects of the active ingredients of HC on the viability of NIH/3T3 and RAW264.7 cells were detected using an MTT assay. The active ingredients of the HC vapor extract included 4-terpineol, α-terpineol, l-bornyl acetate and methyl-n-nonyl ketone. The results of the lung H&E staining, Hyp content, dry/wet weight ratio and VG staining suggested that the HC vapor extract repaired lung injury and reduced RPF in a dose-dependent manner and up-regulated IFN-γ and inhibited the TGF-β1/Smad pathway in vivo. In vitro, it could inhibit the viability of RAW264.7 and NIH/3T3 cells. It also dose-dependently inhibited the expression of TGF-β1 and enhanced the expression of IFN-γ in NIH/3T3. The HC vapor extract inhibited LPS-induced RPF by up-regulating IFN-γ and inhibiting the TGF-β1/Smad pathway. Copyright © 2012 Elsevier B.V. All rights reserved.

Targeted delivery of doxorubicin into tumor cells by nanostructured lipid carriers conjugated to anti-EGFRvIII monoclonal antibody.

PubMed

Abdolahpour, Saeideh; Toliyat, Tayebeh; Omidfar, Kobra; Modjtahedi, Helmout; Wong, Albert J; Rasaee, Mohammad Javad; Kashanian, Susan; Paknejad, Maliheh

2018-02-01

Epidermal growth factor receptor variant III (EGFRvIII) is the most common variant of the EGF receptor in many human tumors. This variant is tumor specific and highly immunogenic, thus, it can be used as a target for targeted drug delivery toward tumor cells. The major aim of this study was to develop an EGFRvIII-mediated drug delivery system by anti-EGFRvIII monoclonal antibody (MAb) conjugated to doxorubicin (Dox)-loaded nanostructured lipid carriers (NLC) to enhance the targeting specificity and cytotoxic effect of Dox on EGFRvIII-overexpressing cell line. In our study, Dox was chosen as a hydrophobic cytotoxic drug and drug-loaded nanostructured lipid carriers (Dox-NLC) was prepared by solvent emulsification/evaporation method. In order to conjugate anti-EGFRvIII MAb to Dox-NLC, DSPE-PEG2000-NHS (1,2-distearoylphosphatidylethanolamine-polyethylene glycol 2000-NHS) was used as a linker. Physicochemical characteristics of antibody conjugated Dox-NLC (MAb-Dox-NLC), including particle size, zeta potential, entrapment efficiency and in vitro Dox release were investigated. Cytotoxicity of MAb-Dox-NLC against NIH-3T3 and HC2 20d2/c (EGFRvIII-transfected NIH-3T3) cell lines was evaluated. The MAb-Dox-NLC appeared to enhance the cytotoxic activity of targeted NLC against HC2 20d2/c cells. The cellular uptake percentage of targeted NLC by HC2 20d2/c cells was higher than that of NIH-3T3 cells, indicating that EGFRvIII can specifically target HC2 20d2/c cells. In conclusion, anti-EGFRvIII MAb-targeted NLC may be considered as an effective nanocarrier for targeted drug delivery.

Correlations between radiation-induced double strand breaks, cell division delay, and cyclin-dependent signaling in x-irradiated NIH3T3 fibroblasts

NASA Astrophysics Data System (ADS)

Cariveau, Mickael J.

2005-07-01

Molecular responses to radiation-induced DNA double strand breaks (DSB) are mediated by the phosphorylation of the histone variant H2AX which forms identifiable gamma-H2AX foci at the site of the DSB. This event is thought to be linked with the down-regulation of signaling proteins contributing to the checkpoints regulating cell cycle progression and, vis-a-vis , the induction of cell division delay. However, it is unclear whether this division delay is directly related to the number of DSB (gamma-H2AX foci) sustained by an irradiated cell and, if so, whether this number drives cells into cell cycle delay or apoptosis. For this reason, studies were conducted in the immortalized NIH/3T3 fibroblast cell in order to establish correlations between the temporal appearance of the gamma-H2AX foci (a DSB) and the expression of the cell cycle regulatory proteins, cyclin E, A, B1, and their cyclin kinase inhibitor, p21. Cell cycle kinetics and flow cytometry were used to establish radiation-induced division delay over a dose range of 1--6 Gy where a mitotic delay of 2.65 min/cGy was established. Correlations between the expression of cyclin E, A, B1, p21, and the generation of DSB were established in NIH/3T3 cells exposed to 2 or 4 Gy x-irradiation. The data suggest that the G1/S and S phase delay (cyclin E and cyclin A protein levels) are dependent on the dose of radiation while the G2/M (cyclin B1 protein levels) delay is dependent on the quantity of DSB sustained by the irradiated cell.

Antisense oligonucleotides suppress cell-volume-induced activation of chloride channels.

PubMed

Gschwentner, M; Nagl, U O; Wöll, E; Schmarda, A; Ritter, M; Paulmichl, M

1995-08-01

Cell volume regulation is an essential feature of most cells. After swelling in hypotonic media, the simultaneous activation of potassium and chloride channels is believed to be the initial, time-determining step in cell volume regulation. The activation of both pathways is functionally linked and enables the cells to lose ions and water, subsequently leading to cell shrinkage and readjustment of the initial volume. NIH 3T3 fibroblasts efficiently regulate their volume after swelling and bear chloride channels that are activated by decreasing extracellular osmolarity. The chloride current elicited in these cells after swelling is reminiscent of the current found in oocytes expressing an outwardly rectifying chloride current termed ICln. Introduction of antisense oligodeoxynucleotides complementary to the first 30 nucleotides of the coding region of the ICln channel into NIH 3T3 fibroblasts suppresses the activation of the swelling-induced chloride current. The experiments directly demonstrate an unambiguous link between a volume-activated chloride current and a cloned protein involved in chloride transport.

Increased NIH 3T3 fibroblast functions on cell culture dishes which mimic the nanometer fibers of natural tissues.

PubMed

Bhardwaj, Garima; Webster, Thomas J

2015-01-01

Traditional flat tissue cell culture dishes have consisted of polystyrene treated with plasma gases for growing, subculturing, and studying cell behavior in vitro. However, increasingly it has been observed that mimicking natural tissue properties (such as chemistry, three-dimensional structure, mechanical properties, etc) in vitro can lead to a better correlation of in vitro to in vivo cellular functions. The following studies compared traditional NIH 3T3 fibroblasts' functions on XanoMatrix scaffolds to standard tissue culture polystyrene. Results found significantly greater fibroblast adhesion and proliferation on XanoMatrix cell culture dishes which mimic the nanoscale geometry of natural tissue fibers with true, tortuous fiber beds creating a robust, consistent, and versatile growth platform. In this manner, this study supports that cell culture dishes which mimic features of natural tissues should be continually studied for a wide range of applications in which mimicking natural cellular functions are important.

WW domain-mediated interaction with Wbp2 is important for the oncogenic property of TAZ

PubMed Central

Chan, S W; Lim, C J; Huang, C; Chong, Y F; Gunaratne, H J; Hogue, K A; Blackstock, W P; Harvey, K F; Hong, W

2011-01-01

The transcriptional co-activators YAP and TAZ are downstream targets inhibited by the Hippo tumor suppressor pathway. YAP and TAZ both possess WW domains, which are important protein–protein interaction modules that mediate interaction with proline-rich motifs, most commonly PPXY. The WW domains of YAP have complex regulatory roles as exemplified by recent reports showing that they can positively or negatively influence YAP activity in a cell and context-specific manner. In this study, we show that the WW domain of TAZ is important for it to transform both MCF10A and NIH3T3 cells and to activate transcription of ITGB2 but not CTGF, as introducing point mutations into the WW domain of TAZ (WWm) abolished its transforming and transcription-promoting ability. Using a proteomic approach, we discovered potential regulatory proteins that interact with TAZ WW domain and identified Wbp2. The interaction of Wbp2 with TAZ is dependent on the WW domain of TAZ and the PPXY-containing C-terminal region of Wbp2. Knockdown of endogenous Wbp2 suppresses, whereas overexpression of Wbp2 enhances, TAZ-driven transformation. Forced interaction of WWm with Wbp2 by direct C-terminal fusion of full-length Wbp2 or its TAZ-interacting C-terminal domain restored the transforming and transcription-promoting ability of TAZ. These results suggest that the WW domain-mediated interaction with Wbp2 promotes the transforming ability of TAZ. PMID:20972459

A homeopathic remedy from arnica, marigold, St. John's wort and comfrey accelerates in vitro wound scratch closure of NIH 3T3 fibroblasts.

PubMed

Hostanska, Katarina; Rostock, Matthias; Melzer, Joerg; Baumgartner, Stephan; Saller, Reinhard

2012-07-18

Drugs of plant origin such as Arnica montana, Calendula officinalis or Hypericum perforatum have been frequently used to promote wound healing. While their effect on wound healing using preparations at pharmacological concentrations was supported by several in vitro and clinical studies, investigations of herbal homeopathic remedies on wound healing process are rare. The objective of this study was to investigate the effect of a commercial low potency homeopathic remedy Similasan® Arnica plus Spray on wound closure in a controlled, blind trial in vitro. We investigated the effect of an ethanolic preparation composed of equal parts of Arnica montana 4x, Calendula officinalis 4x, Hypericum perforatum 4x and Symphytum officinale 6x (0712-2), its succussed hydroalcoholic solvent (0712-1) and unsuccussed solvent (0712-3) on NIH 3T3 fibroblasts. Cell viability was determined by WST-1 assay, cell growth using BrdU uptake, cell migration by chemotaxis assay and wound closure by CytoSelect ™Wound Healing Assay Kit which generated a defined "wound field". All assays were performed in three independent controlled experiments. None of the three substances affected cell viability and none showed a stimulating effect on cell proliferation. Preparation (0712-2) exerted a stimulating effect on fibroblast migration (31.9%) vs 14.7% with succussed solvent (0712-1) at 1:100 dilutions (p < 0.001). Unsuccussed solvent (0712-3) had no influence on cell migration (6.3%; p > 0.05). Preparation (0712-2) at a dilution of 1:100 promoted in vitro wound closure by 59.5% and differed significantly (p < 0.001) from succussed solvent (0712-1), which caused 22.1% wound closure. Results of this study showed that the low potency homeopathic remedy (0712-2) exerted in vitro wound closure potential in NIH 3T3 fibroblasts. This effect resulted from stimulation of fibroblasts motility rather than of their mitosis.

Evidence of Intermediate Hydrogen States in the Formation of a Complex Hydride

DOE PAGES

Sato, Toyoto; Ramirez-Cuesta, Anibal J.; Daemen, Luke L.; ...

2017-12-26

A complex hydride (LaMg 2NiH 7) composed of La 3+, two Mg 2+, [NiH 4] 4– with a covalently bonded hydrogen, and three H – was formed from an intermetallic LaMg 2Ni via an intermediate phase (LaMg 2NiH 4.6) composed of La, Mg, NiH 2, NiH 3 units, and H atoms at tetrahedral sites. The NiH 2 and NiH 3 units in LaMg 2NiH 4.6 were reported as precursors for [NiH 4] 4– in LaMg 2NiH 7 [Miwa et al. J. Phys. Chem. C 2016, 120, 5926–5931]. To further understand the hydrogen states in the precursors (the NiH 2 andmore » NiH 3 units) and H atoms at the tetrahedral sites in the intermediate phase, LaMg 2NiH 4.6, we observed the hydrogen vibrations in LaMg 2NiH 4.6 and LaMg 2NiH 7 by using inelastic neutron scattering. A comparison of the hydrogen vibrations of the NiH 2 and NiH 3 units with that of [NiH 4] 4– shows that the librational modes of the NiH 2 and NiH 3 units were nonexistent; librational modes are characteristic modes for complex anions, such as [NiH 4] 4–. Furthermore, the hydrogen vibrations for the H atoms in the tetrahedral sites showed a narrower wavenumber range than that for H – and a wider range than that for typical interstitial hydrogen. The results indicated the presence of intermediate hydrogen states before the formation of [NiH 4] 4– and H –.« less

Evidence of Intermediate Hydrogen States in the Formation of a Complex Hydride

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sato, Toyoto; Ramirez-Cuesta, Anibal J.; Daemen, Luke L.

A complex hydride (LaMg 2NiH 7) composed of La 3+, two Mg 2+, [NiH 4] 4– with a covalently bonded hydrogen, and three H – was formed from an intermetallic LaMg 2Ni via an intermediate phase (LaMg 2NiH 4.6) composed of La, Mg, NiH 2, NiH 3 units, and H atoms at tetrahedral sites. The NiH 2 and NiH 3 units in LaMg 2NiH 4.6 were reported as precursors for [NiH 4] 4– in LaMg 2NiH 7 [Miwa et al. J. Phys. Chem. C 2016, 120, 5926–5931]. To further understand the hydrogen states in the precursors (the NiH 2 andmore » NiH 3 units) and H atoms at the tetrahedral sites in the intermediate phase, LaMg 2NiH 4.6, we observed the hydrogen vibrations in LaMg 2NiH 4.6 and LaMg 2NiH 7 by using inelastic neutron scattering. A comparison of the hydrogen vibrations of the NiH 2 and NiH 3 units with that of [NiH 4] 4– shows that the librational modes of the NiH 2 and NiH 3 units were nonexistent; librational modes are characteristic modes for complex anions, such as [NiH 4] 4–. Furthermore, the hydrogen vibrations for the H atoms in the tetrahedral sites showed a narrower wavenumber range than that for H – and a wider range than that for typical interstitial hydrogen. The results indicated the presence of intermediate hydrogen states before the formation of [NiH 4] 4– and H –.« less

Foxp3-dependent transformation of human primary CD4+ T lymphocytes by the retroviral protein tax.

PubMed

Chen, Li; Liu, Dan; Zhang, Yang; Zhang, Huan; Cheng, Hua

2015-10-23

The retroviral Tax proteins of human T cell leukemia virus type 1 and 2 (HTLV-1 and -2) are highly homologous viral transactivators. Both viral proteins can immortalize human primary CD4+ memory T cells, but when expressed alone they rarely transform T cells. In the present study, we found that the Tax proteins displayed a differential ability to immortalize human CD4+Foxp3+ T cells with characteristic expression of CTLA-4 and GITR. Because epidermal growth factor receptor (EGFR) was reportedly expressed and activated in a subset of CD4+Foxp3+ T cells, we introduced an activated EGFR into Tax-immortalized CD4+Foxp3+ T cells. We observed that these modified cells were grown independently of exogenous IL-2, correlating with a T cell transformation phenotype. In Tax-immortalized CD4+Foxp3- T cells, ectopic expression of Foxp3 was a prerequisite for Tax transformation of T cells. Accordingly, treatment of the transformed T cells with erlotinib, a selective inhibitor of EGFR, induced degradation of EGFR in lysosome, consequently causing T cell growth inhibition. Further, we identified autophagy as a crucial cellular survival pathway for the transformed T cells. Silencing key autophagy molecules including Beclin1, Atg5 and PI3 kinase class III (PI3KC3) resulted in drastic impairment of T cell growth. Our data, therefore, unveiled a previously unidentified role of Foxp3 in T cell transformation, providing a molecular basis for HTLV-1 transformation of CD4+Foxp3+ T cells. Copyright © 2015 Elsevier Inc. All rights reserved.

Foxp3-dependent Transformation of Human Primary CD4+ T Lymphocytes by the Retroviral Protein Tax

PubMed Central

Chen, Li; Liu, Dan; Zhang, Yang; Zhang, Huan; Cheng, Hua

2015-01-01

The retroviral Tax proteins of human T cell leukemia virus type 1 and 2 (HTLV-1 and -2) are highly homologous viral transactivators. Both viral proteins can immortalize human primary CD4+ memory T cells, but when expressed alone they rarely transform T cells. In the present study, we found that the Tax proteins displayed a differential ability to immortalize human CD4+Foxp3+ T cells with characteristic expression of CTLA-4 and GITR. Because epidermal growth factor receptor (EGFR) was reportedly expressed and activated in a subset of CD4+Foxp3+ T cells, we introduced an activated EGFR into Tax-immortalized CD4+Foxp3+ T cells. We observed that these modified cells were grown independently of exogenous IL-2, correlating with a T cell transformation phenotype. In Tax-immortalized CD4+Foxp3- T cells, ectopic expression of Foxp3 was a prerequisite for Tax transformation of T cells. Accordingly, treatment of the transformed T cells with erlotinib, a selective inhibitor of EGFR, induced degradation of EGFR in lysosome, consequently causing T cell growth inhibition. Further, we identified autophagy as a crucial cellular survival pathway for the transformed T cells. Silencing key autophagy molecules including Beclin1, Atg5 and PI3 kinase class III (PI3KC3) resulted in drastic impairment of T cell growth. Our data, therefore, unveiled a previously unidentified role of Foxp3 in T cell transformation, providing a molecular basis for HTLV-1 transformation of CD4+Foxp3+ T cells. PMID:26381169

Targeting of Several Glycolytic Enzymes Using RNA Interference Reveals Aldolase Affects Cancer Cell Proliferation through a Non-glycolytic Mechanism

PubMed Central

Lew, Carolyn Ritterson; Tolan, Dean R.

2012-01-01

In cancer, glucose uptake and glycolysis are increased regardless of the oxygen concentration in the cell, a phenomenon known as the Warburg effect. Several (but not all) glycolytic enzymes have been investigated as potential therapeutic targets for cancer treatment using RNAi. Here, four previously untargeted glycolytic enzymes, aldolase A, glyceraldehyde 3-phosphate dehydrogenase, triose phosphate isomerase, and enolase 1, are targeted using RNAi in Ras-transformed NIH-3T3 cells. Of these enzymes, knockdown of aldolase causes the greatest effect, inhibiting cell proliferation by 90%. This defect is rescued by expression of exogenous aldolase. However, aldolase knockdown does not affect glycolytic flux or intracellular ATP concentration, indicating a non-metabolic cause for the cell proliferation defect. Furthermore, this defect could be rescued with an enzymatically dead aldolase variant that retains the known F-actin binding ability of aldolase. One possible model for how aldolase knockdown may inhibit transformed cell proliferation is through its disruption of actin-cytoskeleton dynamics in cell division. Consistent with this hypothesis, aldolase knockdown cells show increased multinucleation. These results are compared with other studies targeting glycolytic enzymes with RNAi in the context of cancer cell proliferation and suggest that aldolase may be a useful target in the treatment of cancer. PMID:23093405

Characterization of human palmitoyl-acyl transferase activity using peptides that mimic distinct palmitoylation motifs.

PubMed Central

Varner, Amanda S; Ducker, Charles E; Xia, Zuping; Zhuang, Yan; De Vos, Mackenzie L; Smith, Charles D

2003-01-01

The covalent attachment of palmitate to proteins commonly occurs on cysteine residues near either N-myristoylated glycine residues or C-terminal farnesylated cysteine residues. It therefore seems likely that multiple palmitoyl-acyl transferase (PAT) activities exist to recognize and modify these distinct palmitoylation motifs. To evaluate this possibility, two synthetic peptides representing these palmitoylation motifs, termed MyrGCK(NBD) and FarnCNRas(NBD), were used to characterize PAT activity under a variety of conditions. The human tumour cell lines MCF-7 and Hep-G2 each demonstrated high levels of PAT activity towards both peptides. In contrast, normal mouse fibroblasts (NIH/3T3 cells) demonstrated PAT activity towards the myristoylated substrate peptide but not the farnesylated peptide, while ras -transformed NIH/3T3 cells were able to palmitoylate the FarnCNRas(NBD) peptide. The kinetic parameters for PAT activity were determined using membranes from MCF-7 cells, and indicated that the K (m) values for palmitoyl-CoA were identical for PAT activity towards the two substrate peptides; however, the K (m) for MyrGCK(NBD) was 5-fold lower than the K (m) for FarnCNRas(NBD). PAT activity towards the two substrate peptides was dose-dependently inhibited by 2-bromopalmitate and 3-(1-oxo-hexadecyl)oxiranecarboxamide (16C; IC(50) values of approx. 4 and 1.3 microM, respectively); however, 2-bromopalmitate was found to be uncompetitive with respect to palmitoyl-CoA, whereas 16C was competitive. To seek additional evidence for multiple PATs, the effects of altering the assay conditions on the palmitoylation of MyrGCK(NBD) and FarnCNRas(NBD) were compared. PAT activity towards the two peptide substrates was modulated similarly by changing the ionic strength or incubation temperature, or by the addition of dithiothreitol. In contrast, the enzymic palmitoylation of the two peptides was differentially affected by N -ethylmaleimide and thermal denaturation. Overall, these data demonstrate that the enzymic palmitoylation of farnesyl- and myristoyl-containing peptide substrates can be differentiated, suggesting that multiple motif-specific PATs exist. PMID:12670300

Ascorbate-apatite composite and ascorbate-FGF-2-apatite composite layers formed on external fixation rods and their effects on cell activity in vitro.

PubMed

Wang, Xiupeng; Ito, Atsuo; Sogo, Yu; Li, Xia; Tsurushima, Hideo; Oyane, Ayako

2009-09-01

Ascorbate-apatite and ascorbate-fibroblast growth factor-2 (FGF-2)-apatite composite layers were successfully formed on anodically oxidized Ti rods clinically used for external fixation by a one-step procedure at 25 degrees C, using a metastable supersaturated calcium phosphate solution supplemented with l-ascorbic acid phosphate magnesium salt n-hydrate (AsMg) and FGF-2. The AsMg-apatite and AsMg-FGF-2-apatite composite layers were evaluated in vitro using fibroblastic NIH3T3 and osteoblastic MC3T3-E1 cells. The AsMg-FGF-2-apatite composite layer markedly enhanced the NIH3T3 cell proliferation and procollagen type capital I, Ukrainian gene expression. Without FGF-2, the AsMg-apatite composite layer whose ascorbate content was 3.64+/-1.27microgcm(-2) obviously enhanced osteoblastic proliferation and differentiation. However, the AsMg-FGF-2-apatite composite layers whose FGF-2 contents were from 0.15+/-0.03 to 0.31+/-0.04microgcm(-2) inhibited osteoblastic differentiation in vitro. Thus, the AsMg-FGF-2-apatite composite layer should be precipitated on the surface of external fixators attached to skin and soft tissue. On the other hand, the AsMg-apatite composite layer should be precipitated at the part attached to bone tissue.

Differential expression of the ufo/axl oncogene in human leukemia-lymphoma cell lines.

PubMed

Challier, C; Uphoff, C C; Janssen, J W; Drexler, H G

1996-05-01

The ufo protein (also termed axl) is a member of a new family of receptor tyrosine kinases and is encoded by a transforming gene that was initially isolated from primary human myeloid leukemia cells by DNA-mediated transformation of NIH/3T3 cells. The ligand, Gas6, a protein S-related molecule lacking any known function yet, has recently been identified. We report the expression pattern of ufo mRNA in a panel of 76 human continuous leukemia-lymphoma cell lines. The gene was not expressed in cell lines derived from lymphoid malignancies (n=28), but transcription was seen in 3/11 myeloid, 0/6 monocytic, 9/13 erythroid and 11/18 megakaryocytic cell lines. Several cell lines were treated with phorbol ester leading to significant upregulation of the ufo message in constitutively positive cells. An apparent ufo mRNA overexpression was not found in any of the positive leukemia cell lines, but was identified in the drug-resistant subclones of the cervix carcinoma cell line HeLa. Southern blot analysis of restriction enzyme-digested genomic DNA did not provide evidence for gene amplification, but the HeLa subclones showed banding patterns suggestive of gene rearrangement. Two main ufo mRNA bands of 3.2 and 5.0 kb were identified; no differences in the half-lives (t1/2 = 2.5 h) of these two mRNA species could be identified. In summary, ufo, representing a novel type of receptor tyrosine kinase, is expressed solely in myeloid and erythro-megakaryocytic leukemias but not in lymphoid malignancies. These and previous data suggest an involvement of the ufo receptor tyrosine kinase in normal and malignant myelopoiesis; however, its exact role, if any, and mode of operation in leukemogenesis remains to be determined.

NKX3.1 Genotype and IGF-1 Interact in Prostate Cancer Risk

DTIC Science & Technology

2008-05-01

Cory Abate-Shen, Robert Wood Johnson School of Medicine and Dentistry , New Brunswick, NJ). The data in Figure 2C show that Igfbp-3 mRNA expression was...ImageJ (NIH). P-value is ɘ.002 and was determined by paired t-test using the Prism biostatistics program (Graphpad). 11 Fig 4 NKX3.1 inhibits

Deletion mutants of Harvey ras p21 protein reveal the absolute requirement of at least two distant regions for GTP-binding and transforming activities.

PubMed Central

Lacal, J C; Anderson, P S; Aaronson, S A

1986-01-01

Deletions of small sequences from the viral Harvey ras gene have been generated, and resulting ras p21 mutants have been expressed in Escherichia coli. Purification of each deleted protein allowed the in vitro characterization of GTP-binding, GTPase and autokinase activity of the proteins. Microinjection of the highly purified proteins into quiescent NIH/3T3 cells, as well as transfection experiments utilizing a long terminal repeat (LTR)-containing vector, were utilized to analyze the biological activity of the deleted proteins. Two small regions located at 6-23 and 152-165 residues are shown to be absolutely required for in vitro and in vivo activities of the ras product. By contrast, the variable region comprising amino acids 165-184 was shown not to be necessary for either in vitro or in vivo activities. Thus, we demonstrate that: (i) amino acid sequences at positions 5-23 and 152-165 of ras p21 protein are probably directly involved in the GTP-binding activity; (ii) GTP-binding is required for the transforming activity of ras p21 and by extension for the normal function of the proto-oncogene product; and (iii) the variable region at the C-terminal end of the ras p21 molecule from amino acids 165 to 184 is not required for transformation. Images Fig.2. Fig.4. PMID:3011420

Lycopene-rich extract from red guava (Psidium guajava L.) displays cytotoxic effect against human breast adenocarcinoma cell line MCF-7 via an apoptotic-like pathway.

PubMed

Dos Santos, Raimunda C; Ombredane, Alicia S; Souza, Jéssica Maria T; Vasconcelos, Andreanne G; Plácido, Alexandra; Amorim, Adriany das G N; Barbosa, Eder Alves; Lima, Filipe C D A; Ropke, Cristina D; Alves, Michel M M; Arcanjo, Daniel D R; Carvalho, Fernando A A; Delerue-Matos, Cristina; Joanitti, Graziella A; Leite, José Roberto de S A

2018-03-01

This study investigated a lycopene-rich extract from red guava (LEG) for its chemical composition using spectrophotometry, mass spectrometry, attenuated total reflectance-fourier transform infrared spectroscopy (ATR-FTIR), and computational studies. The cytotoxic activity of LEG and the underlying mechanism was studied in human breast adenocarcinoma cells (MCF-7), murine fibroblast cells (NIH-3T3), BALB/c murine peritoneal macrophages, and sheep blood erythrocytes by evaluating the cell viability with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method and flow cytometry. Spectrophotometry analysis showed that LEG contained 20% of lycopene per extract dry weight. Experimental and theoretical ATR-FTIR suggests the presence of lycopene, whereas MS/MS spectra obtained after fragmentation of the molecular ion [M] +• of 536.4364 show fragment ions at m/z 269.2259, 375.3034, 444.3788, and 467.3658, corroborating the presence of lycopene mostly related to all-trans configuration. Treatment with LEG (1600 to 6.25μg/mL) for 24 and 72h significantly affected the viability of MCF-7 cells (mean half maximal inhibitory concentration [IC 50 ]=29.85 and 5.964μg/mL, respectively) but not NIH-3T3 cells (IC 50 =1579 and 911.5μg/mL, respectively). Furthermore LEG at concentrations from 800 to 6.25μg/mL presented low cytotoxicity against BALB/c peritoneal macrophages (IC 50 ≥800μg/mL) and no hemolytic activity. LEG (400 and 800μg/mL) caused reduction in the cell proliferation and induced cell cycle arrest, DNA fragmentation, modifications in the mitochondrial membrane potential, and morphologic changes related to granularity and size in MCF-7 cells; however, it failed to cause any significant damage to the cell membrane or display necrosis or traditional apoptosis. In conclusion, LEG was able to induce cytostatic and cytotoxic effects on breast cancer cells probably via induction of an apoptotic-like pathway. Copyright © 2017. Published by Elsevier Ltd.

Elevated Cyclic AMP Levels in T Lymphocytes Transformed by Human T-Cell Lymphotropic Virus Type 1▿

PubMed Central

Kress, Andrea K.; Schneider, Grit; Pichler, Klemens; Kalmer, Martina; Fleckenstein, Bernhard; Grassmann, Ralph

2010-01-01

Human T-cell lymphotropic virus type 1 (HTLV-1), the cause of adult T-cell leukemia/lymphoma (ATLL), transforms CD4+ T cells to permanent growth through its transactivator Tax. HTLV-1-transformed cells share phenotypic properties with memory and regulatory T cells (T-reg). Murine T-reg-mediated suppression employs elevated cyclic AMP (cAMP) levels as a key regulator. This led us to determine cAMP levels in HTLV-1-transformed cells. We found elevated cAMP concentrations as a consistent feature of all HTLV-1-transformed cell lines, including in vitro-HTLV-1-transformed, Tax-transformed, and patient-derived cells. In transformed cells with conditional Tax expression, high cAMP levels coincided with the presence of Tax but were lost without it. However, transient ectopic expression of Tax alone was not sufficient to induce cAMP. We found specific downregulation of the cAMP-degrading phosphodiesterase 3B (PDE3B) in HTLV-1-transformed cells, which was independent of Tax in transient expression experiments. This is in line with the notion that PDE3B transcripts and cAMP levels are inversely correlated. Overexpression of PDE3B led to a decrease of cAMP in HTLV-1-transformed cells. Decreased expression of PDE3B was associated with inhibitory histone modifications at the PDE3B promoter and the PDE3B locus. In summary, Tax transformation and its continuous expression contribute to elevated cAMP levels, which may be regulated through PDE3B suppression. This shows that HTLV-1-transformed cells assume biological features of long-lived T-cell populations that potentially contribute to viral persistence. PMID:20573814

Androgen-Induced Cell Migration: Role of Androgen Receptor/Filamin A Association

PubMed Central

Castoria, Gabriella; D'Amato, Loredana; Ciociola, Alessandra; Giovannelli, Pia; Giraldi, Tiziana; Sepe, Leandra; Paolella, Giovanni; Barone, Maria Vittoria; Migliaccio, Antimo; Auricchio, Ferdinando

2011-01-01

Background Androgen receptor (AR) controls male morphogenesis, gametogenesis and prostate growth as well as development of prostate cancer. These findings support a role for AR in cell migration and invasiveness. However, the molecular mechanism involved in AR-mediated cell migration still remains elusive. Methodology/Principal Findings Mouse embryo NIH3T3 fibroblasts and highly metastatic human fibrosarcoma HT1080 cells harbor low levels of transcriptionally incompetent AR. We now report that, through extra nuclear action, AR triggers migration of both cell types upon stimulation with physiological concentrations of the androgen R1881. We analyzed the initial events leading to androgen-induced cell migration and observed that challenging NIH3T3 cells with 10 nM R1881 rapidly induces interaction of AR with filamin A (FlnA) at cytoskeleton. AR/FlnA complex recruits integrin beta 1, thus activating its dependent cascade. Silencing of AR, FlnA and integrin beta 1 shows that this ternary complex controls focal adhesion kinase (FAK), paxillin and Rac, thereby driving cell migration. FAK-null fibroblasts migrate poorly and Rac inhibition by EHT impairs motility of androgen-treated NIH3T3 cells. Interestingly, FAK and Rac activation by androgens are independent of each other. Findings in human fibrosarcoma HT1080 cells strengthen the role of Rac in androgen signaling. The Rac inhibitor significantly impairs androgen-induced migration in these cells. A mutant AR, deleted of the sequence interacting with FlnA, fails to mediate FAK activation and paxillin tyrosine phosphorylation in androgen-stimulated cells, further reinforcing the role of AR/FlnA interaction in androgen-mediated motility. Conclusions/Significance The present report, for the first time, indicates that the extra nuclear AR/FlnA/integrin beta 1 complex is the key by which androgen activates signaling leading to cell migration. Assembly of this ternary complex may control organ development and prostate cancer metastasis. PMID:21359179

The cytotoxic effect of TiF4 and NaF on fibroblasts is influenced by the experimental model, fluoride concentration and exposure time

PubMed Central

Salomão, Priscila Maria Aranda; de Oliveira, Flávia Amadeu; Rodrigues, Paula Danielle; Al-Ahj, Luana Polioni; Gasque, Kellen Cristina da Silva; Jeggle, Pia; Buzalaf, Marilia Afonso Rabelo; de Oliveira, Rodrigo Cardoso; Edwardson, John Michael

2017-01-01

Objective Titanium tetrafluoride (TiF4) has shown promising effect in preventing tooth lesions. Therefore, we compared the cytotoxicity of TiF4 with sodium fluoride (NaF) (already applied in Dentistry) considering different fluoride concentrations, pH values and experimental models. Materials and methods Step 1) NIH/3T3 fibroblasts were exposed to mediums containing NaF or TiF4 (from 0.15 to 2.45% F), both at native and adjusted pH, for 6 h. Step 2) NIH/3T3 were exposed to NaF or TiF4 varnishes with 0.95, 1.95 or 2.45% F (native pH), for 6, 12 or 24 h. We applied MTT (1st and 2nd steps) and Hoescht/PI stain (2nd step) assays. Step 3) NIH/3T3 were exposed to NaF or TiF4 varnish (2.45% F), at native pH, for 6 or 12 h. The cell stiffness was measured by atomic force microscopy (AFM). Results Step 1) All cells exposed to NaF or TiF4 mediums died, regardless of the F concentration and pH. Step 2) Both varnishes, at 1.90 and 2.45% F, reduced cell viability by similar extents (33–86% at 6 h, 35–93% at 12 h, and 87–98% at 24 h) compared with control, regardless of the type of fluoride. Varnishes with 0.95% F did not differ from control. Step 3) TiF4 and NaF reduced cell stiffness to a similar extent, but only TiF4 differed from control at 6 h. Conclusions Based on the results of the 3 experimental steps, we conclude that TiF4 and NaF have similar cytotoxicity. The cytotoxicity was dependent on F concentration and exposure time. This result gives support for testing the effect of TiF4 varnish in vivo. PMID:28614381

A homeopathic remedy from arnica, marigold, St. John’s wort and comfrey accelerates in vitro wound scratch closure of NIH 3T3 fibroblasts

PubMed Central

2012-01-01

Background Drugs of plant origin such as Arnica montana, Calendula officinalis or Hypericum perforatum have been frequently used to promote wound healing. While their effect on wound healing using preparations at pharmacological concentrations was supported by several in vitro and clinical studies, investigations of herbal homeopathic remedies on wound healing process are rare. The objective of this study was to investigate the effect of a commercial low potency homeopathic remedy Similasan® Arnica plus Spray on wound closure in a controlled, blind trial in vitro. Methods We investigated the effect of an ethanolic preparation composed of equal parts of Arnica montana 4x, Calendula officinalis 4x, Hypericum perforatum 4x and Symphytum officinale 6x (0712–2), its succussed hydroalcoholic solvent (0712–1) and unsuccussed solvent (0712–3) on NIH 3T3 fibroblasts. Cell viability was determined by WST-1 assay, cell growth using BrdU uptake, cell migration by chemotaxis assay and wound closure by CytoSelect ™Wound Healing Assay Kit which generated a defined “wound field”. All assays were performed in three independent controlled experiments. Results None of the three substances affected cell viability and none showed a stimulating effect on cell proliferation. Preparation (0712–2) exerted a stimulating effect on fibroblast migration (31.9%) vs 14.7% with succussed solvent (0712–1) at 1:100 dilutions (p < 0.001). Unsuccussed solvent (0712–3) had no influence on cell migration (6.3%; p > 0.05). Preparation (0712–2) at a dilution of 1:100 promoted in vitro wound closure by 59.5% and differed significantly (p < 0.001) from succussed solvent (0712–1), which caused 22.1% wound closure. Conclusion Results of this study showed that the low potency homeopathic remedy (0712–2) exerted in vitro wound closure potential in NIH 3T3 fibroblasts. This effect resulted from stimulation of fibroblasts motility rather than of their mitosis. PMID:22809174

Efficient production of human acidic fibroblast growth factor in pea (Pisum sativum L.) plants by agroinfection of germinated seeds

PubMed Central

2011-01-01

Background For efficient and large scale production of recombinant proteins in plants transient expression by agroinfection has a number of advantages over stable transformation. Simple manipulation, rapid analysis and high expression efficiency are possible. In pea, Pisum sativum, a Virus Induced Gene Silencing System using the pea early browning virus has been converted into an efficient agroinfection system by converting the two RNA genomes of the virus into binary expression vectors for Agrobacterium transformation. Results By vacuum infiltration (0.08 Mpa, 1 min) of germinating pea seeds with 2-3 cm roots with Agrobacteria carrying the binary vectors, expression of the gene for Green Fluorescent Protein as marker and the gene for the human acidic fibroblast growth factor (aFGF) was obtained in 80% of the infiltrated developing seedlings. Maximal production of the recombinant proteins was achieved 12-15 days after infiltration. Conclusions Compared to the leaf injection method vacuum infiltration of germinated seeds is highly efficient allowing large scale production of plants transiently expressing recombinant proteins. The production cycle of plants for harvesting the recombinant protein was shortened from 30 days for leaf injection to 15 days by applying vacuum infiltration. The synthesized aFGF was purified by heparin-affinity chromatography and its mitogenic activity on NIH 3T3 cells confirmed to be similar to a commercial product. PMID:21548923

[Distribution diversity of integrins and calcium channels on major human and mouse host cells of Leptospira species].

PubMed

Li, Cheng-xue; Zhao, Xin; Qian, Jing; Yan, Jie

2012-07-01

To determine the distribution of integrins and calcium channels on major human and mouse host cells of Leptospira species. The expression of β1, β2 and β3 integrins was detected with immunofluorescence assay on the surface of human monocyte line THP-1, mouse mononuclear-macrophage-like cell line J774A.1, human vascular endothelial cell line HUVEC, mouse vascular endothelial cell EOMA, human hepatocyte line L-02, mouse hepatocyte line Hepa1-6, human renal tubular epithelial cell line HEK-293, mouse glomerular membrane epithelial cell line SV40-MES13, mouse collagen blast line NIH/3T3, human and mouse platelets. The distribution of voltage gate control calcium channels Cav3.1, Cav3.2, Cav3.3 and Cav2.3, and receptor gate calcium channels P(2)X(1), P(2)2X(2), P(2)X(3), P(2)X(4), P(2)X(5), P(2)X(6) and P(2)X(7) were determined with Western blot assay. β1 integrin proteins were positively expressed on the membrane surface of J774A.1, THP-1, HUVEC, EOMA, L-02, Hepa1-6 and HEK-239 cells as well as human and mouse platelets. β2 integrin proteins were expressed on the membrane surface of J774A.1, THP-1, HUVEC, EOMA, and NIH/3T3 cells. β3 integrin proteins were expressed on the membrane surface of J774A.1, THP-1, HUVEC, EOMA, Hepa1-6, HEK-239 and NIH/3T3 cells as well as human and mouse platelets. P(2)X(1) receptor gate calcium channel was expressed on the membrane surface of human and mouse platelets, while P(2)X(5) receptor gate calcium channel was expressed on the membrane surface of J774A.1, THP-1, L-02, Hepa1-6, HEK-239 and HUVEC cells. However, the other calcium channels were not detected on the tested cell lines or platelets. There is a large distribution diversity of integrins and calcium channel proteins on the major human and mouse host cells of Leptospira species, which may be associated with the differences of leptospira-induced injury in different host cells.

Induction of mutagenesis and transformation in BALB/c-3T3 clone A31-1 cells by diverse chemical carcinogens

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lubet, R.A.; Kouri, R.E.; Curren, R.A.

1990-01-01

BALB/c-3T3 cells were employed to examine the genotoxic potential of a variety of known chemical carcinogens. BALB/c-3T3 cells displayed a dose-dependent transformation response to a variety of carcinogens (polycyclic hydrocarbons, methylating agents, ethylating agents, aflatoxin B{sub 1} (AFT{sub 1}), and 4-nitroquinoline-N-oxide (4-NQO)). When the ability of these compounds to induce mutagenesis to resistance to the cardiac glycoside ouabain (OUA{sup R}) was examined, the authors found the short chain alkylating agents to be particularly effective mutagens, causing biologic effects at doses below those necessary to induce a transformation response. In contrast, the polycyclic hydrocarbons which were potent transforming agents were weaker,more » albeit significant, mutagens for the OUA{sup R} locus in this system, while AFB{sub 1} was quite weak. Further studies were performed with 5-azacytidine (5-AZA) and the nongenotoxic carcinogen cinnamyl anthranilate (CIN). 5-AZA was a potent transforming agent, but failed to cause mutagenesis. CIN similarly caused in vitro transformation. When a series of eight structurally diverse compounds were examined in both the BALB/c-3T3 and C3H10T1/2 mouse fibroblast transformation systems, the BALB/c-3T3 system was shown to be sensitive to a wide variety of potential carcinogens, whereas the C3H10T1/2 system proved routinely sensitive only to the polycyclic hydrocarbons.« less

Evaluating the Significance of CDK2-PELP1 Axis in Tumorigenesis and Hormone Therapy Resistance

DTIC Science & Technology

2011-02-01

reagents an breast cancer cells MCF7, ZR75, IMR-90, NIH3T3, 93T were obtained from the American Type Culture tion. All stable cell lines were generated...galactosidase activity or tal protein concentration. ime PCR and cell cycle microarray s were harvested with Trizol Reagent (Invitrogen), and NA was isolated...Margue, F Huselstein, G, Grignard , W Dippel, M, Nathan, S Giacchi, R Scheiden. ILK as a potential marker gene to ascertain specific adenocarcinoma

Nckβ Adapter Regulates Actin Polymerization in NIH 3T3 Fibroblasts in Response to Platelet-Derived Growth Factor bb

PubMed Central

Chen, Min; She, Hongyun; Kim, Airie; Woodley, David T.; Li, Wei

2000-01-01

The SH3-SH3-SH3-SH2 adapter Nck represents a two-gene family that includes Nckα (Nck) and Nckβ (Grb4/Nck2), and it links receptor tyrosine kinases to intracellular signaling networks. The function of these mammalian Nck genes has not been established. We report here a specific role for Nckβ in platelet-derived growth factor (PDGF)-induced actin polymerization in NIH 3T3 cells. Overexpression of Nckβ but not Nckα blocks PDGF-stimulated membrane ruffling and formation of lamellipoda. Mutation in either the SH2 or the middle SH3 domain of Nckβ abolishes its interfering effect. Nckβ binds at Tyr-1009 in human PDGF receptor β (PDGFR-β) which is different from Nckα's binding site, Tyr-751, and does not compete with phosphatidylinositol-3 kinase for binding to PDGFR. Microinjection of an anti-Nckβ but not an anti-Nckα antibody inhibits PDGF-stimulated actin polymerization. Constitutively membrane-bound Nckβ but not Nckα blocks Rac1-L62-induced membrane ruffling and formation of lamellipodia, suggesting that Nckβ acts in parallel to or downstream of Rac1. This is the first report of Nckβ's role in receptor tyrosine kinase signaling to the actin cytoskeleton. PMID:11027258

Mutations in the Nucleolar Phosphoprotein, Nucleophosmin, Promote the Expression of the Oncogenic Transcription Factor MEF/ELF4 in Leukemia Cells and Potentiates Transformation*

PubMed Central

Ando, Koji; Tsushima, Hideki; Matsuo, Emi; Horio, Kensuke; Tominaga-Sato, Shinya; Imanishi, Daisuke; Imaizumi, Yoshitaka; Iwanaga, Masako; Itonaga, Hidehiro; Yoshida, Shinichiro; Hata, Tomoko; Moriuchi, Ryozo; Kiyoi, Hitoshi; Nimer, Stephen; Mano, Hiroyuki; Naoe, Tomoki; Tomonaga, Masao; Miyazaki, Yasushi

2013-01-01

Myeloid ELF1-like factor (MEF/ELF4), a member of the ETS transcription factors, can function as an oncogene in murine cancer models and is overexpressed in various human cancers. Here, we report a mechanism by which MEF/ELF4 may be activated by a common leukemia-associated mutation in the nucleophosmin gene. By using a tandem affinity purification assay, we found that MEF/ELF4 interacts with multifactorial protein nucleophosmin (NPM1). Coimmunoprecipitation and GST pull-down experiments demonstrated that MEF/ELF4 directly forms a complex with NPM1 and also identified the region of NPM1 that is responsible for this interaction. Functional analyses showed that wild-type NPM1 inhibited the DNA binding and transcriptional activity of MEF/ELF4 on the HDM2 promoter, whereas NPM1 mutant protein (Mt-NPM1) enhanced these activities of MEF/ELF4. Induction of Mt-NPM1 into MEF/ELF4-overexpressing NIH3T3 cells facilitated malignant transformation. In addition, clinical leukemia samples with NPM1 mutations had higher human MDM2 (HDM2) mRNA expression. Our data suggest that enhanced HDM2 expression induced by mutant NPM1 may have a role in MEF/ELF4-dependent leukemogenesis. PMID:23393136

Mutations in the nucleolar phosphoprotein, nucleophosmin, promote the expression of the oncogenic transcription factor MEF/ELF4 in leukemia cells and potentiates transformation.

PubMed

Ando, Koji; Tsushima, Hideki; Matsuo, Emi; Horio, Kensuke; Tominaga-Sato, Shinya; Imanishi, Daisuke; Imaizumi, Yoshitaka; Iwanaga, Masako; Itonaga, Hidehiro; Yoshida, Shinichiro; Hata, Tomoko; Moriuchi, Ryozo; Kiyoi, Hitoshi; Nimer, Stephen; Mano, Hiroyuki; Naoe, Tomoki; Tomonaga, Masao; Miyazaki, Yasushi

2013-03-29

Myeloid ELF1-like factor (MEF/ELF4), a member of the ETS transcription factors, can function as an oncogene in murine cancer models and is overexpressed in various human cancers. Here, we report a mechanism by which MEF/ELF4 may be activated by a common leukemia-associated mutation in the nucleophosmin gene. By using a tandem affinity purification assay, we found that MEF/ELF4 interacts with multifactorial protein nucleophosmin (NPM1). Coimmunoprecipitation and GST pull-down experiments demonstrated that MEF/ELF4 directly forms a complex with NPM1 and also identified the region of NPM1 that is responsible for this interaction. Functional analyses showed that wild-type NPM1 inhibited the DNA binding and transcriptional activity of MEF/ELF4 on the HDM2 promoter, whereas NPM1 mutant protein (Mt-NPM1) enhanced these activities of MEF/ELF4. Induction of Mt-NPM1 into MEF/ELF4-overexpressing NIH3T3 cells facilitated malignant transformation. In addition, clinical leukemia samples with NPM1 mutations had higher human MDM2 (HDM2) mRNA expression. Our data suggest that enhanced HDM2 expression induced by mutant NPM1 may have a role in MEF/ELF4-dependent leukemogenesis.

Epidermal Growth Factor Receptor Activation in Glioblastoma through Novel Missense Mutations in the Extracellular Domain

PubMed Central

Lee, Jeffrey C; Vivanco, Igor; Beroukhim, Rameen; Huang, Julie H. Y; Feng, Whei L; DeBiasi, Ralph M; Yoshimoto, Koji; King, Jennifer C; Nghiemphu, Phioanh; Yuza, Yuki; Xu, Qing; Greulich, Heidi; Thomas, Roman K; Paez, J. Guillermo; Peck, Timothy C; Linhart, David J; Glatt, Karen A; Getz, Gad; Onofrio, Robert; Ziaugra, Liuda; Levine, Ross L; Gabriel, Stacey; Kawaguchi, Tomohiro; O'Neill, Keith; Khan, Haumith; Liau, Linda M; Nelson, Stanley F; Rao, P. Nagesh; Mischel, Paul; Pieper, Russell O; Cloughesy, Tim; Leahy, Daniel J; Sellers, William R; Sawyers, Charles L; Meyerson, Matthew; Mellinghoff, Ingo K

2006-01-01

Background Protein tyrosine kinases are important regulators of cellular homeostasis with tightly controlled catalytic activity. Mutations in kinase-encoding genes can relieve the autoinhibitory constraints on kinase activity, can promote malignant transformation, and appear to be a major determinant of response to kinase inhibitor therapy. Missense mutations in the EGFR kinase domain, for example, have recently been identified in patients who showed clinical responses to EGFR kinase inhibitor therapy. Methods and Findings Encouraged by the promising clinical activity of epidermal growth factor receptor (EGFR) kinase inhibitors in treating glioblastoma in humans, we have sequenced the complete EGFR coding sequence in glioma tumor samples and cell lines. We identified novel missense mutations in the extracellular domain of EGFR in 13.6% (18/132) of glioblastomas and 12.5% (1/8) of glioblastoma cell lines. These EGFR mutations were associated with increased EGFR gene dosage and conferred anchorage-independent growth and tumorigenicity to NIH-3T3 cells. Cells transformed by expression of these EGFR mutants were sensitive to small-molecule EGFR kinase inhibitors. Conclusions Our results suggest extracellular missense mutations as a novel mechanism for oncogenic EGFR activation and may help identify patients who can benefit from EGFR kinase inhibitors for treatment of glioblastoma. PMID:17177598

The Fn14 cytoplasmic tail binds tumour-necrosis-factor-receptor-associated factors 1, 2, 3 and 5 and mediates nuclear factor-kappaB activation.

PubMed Central

Brown, Sharron A N; Richards, Christine M; Hanscom, Heather N; Feng, Sheau-Line Y; Winkles, Jeffrey A

2003-01-01

Fn14 is a growth-factor-inducible immediate-early-response gene encoding a 102-amino-acid type I transmembrane protein. The human Fn14 protein was recently identified as a cell-surface receptor for the tumour necrosis factor (TNF) superfamily member named TWEAK (TNF-like weak inducer of apoptosis). In the present paper, we report that the human TWEAK extracellular domain can also bind the murine Fn14 protein. Furthermore, site-specific mutagenesis and directed yeast two-hybrid interaction assays revealed that the TNFR-associated factor (TRAF) 1, 2, 3 and 5 adaptor molecules bind the murine Fn14 cytoplasmic tail at an overlapping, but non-identical, amino acid sequence motif. We also found that TWEAK treatment of quiescent NIH 3T3 cells stimulates inhibitory kappaBalpha phosphorylation and transcriptional activation of a nuclear factor-kappaB (NF-kappaB) enhancer/luciferase reporter construct. Fn14 overexpression in transiently transfected NIH 3T3 cells also promotes NF-kappaB activation, and this cellular response requires an intact TRAF binding site. These results indicate that Fn14 is a functional TWEAK receptor that can associate with four distinct TRAF family members and stimulate the NF-kappaB transcription factor signalling pathway. PMID:12529173

The NIH 3D Print Exchange: A Public Resource for Bioscientific and Biomedical 3D Prints.

PubMed

Coakley, Meghan F; Hurt, Darrell E; Weber, Nick; Mtingwa, Makazi; Fincher, Erin C; Alekseyev, Vsevelod; Chen, David T; Yun, Alvin; Gizaw, Metasebia; Swan, Jeremy; Yoo, Terry S; Huyen, Yentram

2014-09-01

The National Institutes of Health (NIH) has launched the NIH 3D Print Exchange, an online portal for discovering and creating bioscientifically relevant 3D models suitable for 3D printing, to provide both researchers and educators with a trusted source to discover accurate and informative models. There are a number of online resources for 3D prints, but there is a paucity of scientific models, and the expertise required to generate and validate such models remains a barrier. The NIH 3D Print Exchange fills this gap by providing novel, web-based tools that empower users with the ability to create ready-to-print 3D files from molecular structure data, microscopy image stacks, and computed tomography scan data. The NIH 3D Print Exchange facilitates open data sharing in a community-driven environment, and also includes various interactive features, as well as information and tutorials on 3D modeling software. As the first government-sponsored website dedicated to 3D printing, the NIH 3D Print Exchange is an important step forward to bringing 3D printing to the mainstream for scientific research and education.

Differentially expressed proteins in nitric oxide-stimulated NIH/3T3 fibroblasts: implications for inhibiting cancer development.

PubMed

Shim, Dong Hwi; Lim, Joo Weon; Kim, Hyeyoung

2015-03-01

Recent evidence shows that nitric oxide (NO) may exhibit both pro-cancer and anti-cancer activities. The present study aimed to determine the differentially expressed proteins in NO-treated NIH/3T3 fibroblasts in order to investigate whether NO induces proteins with pro-cancer or anti-cancer effects. The cells were treated with 300 μM of an NO donor 3,3-bis-(aminoethyl)-1-hydroxy-2-oxo-1-triazene (NOC-18) for 12 h. The changed protein patterns, which were separated by two-dimensional electrophoresis using pH gradients of 4-7, were conclusively identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis of the peptide digests. Seventeen differentially expressed proteins were identified in NOC-18-treated cells. Nine proteins [vinculin protein, keratin 19, ubiquitous tropomodulin, F-actin capping protein (α1 subunit), tropomyosin 3, 26S proteasome-associated pad1 homolog, T-complex protein 1 (ε subunit) N(G)-dimethylarginine dimethylaminohydrolase, and heat shock protein 90] were increased and eight proteins (heat shock protein 70, glucosidase II, lamin B1, calreticulin, nucleophosmin 1, microtubule-associated protein retinitis pigmentosa/end binding family member 1, 150 kD oxygen-regulated protein precursor, and heat shock 70-related protein albino or pale green 2) were decreased by NOC-18 in the cells. Thirteen proteins are related to the suppression of cancer cell proliferation, invasion, and metastasis while two proteins (heat shock protein 90 and N(G)-dimethylarginine dimethylaminohydrolase) are related to carcinogenesis. The functions of 150 kD oxygen-regulated protein precursor and T-complex protein 1 (ε subunit) are unknown in relation to carcinogenesis. Most proteins differentially expressed by NOC-18 are involved in inhibiting cancer development.

Novel synergistic mechanism for sst2 somatostatin and TNFalpha receptors to induce apoptosis: crosstalk between NF-kappaB and JNK pathways.

PubMed

Guillermet-Guibert, J; Saint-Laurent, N; Davenne, L; Rochaix, P; Cuvillier, O; Culler, M D; Pradayrol, L; Buscail, L; Susini, C; Bousquet, C

2007-02-01

Somatostatin is a multifunctional hormone that modulates cell proliferation, differentiation and apoptosis. Mechanisms for somatostatin-induced apoptosis are at present mostly unsolved. Therefore, we investigated whether somatostatin receptor subtype 2 (sst2) induces apoptosis in the nontransformed murine fibroblastic NIH3T3 cells. Somatostatin receptor subtype 2 expression induced an executioner caspase-mediated apoptosis through a tyrosine phosphatase SHP-1 (Src homology domain phosphatase-1)-dependent stimulation of nuclear factor kappa B (NF-kappaB) activity and subsequent inhibition of the mitogen-activated protein kinase JNK. Tumor necrosis factor alpha (TNFalpha) stimulated both NF-kappaB and c-Jun NH2-terminal kinase (JNK) activities, which had opposite action on cell survival. Importantly, sst2 sensitized NIH3T3 cells to TNFalpha-induced apoptosis by (1) upregulating TNFalpha receptor protein expression, and sensitizing to TNFalpha-induced caspase-8 activation; (2) enhancing TNFalpha-mediated activation of NF-kappaB, resulting in JNK inhibition and subsequent executioner caspase activation and cell death. We have here unraveled a novel signaling mechanism for a G protein-coupled receptor, which directly triggers apoptosis and crosstalks with a death receptor to enhance death ligand-induced apoptosis.

Myostatin regulates proliferation and extracellular matrix mRNA expression in NIH3T3 fibroblasts.

PubMed

Z Hosaka, Yoshinao; Ishibashi, Mika; Wakamatsu, Jun-Ichi; Uehara, Masato; Nishimura, Takanori

2012-12-01

The aim of this study was to clarify the effects of myostatin, which is a negative regulator of skeletal muscle mass, on the proliferation of NIH3T3 fibroblasts and the synthesis of extracellular matrix (ECM) by them. A proliferation assay revealed that myostatin attenuated cell growth at any of the doses used. High doses of myostatin strongly inhibited cell proliferation. Moreover, myostatin receptor, activin receptor type-2B (ActRIIB), was found to be distributed on cells and it was also clarified that myostatin increased the expression of cyclin-dependent kinase inhibitor p21 (p21). These results suggested that a high dose of myostatin inhibits fibroblast proliferation by the same mechanism as that for inhibition of myoblast proliferation. We then examined the effects of myostatin on the mRNA expression of ECM molecules (decorin, biglycan, type I collagen, type III collagen, type IV collagen and type V collagen) by real-time PCR. Real-time PCR showed that myostatin increased the mRNA of decorin, biglycan and collagen (types I, IV and V) in fibroblasts. The results suggest that myostatin regulates ECM synthesis in cultured fibroblasts.

Robust synchronization of coupled circadian and cell cycle oscillators in single mammalian cells.

PubMed

Bieler, Jonathan; Cannavo, Rosamaria; Gustafson, Kyle; Gobet, Cedric; Gatfield, David; Naef, Felix

2014-07-15

Circadian cycles and cell cycles are two fundamental periodic processes with a period in the range of 1 day. Consequently, coupling between such cycles can lead to synchronization. Here, we estimated the mutual interactions between the two oscillators by time-lapse imaging of single mammalian NIH3T3 fibroblasts during several days. The analysis of thousands of circadian cycles in dividing cells clearly indicated that both oscillators tick in a 1:1 mode-locked state, with cell divisions occurring tightly 5 h before the peak in circadian Rev-Erbα-YFP reporter expression. In principle, such synchrony may be caused by either unidirectional or bidirectional coupling. While gating of cell division by the circadian cycle has been most studied, our data combined with stochastic modeling unambiguously show that the reverse coupling is predominant in NIH3T3 cells. Moreover, temperature, genetic, and pharmacological perturbations showed that the two interacting cellular oscillators adopt a synchronized state that is highly robust over a wide range of parameters. These findings have implications for circadian function in proliferative tissues, including epidermis, immune cells, and cancer. © 2014 The Authors. Published under the terms of the CC BY 4.0 license.

Magnetic nanoparticle-loaded electrospun polymeric nanofibers for tissue engineering.

PubMed

Zhang, Heng; Xia, JiYi; Pang, XianLun; Zhao, Ming; Wang, BiQiong; Yang, LingLin; Wan, HaiSu; Wu, JingBo; Fu, ShaoZhi

2017-04-01

Magnetic nanoparticles have been one of the most attractive nanomaterials for various biomedical applications including magnetic resonance imaging (MRI), diagnostic contrast enhancement, magnetic cell separation, and targeted drug delivery. Three-dimensional (3-D) fibrous scaffolds have broad application prospects in the biomedical field, such as drug delivery and tissue engineering. In this work, a novel three-dimensional composite membrane composed of the tri-block copolymer poly(ε-caprolactone)-poly(ethylene glycol)-poly(ε-caprolactone) (PCL-PEG-PCL, PCEC) and magnetic iron oxide nanoparticles (Fe 3 O 4 NPs) were fabricated using electrospinning technology. The physico-chemical properties of the PCEC/Fe 3 O 4 membranes were investigated by Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD) and differential scanning calorimetry (DSC). Morphological observation using scanning electron microscopy (SEM) showed that the composite fibers containing 5% Fe 3 O 4 nanoparticles had a diameter of 250nm. In vitro cell culture of NIH 3T3 cells on the PCEC/Fe 3 O 4 membranes showed that the PCEC/Fe 3 O 4 fibers might be a suitable scaffold for cell adhesion. Moreover, MTT analysis also demonstrated that the membranes possessed lower cytotoxicity. Therefore, this study revealed that the magnetic PCEC/Fe 3 O 4 fibers might have great potential for using in skin tissue engineering. Copyright © 2016 Elsevier B.V. All rights reserved.

Insulation of the Chicken β-Globin Chromosomal Domain from a Chromatin-Condensing Protein, MENT

PubMed Central

Istomina, Natalia E.; Shushanov, Sain S.; Springhetti, Evelyn M.; Karpov, Vadim L.; A. Krasheninnikov, Igor; Stevens, Kimberly; Zaret, Kenneth S.; Singh, Prim B.; Grigoryev, Sergei A.

2003-01-01

Active genes are insulated from developmentally regulated chromatin condensation in terminally differentiated cells. We mapped the topography of a terminal stage-specific chromatin-condensing protein, MENT, across the active chicken β-globin domain. We observed two sharp transitions of MENT concentration coinciding with the β-globin boundary elements. The MENT distribution profile was opposite to that of acetylated core histones but correlated with that of histone H3 dimethylated at lysine 9 (H3me2K9). Ectopic MENT expression in NIH 3T3 cells caused a large-scale and specific remodeling of chromatin marked by H3me2K9. MENT colocalized with H3me2K9 both in chicken erythrocytes and NIH 3T3 cells. Mutational analysis of MENT and experiments with deacetylase inhibitors revealed the essential role of the reaction center loop domain and an inhibitory affect of histone hyperacetylation on the MENT-induced chromatin remodeling in vivo. In vitro, the elimination of the histone H3 N-terminal peptide containing lysine 9 by trypsin blocked chromatin self-association by MENT, while reconstitution with dimethylated but not acetylated N-terminal domain of histone H3 specifically restored chromatin self-association by MENT. We suggest that histone H3 modification at lysine 9 directly regulates chromatin condensation by recruiting MENT to chromatin in a fashion that is spatially constrained from active genes by gene boundary elements and histone hyperacetylation. PMID:12944473

Artemisia asiatica ethanol extract exhibits anti-photoaging activity.

PubMed

Jeong, Deok; Lee, Jongsung; Jeong, Seong-Gu; Hong, Yo Han; Yoo, Sulgi; Han, Sang Yun; Kim, Ji Hye; Kim, Sunggyu; Kim, Jin Sic; Chung, Young Soo; Kim, Jong-Hoon; Yi, Young-Su; Cho, Jae Youl

2018-06-28

Artemisia asiatica Nakai is a traditional herbal plant that has long been used in anti-inflammatory, anti-infective and skin protective remedies. In this study, traditionally known skin-protective activity of Artemisia asiatica Nakai was examined with its ethanol extract (Aa-EE) under various photoaging conditions using skin-originated cells, and the underlying mechanism was also examined using various types of cells. Effects of Aa-EE on cell viability, photocytotoxicity, and expression of matrix metalloproteinases (MMPs), cyclooxygenase (COX)-2, and moisturizing factors were measured in B16F10, HEK293, NIH3T3, and HaCaT cells under untreated and ultraviolet B (UVB)-irradiation conditions. Anti-melanogenic effect of Aa-EE was also examined by measuring both melanin content in B16F10 cells and tyrosinase activity. Anti-photoaging mechanism of Aa-EE was explored by determining the activation levels of signaling molecules by immunoblotting analysis. Aa-EE protected HaCaT cells from UVB irradiation-induced death. Aa-EE increased the expression of a type 1 pro-collagen gene and decreased the expression of matrix metalloproteinases, and COX-2 in NIH3T3 cells induced by UVB. Aa-EE increased the expression of transglutamase-1, hyaluronic acid synthase (HAS)-2, and HAS-3 in HaCaT cells and decreased the production of melanin in α-melanocyte-stimulating hormone-stimulated B16F10 cells by suppressing tyrosinase activity and the expression of tyrosinase, microphthalmia-associated transcription factor, tyrosinase-related protein (TRP)-1 and TRP-2. The results suggest that Aa-EE could be skin-protective remedy with anti-photoaging, anti-apoptotic, skin remodeling, moisturizing, and anti-melanogenesis properties. Copyright © 2018 Elsevier B.V. All rights reserved.

Integration of growth factor signals at the c-fos serum response element.

PubMed

Price, M A; Hill, C; Treisman, R

1996-04-29

A transcription factor ternary complex composed of serum response factor (SRF) and a second factor, ternary complex factor (TCF), mediates the response of the c-fos Serum Response Element to growth factors and mitogens. In NIH3T3 fibroblasts, TCF binding is required for transcriptional activation by the SRE in response to activation of the Ras-Raf-ERK pathway. We compared the properties of three members of the TCF family, Elk-1, SAP-1 and SAP-2 (ERP/NET). Although all the proteins contain sequences required for ternary complex formation with SRF, only Elk-1 and SAP-1 appear to interact with the c-fos SRE efficiently in vivo. Each TCF contains a C-terminal activation domain capable of transcriptional activation in response to activation of the Ras-Raf-ERK pathway, and this is dependent on the integrity of S/T-P motifs conserved between all the TCF family members. In contrast, activation of the SRE by whole serum and the mitogenic phospholipid LPA requires SRF binding alone. Constitutively activated members of the Rho subfamily of Ras-like GTPases are also capable of inducing activation of the SRE in the absence of TCF; unlike activated Ras itself, these proteins do not activate the TCFs in NIH3T3 cells. At the SRE, SRF- and TCF-linked signalling pathways act synergistically to potentiate transcription.

Host range and cell cycle activation properties of polyomavirus large T-antigen mutants defective in pRB binding

DOE Office of Scientific and Technical Information (OSTI.GOV)

Freund, R.; Bauer, P.H.; Benjamin, T.L.

1994-11-01

The authors have examined the growth properties of polyomavirus large T-antigen mutants that ar unable to bind pRB, the product of the retinoblastoma tumor suppressor gene. These mutants grow poorly on primary mouse cells yet grow well on NIH 3T3 and other established mouse cell lines. Preinfection of primary baby mouse kidney (BMK) epithelial cells with wild-type simian virus 40 renders these cells permissive to growth of pRB-binding polyomavirus mutants. Conversely, NIH 3T3 cells transfected by and expressing wild-type human pRB become nonpermissive. Primary fibroblasts for mouse embryos that carry a homozygous knockout of the RB gene are permissive, whilemore » those from normal littermates are nonpermissive. The host range of polyomavirus pRB-binding mutants is thus determined by expression or lack of expression of functional pRB by the host. These results demonstrate the importance of pRB binding by large T antigen for productive viral infection in primary cells. Failure of pRB-binding mutants to grow well in BMK cells correlates with their failure to induce progression from G{sub 0} or G{sub 1} through the S phase of the cell cycle. Time course studies show delayed synthesis and lower levels of accumulation of large T antigen, viral DNA, and VP1 in mutant compared with wild-type virus-infected BMK cells. These results support a model in which productive infection by polyomavirus in normal mouse cells is tightly coupled to the induction and progression of the cell cycle. 48 refs., 6 figs., 5 tabs.« less

Barium promotes anchorage-independent growth and invasion of human HaCaT keratinocytes via activation of c-SRC kinase.

PubMed

Thang, Nguyen Dinh; Yajima, Ichiro; Kumasaka, Mayuko Y; Ohnuma, Shoko; Yanagishita, Takeshi; Hayashi, Rumiko; Shekhar, Hossain U; Watanabe, Daisuke; Kato, Masashi

2011-01-01

Explosive increases in skin cancers have been reported in more than 36 million patients with arsenicosis caused by drinking arsenic-polluted well water. This study and previous studies showed high levels of barium as well as arsenic in the well water. However, there have been no reports showing a correlation between barium and cancer. In this study, we examined whether barium (BaCl(2)) may independently have cancer-related effects on human precancerous keratinocytes (HaCaT). Barium (5-50 µM) biologically promoted anchorage-independent growth and invasion of HaCaT cells in vitro. Barium (5 µM) biochemically enhanced activities of c-SRC, FAK, ERK and MT1-MMP molecules, which regulate anchorage-independent growth and/or invasion. A SRC kinase specific inhibitor, protein phosphatase 2 (PP2), blocked barium-mediated promotion of anchorage-independent growth and invasion with decreased c-SRC kinase activity. Barium (2.5-5 µM) also promoted anchorage-independent growth and invasion of fibroblasts (NIH3T3) and immortalized nontumorigenic melanocytes (melan-a), but not transformed cutaneous squamous cell carcinoma (HSC5 and A431) and malignant melanoma (Mel-ret) cells, with activation of c-SRC kinase. Taken together, our biological and biochemical findings newly suggest that the levels of barium shown in drinking well water independently has the cancer-promoting effects on precancerous keratinocytes, fibroblast and melanocytes in vitro.

Barium Promotes Anchorage-Independent Growth and Invasion of Human HaCaT Keratinocytes via Activation of c-SRC Kinase

PubMed Central

Thang, Nguyen Dinh; Yajima, Ichiro; Kumasaka, Mayuko Y.; Ohnuma, Shoko; Yanagishita, Takeshi; Hayashi, Rumiko; Shekhar, Hossain U.; Watanabe, Daisuke; Kato, Masashi

2011-01-01

Explosive increases in skin cancers have been reported in more than 36 million patients with arsenicosis caused by drinking arsenic-polluted well water. This study and previous studies showed high levels of barium as well as arsenic in the well water. However, there have been no reports showing a correlation between barium and cancer. In this study, we examined whether barium (BaCl2) may independently have cancer-related effects on human precancerous keratinocytes (HaCaT). Barium (5–50 µM) biologically promoted anchorage-independent growth and invasion of HaCaT cells in vitro. Barium (5 µM) biochemically enhanced activities of c-SRC, FAK, ERK and MT1-MMP molecules, which regulate anchorage-independent growth and/or invasion. A SRC kinase specific inhibitor, protein phosphatase 2 (PP2), blocked barium-mediated promotion of anchorage-independent growth and invasion with decreased c-SRC kinase activity. Barium (2.5–5 µM) also promoted anchorage-independent growth and invasion of fibroblasts (NIH3T3) and immortalized nontumorigenic melanocytes (melan-a), but not transformed cutaneous squamous cell carcinoma (HSC5 and A431) and malignant melanoma (Mel-ret) cells, with activation of c-SRC kinase. Taken together, our biological and biochemical findings newly suggest that the levels of barium shown in drinking well water independently has the cancer-promoting effects on precancerous keratinocytes, fibroblast and melanocytes in vitro. PMID:22022425

Multifunctional quantum dot-polypeptide hybrid nanogel for targeted imaging and drug delivery

NASA Astrophysics Data System (ADS)

Yang, Jie; Yao, Ming-Hao; Wen, Lang; Song, Ji-Tao; Zhang, Ming-Zhen; Zhao, Yuan-Di; Liu, Bo

2014-09-01

A new type of multifunctional quantum dot (QD)-polypeptide hybrid nanogel with targeted imaging and drug delivery properties has been developed by metal-affinity driven self-assembly between artificial polypeptides and CdSe-ZnS core-shell QDs. On the surface of QDs, a tunable sandwich-like microstructure consisting of two hydrophobic layers and one hydrophilic layer between them was verified by capillary electrophoresis, transmission electron microscopy, and dynamic light scattering measurements. Hydrophobic and hydrophilic drugs can be simultaneously loaded in a QD-polypeptide nanogel. In vitro drug release of drug-loaded QD-polypeptide nanogels varies strongly with temperature, pH, and competitors. A drug-loaded QD-polypeptide nanogel with an arginine-glycine-aspartic acid (RGD) motif exhibited efficient receptor-mediated endocytosis in αvβ3 overexpressing HeLa cells but not in the control MCF-7 cells as analyzed by confocal microscopy and flow cytometry. In contrast, non-targeted QD-polypeptide nanogels revealed minimal binding and uptake in HeLa cells. Compared with the original QDs, the QD-polypeptide nanogels showed lower in vitro cytotoxicity for both HeLa cells and NIH 3T3 cells. Furthermore, the cytotoxicity of the targeted QD-polypeptide nanogel was lower for normal NIH 3T3 cells than that for HeLa cancer cells. These results demonstrate that the integration of imaging and drug delivery functions in a single QD-polypeptide nanogel has the potential for application in cancer diagnosis, imaging, and therapy.A new type of multifunctional quantum dot (QD)-polypeptide hybrid nanogel with targeted imaging and drug delivery properties has been developed by metal-affinity driven self-assembly between artificial polypeptides and CdSe-ZnS core-shell QDs. On the surface of QDs, a tunable sandwich-like microstructure consisting of two hydrophobic layers and one hydrophilic layer between them was verified by capillary electrophoresis, transmission electron microscopy, and dynamic light scattering measurements. Hydrophobic and hydrophilic drugs can be simultaneously loaded in a QD-polypeptide nanogel. In vitro drug release of drug-loaded QD-polypeptide nanogels varies strongly with temperature, pH, and competitors. A drug-loaded QD-polypeptide nanogel with an arginine-glycine-aspartic acid (RGD) motif exhibited efficient receptor-mediated endocytosis in αvβ3 overexpressing HeLa cells but not in the control MCF-7 cells as analyzed by confocal microscopy and flow cytometry. In contrast, non-targeted QD-polypeptide nanogels revealed minimal binding and uptake in HeLa cells. Compared with the original QDs, the QD-polypeptide nanogels showed lower in vitro cytotoxicity for both HeLa cells and NIH 3T3 cells. Furthermore, the cytotoxicity of the targeted QD-polypeptide nanogel was lower for normal NIH 3T3 cells than that for HeLa cancer cells. These results demonstrate that the integration of imaging and drug delivery functions in a single QD-polypeptide nanogel has the potential for application in cancer diagnosis, imaging, and therapy. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr03058c

A Novel Therapeutic Vaccine for Metastatic Mammary Carcinoma: Focusing MHC/Peptide Complexes to Lipid Rafts

DTIC Science & Technology

2006-11-01

reportable outcomes). Briefly, the T cell lymphoma EL4 and the immortalized fibroblast cell line DAP (both expressing ova) were used to measure...and Use Committee. Cells, transfections, and antibodies B16.BL6 8.2, A20, EL4 and EL4 /ova were cultured as described (20-22). NIH3T3 cells were...types can donate MHC class I molecules to DC. To determine if the levels of MHC class I on the donor cell affected the efficiency of transfer, EL4 /ova

Dual functional NaYF4:Yb3+, Er3+@NaYF4:Yb3+, Nd3+ core-shell nanoparticles for cell temperature sensing and imaging

NASA Astrophysics Data System (ADS)

Shi, Zengliang; Duan, Yue; Zhu, Xingjun; Wang, Qiwei; Li, DongDong; Hu, Ke; Feng, Wei; Li, Fuyou; Xu, Chunxiang

2018-03-01

Lanthanide-doped up-conversion nanoparticles (UCNPs) provide a remote temperature sensing approach to monitoring biological microenvironments. In this research, the UCNPs of NaYF4:Yb3+, Er3+@NaYF4:Yb3+, Nd3+ with hexagonal (β)-phase were synthesized and applied in cell temperature sensing as well as imaging after surface modification with meso-2, 3-dimercaptosuccinic acid. In the core-shell UCNPs, Yb3+ ions were introduced as energy transfer media between sensitizers of Nd3+ and activators of Er3+ to improve Er3+emission and prevent their quenching behavior due to multiple energy levels of Nd3+. Under the excitations of 808 nm and 980 nm lasers, the NaYF4:Yb3+, Er3+@NaYF4:Yb3+, Nd3+ nanoparticles exhibited an efficient green band with two emission peaks at 525 nm and 545 nm, respectively, which originated from the transitions of 2H11/2 → 4I15/2 and 4S3/2 → 4I15/2 for Er3+ ions. We demonstrate that an occurrence of good logarithmic linearity exists between the intensity ratio of these two emission peaks and the reciprocal of the inside or outside temperature of NIH-3T3 cells. A better thermal stability is proved through temperature-dependent spectra with a heating-cooling cycle. The obtained viability of NIH-3T3 cells is greater than 90% after incubations of about 12 and 24 (h), and they possess a lower cytotoxicity of UCNPs. This work provides a method for monitoring the cell temperature and its living state from multiple dimensions including temperature response, cell images and visual up-conversion fluorescent color.

Improved l1-SPIRiT using 3D walsh transform-based sparsity basis.

PubMed

Feng, Zhen; Liu, Feng; Jiang, Mingfeng; Crozier, Stuart; Guo, He; Wang, Yuxin

2014-09-01

l1-SPIRiT is a fast magnetic resonance imaging (MRI) method which combines parallel imaging (PI) with compressed sensing (CS) by performing a joint l1-norm and l2-norm optimization procedure. The original l1-SPIRiT method uses two-dimensional (2D) Wavelet transform to exploit the intra-coil data redundancies and a joint sparsity model to exploit the inter-coil data redundancies. In this work, we propose to stack all the coil images into a three-dimensional (3D) matrix, and then a novel 3D Walsh transform-based sparsity basis is applied to simultaneously reduce the intra-coil and inter-coil data redundancies. Both the 2D Wavelet transform-based and the proposed 3D Walsh transform-based sparsity bases were investigated in the l1-SPIRiT method. The experimental results show that the proposed 3D Walsh transform-based l1-SPIRiT method outperformed the original l1-SPIRiT in terms of image quality and computational efficiency. Copyright © 2014 Elsevier Inc. All rights reserved.

The arginine metabolite agmatine protects mitochondrial function and confers resistance to cellular apoptosis

PubMed Central

Arndt, Mary Ann; Battaglia, Valentina; Parisi, Eva; Lortie, Mark J.; Isome, Masato; Baskerville, Christopher; Pizzo, Donald P.; Ientile, Riccardo; Colombatto, Sebastiano; Toninello, Antonio; Satriano, Joseph

2009-01-01

Agmatine, an endogenous metabolite of arginine, selectively suppresses growth in cells with high proliferative kinetics, such as transformed cells, through depletion of intracellular polyamine levels. In the present study, we depleted intracellular polyamine content with agmatine to determine if attrition by cell death contributes to the growth-suppressive effects. We did not observe an increase in necrosis, DNA fragmentation, or chromatin condensation in Ha-Ras-transformed NIH-3T3 cells administered agmatine. In response to Ca2+-induced oxidative stress in kidney mitochondrial preparations, agmatine demonstrated attributes of a free radical scavenger by protecting against the oxidation of sulfhydryl groups and decreasing hydrogen peroxide content. The functional outcome was a protective effect against Ca2+-induced mitochondrial swelling and mitochondrial membrane potential collapse. We also observed decreased expression of proapoptotic Bcl-2 family members and of execution caspase-3, implying antiapoptotic potential. Indeed, we found that apoptosis induced by camptothecin or 5-fluorourocil was attenuated in cells administered agmatine. Agmatine may offer an alternative to the ornithine decarboxylase inhibitor difluoromethyl ornithine for depletion of intracellular polyamine content while avoiding the complications of increasing polyamine import and reducing the intracellular free radical scavenger capacity of polyamines. Depletion of intracellular polyamine content with agmatine suppressed cell growth, yet its antioxidant capacity afforded protection from mitochondrial insult and resistance to cellular apoptosis. These results could explain the beneficial outcomes observed with agmatine in models of injury and disease. PMID:19321739

Effects of diamines on ornithine decarboxylase activity in control and virally transformed mouse fibroblasts.

PubMed Central

Bethell, D R; Pegg, A E

1979-01-01

1. The induction of ornithine decarboxylase activity in mouse 3T3 fibroblasts or an SV-40 transformed 3T3 cell line by serum was prevented by addition of the naturally occurring polyamines putrescine (butane-1,4-diamine) and spermidine. Much higher concentrations of these amines were required to fully suppress ornithine decarboxylase activity in the transformed SV-3T3 cells than in the 3T3 fibroblasts. 2. Synthetic alpha omega-diamines with 3--12 carbon atoms also prevented the increase in ornithine decarboxylase activity induced by serum in these cells. The longer chain diamines were somewhat more potent than propane-1,3-diamine in this effect, but the synthetic diamines were less active than putrescine in the 3T3 cells. There was little difference between the responses of 3T3 and SV-3T3 cells to the synthetic diamines propane-1,3-diamine and heptane-1,7-diamine. 3. These results are discussed in relation to the control of polyamine synthesis in mammalian cells. PMID:486108

Hyaluronic acid oligosaccharide modified redox-responsive mesoporous silica nanoparticles for targeted drug delivery.

PubMed

Zhao, Qinfu; Geng, Hongjian; Wang, Ying; Gao, Yikun; Huang, Jiahao; Wang, Yan; Zhang, Jinghai; Wang, Siling

2014-11-26

A redox-responsive delivery system based on colloidal mesoporous silica (CMS) has been developed, in which 6-mercaptopurine (6-MP) was conjugated to vehicles by cleavable disulfide bonds. The oligosaccharide of hyaluronic acid (oHA) was modified on the surface of CMS by disulfide bonds as a targeting ligand and was able to increase the stability and biocompatibility of CMS under physiological conditions. In vitro release studies indicated that the cumulative release of 6-MP was less than 3% in the absence of glutathione (GSH), and reached nearly 80% within 2 h in the presence of 3 mM GSH. Confocal microscopy and fluorescence-activated cell sorter (FACS) methods were used to evaluate the cellular uptake performance of fluorescein isothiocyanate (FITC) labeled CMS, with and without oHA modification. The CMS-SS-oHA exhibited a higher cellular uptake performance via CD44 receptor-mediated endocytosis in HCT-116 (CD44 receptor-positive) cells than in NIH-3T3 (CD44 receptor-negative) cells. 6-MP loaded CMS-SS-oHA exhibited greater cytotoxicity against HCT-116 cells than NIH-3T3 cells due to the enhanced cell uptake behavior of CMS-SS-oHA. This study provides a novel strategy to covalently link bioactive drug and targeting ligand to the interiors and exteriors of mesoporous silica to construct a stimulus-responsive targeted drug delivery system.

Induction of G2/M arrest and apoptosis through mitochondria pathway by a dimer sesquiterpene lactone from Smallanthus sonchifolius in HeLa cells.

PubMed

Kitai, Yurika; Zhang, Xia; Hayashida, Yushi; Kakehi, Yoshiyuki; Tamura, Hirotoshi

2017-07-01

Dimer sesquiterpene lactones (SLs), uvedafolin and enhydrofolin, against four monomer SLs isolated from yacon, Smallanthus sonchifolius, leaf were the most cytotoxic substances on HeLa cells (IC 50 values 2.96-3.17 μM at 24 hours). However, the cytotoxic mechanism of dimer SL has not been elucidated yet. Therefore, in this study, we clarified the in vitro cytotoxic mechanism of uvedafolin on the HeLa cells, and evaluated the cytotoxicity against NIH/3T3 cells which were used as normal cells. In consequence, the dimer SLs had low toxicity for the NIH/3T3 cells (IC 50 4.81-4.98 μM at 24 hours) and then the uvedafolin mediated cell cycle arrest at the G 2 /M phase and induced apoptosis on the HeLa cells evidenced by appearance of a subG1 peak. Uvedafolin induced apoptosis was attributed to caspase-9 and caspase-3/7 activities. An effectively induced apoptosis pathway was demonstrated from mitochondria membrane potential change and cytochrome c release to cytosol. These results reveal that uvedafolin induced apoptosis via the mitochondria pathway. The present results indicate the potential of uvedafolin as a leading compound of new anticancer agents. Copyright © 2016. Published by Elsevier B.V.

Imaging, Spectroscopic, Mechanical and Biocompatibility Studies of Electrospun Tecoflex® EG 80A Nanofibers and Composites Thereof Containing Multiwalled Carbon Nanotubes

PubMed Central

Macossay, Javier; Sheikh, Faheem A.; Cantu, Travis; Eubanks, Thomas M.; Salinas, M. Esther; Farhangi, Chakavak S.; Ahmad, Hassan; Hassan, M. Shamshi; Khil, Myung-seob; Maffi, Shivani K.; Kim, Hern; Bowlin, Gary l.

2014-01-01

The present study discusses the design, development and characterization of electrospun Tecoflex® EG 80A class of polyurethane nanofibers and the incorporation of multiwalled carbon nanotubes (MWCNTs) to these materials. Scanning electron microscopy results confirmed the presence of polymer nanofibers, which showed a decrease in fiber diameter at 0.5% wt. and 1% wt. MWCNTs loadings, while transmission electron microscopy showed evidence of the MWCNTs embedded within the polymer matrix. The fourier transform infrared spectroscopy and Raman spectroscopy were used to elucidate the polymer-MWCNTs intermolecular interactions, indicating that the C-N and N-H bonds in polyurethanes are responsible for the interactions with MWCNTs. Furthermore, tensile testing indicated an increase in the Young’s modulus of the nanofibers as the MWCNTs concentration was increased. Finally, NIH 3T3 fibroblasts were seeded on the obtained nanofibers, demonstrating cell biocompatibility and proliferation. Therefore, the results indicate the successful formation of polyurethane nanofibers with enhanced mechanical properties, and demonstrate their biocompatibility, suggesting their potential application in biomedical areas. PMID:25435600

Imaging, spectroscopic, mechanical and biocompatibility studies of electrospun Tecoflex® EG 80A nanofibers and composites thereof containing multiwalled carbon nanotubes

NASA Astrophysics Data System (ADS)

Macossay, Javier; Sheikh, Faheem A.; Cantu, Travis; Eubanks, Thomas M.; Salinas, M. Esther; Farhangi, Chakavak S.; Ahmad, Hassan; Hassan, M. Shamshi; Khil, Myung-seob; Maffi, Shivani K.; Kim, Hern; Bowlin, Gary l.

2014-12-01

The present study discusses the design, development, and characterization of electrospun Tecoflex® EG 80A class of polyurethane nanofibers and the incorporation of multiwalled carbon nanotubes (MWCNTs) to these materials. Scanning electron microscopy results confirmed the presence of polymer nanofibers, which showed a decrease in fiber diameter at 0.5% wt. and 1% wt. MWCNTs loadings, while transmission electron microscopy showed evidence of the MWCNTs embedded within the polymer matrix. The Fourier transform infrared spectroscopy and Raman spectroscopy were used to elucidate the polymer-MWCNTs intermolecular interactions, indicating that the C-N and N-H bonds in polyurethanes are responsible for the interactions with MWCNTs. Furthermore, tensile testing indicated an increase in the Young's modulus of the nanofibers as the MWCNTs concentration was increased. Finally, NIH 3T3 fibroblasts were seeded on the obtained nanofibers, demonstrating cell biocompatibility and proliferation. Therefore, the results indicate the successful formation of polyurethane nanofibers with enhanced mechanical properties, and demonstrate their biocompatibility, suggesting their potential application in biomedical areas.

Imaging, Spectroscopic, Mechanical and Biocompatibility Studies of Electrospun Tecoflex® EG 80A Nanofibers and Composites Thereof Containing Multiwalled Carbon Nanotubes.

PubMed

Macossay, Javier; Sheikh, Faheem A; Cantu, Travis; Eubanks, Thomas M; Salinas, M Esther; Farhangi, Chakavak S; Ahmad, Hassan; Hassan, M Shamshi; Khil, Myung-Seob; Maffi, Shivani K; Kim, Hern; Bowlin, Gary L

2014-12-01

The present study discusses the design, development and characterization of electrospun Tecoflex ® EG 80A class of polyurethane nanofibers and the incorporation of multiwalled carbon nanotubes (MWCNTs) to these materials. Scanning electron microscopy results confirmed the presence of polymer nanofibers, which showed a decrease in fiber diameter at 0.5% wt. and 1% wt. MWCNTs loadings, while transmission electron microscopy showed evidence of the MWCNTs embedded within the polymer matrix. The fourier transform infrared spectroscopy and Raman spectroscopy were used to elucidate the polymer-MWCNTs intermolecular interactions, indicating that the C-N and N-H bonds in polyurethanes are responsible for the interactions with MWCNTs. Furthermore, tensile testing indicated an increase in the Young's modulus of the nanofibers as the MWCNTs concentration was increased. Finally, NIH 3T3 fibroblasts were seeded on the obtained nanofibers, demonstrating cell biocompatibility and proliferation. Therefore, the results indicate the successful formation of polyurethane nanofibers with enhanced mechanical properties, and demonstrate their biocompatibility, suggesting their potential application in biomedical areas.

Hypotonic activation of short ClC3 isoform is modulated by direct interaction between its cytosolic C-terminal tail and subcortical actin filaments.

PubMed

McCloskey, Diana T; Doherty, Lynda; Dai, Yan-Ping; Miller, Lisa; Hume, Joseph R; Yamboliev, Ilia A

2007-06-08

Short ClC3 isoform (sClC3) functions as a volume-sensitive outwardly rectifying anion channel (VSOAC) in some cell types. In previous studies, we have shown that the hypotonic activation of sClC3 is linked to cell swelling-mediated remodeling of the actin cytoskeleton. In the present study, we have tested the hypothesis that the cytosolic tails of sClC3 bind to actin directly and that binding modulates the hypotonic activation of the channel. Co-sedimentation assays in vitro demonstrated a strong binding between the glutathione S-transferase-fused cytosolic C terminus of sClC3 (GST-sClC3-CT) to filamentous actin (F-actin) but not to globular monomeric actin (G-actin). The GST-fused N terminus (GST-sClC3-NT) exhibited low binding affinity to both G- and F-actin. Co-sedimentation experiments with progressively truncated GST-sClC3-CT indicated that the F-actin binding region is located between amino acids 690 and 760 of sClC3. Two synthetic peptides mapping basic clusters of the cytosolic sClC3-CT (CTP2, isoleucine 716 to leucine 734; and CTP3, proline 688 to proline 709) prevented binding of GST-sClC3-CT to F-actin in vitro. Dialysis into NIH/3T3 cells of these two peptides (but not of synthetic peptide CTP1 (isoleucine 737 to glutamine 748)) reduced the maximal current density by 60 and 38%, respectively. Based on these results, we have concluded that, by direct interaction with subcortical actin filaments, sClC3 contributes to the hypotonic stress-induced VSOACs in NIH/3T3 cells.

3 Tools to Help You Quit | NIH MedlinePlus the Magazine

MedlinePlus

... and long-term challenges, start by examining your smoking habits. Track how many cigarettes you smoke a day and what you are doing when you light up. Look for patterns. You may discover triggers you weren't aware of. Smoking at certain times or in varying circumstances may ...

Wound-healing promoting effect of total tannins from Entada phaseoloides (L.) Merr. in rats.

PubMed

Su, Xiaowen; Liu, Xinguang; Wang, Shouyu; Li, Bin; Pan, Taowen; Liu, Dingrui; Wang, Fei; Diao, Yunpeng; Li, Kun

2017-06-01

The healing of wounds has always provided challenges for the medical community whether chronic or acute. Modern and traditional medicine has proved that herbal medicine shown superiority over chemical drugs. Herein, we report an Entada phaseoloides (L.) Merr. extract with a total tannin content of 76.18% showed wound-healing promoting effect in rat model. We found significantly accelerated wound closure already on day 7 in animals treated with total Entada phaseoloides (L.) Merr. tannins (TEPT) as compared to vaseline treated controls (p<0.05). At day 15, histologically, the wounds in animals treated with TEPT were completely closed as compared to controls. In vitro, TEPT promotes fibroblast proliferation and migration into wounds of NIH3T3 with concentration range of 9.38-37.50μg/ml. TEPT also had an inhibitory action against Staphylococcus aureus with MBC of 1.5mg/ml and the result was further proved by transmission electron microscope. Thus, TEPT could promote wound shrinkage, improve healing rate and promote healing of infectious wounds in rats. And this effect may due to antibacterial activities and NIH3T3 cell pro-proliferative effect of the tannins compounds, which indicating that TEPT can be used as efficient treatment in traumatic injury. Copyright © 2016 Elsevier Ltd and ISBI. All rights reserved.

Tunable swelling of polyelectrolyte multilayers in cell culture media for modulating NIH-3T3 cells adhesion.

PubMed

Qi, Wei; Cai, Peng; Yuan, Wenjing; Wang, Hua

2014-11-01

For polyelectrolyte multilayers (PEMs) assembled by the layer-by-layer (LbL) assembly technique, their nanostructure and properties can be governed by many parameters during the building process. Here, it was demonstrated that the swelling of the PEMs containing poly(diallyldimethylammonium chloride) (PDDA) and poly(sodium 4-styrenesulfonate) (PSS) in cell culture media could be tuned with changing supporting salt solutions during the assembly process. Importantly, the influence of the PEMs assembled in different salt solutions on NIH-3T3 cell adhesion was observable. Specifically, the cells could possess a higher affinity for the films assembled in low salt concentration (i.e. 0.15M NaCl) or no salt, the poorly swelling films in cell culture media, which was manifested by the large cell spreading area and focal adhesions. In contrast, those were assembled in higher salt concentration, highly swelling films in cell culture media, were less attractive for the fibroblasts. As a result, the cell adhesion behaviors may be manipulated by tailoring the physicochemical properties of the films, which could be performed by changing the assembly conditions such as supporting salt concentration. Such a finding might promise a great potential in designing desired biomaterials for tissue engineering and regenerative medicine. © 2014 Wiley Periodicals, Inc.

Chemical sporulation and germination: cytoprotective nanocoating of individual mammalian cells with a degradable tannic acid-FeIII complex

NASA Astrophysics Data System (ADS)

Lee, Juno; Cho, Hyeoncheol; Choi, Jinsu; Kim, Doyeon; Hong, Daewha; Park, Ji Hun; Yang, Sung Ho; Choi, Insung S.

2015-11-01

Individual mammalian cells were coated with cytoprotective and degradable films by cytocompatible processes maintaining the cell viability. Three types of mammalian cells (HeLa, NIH 3T3, and Jurkat cells) were coated with a metal-organic complex of tannic acid (TA) and ferric ion, and the TA-FeIII nanocoat effectively protected the coated mammalian cells against UV-C irradiation and a toxic compound. More importantly, the cell proliferation was controlled by programmed formation and degradation of the TA-FeIII nanocoat, mimicking the sporulation and germination processes found in nature.Individual mammalian cells were coated with cytoprotective and degradable films by cytocompatible processes maintaining the cell viability. Three types of mammalian cells (HeLa, NIH 3T3, and Jurkat cells) were coated with a metal-organic complex of tannic acid (TA) and ferric ion, and the TA-FeIII nanocoat effectively protected the coated mammalian cells against UV-C irradiation and a toxic compound. More importantly, the cell proliferation was controlled by programmed formation and degradation of the TA-FeIII nanocoat, mimicking the sporulation and germination processes found in nature. Electronic supplementary information (ESI) available: Experimental details, LSCM images, and SEM and TEM images. See DOI: 10.1039/c5nr05573c

Binding efficiency of recombinant collagen-binding basic fibroblast growth factors (CBD-bFGFs) and their promotion for NIH-3T3 cell proliferation.

PubMed

Wu, Zhenxu; Zhou, Yulai; Chen, Li; Hu, Mingxin; Wang, Yu; Li, Linlong; Wang, Zongliang; Zhang, Peibiao

2018-03-01

The recombinant basic fibroblast growth factor (bFGF) containing collagen-binding domain (CBD) has been found to be a potential therapeutic factor in tissue regeneration. However, its binding efficiency and quantification remain uncertain. In this research, massive recombinant bFGFs with good bioactivity for enhancing the proliferation of NIH-3T3 cells were achieved. An ELISA-based quantitative method was set up to investigate the binding efficiency of CBD-bFGFs on collagen films. It indicated that the CBDs significantly increased the collagen-binding ability of bFGF (P < .05), with the optimum binding condition first determined to be in the pH range of 7.5-9.5 (P < .05). Then, the relevant equations to calculate the binding density of bFGF, C-bFGF, and V-bFGF were acquired. Analysis confirmed that the bioactivity of immobilized bFGFs was well correlated with the density of growth factor on collagen films. Based on this research, the density of growth factor is a logical and applicable dosage unit for quantification of binding efficiency of growth factors, rather than traditional concentration of soluble growth factors in tissue engineering applications. © 2018 Wiley Periodicals, Inc.

Improved penetration of wild ginseng extracts into the skin using low-temperature atmospheric pressure plasma

NASA Astrophysics Data System (ADS)

Nam, Seoul Hee; Hae Choi, Jeong; Song, Yeon Suk; Lee, Hae-June; Hong, Jin-Woo; Kim, Gyoo Cheon

2018-04-01

Wild ginseng (WG) is a well-known traditional medicinal plant that grows in natural environments in deep mountains. WG has been thought to exert potent physiological and medicinal effects, and, recently, its use in skin care has attracted much interest. This study investigated the efficient penetration of WG extracts into the skin by means of low-temperature atmospheric pressure plasma (LTAPP), and its effects on the skin at the cellular and tissue levels. NIH3T3 mouse embryonic fibroblasts and HRM-2 hairless mice were used to confirm the improved absorption of WG extracts into the skin using LTAPP. The gene expression levels in NIH3T3 cells and morphological changes in skin tissues after WG treatment were monitored using both in vitro and in vivo experiments. Although WG extracts did not show any significant effects on proliferative activity and cytotoxicity, at a concentration of 1:800, it significantly increased the expression of fibronectin and vascular endothelial growth factor. In the in vivo study, the combinational treatment of LTAPP and WG markedly induced the expression of fibronectin and integrin α6, and it thickened. Our results showed that LTAPP treatment safely and effectively accelerated the penetration of the WG extracts into the skin, thereby increasing the effects of WG on the skin.

Analyses of variant human papillomavirus type-16 E5 proteins for their ability to induce mitogenesis of murine fibroblasts

PubMed Central

Nath, Rahul; Mant, Christine A; Kell, Barbara; Cason, John; Bible, Jon M

2006-01-01

Background Human papillomavirus type 16 (HPV-16) E5 protein co-operates with epidermal growth factor to stimulate mitogenesis of murine fibroblasts. Currently, little is known about which viral amino acids are involved in this process. Using sequence variants of HPV-16 E5 we have investigated their effects upon E5 transcription, cell-cycling and cell-growth of murine fibroblasts. Results We demonstrate that: (i) introduction of Thr64 into the reference E5 sequence of HPV-16 abrogates mitogenic activity: both were poorly transcribed in NIH-3T3 cells; (ii) substitution of Leu44Val65 or, Thr37Leu44Val65 into the HPV-16 E5 reference backbone resulted in high transcription in NIH-3T3 cells, enhanced cell-cycle progression and high cell-growth; and, (iii) inclusion of Tyr8 into the Leu44Val65 backbone inhibited E5 induced cell-growth and repression of p21 expression, despite high transcription levels. Conclusion The effects of HPV-16 E5 variants upon mitosis help to explain why Leu44Val65 HPV-16 E5 variants are most prevalent in 'wild' pathogenic viral populations in the UK. PMID:16899131

Enhanced fluorescence detection using liquid-liquid extraction in a microfluidic droplet system.

PubMed

Chen, Yan-Yu; Chen, Zhao-Ming; Wang, Hsiang-Yu

2012-11-07

Reducing the fluorescence background in microfluidic assays is important in obtaining accurate outcomes and enhancing the quality of detections. This study demonstrates an integrated process including cell labelling, fluorescence background reduction, and biomolecule detection using liquid-liquid extraction in a microfluidic droplet system. The cellular lipids in Chlorella vulgaris and NIH/3T3 cells were labelled with a hydrophobic dye, Nile red, to investigate the performance of the proposed method. The fluorescence background of the lipid detection can be reduced by 85% and the removal efficiency increased with the volume of continuous phase surrounding a droplet. The removal rate of the fluorescence background increased as the surface area to volume ratio of a droplet increased. Before Nile red was removed from the droplet, the signal to noise ratio was as low as 1.30 and it was difficult to distinguish cells from the background. Removing Nile red increased the signal to noise ratio to 22 and 34 for Chlorella vulgaris and NIH/3T3, respectively, and these were 17 fold and 10 fold of the values before extraction. The proposed method successfully demonstrates the enhancement of fluorescence detection of cellular lipids and has great potential in improving other fluorescence-based detections in microfluidic systems.

Magnetic Beads Enhance Adhesion of NIH 3T3 Fibroblasts: A Proof-of-Principle In Vitro Study for Implant-Mediated Long-Term Drug Delivery to the Inner Ear

PubMed Central

Aliuos, Pooyan; Schulze, Jennifer; Schomaker, Markus; Reuter, Günter; Stolle, Stefan R. O.; Werner, Darja; Ripken, Tammo; Lenarz, Thomas; Warnecke, Athanasia

2016-01-01

Introduction Long-term drug delivery to the inner ear may be achieved by functionalizing cochlear implant (CI) electrodes with cells providing neuroprotective factors. However, effective strategies in order to coat implant surfaces with cells need to be developed. Our vision is to make benefit of electromagnetic field attracting forces generated by CI electrodes to bind BDNF-secreting cells that are labelled with magnetic beads (MB) onto the electrode surfaces. Thus, the effect of MB-labelling on cell viability and BDNF production were investigated. Materials and Methods Murine NIH 3T3 fibroblasts—genetically modified to produce BDNF—were labelled with MB. Results Atomic force and bright field microscopy illustrated the internalization of MB by fibroblasts after 24 h of cultivation. Labelling cells with MB did not expose cytotoxic effects on fibroblasts and allowed adhesion on magnetic surfaces with sufficient BDNF release. Discussion Our data demonstrate a novel approach for mediating enhanced long-term adhesion of BDNF-secreting fibroblasts on model electrode surfaces for cell-based drug delivery applications in vitro and in vivo. This therapeutic strategy, once transferred to cells suitable for clinical application, may allow the biological modifications of CI surfaces with cells releasing neurotrophic or other factors of interest. PMID:26918945

Augmentor α and β (FAM150) are ligands of the receptor tyrosine kinases ALK and LTK: Hierarchy and specificity of ligand-receptor interactions.

PubMed

Reshetnyak, Andrey V; Murray, Phillip B; Shi, Xiarong; Mo, Elizabeth S; Mohanty, Jyotidarsini; Tome, Francisco; Bai, Hanwen; Gunel, Murat; Lax, Irit; Schlessinger, Joseph

2015-12-29

Receptor tyrosine kinases (RTKs) are a class of cell surface receptors that, upon ligand binding, stimulate a variety of critical cellular functions. The orphan receptor anaplastic lymphoma kinase (ALK) is one of very few RTKs that remain without a firmly established protein ligand. Here we present a novel cytokine, FAM150B, which we propose naming augmentor-α (AUG-α), as a ligand for ALK. AUG-α binds ALK with high affinity and activates ALK in cells with subnanomolar potency. Detailed binding experiments using cells expressing ALK or the related receptor leukocyte tyrosine kinase (LTK) demonstrate that AUG-α binds and robustly activates both ALK and LTK. We show that the previously established LTK ligand FAM150A (AUG-β) is specific for LTK and only weakly binds to ALK. Furthermore, expression of AUG-α stimulates transformation of NIH/3T3 cells expressing ALK, induces IL-3 independent growth of Ba/F3 cells expressing ALK, and is expressed in neuroblastoma, a cancer partly driven by ALK. These experiments reveal the hierarchy and specificity of two cytokines as ligands for ALK and LTK and set the stage for elucidating their roles in development and disease states.

Proinflammatory cytokines cause FAT10 upregulation in cancers of liver and colon.

PubMed

Lukasiak, S; Schiller, C; Oehlschlaeger, P; Schmidtke, G; Krause, P; Legler, D F; Autschbach, F; Schirmacher, P; Breuhahn, K; Groettrup, M

2008-10-09

The mRNA of the ubiquitin-like modifier FAT10 has been reported to be overexpressed in 90% of hepatocellular carcinoma (HCC) and in over 80% of colon, ovary and uterus carcinomas. Elevated FAT10 expression in malignancies was attributed to transcriptional upregulation upon the loss of p53. Moreover, FAT10 induced chromosome instability in long-term in vitro culture, which led to the hypothesis that FAT10 might be involved in carcinogenesis. In this study we show that interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha synergistically upregulated FAT10 expression in liver and colon cancer cells 10- to 100-fold. Real-time RT-PCR revealed that FAT10 mRNA was significantly overexpressed in 37 of 51 (72%) of human HCC samples and in 8 of 15 (53%) of human colon carcinomas. The FAT10 cDNA sequences in HCC samples were not mutated and intact FAT10 protein was detectable. FAT10 expression in both cancer tissues correlated with expression of the IFN-gamma- and TNF-alpha-dependent proteasome subunit LMP2 strongly suggesting that proinflammatory cytokines caused the joint overexpression of FAT10 and LMP2. NIH3T3 transformation assays revealed that FAT10 had no transforming capability. Taken together, FAT10 qualifies as a marker for an interferon response in HCC and colon carcinoma but is not significantly overexpressed in cancers lacking a proinflammatory environment.

MicroRNA-29b Regulates the Expression Level of Human Progranulin, a Secreted Glycoprotein Implicated in Frontotemporal Dementia

PubMed Central

Jiao, Jian; Herl, Lauren D.; Farese, Robert V.; Gao, Fen-Biao

2010-01-01

Progranulin deficiency is thought to cause some forms of frontotemporal dementia (FTD), a major early-onset age-dependent neurodegenerative disease. How progranulin (PGRN) expression is regulated is largely unknown. We identified an evolutionarily conserved binding site for microRNA-29b (miR-29b) in the 3′ untranslated region (3′UTR) of the human PGRN (hPGRN) mRNA. miR-29b downregulates the expression of luciferase through hPGRN or mouse PGRN (mPGRN) 3′UTRs, and the regulation was abolished by mutations in the miR-29b binding site. To examine the direct effect of manipulating endogenous miR-29b on hPGRN expression, we established a stable NIH3T3 cell line that expresses hPGRN under the control of the cytomegalovirus promoter. Ectopic expression of miR-29b decreased hPGRN expression at the both mRNA and protein levels. Conversely, knockdown of endogenous miR-29b with locked nucleic acid increased the production and secretion of hPGRN in NIH3T3 cells. Endogenous hPGRN in HEK 293 cells was also regulated by miR-29b. These findings identify miR-29b as a novel posttranscriptional regulator of PGRN expression, raising the possibility that miR-29b or other miRNAs might be targeted therapeutically to increase hPGRN levels in some FTD patients. PMID:20479936

Laser processing of polymer constructs from poly(3-hydroxybutyrate).

PubMed

Volova, T G; Tarasevich, A A; Golubev, A I; Boyandin, A N; Shumilova, A A; Nikolaeva, E D; Shishatskaya, E I

2015-01-01

CO2 laser radiation was used to process poly(3-hydroxybutyrate) constructs - films and 3D pressed plates. Laser processing increased the biocompatibility of unperforated films treated with moderate uniform radiation, as estimated by the number and degree of adhesion of NIH 3T3 mouse fibroblast cells. The biocompatibility of perforated films modified in the pulsed mode did not change significantly. At the same time, pulsed laser processing of the 3D plates produced perforated scaffolds with improved mechanical properties and high biocompatibility with bone marrow-derived multipotent, mesenchymal stem cells, which show great promise for bone regeneration.

The Use of Xanthan Gum as Vaccine Adjuvant: An Evaluation of Immunostimulatory Potential in BALB/c Mice and Cytotoxicity In Vitro

PubMed Central

Oliveira, Thaís Larré; Collares, Thaís Farias; Monte, Leonardo Garcia; Inda, Guilherme Roig; Moreira, Angelita da Silveira

2017-01-01

The successful production of new, safe, and effective vaccines that generate immunological memory is directly related to adjuvant feature, which is responsible for increasing and/or modulating the immune response. Several compounds display adjuvant activity, including carbohydrates. These compounds play important roles in the immune response, as well as having biocompatible properties in vaccine formulations. One such carbohydrate is xanthan gum, a polysaccharide that is produced by the plant-pathogenic bacterium Xanthomonas spp., which has adjuvant attributes. This study evaluated the immune response induced by xanthan gum associated with ovalbumin in BALB/c mice, which were subcutaneously immunized, in terms of antibody production (IgG1, IgG2a, IgG2b, and IgG3), and assessed the levels of IFN-γ in the splenocyte culture using indirect ELISA. Furthermore, we investigated in vitro cytotoxicity of xanthan in the embryo fibroblasts cell line of the NIH/3T3 mouse by MTT assay and propidium iodide uptake assay. The mice immunized with ovalbumin plus xanthan gum exhibited higher antibody IgG1 responses than control groups. Furthermore, the xanthan polysaccharide was capable of increasing the immunogenicity of antigens by producing IFN-γ and did not exhibit cytotoxicity effects in NIH/3T3 mouse fibroblast cells, considered a promising candidate for vaccine adjuvant. PMID:28555192

Interactions of polymeric drug carriers with DDT reduce their combined cytotoxicity.

PubMed

Zhang, Xuejiao; Lei, Lei; Zhang, Haiyan; Zhang, Siyu; Xing, Weiwei; Wang, Jin; Li, Haibo; Zhao, Qing; Xing, Baoshan

2018-06-07

Attention has been paid to the environmental distribution and fate of nanomedicines. However, their effects on the toxicity of environmental pollutants are lack of knowledge. In this study, the negatively charged poly (ethylene glycol)-b-poly (L-lactide-co-glycolide) (mPEG-PLA) and positively charged polyethyleneimine-palmitate (PEI-PA) nanomicelles were synthesized and served as model drug carriers to study the interaction and combined toxicity with dichlorodiphenyltrichloroethane (DDT). DDT exerted limited effect on the biointerfacial behavior of mPEG-PLA nanomicelles, whereas it significantly mitigated the attachment of PEI-PA nanomicelles on the model cell membrane as monitored by quartz crystal microbalance with dissipation (QCM-D). The cytotoxicity of DDT towards NIH 3T3 cells was greatly decreased by either co-treatment or pre-treatment with the nanomicelles according to the results of real-time cell analysis (RTCA). The cell viability of NIH 3T3 exposed to DDT was increased up to 90% by the co-treatment with mPEG-PLA nanomicelles. Three possible reasons were proposed: (1) decreased amount of free DDT in the cell culture medium due to the partitioning of DDT into nanomicelles; (2) mitigated cellular uptake of nanomicelle-DDT complexes due to the complex agglomeration or electrostatic repulsion between complexes and cell membrane; (3) detoxification effect in the lysosome upon endocytosis of nanomicelle-DDT complexes. Copyright © 2018 Elsevier Ltd. All rights reserved.

Optical spectrum measurement of a cell-adhered microcavity for the cell-cycle analysis applications

NASA Astrophysics Data System (ADS)

Saito, Ryusuke; Terakawa, Mitsuhiro; Tanabe, Takasumi

2015-03-01

We build a setup and demonstrate successful measurement of the transmittance spectrum of a whispering gallery mode silica optical microcavity in which NIH 3T3 cells adhered on the top surface to achieve real-time and label-free measurement of the cell cycle. Label-free measurement is expected to prevent the cells to exhibit secondary effect. We build a system that enables the control of the gap distance between the microcavity and the tapered fiber, both of which are placed in the cell culture medium. The optimization of the tapered fiber diameter is the key to measure the spectrum of a microcavity in liquid. A swept wavelength laser light at a wavelength of 766 to 780 nm is used for the measurement. The cavity exhibit a Q of 1 . 0 ×106 in air, where the value is 1 . 0 ×105 in the medium and drops to 3 . 1 ×104 after the cell-adhesion. Still the Q of the microcavity is sufficiently high to detect the change at the cavity surface. Indeed we observe slight spectrum shift toward a longer wavelength, which we believe is due to the adherence of NIH 3T3 cells on the silica microcavity.The successful measurement of the transmittance spectrum of a microcavity in cell culture medium is the first step to realize the analysis of the cell-cycle based on microcavity system.

A novel RET rearrangement (ACBD5/RET) by pericentric inversion, inv(10)(p12.1;q11.2), in papillary thyroid cancer from an atomic bomb survivor exposed to high-dose radiation.

PubMed

Hamatani, Kiyohiro; Eguchi, Hidetaka; Koyama, Kazuaki; Mukai, Mayumi; Nakachi, Kei; Kusunoki, Yoichiro

2014-11-01

During analysis of RET/PTC rearrangements in papillary thyroid cancer (PTC) among atomic bomb survivors, a cDNA fragment of a novel type of RET rearrangement was identified in a PTC patient exposed to a high radiation dose using the improved 5' RACE method. This gene resulted from the fusion of the 3' portion of RET containing tyrosine kinase domain to the 5' portion of the acyl-coenzyme A binding domain containing 5 (ACBD5) gene, by pericentric inversion inv(10)(p12.1;q11.2); expression of the fusion gene was confirmed by RT-PCR. ACBD5 gene is ubiquitously expressed in various human normal tissues including thyroid. Full-length cDNA of the ACBD5-RET gene was constructed and then examined for tumorigenicity. Enhanced phosphorylation of ERK proteins in the MAPK pathway was observed in NIH3T3 cells transfected with expression vector encoding the full-length ACBD5/RET cDNA, while this was not observed in the cells transfected with empty expression vector. Stable NIH3T3 transfectants with ACBD5-RET cDNA induced tumor formation after their injection into nude mice. These findings suggest that the ACBD5-RET rearrangement is causatively involved in the development of PTC.

Apoptosis induced by the myelodysplastic syndrome-associated NPM-MLF1 chimeric protein.

PubMed

Yoneda-Kato, N; Fukuhara, S; Kato, J

1999-06-24

The NPM-MLF1 chimeric protein is produced by the t(3;5)(q25.1;q34) chromosomal translocation, which is associated with myelodysplastic syndrome (MDS) prior to progression into acute myeloid leukemia (AML). Here we report that K562 human leukemia cells ectopically expressing NPM-MLF1, but not those with wild-type MLF1, were gradually eliminated from the culture by undergoing apoptosis. NIH3T3 mouse fibroblasts engineered to overexpress NPM-MLF1 grew normally but serum deprivation triggered apoptotic cell death with slower kinetics than did other well-known apoptotic inducers such as c-Myc or E2F-1. Quantitative analysis of apoptotic induction confirmed that, neither NPM nor MLF1, but the NPM-MLF1 fusion protein was able to induce apoptosis. Analyses using a variety of deletion mutants of NPM-MLF1 revealed that induction of apoptosis required the N-terminal domain of MLF1 and the NPM domain containing nuclear localization signal and that removal of the NPM dimerization domain markedly impaired the ability to induce apoptosis. Co-expression of Bcl-2 rescued NIH3T3 fibroblasts from NPM-MLF1-mediated cell death without affecting the expression level or the subcellular localization of NPM-MLF1 and enabled cells to progress into S phase in low serum. These findings provide an NPM-MLF1-mediated novel mechanism of apoptotic induction and imply that NPM-MLFI in collaboration with anti-apoptotic oncoproteins may play an important role in multi-step progression from MDS to AML.

Clinical response of the novel activating ALK-I1171T mutation in neuroblastoma to the ALK inhibitor ceritinib.

PubMed

Guan, Jikui; Fransson, Susanne; Siaw, Joachim Tetteh T; Treis, Diana; Van den Eynden, Jimmy; Chand, Damini; Umapathy, Ganesh; Svenberg, Petter; Ruuth, Kristina; Wessman, Sandra; Shamikh, Alia; Jacobsson, Hans; Gordon, Lena; Stenman, Jakob; Larsson, Erik; Svensson, Par-Johan; Hansson, Magnus; Martinsson, Tommy; Kogner, Per; Palmer, Ruth H; Hallberg, Bengt

2018-06-15

Tumors with Anaplastic Lymphoma Kinase (ALK) fusion rearrangements, including non-small cell lung cancer and anaplastic large cell lymphoma, are highly sensitive to ALK tyrosine kinase inhibitors (TKIs), underscoring the notion that such cancers are addicted to ALK activity. While mutations in ALK are heavily implicated in childhood neuroblastoma, response to the ALK TKI crizotinib has been disappointing. Embryonal tumors in patients with DNA repair defects such as Fanconi anemia (FA) often have a poor prognosis, due to lack of therapeutic options. Here we report a child with underlying FA and ALK mutant high-risk neuroblastoma responding strongly to precision therapy with the ALK TKI ceritinib. Conventional chemotherapy treatment caused severe, life-threatening toxicity. Genomic analysis of the initial biopsy identified germ-line FANCA mutations as well as a novel ALK-I1171T variant. ALK-I1171T generates a potent gain-of-function mutant, as measured in PC12 cell neurite outgrowth and NIH3T3 transformation. Pharmacological inhibition profiling of ALK-I1171T in response to various ALK TKIs identified an 11-fold improved inhibition of ALK-I1171T with ceritinib when compared with crizotinib. Immunoaffinity-coupled LC-MS/MS phosphoproteomics analysis indicated a decrease in ALK signaling in response to ceritinib. Ceritinib was therefore selected for treatment in this child. Mono-therapy with ceritinib was well tolerated and resulted in normalized catecholamine markers and tumor shrinkage. After 7.5 months treatment, residual primary tumor was surgically removed and exhibited hallmarks of differentiation together with reduced Ki67 levels. Clinical follow-up after 21 months treatment revealed complete clinical remission including all metastatic sites. Therefore, ceritinib presents a viable therapeutic option for ALK-positive neuroblastoma. Cold Spring Harbor Laboratory Press.

Synthesis of Bioactive Chlorogenic Acid-Silica Hybrid Materials via the Sol–Gel Route and Evaluation of Their Biocompatibility

PubMed Central

Pacifico, Severina

2017-01-01

Natural phenol compounds are gaining a great deal of attention because of their potential use as prophylactic and therapeutic agents in many diseases, as well as in applied science for their preventing role in oxidation deterioration. With the aim to synthetize new phenol-based materials, the sol–gel method was used to embed different content of the phenolic antioxidant chlorogenic acid (CGA) within silica matrices to obtain organic-inorganic hybrid materials. Fourier transform infrared (FTIR) measurements were used to characterize the prepared materials. The new materials were screened for their bioactivity and antioxidant potential. To this latter purpose, direct DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS (2,2′-azinobis-(3-ethylbenzothiazolin-6-sulfonic acid) methods were applied: radical scavenging capability appeared strongly dependent on the phenol amount in investigated hybrids, and became pronounced, mainly toward the ABTS radical cation, when materials with CGA content equal to 15 wt% and 20 wt% were analyzed. The in vitro biocompatibility of the synthetized materials was estimated by using the MTT assay towards fibroblast NIH 3T3 cells, human keratinocyte HaCaT cells, and the neuroblastoma SH-SY5Y cell line. As cell viability and morphology of tested cell lines seemed to be unaffected by new materials, the attenuated total reflectance (ATR)-FTIR method was applied to deeply measure the effects of the hybrids in the three different cell lines. PMID:28773198

Synthesis of Bioactive Chlorogenic Acid-Silica Hybrid Materials via the Sol-Gel Route and Evaluation of Their Biocompatibility.

PubMed

Catauro, Michelina; Pacifico, Severina

2017-07-21

Natural phenol compounds are gaining a great deal of attention because of their potential use as prophylactic and therapeutic agents in many diseases, as well as in applied science for their preventing role in oxidation deterioration. With the aim to synthetize new phenol-based materials, the sol-gel method was used to embed different content of the phenolic antioxidant chlorogenic acid (CGA) within silica matrices to obtain organic-inorganic hybrid materials. Fourier transform infrared (FTIR) measurements were used to characterize the prepared materials. The new materials were screened for their bioactivity and antioxidant potential. To this latter purpose, direct DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS (2,2'-azinobis-(3-ethylbenzothiazolin-6-sulfonic acid) methods were applied: radical scavenging capability appeared strongly dependent on the phenol amount in investigated hybrids, and became pronounced, mainly toward the ABTS radical cation, when materials with CGA content equal to 15 wt% and 20 wt% were analyzed. The in vitro biocompatibility of the synthetized materials was estimated by using the MTT assay towards fibroblast NIH 3T3 cells, human keratinocyte HaCaT cells, and the neuroblastoma SH-SY5Y cell line. As cell viability and morphology of tested cell lines seemed to be unaffected by new materials, the attenuated total reflectance (ATR)-FTIR method was applied to deeply measure the effects of the hybrids in the three different cell lines.

The combi-targeting concept: synthesis of stable nitrosoureas designed to inhibit the epidermal growth factor receptor (EGFR).

PubMed

Domarkas, Juozas; Dudouit, Fabienne; Williams, Christopher; Qiyu, Qiu; Banerjee, Ranjita; Brahimi, Fouad; Jean-Claude, Bertrand Jacques

2006-06-15

According to the "combi-targeting" concept, the EGFR tyrosine kinase (TK) inhibitory potency of compounds termed "combi-molecules" is critical for selective growth inhibition of tumor cells with disordered expression of EGFR or its closest family member erbB2. Here we report on the optimization of the EGFR TK inhibitory potency of the combi-molecules of the nitrosourea class by comparison with their aminoquinazoline and ureidoquinazoline precursors. This led to the discovery of a new structural parameter that influences their EGFR TK inhibitory potency, i.e., the torsion angle between the plane of the quinazoline ring and the ureido or the nitrosoureido moiety of the synthesized drugs. Compounds (3'-Cl and Br series) with small angles (0.5-3 degrees ) were generally stronger EGFR TK inhibitors than those with large angles (18-21 degrees ). This was further corroborated by ligand-receptor van der Waals interaction calculations that showed significant binding hindrance imposed by large torsion angles in the narrow ATP cleft of EGFR. Selective antiproliferative studies in a pair of mouse fibroblast NIH3T3 cells, one of which NIH3T3/neu being transfected with the erbB2 oncogene, showed that IC(50) values for inhibition of EGFR TK could be good predictors of their selective potency against the serum-stimulated growth of the erbB2-tranfected cell line (Pearson r = 0.8). On the basis of stability (t(1/2)), EGFR TK inhibitory potency (IC(50)), and selective erbB2 targeting, compound 23, a stable nitrosourea, was considered to have the structural requirements for further development.

An Overview of the Common Fluid Models Used in Fluid-Structure Interactions

DTIC Science & Technology

1991-08-05

inverse transform of (63) and us- ing the relation f (t - a) = exp (iwa)f(0) (64) for arbitrary (reasonably behaved) f, with f = C and a = r/c since...lri( r - ) - F -la* exp(ikr)dS’ using (53). The inverse transform of this equation gives 3 rV (Xi, t) = 2Vi c + T r3’fV+(- C (an ))dS’ (72) upon...T, when it exists, has the property rl-Tf= T=rf = f for a generic function f, the Laplace transform and inverse transform being examples of such a T

Cloning and expression of sheep renal K-CI cotransporter-1.

PubMed

Zhang, Jin J; Misri, Sandeep; Adragna, Norma C; Gagnon, Kenneth B E; Fyffe, Robert E W; Lauf, Peter K

2005-01-01

Sheep K-Cl cotransporter-1(shKCC1) cDNA was cloned from kidney by RT-PCR with an open reading frame of 3258 base pairs exhibiting 92%, 90%, 88% and 87% identity with pig, rabbit and human, rat and mouse KCC1 cDNAs, respectively, encoding an approximately 122 kDa polypeptide of 1086-amino acids. Hydropathy analysis reveals the familiar KCC1 topology with 12 transmembrane domains (TMDs) and the hydrophilic NH2-terminal (NTD) and COOH-terminal (CTD) domains both at the cytoplasmic membrane face. However, shKCC1 has two rather than one large extracellular loops (ECL): ECL3 between TMDs 5 and 6, and ECL6, between TMDs 11 and 12. The translated shKCC1 protein differs in 12 amino acid residues from other KCC1s, mainly within the NTD, ECL3, ICL4, ECL6, and CTD. Notably, a tyrosine residue at position 996 replaces aspartic acid conserved in all other species. Human embryonic kidney (HEK293) cells and mouse NIH/3T3 fibroblasts, transiently transfected with shKCCI-cDNA, revealed the glycosylated approximately 150 kDa proteins by Western blots and positive immunofluorescence-staining with polyclonal rabbit anti-ratKCC1 antibodies. ShKCC1 was functionally expressed in NIH/3T3 cells by an elevated basal Cl-dependent K influx measured with Rb as K-congener that was stimulated three-fold by the KCC-activator N-ethylmaleimide. Copyright (c) 2005 S. Karger AG, Basel.

Cyto/hemocompatible magnetic hybrid nanoparticles (Ag2S-Fe3O4) with luminescence in the near-infrared region as promising theranostic materials.

PubMed

Hocaoglu, Ibrahim; Asik, Didar; Ulusoy, Gulen; Grandfils, Christian; Ojea-Jimenez, Isaac; Rossi, François; Kiraz, Alper; Doğan, Nurcan; Acar, Havva Yagci

2015-09-01

Small hybrid nanoparticles composed of highly biocompatible Ag2S quantum dots (QD) emitting in the near-infrared region and superparamagnetic iron oxide (SPION) are produced in a simple extraction method utilizing ligand exchange mechanism. Hybrid nanoparticles luminesce at the same wavelength as the parent QD, therefore an array of hybrid nanoparticles with emission between 840 and 912nm were easily produced. Such hybrid structures have (1) strong luminescence in the medical imaging window eliminating the autofluoresence of cells as effective optical probes, (2) strong magnetic response for magnetic targeting and (3) good cyto/hemocompatibility. An interesting size dependent cytotoxicity behavior was observed in HeLa and NIH/3T3 cell lines: smallest particles are internalized significantly more by both of the cell lines, yet showed almost no significant cytotoxicity in HeLa between 10 and 25μg/mL Ag concentration but were most toxic in NIH/3T3 cells. Cell internalization and hence the cytotoxicity enhanced when cells were incubated with the hybrid nanoparticles under magnetic field, especially with the hybrid nanoparticles containing larger amounts of SPION in the hybrid composition. These results prove them as effective optical imaging agents and magnetic delivery vehicles. Combined with the known advantages of SPIONs as a contrast agent in MRI, these particles are a step forward for new theranostics for multimode imaging and magnetic targeting. Copyright © 2015 Elsevier B.V. All rights reserved.

Testosterone and Chronic Prostatitis/Chronic Pelvic Pain Syndrome: A Propensity Score-Matched Analysis.

PubMed

Lee, Jun Ho; Lee, Sung Won

2016-07-01

A recent study investigated the role of testosterone (T) in chronic prostatitis or chronic pelvic pain syndrome (CP/CPPS). However, only a small amount of data is available to date, and the results are inconsistent. To evaluate the relation between total T (TT) and CP/CPPS. We conducted a propensity-matched study by identifying men with a TT level lower than 3.5 ng/mL among 8,336 men in their 40s and 50s. A control group of men with a TT level of at least 3.5 ng/mL matched for age, metabolic syndrome, and body mass index at a 5:1 ratio was selected for comparison. Using the same cohort and methods, another case group (TT < 3.0 ng/mL) and control group (TT ≥ 3.0 ng/mL) were selected. The National Institutes of Health-Chronic Prostatitis Symptom Index (NIH-CPSI) was administered. A χ(2) test, a t-test and logistic regression analyses were used to evaluate the relation between TT and prostatitis-like symptoms. Association of TT with NIH-CPSI score. After propensity score matching, 948 cases (TT < 3.5 ng/mL) and 4,740 controls (TT ≥ 3.5 ng/mL) were included. The ratio of mild and moderate to severe prostatitis-like symptoms was higher in the case group than in the control group (24.0% vs 27.4%, P = .001). The ratio of moderate to severe prostatitis-like symptoms also was higher in the case group than in the control group (6.2% vs 9.2%, P = .028). The pain domain of the NIH-CPSI, quality of life, and total NIH-CPSI scores also were higher in the case group. Ratios of severe lower urinary tract symptoms (12.6% vs 15.1%, P = .044) to maximal flow rate no higher than 10 mL/sec (3.8% vs 5.3%, P = .044) and postvoid residual urine volume of at least 100 mL (4.0% vs 5.6%, P = .035), which suggest high pressure in the prostate urethra, were higher in the case group. After adjusting for voided volume during uroflowmetry and total prostate volume, the relations of a TT level lower than 3.5 ng/mL to a maximal flow rate no higher than 10 mL/sec (odds ratio = 1.402, 95% CI = 1.017-1.934, P = .039) and to a postvoid residual urine volume of at least 100 mL (odds ratio = 1.410, 95% CI = 1.031-1.927, P = .031) were maintained. Using the cutoff TT value of 3.0 ng/mL, 437 cases (TT < 3.0 ng/mL) and 2,185 controls (TT ≥ 3.0 ng/mL) were included. The result of the 3.0-ng/mL cutoff value for TT showed a higher incidence of prostatitis-like symptoms in the group with a TT level lower than 3.0 ng/mL, but this was not statistically significant. Low TT level (<3.5 ng/mL) was significantly correlated with prostatitis-like symptoms in this study. Copyright © 2016. Published by Elsevier Inc.

Mammalian knock out cells reveal prominent roles for atlastin GTPases in ER network morphology.

PubMed

Zhao, Guohua; Zhu, Peng-Peng; Renvoisé, Benoît; Maldonado-Báez, Lymarie; Park, Seong Hee; Blackstone, Craig

2016-11-15

Atlastins are large, membrane-bound GTPases that participate in the fusion of endoplasmic reticulum (ER) tubules to generate the polygonal ER network in eukaryotes. They also regulate lipid droplet size and inhibit bone morphogenetic protein (BMP) signaling, though mechanisms remain unclear. Humans have three atlastins (ATL1, ATL2, and ATL3), and ATL1 and ATL3 are mutated in autosomal dominant hereditary spastic paraplegia and hereditary sensory neuropathies. Cellular investigations of atlastin orthologs in most yeast, plants, flies and worms are facilitated by the presence of a single or predominant isoform, but loss-of-function studies in mammalian cells are complicated by multiple, broadly-expressed paralogs. We have generated mouse NIH-3T3 cells lacking all three mammalian atlastins (Atl1/2/3) using CRISPR/Cas9-mediated gene knockout (KO). ER morphology is markedly disrupted in these triple KO cells, with prominent impairment in formation of three-way ER tubule junctions. This phenotype can be rescued by expression of distant orthologs from Saccharomyces cerevisiae (Sey1p) and Arabidopsis (ROOT HAIR DEFECTIVE3) as well as any one of the three human atlastins. Minimal, if any, changes are observed in the morphology of mitochondria and the Golgi apparatus. Alterations in BMP signaling and increased sensitivity to ER stress are also noted, though effects appear more modest. Finally, atlastins appear required for the proper differentiation of NIH-3T3 cells into an adipocyte-like phenotype. These findings have important implications for the pathogenesis of hereditary spastic paraplegias and sensory neuropathies associated with atlastin mutations. Published by Elsevier Inc.

The diffusion tensor imaging (DTI) component of the NIH MRI study of normal brain development (PedsDTI).

PubMed

Walker, Lindsay; Chang, Lin-Ching; Nayak, Amritha; Irfanoglu, M Okan; Botteron, Kelly N; McCracken, James; McKinstry, Robert C; Rivkin, Michael J; Wang, Dah-Jyuu; Rumsey, Judith; Pierpaoli, Carlo

2016-01-01

The NIH MRI Study of normal brain development sought to characterize typical brain development in a population of infants, toddlers, children and adolescents/young adults, covering the socio-economic and ethnic diversity of the population of the United States. The study began in 1999 with data collection commencing in 2001 and concluding in 2007. The study was designed with the final goal of providing a controlled-access database; open to qualified researchers and clinicians, which could serve as a powerful tool for elucidating typical brain development and identifying deviations associated with brain-based disorders and diseases, and as a resource for developing computational methods and image processing tools. This paper focuses on the DTI component of the NIH MRI study of normal brain development. In this work, we describe the DTI data acquisition protocols, data processing steps, quality assessment procedures, and data included in the database, along with database access requirements. For more details, visit http://www.pediatricmri.nih.gov. This longitudinal DTI dataset includes raw and processed diffusion data from 498 low resolution (3 mm) DTI datasets from 274 unique subjects, and 193 high resolution (2.5 mm) DTI datasets from 152 unique subjects. Subjects range in age from 10 days (from date of birth) through 22 years. Additionally, a set of age-specific DTI templates are included. This forms one component of the larger NIH MRI study of normal brain development which also includes T1-, T2-, proton density-weighted, and proton magnetic resonance spectroscopy (MRS) imaging data, and demographic, clinical and behavioral data. Published by Elsevier Inc.

Synthesis, maturation and extracellular release of procathepsin D as influenced by cell proliferation or transformation.

PubMed

Isidoro, C; Demoz, M; De Stefanis, D; Baccino, F M; Bonelli, G

1995-12-11

The relationship between cell growth and intra- and extracellular accumulation of cathepsin D (CD), a lysosomal endopeptidase involved in cell protein breakdown, was examined in cultures of normal and transformed BALB/c mouse 3T3 fibroblasts grown at various cell densities. In crowded cultures of normal 3T3 cells (doubling time, Td, 53 hr) intracellular CD activity was 2-fold higher than in sparse, rapidly-growing (Td, 27 hr) cultures. In uncrowded (Td, 18 hr) and crowded (Td, 32 hr) cultures of benzo[a]pyrene-transformed cells intracellular CD levels were one third and two thirds, respectively, of those measured in hyperconfluent 3T3 cultures. Regardless of cell density, SV-40-virus-transformed cells (Td, 12 hr) contained one third of CD levels found in hyperconfluent 3T3 cells. Both transformed cell lines released into the medium a higher proportion of CD, compared with their untransformed counterpart, yet the amount secreted was not sufficient to account for the reduced intracellular level of the enzyme. Serum withdrawal induced a marked increase of both intra- and extracellular levels of CD activity. In both normal and virally or chemically transformed 3T3 cells CD comprised a precursor (52 kDa) and processed mature polypeptides; the latter were mostly represented by a 48-kDa peptide, but a minor part was in a double-chain form (31 and 16 kDa respectively). The proportion of mature enzyme vs. precursor was much higher in confluent, slowly-growing cells than in fast-growing cells, whether normal or transformed. In the latter, conversion of mature 48-kDa peptide into the double-chain form occurred more efficiently.

Transcriptional and translational control of ornithine decarboxylase during Ras transformation.

PubMed Central

Shantz, Lisa M

2004-01-01

ODC (ornithine decarboxylase) activity is induced following ras activation. However, the Ras effector pathways responsible are unknown. These experiments used NIH-3T3 cells expressing partial-loss-of-function Ras mutants to activate selectively pathways downstream of Ras and examined the contribution of each pathway to ODC induction. Overexpression of Ras12V, a constitutively active mutant, resulted in ODC activities up to 20-fold higher than controls. Stable transfections of Ras partial-loss-of-function mutants and constitutively active forms of MEK (MAPK kinase) and Akt indicated that activation of more than one Ras effector pathway is necessary for the complete induction of ODC activity. The increase in ODC activity in Ras12V-transformed cells is not owing to a substantial change in ODC protein half-life, which increased by <2-fold. Northern-blot analysis and reporter assays suggested that the mechanism of ODC induction involves both a modest increase in the transcription of ODC mRNA and a much more considerable increase in the translation of mRNA into protein. ODC transcription was controlled through a pathway dependent on Raf/MEK/ERK (where ERK stands for extracellular-signal-regulated kinase) activation, whereas activation of the phosphoinositide 3-kinase and the Raf/MEK/ERK pathways were necessary for translational regulation of ODC. The increase in ODC synthesis was accompanied by changes in phosphorylation of eukaryotic initiation factor 4E and its binding protein 4E-BP1. Results show that the phosphoinositide 3-kinase pathway regulates phosphorylation of both proteins, whereas the Raf/MEK/ERK pathway affects only the eukaryotic initiation factor 4E phosphorylation. PMID:14519103

A Constant Percentage Bandwidth Transform for Acoustic Signal Processing

DTIC Science & Technology

1980-01-01

t)eJwt d . 27Th (0) Equation 2.9 is not, however, the most general form for short-time Fourier synthesis, but is in fact a particular case of the...form of the analysis integral. F(Wk? t)eJwkt = f(t) * h( t)’ukt (3.4) Fourier transforming both sides of this equation (and invoking the convolution...properties. In what follows, define 38 f(t) - F(w,t) (3.24) to be an equivalent statement to equation 3.1. 3.6.1 Linearity Property If F1 w,t) and F2 (w

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The transforming power of early career acute care surgery research scholarships on academic productivity.

PubMed

Zarzaur, Ben L; Valsangkar, Nakul; Feliciano, David F; Koniaris, Leonidas G

2016-07-01

More than 75% of respondents to an Eastern Association for the Surgery of Trauma survey felt that barriers to research had increased and that acute care surgeon (ACS) academic productivity had decreased. Recent data confirm this impression and show lower academic productivity of junior ACS faculty compared with peers in other general surgical fields. The purpose of this study was to determine if early career acute care surgery research scholarships are associated with improved ACS academic productivity. Faculty data at the Top 55 National Institutes of Health (NIH)-funded departments of surgery (Top 55) were obtained using SCOPUS, NIH, department, and professional society databases. Academic productivity was measured using total publications, citations, and the Hirsch index. Scholarship recipients from the American Association for the Surgery of Trauma and Eastern Association for the Surgery of Trauma were identified. A total of 4,101 surgical faculty (8.3% ACS) who belong to the Top 55 NIH-funded departments of surgery and 85 scholarship recipients were identified. After merging, 34 scholarship recipients (40%) were current faculty at a Top 55 NIH-funded department of surgery, and 24 of those (71%) were ACS faculty. Scholarship recipients had higher median total publications compared with nonrecipients at assistant and associate ranks but not at full professor rank. For all ranks, scholarship recipients were more likely to have NIH funding compared with nonrecipients (33% vs. 11%, p < 0.05). On multivariable analysis, only NIH funding was associated with increased total publications, with an average of 89 more publications over a career (p < 0.05). Research scholarships granted by acute care surgery professional organizations remain largely among ACS faculty in Top 55 NIH-funded departments of surgery. Among junior ACS faculty, recipients are associated with increased academic productivity and NIH funding. To fill the academic productivity gap among junior ACSs, professional organizations should consider increasing research funding scholarships for promising investigators.

Size- and dose-dependent toxicity of cellulose nanocrystals (CNC) on human fibroblasts and colon adenocarcinoma.

PubMed

Hanif, Zahid; Ahmed, Farrukh R; Shin, Seung Won; Kim, Young-Kee; Um, Soong Ho

2014-07-01

A controlled preparation of cellulose nanocrystals of different sizes and shapes has been carried out by acid hydrolysis of microcrystalline cellulose. The size- and concentration-dependent toxicity effects of the resulting cellulose nanocrystals were evaluated against two different cell lines, NIH3T3 murine embryo fibroblasts and HCT116 colon adenocarcinoma. It could serve as a therapeutic platform for cancer treatment. Copyright © 2014 Elsevier B.V. All rights reserved.

Programmed packaging of multicomponent envelope-type nanoparticle system for gene delivery

NASA Astrophysics Data System (ADS)

Pozzi, Daniela; Marianecci, Carlotta; Carafa, Maria; Marchini, Cristina; Montani, Maura; Amici, Augusto; Caracciolo, Giulio

2010-05-01

A programmed packaging strategy to develop a multicomponent envelope-type nanoparticle system (MENS) is presented. To this end, we took specific advantage of using in-house tailored liposomes that have been recently shown to exhibit intrinsic endosomal rupture properties that allow plasmid DNA to escape from endosomes and to enter the nucleus with extremely high efficiency. Transfection efficiency experiments on NIH 3T3 mouse fibroblasts indicate that MENS is a promising transfection candidate.

Cytotoxic Activity of Kenaf Seed Oils from Supercritical Carbon Dioxide Fluid Extraction towards Human Colorectal Cancer (HT29) Cell Lines.

PubMed

Abd Ghafar, Siti Aisyah; Ismail, Maznah; Saiful Yazan, Latifah; Fakurazi, Sharida; Ismail, Norsharina; Chan, Kim Wei; Md Tahir, Paridah

2013-01-01

Kenaf (Hibiscus cannabinus) from the family Malvaceae, is a valuable fiber plant native to India and Africa and is currently planted as the fourth commercial crop in Malaysia. Kenaf seed oil contains alpha-linolenic acid, phytosterol such as β -sitosterol, vitamin E, and other antioxidants with chemopreventive properties. Kenaf seeds oil (KSO) was from supercritical carbon dioxide extraction fluid (SFE) at 9 different permutations of parameters based on range of pressures from 200 to 600 bars and temperature from 40 to 80°C. They were 200/40, 200/60, 200/80, 400/40, 400/60, 400/80, 600/40, 600/60, and 600/80. Extraction from 9 parameters of KSO-SFE was screened for cytotoxicity towards human colorectal cancer cell lines (HT29) and mouse embryonic fibroblast (NIH/3T3) cell lines using MTS assay. KSO-SFE at 600/40 showed the strongest cytotoxicity towards HT29 with IC50 of 200 µg/mL. The IC50 for NIH/3T3 was not detected even at highest concentration employed. Cell cycle analysis showed a significant increase in the accumulation of KSO-SFE-treated cells at sub-G1 phase, indicating the induction of apoptosis by KSO-SFE. Further apoptosis induction was confirmed by Annexin V/PI and AO/PI staining.

Cytotoxic Activity of Kenaf Seed Oils from Supercritical Carbon Dioxide Fluid Extraction towards Human Colorectal Cancer (HT29) Cell Lines

PubMed Central

Abd Ghafar, Siti Aisyah; Ismail, Maznah; Saiful Yazan, Latifah; Fakurazi, Sharida; Chan, Kim Wei; Md Tahir, Paridah

2013-01-01

Kenaf (Hibiscus cannabinus) from the family Malvaceae, is a valuable fiber plant native to India and Africa and is currently planted as the fourth commercial crop in Malaysia. Kenaf seed oil contains alpha-linolenic acid, phytosterol such as β-sitosterol, vitamin E, and other antioxidants with chemopreventive properties. Kenaf seeds oil (KSO) was from supercritical carbon dioxide extraction fluid (SFE) at 9 different permutations of parameters based on range of pressures from 200 to 600 bars and temperature from 40 to 80°C. They were 200/40, 200/60, 200/80, 400/40, 400/60, 400/80, 600/40, 600/60, and 600/80. Extraction from 9 parameters of KSO-SFE was screened for cytotoxicity towards human colorectal cancer cell lines (HT29) and mouse embryonic fibroblast (NIH/3T3) cell lines using MTS assay. KSO-SFE at 600/40 showed the strongest cytotoxicity towards HT29 with IC50 of 200 µg/mL. The IC50 for NIH/3T3 was not detected even at highest concentration employed. Cell cycle analysis showed a significant increase in the accumulation of KSO-SFE-treated cells at sub-G1 phase, indicating the induction of apoptosis by KSO-SFE. Further apoptosis induction was confirmed by Annexin V/PI and AO/PI staining. PMID:23606884

A QUICK Screen for Lrrk2 Interaction Partners – Leucine-rich Repeat Kinase 2 is Involved in Actin Cytoskeleton Dynamics*

PubMed Central

Meixner, Andrea; Boldt, Karsten; Van Troys, Marleen; Askenazi, Manor; Gloeckner, Christian J.; Bauer, Matthias; Marto, Jarrod A.; Ampe, Christophe; Kinkl, Norbert; Ueffing, Marius

2011-01-01

Mutations in human leucine-rich repeat kinase 2 (Lrrk2), a protein of yet unknown function, are linked to Parkinson's disease caused by degeneration of midbrain dopaminergic neurons. The protein comprises several domains including a GTPase and a kinase domain both affected by several pathogenic mutations. To elucidate the molecular interaction network of endogenous Lrrk2 under stoichiometric constraints, we applied QUICK (quantitative immunoprecipitation combined with knockdown) in NIH3T3 cells. The identified interactome reveals actin isoforms as well as actin-associated proteins involved in actin filament assembly, organization, rearrangement, and maintenance, suggesting that the biological function of Lrrk2 is linked to cytoskeletal dynamics. In fact, we demonstrate Lrrk2 de novo binding to F-actin and its ability to modulate its assembly in vitro. When tested in intact cells, knockdown of Lrrk2 causes morphological alterations in NIH3T3 cells. In developing dopaminergic midbrain primary neurons, Lrrk2 knockdown results in shortened neurite processes, indicating a physiological role of Lrrk2 in cytoskeletal organization and dynamics of dopaminergic neurons. Hence, our results demonstrate that molecular interactions as well as the physiological function of Lrrk2 are closely related to the organization of the actin-based cytoskeleton, a crucial feature of neuronal development and neuron function. PMID:20876399

Identification and Analysis of Expression of Novel MicroRNAs of Murine Gammaherpesvirus 68▿ †

PubMed Central

Zhu, Jia Yun; Strehle, Martin; Frohn, Anne; Kremmer, Elisabeth; Höfig, Kai P.; Meister, Gunter; Adler, Heiko

2010-01-01

Murine gammaherpesvirus 68 (MHV-68) is closely related to Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) and provides a small-animal model with which to study the pathogenesis of gammaherpesvirus (γHV) infections. To completely explore the potential of the MHV-68 system for the investigation of γHV microRNAs (miRNAs), it would be desirable to know the number and expression patterns of all miRNAs encoded by MHV-68. By deep sequencing of small RNAs, we systematically investigated the expression profiles of MHV-68 miRNAs in both lytically and persistently infected cells. In addition to the nine known MHV-68 miRNAs, we identified six novel MHV-68 miRNA genes and analyzed the expression levels of all MHV-68 miRNAs. Furthermore, we also characterized the cellular miRNA expression signatures in MHV-68-infected versus noninfected NIH 3T3 fibroblasts and in 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-treated versus nontreated S11 cells. We found that mmu-mir-15b and mmu-mir-16 are highly upregulated upon MHV-68 infection of NIH 3T3 cells, indicating a potential role for cellular miRNAs during MHV-68 infection. Our data will aid in the full exploration of the functions of γHV miRNAs. PMID:20668074

Comparative study on the physicochemical and functional properties of the mucilage in the carpel of Nymphaea odorata using ultrasonic and classical heating extractions.

PubMed

Wu, WeiZhi; Tu, ChinWei; Yang, WenJen; Wang, HengLong; Chang, ChaoLin; Chung, JengDer; Lu, MeiKuang; Liao, WeiTung

2018-02-21

The cooked carpel of Nymphaea odorata has a large amount of transparent mucilage; however, the basic characteristics of this mucilage have not yet been reported. This study compared the physicochemical and functional properties of this mucilage obtained using conventional hot water extraction (HWM) and ultrasonic-assisted extraction (UAM). Neither HWM nor UAM affected the viability of mouse skin fibroblasts (NIH/3T3) below 100 μg/mL. UAM had a higher yield production, phenol concentration, and in vitro antioxidant activity, but lower viscosity and water-holding capacity than for HWM. The Fourier transform infrared spectra revealed that the dialyzed HWM and UAM, named HWMD and UAMD, respectively, appeared major spectral differences at 1730 cm -1 and 1605 cm -1 , implying that the degree of methylation was different between HWMD and UAMD. Compared to HWMD, UAMD in low-molecular weight polysaccharides increased. Evidently, the basic characteristics of native mucilage in the carpel of N. odorata were greatly changed by various extractions. Nevertheless, sugar analysis indicated that glucuronic acid was the mainly composition of HWMD and UAMD. Copyright © 2017. Published by Elsevier B.V.

Membrane-bound LERK2 ligand can signal through three different Eph-related receptor tyrosine kinases.

PubMed Central

Brambilla, R; Schnapp, A; Casagranda, F; Labrador, J P; Bergemann, A D; Flanagan, J G; Pasquale, E B; Klein, R

1995-01-01

The Eph-related family of receptor tyrosine kinases consists of at least 13 members, several of which display distinctive expression patterns in the developing and adult nervous system. Recently, a small family of ligands, structurally related to the B61 protein, was identified. Binding of these ligands to Eph-related receptors did not, however, elicit measurable biological signals in cultured cells. In order to study functional interactions between B61-related ligands and Eph-related receptors, we constructed chimeric receptors, containing an Eph-related ectodomain and the cytoplasmic domain of the TrkB neurotrophin receptor. Expression and activation of such chimeric receptors in NIH 3T3 cells induced transformation in focus formation assays. Membrane-bound LERK2 ligand is shown to signal through three different Eph-related receptors, namely Cek5, Cek10 and Elk. LERK2, however, fails to interact functionally with the Cek9 receptor. Quantitative analysis including binding assays indicates that Cek10 is the preferred LERK2 receptor. Preliminary mutagenesis of the LERK2 protein suggests a negative regulatory role for its cytoplasmic domain in LERK2 signaling. Images PMID:7621826

Plasma Shh levels reduced in pancreatic cancer patients

PubMed Central

El-Zaatari, Mohamad; Daignault, Stephanie; Tessier, Art; Kelsey, Gail; Travnikar, Lisa A.; Cantu, Esperanza F.; Lee, Jamie; Plonka, Caitlyn M.; Simeone, Diane M.; Anderson, Michelle A.; Merchant, Juanita L.

2012-01-01

Objectives Normally, sonic hedgehog (Shh) is expressed in the pancreas during fetal development and transiently after tissue injury. Although pancreatic cancers express Shh, it is not known if the protein is secreted into the blood and whether its plasma levels change with pancreatic transformation. The goal of this study was to develop an ELISA to detect human Shh in blood, and determine the levels in subjects with and without pancreatic cancer. Methods A human Shh ELISA assay was developed, and plasma Shh levels were measured in blood samples from normal volunteers and subjects with pancreatitis or pancreatic cancer. The biological activity of plasma Shh was tested using NIH-3T3 cells. Results The average levels of Shh in human blood were lower in pancreatitis and pancreatic cancer patients than in normal individuals. Hematopoietic cells did not express Shh suggesting that Shh is secreted into the bloodstream. Plasma fractions enriched for Shh did not induce Gli-1 mRNA suggesting that the protein was not biologically active. Conclusions Shh is secreted from tissues and organs into the circulation but its activity is blocked by plasma proteins. Reduced plasma levels were found in pancreatic cancer patients, but alone were not sufficient to predict pancreatic cancer. PMID:22513293

Plasma Shh levels reduced in pancreatic cancer patients.

PubMed

El-Zaatari, Mohamad; Daignault, Stephanie; Tessier, Art; Kelsey, Gail; Travnikar, Lisa A; Cantu, Esperanza F; Lee, Jamie; Plonka, Caitlyn M; Simeone, Diane M; Anderson, Michelle A; Merchant, Juanita L

2012-10-01

Normally, sonic hedgehog (Shh) is expressed in the pancreas during fetal development and transiently after tissue injury. Although pancreatic cancers express Shh, it is not known if the protein is secreted into the blood and whether its plasma levels change with pancreatic transformation. The goal of this study was to develop an enzyme-linked immunosorbent assay to detect human Shh in blood and determine its levels in subjects with and without pancreatic cancer. A human Shh enzyme-linked immunosorbent assay was developed, and plasma Shh levels were measured in blood samples from healthy subjects and patients with pancreatitis or pancreatic cancer. The biological activity of plasma Shh was tested using NIH-3T3 cells. The mean levels of Shh in human blood were lower in patients with pancreatitis and pancreatic cancer than in healthy subjects. Hematopoietic cells did not express Shh, suggesting that Shh is secreted into the bloodstream. Plasma fractions enriched with Shh did not induce Gli-1 messenger RNA, suggesting that the protein was not biologically active. Shh is secreted from tissues and organs into the circulation, but its activity is blocked by plasma proteins. Reduced plasma levels were found in pancreatic cancer patients, but alone were not sufficient to predict pancreatic cancer.

Safety of the trivalent, cold-adapted influenza vaccine (CAIV-T) in children.

PubMed

Piedra, Pedro A

2002-04-01

The trivalent, cold-adapted influenza vaccine (CAIV-T, FluMist, Aviron, Mountain View, CA) is a live attenuated influenza virus vaccine that is administered by nasal spray. CAIV-T is efficacious in preventing influenza virus infection. The vaccine was submitted to the Food and Drug Administration for licensure in healthy children and adults. Universal immunization is being considered in children, and an effective vaccine with minimal adverse reactions is thus required. The published studies on the safety of CAIV-T in children reviewed in this article were clinical trials sponsored by the National Institutes of Health (NIH) conducted in children from 1975 to 1991, clinical trials from 1991 to 1993 sponsored by a cooperative agreement between NIH and Wyeth-Ayerst Research, and clinical trials from 1995 to the present sponsored by a cooperative agreement between NIH and Aviron. Safety assessments included the occurrence of: 1) specific influenza-like symptoms, unexpected symptoms, and use of medications within the first 10 days after vaccination; 2) acute illness and use of medication within 11 to 42 days postvaccination; 3) serious adverse events and rare events within 42 days after vaccination; 4) healthcare utilization within 14 days after vaccination; and 5) acute respiratory symptoms with annual sequential vaccine doses. CAIV-T was safe and well-tolerated. Transient, mild respiratory symptoms were observed in a minority (10%-15%) of children and primarily with the first CAIV-T dose. Vomiting and abdominal pain occurred in fewer than 2 percent of CAIV-T recipients. The gastrointestinal symptoms were mild and of short duration. An excess of illness or use of medication was not observed after the 10th day of vaccination. Sequential annual doses of CAIV-T were well-tolerated and not associated with increased reactogenicity. CAIV-T did not cause an increase in healthcare utilization. Thus CAIV-T is safe in healthy children and should complement the use of inactivated influenza vaccine, trivalent (IIV-T) in children with underlying chronic conditions.

Enantioselective cellular localisation of europium(iii) coordination complexes† †Electronic supplementary information (ESI) available: Complex synthesis and characterisation, selected cell images showing correspondence with commercial lysosomal or mitochondrial stains and spectral imaging profiles. See DOI: 10.1039/c7sc04422d

PubMed Central

Frawley, Andrew T.; Linford, Holly V.; Starck, Matthieu; Pal, Robert

2017-01-01

The selective mitochondrial localisation of the Λ enantiomer of three different emissive europium(iii) complexes in NIH 3T3 and MCF7 cells contrasts with the behaviour of the Δ enantiomer, for which a predominant lysosomal localisation was observed by confocal microscopy. In each case, cell uptake occurs via macropinocytosis. PMID:29675151

Rho A and the Rho kinase pathway regulate fibroblast contraction: Enhanced contraction in constitutively active Rho A fibroblast cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nobe, Koji, E-mail: kojinobe@pharm.showa-u.ac.jp; Nobe, Hiromi; Department of Physical Therapy, Bunkyo-Gakuin University

Research highlights: {yields} Mechanisms of fibroblast cell contraction in collagen matrix. {yields} Assessed an isometric force development using 3D-reconstituted-fibroblast fiber. {yields} Constitutively active Rho A induced the over-contraction of fibroblast cells. {yields} Rho A and Rho kinase pathway has a central role in fibroblast cell contraction. -- Abstract: Fibroblast cells play a central role in the proliferation phase of wound healing processes, contributing to force development. The intracellular signaling pathways regulating this non-muscle contraction are only partially understood. To study the relations between Rho A and contractile responses, constitutively active Rho A (CA-Rho A) fibroblast cells were reconstituted into fibersmore » and the effects of calf serum (CS) on isometric force were studied. CS-induced force in CA-Rho A fibroblast fibers was twice as large as that in wild type (NIH 3T3) fibroblast fibers. During this response, the translocation of Rho A from the cytosol to the membrane was detected by Rho A activity assays and Western blot analysis. Pre-treatment with a Rho specific inhibitor (C3-exoenzyme) suppressed translocation as well as contraction. These results indicate that Rho A activation is essential for fibroblast contraction. The Rho kinase inhibitor ( (Y27632)) inhibited both NIH 3T3 and CA-Rho A fibroblast fiber contractions. Activation of Rho A is thus directly coupled with Rho kinase activity. We conclude that the translocation of Rho A from the cytosol to the membrane and the Rho kinase pathway can regulate wound healing processes mediated by fibroblast contraction.« less

Induction of morphological transformation in mouse C3H/10T1/2 clone 8 cells and chromosomal damage in hamster A(T1)C1-3 cells by cancer chemotherapeutic agents.

PubMed

Benedict, W F; Banerjee, A; Gardner, A; Jones, P A

1977-07-01

Various cancer chemotherapeutic agents including alkylating agents, antimetabolites, and antibiotics or natural products were studied for their ability to produce morphological transformation in the C3H/10T1/2 clone 8 mouse cell line and chromosomal damage in the A(T1)C1-3 hamster cell line following a 24-hr exposure of each agent at different concentrations. Those drugs that were known to be carcinogenic in vivo also produced morphological transformation and chromosomal damage, whereas those agents that have not been shown to be carcinogenic in vivo produced neither transformation nor chromosomal lesions. The concentrations used for these studies were in general similar to those actually reached in the plasma of patients treated with these same drugs for malignant, as well as certain nonmalignant, conditions.

EGFR Exon 18 Mutations in Lung Cancer: Molecular Predictors of Augmented Sensitivity to Afatinib or Neratinib as Compared with First- or Third-Generation TKIs.

PubMed

Kobayashi, Yoshihisa; Togashi, Yosuke; Yatabe, Yasushi; Mizuuchi, Hiroshi; Jangchul, Park; Kondo, Chiaki; Shimoji, Masaki; Sato, Katsuaki; Suda, Kenichi; Tomizawa, Kenji; Takemoto, Toshiki; Hida, Toyoaki; Nishio, Kazuto; Mitsudomi, Tetsuya

2015-12-01

Lung cancers harboring common EGFR mutations respond to EGFR tyrosine kinase inhibitors (TKI), whereas exon 20 insertions (Ins20) are resistant to them. However, little is known about mutations in exon 18. Mutational status of lung cancers between 2001 and 2015 was reviewed. Three representative mutations in exon 18, G719A, E709K, and exon 18 deletion (Del18: delE709_T710insD) were retrovirally introduced into Ba/F3 and NIH/3T3 cells. The 90% inhibitory concentrations (IC90s) of first-generation (1G; gefitinib and erlotinib), second-generation (2G; afatinib, dacomitinib, and neratinib), and third-generation TKIs (3G; AZD9291 and CO1686) were determined. Among 1,402 EGFR mutations, Del19, L858R, and Ins20 were detected in 40%, 47%, and 4%, respectively. Exon 18 mutations, including G719X, E709X, and Del18, were present in 3.2%. Transfected Ba/F3 cells grew in the absence of IL3, and NIH/3T3 cells formed foci with marked pile-up, indicating their oncogenic abilities. IC90s of 1G and 3G TKIs in G719A, E709K, and Del18 were much higher than those in Del19 (by >11-50-fold), whereas IC90s of afatinib were only 3- to 7-fold greater than those for Del19. Notably, cells transfected with G719A and E709K exhibited higher sensitivity to neratinib (by 5-25-fold) than those expressing Del19. Patients with lung cancers harboring G719X exhibited higher response rate to afatinib or neratinib (∼ 80%) than to 1G TKIs (35%-56%) by compilation of data in the literature. Lung cancers harboring exon 18 mutations should not be overlooked in clinical practice. These cases can be best treated with afatinib or neratinib, although the currently available in vitro diagnostic kits cannot detect all exon 18 mutations. ©2015 American Association for Cancer Research.

NIH Research: Cancers: A "Constellation" of Diseases | NIH MedlinePlus the Magazine

MedlinePlus

... Dr. Varmus received early training in genetics and molecular biology at the National Cancer Institute (NCI) in the ... now new technologies for genomic analysis. Progress in molecular biology has transformed our ability to understand the broken ...

MDC9, a widely expressed cellular disintegrin containing cytoplasmic SH3 ligand domains

PubMed Central

1996-01-01

Cellular disintegrins are a family of proteins that are related to snake venom integrin ligands and metalloproteases. We have cloned and sequenced the mouse and human homologue of a widely expressed cellular disintegrin, which we have termed MDC9 (for metalloprotease/disintegrin/cysteine-rich protein 9). The deduced mouse and human protein sequences are 82% identical. MDC9 contains several distinct protein domains: a signal sequence is followed by a prodomain and a domain with sequence similarity to snake venom metalloproteases, a disintegrin domain, a cysteine-rich region, an EGF repeat, a membrane anchor, and a cytoplasmic tail. The cytoplasmic tail of MDC9 has two proline-rich sequences which can bind the SH3 domain of Src, and may therefore function as SH3 ligand domains. Western blot analysis shows that MDC9 is an approximately 84-kD glycoprotein in all mouse tissues examined, and in NIH 3T3 fibroblast and C2C12 myoblast mouse cell lines. MDC9 can be both cell surface biotinylated and 125I-labeled in NIH 3T3 mouse fibroblasts, indicating that the protein is present on the plasma membrane. Expression of MDC9 in COS-7 cells yields an 84-kD protein, and immunofluorescence analysis of COS-7 cells expressing MDC9 shows a staining pattern that is consistent with a plasma membrane localization. The apparent molecular mass of 84 kD suggests that MDC9 contains a membrane-anchored metalloprotease and disintegrin domain. We propose that MDC9 might function as a membrane-anchored integrin ligand or metalloprotease, or that MDC9 may combine both activities in one protein. PMID:8647900

RGD/TAT-functionalized chitosan-graft-PEI-PEG gene nanovector for sustained delivery of NT-3 for potential application in neural regeneration.

PubMed

Wu, Dongni; Zhang, Yongnu; Xu, Xiaoting; Guo, Ting; Xie, Deming; Zhu, Rong; Chen, Shengfeng; Ramakrishna, Seeram; He, Liumin

2018-05-01

In this study, we prepared a multifunctional gene delivery nanovector containing a chitosan (CS) backbone and polyethylenimine (PEI) arms with arginine-glycine-aspartate (RGD)/twin-arginine translocation (TAT) conjugated via polyethylene glycol (PEG). Branched PEI, with a molecular weight of 2000 Da, was used to achieve a balance between biocompatibility and transfection efficiency, whereas RGD/TAT peptides were conjugated for enhanced targeting ability and cellular uptake. Synthesis of the copolymers was confirmed by characterizing the chemical structure with 1 H nuclear magnetic resonance and Fourier Transform Infrared Spectroscopy (FTIR). The nanovector was biocompatible with cells and showed excellent capability for DNA condensation; the resulting complexes with DNA were well-formed, and possessed small particle size and reasonable positive charge. Higher gene transfection efficiency, compared to that achieved with PEI (25 kDa), was confirmed in tumor (HeLa cells) and normal cells (293T and NIH 3T3 cells). More importantly, the cells transfected with the chitosan-graft-PEI-PEG/pCMV-EGFP-Ntf3 complex produced sustained neurotrophin-3 with a linear increase in cumulative concentration, which induced neuronal differentiation of neural stem cell and promoted neurite outgrowth. These findings suggested that our multifunctional copolymers might be ideal nanovectors for engineering cells via gene transfection, and could potentially be applied in tumor therapy and regenerative medicine. We successfully prepared a multifunctional gene delivery nanovector containing branched PEI with a molecular weight of 2000 Da to balance between biocompatibility and transfection efficiency, and RGD/TAT peptides for enhanced targeting ability and cellular uptake. The well-formed CPPP/DNA complexes of small particle size and reasonable positive charges potentially enhanced gene transfection in both tumor and normal cells. More importantly, the CPPP/pCMV-EGFP-Ntf3 complex-transfected 293T cells could produce sustained NT-3 with a constant ratio, which induced neuron differentiation of NSC and promoted neurite outgrowth. Therefore, our study provided an effective strategy for producing neurotrophins by engineering cells with gene delivery, which deserved wide investigation and potential application in regenerative medicine. Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Caveolin-1 interacts with the Gag precursor of murine leukaemia virus and modulates virus production

PubMed Central

Yu, Zheng; Beer, Christiane; Koester, Mario; Wirth, Manfred

2006-01-01

Background Retroviral Gag determines virus assembly at the plasma membrane and the formation of virus-like particles in intracellular multivesicular bodies. Thereby, retroviruses exploit by interaction with cellular partners the cellular machineries for vesicular transport in various ways. Results The retroviral Gag precursor protein drives assembly of murine leukaemia viruses (MLV) at the plasma membrane (PM) and the formation of virus like particles in multivesicular bodies (MVBs). In our study we show that caveolin-1 (Cav-1), a multifunctional membrane-associated protein, co-localizes with Gag in a punctate pattern at the PM of infected NIH 3T3 cells. We provide evidence that Cav-1 interacts with the matrix protein (MA) of the Gag precursor. This interaction is mediated by a Cav-1 binding domain (CBD) within the N-terminus of MA. Interestingly, the CBD motif identified within MA is highly conserved among most other γ-retroviruses. Furthermore, Cav-1 is incorporated into MLV released from NIH 3T3 cells. Overexpression of a GFP fusion protein containing the putative CBD of the retroviral MA resulted in a considerable decrease in production of infectious retrovirus. Moreover, expression of a dominant-negative Cav-1 mutant affected retroviral titres significantly. Conclusion This study demonstrates that Cav-1 interacts with MLV Gag, co-localizes with Gag at the PM and affects the production of infectious virus. The results strongly suggest a role for Cav-1 in the process of virus assembly. PMID:16956408

Intracellular dynamics of cationic and anionic polystyrene nanoparticles without direct interaction with mitotic spindle and chromosomes.

PubMed

Liu, Yuexian; Li, Wei; Lao, Fang; Liu, Ying; Wang, Liming; Bai, Ru; Zhao, Yuliang; Chen, Chunying

2011-11-01

The fate of nanomaterials with different sizes and charges in mitotic cells is of great importance but seldom explored. Herein we investigate the intracellular fate of negatively charged carboxylated polystyrene (COOH-PS) and positively charged amino-modified polystyrene (NH(2)-PS) nanoparticles of three different diameters (50, 100 and 500 nm) on cancer HeLa cells and normal NIH 3T3 cells during the cell cycles. The results showed that all the fluorescent PS nanoparticles differing in size and/or charge did not interact with chromosome reorganization and cytoskeleton assembly during the mitotic process in live cells. They neither disturbed chromosome reorganization nor affected the cytoskeleton reassembly in both normal and cancer cells. However, NH(2)-PS at the size of 50 nm caused G1 phase delay and a decrease of cyclin (D, E) expression, respectively. Moreover, NH(2)-PS displayed higher cellular toxicity and NH(2)-PS of 50 nm disturbed the integrity of cell membranes. Both cationic and anionic PS nanoparticles had a more pronounced effect on normal NIH 3T3 cells than cancer HeLa cell. Our research provides insight into the dynamic fate, intracellular behavior, and the effects of nanoparticles on spindle and chromosomes during cell division, which will enable the optimization of design and selection of much safer nanoparticles for lower risk to human health and widely medical applications. Copyright © 2011 Elsevier Ltd. All rights reserved.

Antibacterial and cytotoxic activity of isoprenylated coumarin mammea A/AA isolated from Mammea africana.

PubMed

Canning, Corene; Sun, Shi; Ji, Xiangming; Gupta, Smiti; Zhou, Kequan

2013-05-02

The stem bark of Mammea africana is widely distributed in tropical Africa and commonly used in traditional medicine. This study aims to identify the active compound in Mammea africana and to evaluate its antimicrobial and antiproliferative activity. Methanol extract from the bark of the Mammea africana was separated by liquid-liquid extraction, followed by open column chromatography. A principal antimicrobial compound was purified by high performance liquid chromatography (HPLC) and its structure was elucidated by nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (MS). The antibacterial activity of the purified compound was determined using the broth microdilution method against 7 common pathogenic bacteria. The compound was also evaluated for cytotoxicity by cell proliferation assay (MTS) using the mouse embryonic fibroblast cell line NIH 3T3 and the non-small cell lung cancer cell line A549. The purified active compound was determined to be mammea A/AA and was found to be highly active against Campylobacter jejuni (MIC=0.5 μg/ml), Streptococcus pneumoniae (MIC=0.25 μg/ml), and Clostridium difficile (MIC=0.25 μg/ml). The compound exhibited significant antiproliferative activities against both NIH 3T3 and A549 cell lines. Mammea A/AA isolated from Mammea africana exerts specific inhibitory activity against Campylobacter jejuni, Streptococcus pneumoniae, and Campylobacter difficile. Mammea A/AA was also found to exhibit significant cytotoxicity against both cancer and normal cell lines. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Carbon nanoparticles for gene transfection in eukaryotic cell lines.

PubMed

Zanin, H; Hollanda, L M; Ceragioli, H J; Ferreira, M S; Machado, D; Lancellotti, M; Catharino, R R; Baranauskas, V; Lobo, A O

2014-06-01

For the first time, oxygen terminated cellulose carbon nanoparticles (CCN) was synthesised and applied in gene transfection of pIRES plasmid. The CCN was prepared from catalytic of polyaniline by chemical vapour deposition techniques. This plasmid contains one gene that encodes the green fluorescent protein (GFP) in eukaryotic cells, making them fluorescent. This new nanomaterial and pIRES plasmid formed π-stacking when dispersed in water by magnetic stirring. The frequencies shift in zeta potential confirmed the plasmid strongly connects to the nanomaterial. In vitro tests found that this conjugation was phagocytised by NG97, NIH-3T3 and A549 cell lines making them fluorescent, which was visualised by fluorescent microscopy. Before the transfection test, we studied CCN in cell viability. Both MTT and Neutral Red uptake tests were carried out using NG97, NIH-3T3 and A549 cell lines. Further, we use metabolomics to verify if small amounts of nanomaterial would be enough to cause some cellular damage in NG97 cells. We showed two mechanisms of action by CCN-DNA complex, producing an exogenous protein by the transfected cell and metabolomic changes that contributed by better understanding of glioblastoma, being the major finding of this work. Our results suggested that this nanomaterial has great potential as a gene carrier agent in non-viral based therapy, with low cytotoxicity, good transfection efficiency, and low cell damage in small amounts of nanomaterials in metabolomic tests. Copyright © 2014 Elsevier B.V. All rights reserved.

Absence of PDGF-induced, PKC-independent c-fos expression in a chemically transformed C3H/10T1/2 cell clone.

PubMed

Vassbotn, F S; Skar, R; Holmsen, H; Lillehaug, J R

1992-09-01

The effect of platelet-derived growth factor (PDGF) on c-fos mRNA transcription was studied in the immortalized mouse embryo fibroblast C3H/10T1/2 Cl 8 (10T1/2) cells and the chemically transformed, tumorigenic subclone C3H/10T1/2 Cl 16 (Cl 16). In the 10T1/2 cells as well as the Cl 16 subclone, the dose-dependent PDGF stimulation of c-fos mRNA synthesis was similar in both logarithmically growing and confluent cultures. c-fos mRNA was induced severalfold by 12-O-tetradecanoylphorbol-13-acetate (TPA) in both 10T1/2 and Cl 16. Down-regulation of protein kinase C (PKC) activity by TPA pretreatment inhibited PDGF-stimulated c-fos mRNA expression in Cl 16 cells but did not affect this induction in the 10T1/2 cells. This inhibition was not a general phenomenon of 3-methylcholanthrene-mediated transformation of 10T1/2 cells since experiments with another transformed 10T1/2 cell clone, C3H/10T1/2 TPA 482, gave qualitatively the same results as the 10T1/2 cells. Receptor binding experiments showed that the nontransformed and transformed cells had a comparable number of PDGF receptors, 1.3 x 10(5) and 0.7 x 10(5) receptors per cell, respectively. Furthermore, cAMP-induced c-fos expression induced by forskolin is formerly shown to be independent of PKC down-regulation. In our experiments, forskolin induced c-fos expression in both clones. However, PKC down-regulation inhibited the forskolin-induced c-fos expression in Cl 16 cells. This apparently demonstrates cross talk between PKC and PKA in the c-fos induction pathway. The present results provide evidence for an impaired mechanism for activating c-fos expression through PKC-independent, PDGF-induced signal transduction in the chemically transformed Cl 16 fibroblasts compared to that in nontransformed 10T1/2 cells.

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Effect of cell passage on the susceptibility of BALB/3T3 clone A31-1-1 cells to 3-methylcholanthrene-induced morphological transformation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sheu, C.W.; Moreland, F.M.; Dunkel, V.C.

1987-01-01

The response of BALB/3T3 clone A31-1-1 cells to chemically induced morphological transformation was evaluated using 3-methylcholanthrene (MCA). Stock cultures were initiated from cryopreserved cells, grown in T25 flasks containing 5 ml of medium, and replated at subconfluency. Serially transferred cells were then subjected to transformation assay. After 24-hr seeding, cells were incubated 48 hr with MCA in a 5% CO/sub 2/ incubator. They were then rinsed and incubated for an additional 4 weeks with twice weekly medium change. Type III foci were scored after fixation and staining with Giemsa. With serial passage from the frozen state, cells of passages 3-14more » had a low level of spontaneous transformation; zero to 6 type III foci per 20 dishes were counted. In the MCA-treated cultures the number of transformed foci, however, increased with passage. Such passage-related sensitivity to MCA was demonstrated for cells cultured in two batches of sera: one from MA Bioproducts (Lot no. 2E052) and the other from Armour Pharmaceuticals (Lot no. Y65801). The passage-related increase in number of transformed foci was not related to doubling time, cloning efficiency, or MCA-induced growth inhibition.« less

Revisiting overexpression of a heterologous β-glucosidase in Trichoderma reesei: fusion expression of the Neosartorya fischeri Bgl3A to cbh1 enhances the overall as well as individual cellulase activities.

PubMed

Xue, Xianli; Wu, Yilan; Qin, Xing; Ma, Rui; Luo, Huiying; Su, Xiaoyun; Yao, Bin

2016-07-11

The filamentous fungus Trichoderma reesei has the capacity to secret large amounts of cellulase and is widely used in a variety of industries. However, the T. reesei cellulase is weak in β-glucosidase activity, which results in accumulation of cellobiose inhibiting the endo- and exo-cellulases. By expressing an exogenous β-glucosidase gene, the recombinant T. reesei cellulase is expected to degrade cellulose into glucose more efficiently. The thermophilic β-glucosidase NfBgl3A from Neosartorya fischeri is chosen for overexpression in T. reesei due to its robust activity. In vitro, the Pichia pastoris-expressed NfBgl3A aided the T. reesei cellulase in releasing much more glucose with significantly lower amounts of cellobiose from crystalline cellulose. The NfBgl3A gene was hence fused to the cbh1 structural gene and assembled between the strong cbh1 promoter and cbh1 terminator to obtain pRS-NfBgl3A by using the DNA assembler method. pRS-NfBgl3A was transformed into the T. reesei uridine auxotroph strain TU-6. Six positive transformants showed β-glucosidase activities of 2.3-69.7 U/mL (up to 175-fold higher than that of wild-type). The largely different β-glucosidase activities in the transformants may be ascribed to the gene copy numbers of NfBgl3A or its integration loci. The T. reesei-expressed NfBgl3A showed highly similar biochemical properties to that expressed in P. pastoris. As expected, overexpression of NfBgl3A enhanced the overall cellulase activity of T. reesei. The CBHI activity in all transformants increased, possibly due to the extra copies of cbh1 gene introduced, while the endoglucanase activity in three transformants also largely increased, which was not observed in any other studies overexpressing a β-glucosidase. NfBgl3A had significant transglycosylation activity, generating sophorose, a potent cellulase inducer, and other oligosaccharides from glucose and cellobiose. We report herein the successful overexpression of a thermophilic N. fischeri β-glucosidase in T. reesei. In the same time, the fusion of NfBgl3A to the cbh1 gene introduced extra copies of the cellobiohydrolase 1 gene. As a result, we observed improved β-glucosidase and cellobiohydrolase activity as well as the overall cellulase activity. In addition, the endoglucanase activity also increased in some of the transformants. Our results may shed light on design of more robust T. reesei cellulases.

Inhibition of the Human ABC Efflux Transporters P-gp and ...

EPA Pesticide Factsheets

High body burdens of polybrominated diphenyl ethers (PBDEs) in infants and young children have led to increased concern over their potential impact on human development. PBDE exposure can alter the expression of genes involved in thyroid homeostasis, including those of ATP-binding cassette (ABC) transporters, which mediate cellular xenobiotic efflux. However, little information exists on how PBDEs interact with ABC transporters such as P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP). The purpose of this study was to evaluate the interactions of 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) and its hydroxylated metabolite 6-OH-BDE-47 with P-gp and BCRP, using human MDR1- and BCRP-expressing membrane vesicles and stably transfected NIH-3T3-MDR1 and MDCK-BCRP cells. In P-gp membranes, BDE-47 did not affect P-gp activity; however, 6-OH-BDE-47 inhibited P-gp activity at low µM concentrations (IC50 = 11.7 µM). In BCRP membranes, BDE-47 inhibited BCRP activity; however, 6-OH-BDE-47 was a stronger inhibitor [IC50 = 45.9 µM (BDE-47) vs. IC50 = 9.4 µM (6-OH-BDE-47)]. Intracellular concentrations of known P-gp and BCRP substrates [(3H)-paclitaxel and (3H)-prazosin, respectively] were significantly higher (indicating less efflux) in NIH-3T3-MDR1 and MDCK-BCRP cells in the presence of 6-OH-BDE-47, but not BDE-47. Collectively, our results indicate that the BDE-47 metabolite 6-OH-BDE-47 is an inhibitor of both P-gp and BCRP efflux activity.

An Integrin from Shrimp Litopenaeus vannamei Mediated Microbial Agglutination and Cell Proliferation

PubMed Central

Zhang, Ying; Wang, Leilei; Wang, Lingling; Wu, Ning; Zhou, Zhi; Song, Linsheng

2012-01-01

Background Integrins are a family of adhesion receptors which regulate cell proliferation, differentiation, leukocyte migration, and complement receptor-dependent phagocytosis. In invertebrates, as a cell adhesion receptor, β integrins play an important role for the balanced activation of immune defense responses especially during the encounter of infections. The present study attempts to characterize the immune functions of shrimp integrin (LvIntegrin) to have better understanding on the immune system and its regulation mechanisms in shrimps. Methodology A shrimp integrin was identified from the Pacific white shrimp Litopenaeus vannamei (designated as LvIntegrin). Its full-length cDNA was of 2621 bp with an open reading frame (ORF) of 2439 bp encoding a polypeptide of 812 amino acids. The mRNA expression of LvIntegrin was significantly up-regulated at 3, 6 and 12 h after Listonella anguillarum challenge. The cDNA fragment encoding β integrin domains (βA and hybrid domain) of LvIntegrin was recombined and expressed in Escherichia coli BL21(DE3)-pLysS. The recombinant protein (rLvIntegrin) could significantly agglutinate the tested microbe including E. coli JM109, L. anguillarum, Micrococcus luteus and Candida dattiladattila in the presence of divalent cations. Moreover, when NIH3T3 cells were cultured with rLvIntegrin, the proliferation rate increased significantly in a dose-dependent manner. Conclusions LvIntegrin, a shrimp β integrin was identified from L. vannamei, shared several highly conserved features. LvIntegrin exhibited broad-spectrum agglutination activity towards both bacteria and fungi and could improve the proliferation of NIH3T3 cells, indicating that LvIntegrin is involved in the immune response against microbe challenge and regulation of cell proliferation as a cell adhesion receptor in shrimp. PMID:22792387

An integrin from shrimp Litopenaeus vannamei mediated microbial agglutination and cell proliferation.

PubMed

Zhang, Ying; Wang, Leilei; Wang, Lingling; Wu, Ning; Zhou, Zhi; Song, Linsheng

2012-01-01

Integrins are a family of adhesion receptors which regulate cell proliferation, differentiation, leukocyte migration, and complement receptor-dependent phagocytosis. In invertebrates, as a cell adhesion receptor, β integrins play an important role for the balanced activation of immune defense responses especially during the encounter of infections. The present study attempts to characterize the immune functions of shrimp integrin (LvIntegrin) to have better understanding on the immune system and its regulation mechanisms in shrimps. A shrimp integrin was identified from the Pacific white shrimp Litopenaeus vannamei (designated as LvIntegrin). Its full-length cDNA was of 2621 bp with an open reading frame (ORF) of 2439 bp encoding a polypeptide of 812 amino acids. The mRNA expression of LvIntegrin was significantly up-regulated at 3, 6 and 12 h after Listonella anguillarum challenge. The cDNA fragment encoding β integrin domains (βA and hybrid domain) of LvIntegrin was recombined and expressed in Escherichia coli BL21(DE3)-pLysS. The recombinant protein (rLvIntegrin) could significantly agglutinate the tested microbe including E. coli JM109, L. anguillarum, Micrococcus luteus and Candida dattiladattila in the presence of divalent cations. Moreover, when NIH3T3 cells were cultured with rLvIntegrin, the proliferation rate increased significantly in a dose-dependent manner. LvIntegrin, a shrimp β integrin was identified from L. vannamei, shared several highly conserved features. LvIntegrin exhibited broad-spectrum agglutination activity towards both bacteria and fungi and could improve the proliferation of NIH3T3 cells, indicating that LvIntegrin is involved in the immune response against microbe challenge and regulation of cell proliferation as a cell adhesion receptor in shrimp.

Availability Equations for Redundant Systems, Both Single and Multiple Repair Capability

DTIC Science & Technology

1987-10-01

roots of S2StAg)X AL 2 Having the denominator factored enables- Partial fraction expansion and hence finding the inverse transform . The Inverse ...S1 : Xl Tp S2 = x2 ŗ S3 x T The inverse transform for P0 is: Pot l = 6X3 x Sit Spt S t Vs S-)(S2S ’-SS(SS-S )e + iS 3 Si-S 3( e. _S(S 1 -S,)e...roots of S3 + S2 (6\\+6ý0+SMll(\\+2 )+6(1.+-03 S1 -(\\ + • S 2 : -2(X) + • S3 -3(X +0) "The Inverse transform is the same as for Case 3 except for the

Lost in Translation: NIH Funding for Family Medicine Research Remains Limited.

PubMed

Cameron, Brianna J; Bazemore, Andrew W; Morley, Christopher P

2016-01-01

Departments of Family Medicine (DFMs) in the United States consistently received around 0.2% of total research funding dollars and 0.3% of all awards awarded by the National Institutes of Health (NIH) across the years 2002 to 2014. We used the NIH Reporter tool to quantify the amount of funding and the number of grants received by DFMs from the NIH from 2002 to 2014, using criteria similar to those applied by previous researchers. NIH funding to DFMs as remained fairly consistent across the time period, at roughly 0.2% of total NIH funding and 0.3% of total grants awarded. Changing these proportions will likely require considerable effort to build research capacity within DFMs and their frontline practice research networks, and to shift policymaker and funder perceptions of the value of the FM research enterprise. © Copyright 2016 by the American Board of Family Medicine.

Investigation of the anticoagulant and antithrombotic effects of chlorogenic acid.

PubMed

Choi, Jun-Hui; Kim, Seung

2017-03-01

Thrombosis is a leading cause of morbidity and mortality throughout the world. Thrombolytic agents are important for both the prevention and treatment of thrombosis. Fibrin clot and turbidity assays revealed that it was able to inhibit the formation of fibrin clot. Chlorogenic acid degraded blood clot and inhibited the enzymatic activity of procoagulant proteases, thrombin, activated factor X (FXa), and activated factor XIII (FXIIIa). Chlorogenic acid was found to delay activated partial thromboplastin time, prothrombin time, and thrombin time. PFA-100 assays showed that it prolonged the closure time of citrated whole human blood. It demonstrated the antithrombotic effect in collagen and epinephrine-induced acute thromboembolism mice model. These antithrombotic profiles together with its anticoagulant and platelet disaggregation properties, and lack of toxicity to NIH-3T3 and 3T3-L1 cells, make it a potential agent for thrombotic treatment and prevention. © 2016 Wiley Periodicals, Inc.

The Science of Eliminating Health Disparities: Summary and Analysis of the NIH Summit Recommendations

PubMed Central

Rhee, Kyu B.; Williams, Kester; Sanchez, Idalia; Sy, Francisco S.; Stinson, Nathaniel; Ruffin, John

2010-01-01

In December 2008, the National Institutes of Health (NIH) sponsored the first NIH Summit showcasing its investment and contribution to health disparities research and unveiling a framework for moving this important field forward. The Summit, titled “The Science of Eliminating Health Disparities,” drew on extensive experience of experts leading health disparities research transformation in diverse fields. The Summit also provided a historic educational opportunity to contribute to health care reform. The theme, addressing disparities through integration of science, practice, and policy, introduced a paradigm for advancing research through transformational, translational, and transdisciplinary research. Engaging active participation throughout the Summit generated recommendations bridging science, practice, and policy, including action on social determinants of health, community engagement, broad partnerships, capacity-building, and media outreach. PMID:20147660

Polycomb (PcG) Proteins, BMI1 and SUZ12, Regulate Arsenic-induced Cell Transformation*

PubMed Central

Kim, Hong-Gyum; Kim, Dong Joon; Li, Shengqing; Lee, Kun Yeong; Li, Xiang; Bode, Ann M.; Dong, Zigang

2012-01-01

Inorganic arsenic is a well-documented human carcinogen associated with cancers of the skin, lung, liver, and bladder. However, the underlying mechanisms explaining the tumorigenic role of arsenic are not well understood. The present study explored a potential mechanism of cell transformation induced by arsenic exposure. Exposure to a low dose (0.5 μm) of arsenic trioxide (As2O3) caused transformation of BALB/c 3T3 cells. In addition, in a xenograft mouse model, tumor growth of the arsenic-induced transformed cells was dramatically increased. In arsenic-induced transformed cells, polycomb group (PcG) proteins, including BMI1 and SUZ12, were activated resulting in enhanced histone H3K27 tri-methylation levels. On the other hand, tumor suppressor p16INK4a and p19ARF mRNA and protein expression were dramatically suppressed. Introduction of small hairpin (sh) RNA-BMI1 or -SUZ12 into BALB/c 3T3 cells resulted in suppression of arsenic-induced transformation. Histone H3K27 tri-methylation returned to normal in BMI1- or SUZ12-knockdown BALB/c 3T3 cells compared with BMI1- or SUZ12-wildtype cells after arsenic exposure. As a consequence, the expression of p16INK4a and p19ARF was recovered in arsenic-treated BMI1- or SUZ12-knockdown cells. Thus, arsenic-induced cell transformation was blocked by inhibition of PcG function. Taken together, these results strongly suggest that the polycomb proteins, BMI1 and SUZ12 are required for cell transformation induced by organic arsenic exposure. PMID:22843710

The Colony-Stimulating Factor 3 Receptor T640N Mutation Is Oncogenic, Sensitive to JAK Inhibition, and Mimics T618I.

PubMed

Maxson, Julia E; Luty, Samuel B; MacManiman, Jason D; Paik, Jason C; Gotlib, Jason; Greenberg, Peter; Bahamadi, Swaleh; Savage, Samantha L; Abel, Melissa L; Eide, Christopher A; Loriaux, Marc M; Stevens, Emily A; Tyner, Jeffrey W

2016-02-01

Colony-stimulating factor 3 receptor (CSF3R) mutations have been identified in the majority of chronic neutrophilic leukemia (CNL) and a smaller percentage of atypical chronic myeloid leukemia (aCML) cases. Although CSF3R point mutations (e.g., T618I) are emerging as key players in CNL/aCML, the significance of rarer CSF3R mutations is unknown. In this study, we assess the importance of the CSF3R T640N mutation as a marker of CNL/aCML and potential therapeutic target. Sanger sequencing of leukemia samples was performed to identify CSF3R mutations in CNL and aCML. The oncogenicity of the CSF3R T640N mutation relative to the T618I mutation was assessed by cytokine independent growth assays and by mouse bone marrow transplant. Receptor dimerization and O-glycosylation of the mutants was assessed by Western blot, and JAK inhibitor sensitivity was assessed by colony assay. Here, we identify a CSF3R T640N mutation in two patients with CNL/aCML, one of whom was originally diagnosed with MDS and acquired the T640N mutation upon evolution of disease to aCML. The T640N mutation is oncogenic in cellular transformation assays and an in vivo mouse bone marrow transplantation model. It exhibits many similar phenotypic features to T618I, including ligand independence and altered patterns of O-glycosylation--despite the transmembrane location of T640 preventing access by GalNAc transferase enzymes. Cells transformed by the T640N mutation are sensitive to JAK kinase inhibition to a similar degree as cells transformed by CSF3R T618I. Because of its similarities to CSF3R T618I, the T640N mutation likely has diagnostic and therapeutic relevance in CNL/aCML. ©2015 American Association for Cancer Research.

Advance Care Plan: A Checklist for the Future | NIH MedlinePlus the Magazine

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EVALUATION OF BENZO[C]CHRYSENE DIHYDRODIOLS IN THE MORPHOLOGICAL CELL TRANSFORMATION OF MOUSE EMBRYO FIBROBLAST C3H10T1/2CL8 CELLS

EPA Science Inventory

EVALUATION OF BENZO[c]CHRYSENE DIHYDRODIOLS IN THE MORPHOLOGICAL CELL TRANSFORMATION OF MOUSE EMBRYO FIBROBLAST C3H10T?CL8 CELLS

Abstract The morphological cell transforming activities of three dihydrodiols of benzo[c]chrysene (B[c]C), trans-B[c]C-7,8-diol, trans-B[c]C-9...

[Transgenic wheat (Triticum aestivum L.) with increased resistance to the storage pest obtained by Agrobacterium tumefaciens--mediated].

PubMed

Bi, Rui-Ming; Jia, Hai-Yan; Feng, De-Shun; Wang, Hong-Gang

2006-05-01

The transgenic wheat of improved resistance to the storage pest was production. We have introduced the cowpea trypsin inhibitor gene (CpTI) into cultured embryonic callus cells of immature embryos of wheat elite line by Agrobacterium-mediated method. Independent plantlets were obtained from the kanamycin-resistant calli after screening. PCR and real time PCR analysis, PCR-Southern and Southern blot hybridization indicated that there were 3 transgenic plants viz. transformed- I, II and III (T- I, T-II and T-III). The transformation frequencies were obviously affected by Agrobacterium concentration, the infection duration and transformation treatment. The segregations of CpTI in the transgenic wheat progenies were not easily to be elucidated, and some transgenic wheat lines (T- I and T-III) showed Mendelian segregations. The determinations of insect resistance to the stored grain insect of wheat viz. the grain moth (Sitotroga cerealella Olivier) indicated that the 3 transgenic wheat progeny seeds moth-resistance was improved significantly. The seed moth-eaten ratio of T- I, T-II, T-III and nontransformed control was 19.8%, 21.9%, 32.9% and 58.3% respectively. 3 transgenic wheat T1 PCR-positive plants revealed that the 3 transgenic lines had excellent agronomic traits. They supplied good germplasm resource of insect-resistance for wheat genetic improvement.

Thermodynamic Characteristics of Reactions of the Formation of Complexes between Triglycine and Ni2+ Ions in Aqueous Solution

NASA Astrophysics Data System (ADS)

Gorboletova, G. G.; Metlin, A. A.; Bychkova, S. A.

2018-05-01

Thermal effects of reactions of the formation of complexes between Ni(II) and triglycine are determined via direct calorimetry in aqueous solutions at 298.15 K and ionic strengths of 0.2, 0.5, and 1.0 (KNO3). Standard thermodynamic characteristics (Δr H°, Δr G°, Δr S°) of complexing processes in the investigated systems are calculated. The structures of triglycinate complexes NiL+, NiH-1L, NiL2, NiH-2L2- 2, NiL- 3, and NiH-3L4- 3 are introduced to compare the obtained values and data on the thermodynamics of triglycinate complexes of Ni(II).

Safety of tPA in stroke mimics and neuroimaging-negative cerebral ischemia(Podcast)(e–Pub ahead of print)(CME)

PubMed Central

Chernyshev, O.Y.; Martin-Schild, S.; Albright, K.C.; Barreto, A.; Misra, V.; Acosta, I.; Grotta, J.C.; Savitz, S.I.

2010-01-01

Background: Patients with acute neurologic symptoms may have other causes simulating ischemic stroke, called stroke mimics (SM), but they may also have averted strokes that do not appear as infarcts on neuroimaging, which we call neuroimaging-negative cerebral ischemia (NNCI). We determined the safety and outcome of IV thrombolysis within 3 hours of symptom onset in patients with SM and NNCI. Methods: Patients treated with IV tissue plasminogen activator (tPA) within 3 hours of symptom onset were identified from our stroke registry from June 2004 to October 2008. We collected admission NIH Stroke Scale (NIHSS) score, modified Rankin score (mRS), length of stay (LOS), symptomatic intracerebral hemorrhage (sICH), and discharge diagnosis. Results: Among 512 treated patients, 21% were found not to have an infarct on follow-up imaging. In the SM group (14%), average age was 55 years, median admission NIHSS was 7, median discharge NIHSS was 0, median LOS was 3 days, and there were no instances of sICH. The most common etiologies were seizure, complicated migraine, and conversion disorder. In the NNCI group (7%), average age was 61 years, median admission NIHSS was 7, median discharge NIHSS was 0, median LOS was 3 days, and there were no instances of sICH. Nearly all SM (87%) and NNCI (91%) patients were functionally independent on discharge (mRS 0–1). Conclusions: Our data support the safety of administering IV tissue plasminogen activator to patients with suspected acute cerebral ischemia within 3 hours of symptom onset, even when the diagnosis ultimately is found not to be stroke or imaging does not show an infarct. GLOSSARY AIS = acute ischemic stroke; CI = confidence interval; DWI = diffusion-weighted imaging; ED = emergency department; LOS = length of stay; mRS = modified Rankin score; NIHSS = NIH Stroke Scale; NNCI = neuroimaging-negative cerebral ischemia; OR = odds ratio; sICH = symptomatic intracerebral hemorrhage; SM = stroke mimics; tPA = tissue plasminogen activator. PMID:20335564

The uPA/uPAR system regulates the bioavailability of PDGF-DD: implications for tumour growth.

PubMed

Ehnman, M; Li, H; Fredriksson, L; Pietras, K; Eriksson, U

2009-01-29

Members of the platelet-derived growth factor (PDGF) family are mitogens for cells of mesenchymal origin and have important functions during embryonic development, blood vessel maturation, fibrotic diseases and cancer. In contrast to the two classical PDGFs, the novel and less well-characterized members, PDGF-CC and PDGF-DD, are latent factors that need to be processed extracellularly by activating proteases, before they can mediate PDGF receptor activation. Here, we elucidate the structural requirements for urokinase plasminogen activator (uPA)-mediated activation of PDGF-DD, as well as the intricate interplay with uPA receptor (uPAR) signalling. Furthermore, we show that activated PDGF-DD, in comparison to latent, more potently transforms NIH/3T3 cells in vitro. Conversely, xenograft studies in nude mice demonstrate that cells expressing latent PDGF-DD are more tumorigenic than those expressing activated PDGF-DD. These findings imply that a fine-tuned proteolytic activation, in the local milieu, controls PDGF-DD bioavailability. Moreover, we suggest that proteolytic activation of PDGF-DD reveals a retention motif mediating interactions with pericellular components. Our proposed mechanism, where uPA not only generates active PDGF-DD, but also regulates its spatial distribution, provides novel insights into the biological function of PDGF-DD.

Centrosomal Nlp is an oncogenic protein that is gene-amplified in human tumors and causes spontaneous tumorigenesis in transgenic mice.

PubMed

Shao, Shujuan; Liu, Rong; Wang, Yang; Song, Yongmei; Zuo, Lihui; Xue, Liyan; Lu, Ning; Hou, Ning; Wang, Mingrong; Yang, Xiao; Zhan, Qimin

2010-02-01

Disruption of mitotic events contributes greatly to genomic instability and results in mutator phenotypes. Indeed, abnormalities of mitotic components are closely associated with malignant transformation and tumorigenesis. Here we show that ninein-like protein (Nlp), a recently identified BRCA1-associated centrosomal protein involved in microtubule nucleation and spindle formation, is an oncogenic protein. Nlp was found to be overexpressed in approximately 80% of human breast and lung carcinomas analyzed. In human lung cancers, this deregulated expression was associated with NLP gene amplification. Further analysis revealed that Nlp exhibited strong oncogenic properties; for example, it conferred to NIH3T3 rodent fibroblasts the capacity for anchorage-independent growth in vitro and tumor formation in nude mice. Consistent with these data, transgenic mice overexpressing Nlp displayed spontaneous tumorigenesis in the breast, ovary, and testicle within 60 weeks. In addition, Nlp overexpression induced more rapid onset of radiation-induced lymphoma. Furthermore, mouse embryonic fibroblasts (MEFs) derived from Nlp transgenic mice showed centrosome amplification, suggesting that Nlp overexpression mimics BRCA1 loss. These findings demonstrate that Nlp abnormalities may contribute to genomic instability and tumorigenesis and suggest that Nlp might serve as a potential biomarker for clinical diagnosis and therapeutic target.

Centrosomal Nlp is an oncogenic protein that is gene-amplified in human tumors and causes spontaneous tumorigenesis in transgenic mice

PubMed Central

Shao, Shujuan; Liu, Rong; Wang, Yang; Song, Yongmei; Zuo, Lihui; Xue, Liyan; Lu, Ning; Hou, Ning; Wang, Mingrong; Yang, Xiao; Zhan, Qimin

2010-01-01

Disruption of mitotic events contributes greatly to genomic instability and results in mutator phenotypes. Indeed, abnormalities of mitotic components are closely associated with malignant transformation and tumorigenesis. Here we show that ninein-like protein (Nlp), a recently identified BRCA1-associated centrosomal protein involved in microtubule nucleation and spindle formation, is an oncogenic protein. Nlp was found to be overexpressed in approximately 80% of human breast and lung carcinomas analyzed. In human lung cancers, this deregulated expression was associated with NLP gene amplification. Further analysis revealed that Nlp exhibited strong oncogenic properties; for example, it conferred to NIH3T3 rodent fibroblasts the capacity for anchorage-independent growth in vitro and tumor formation in nude mice. Consistent with these data, transgenic mice overexpressing Nlp displayed spontaneous tumorigenesis in the breast, ovary, and testicle within 60 weeks. In addition, Nlp overexpression induced more rapid onset of radiation-induced lymphoma. Furthermore, mouse embryonic fibroblasts (MEFs) derived from Nlp transgenic mice showed centrosome amplification, suggesting that Nlp overexpression mimics BRCA1 loss. These findings demonstrate that Nlp abnormalities may contribute to genomic instability and tumorigenesis and suggest that Nlp might serve as a potential biomarker for clinical diagnosis and therapeutic target. PMID:20093778

Fibroblast growth factor 8 is expressed at higher levels in lactating human breast and in breast cancer.

PubMed

Zammit, C; Coope, R; Gomm, J J; Shousha, S; Johnston, C L; Coombes, R C

2002-04-08

Fibroblast growth factor 8 can transform NIH3T3 cells and its expression has been found to be associated with breast and prostate cancer. Following our finding that fibroblast growth factor 8 mRNA expression is increased in breast cancer, we have undertaken an immunohistochemistry study of fibroblast growth factor 8 expression in a series of human breast tissues and other normal tissues. Our findings confirm increased expression of fibroblast growth factor 8 in malignant breast tissue but also show significant fibroblast growth factor 8 expression in non-malignant breast epithelial cells. No significant difference in fibroblast growth factor 8 expression was found between different grades of ductal carcinoma, lobular carcinoma and ductal carcinoma in-situ or cancer of different oestrogen receptor, progesterone receptor or nodal status. The highest levels of fibroblast growth factor 8 expression were found in lactating breast tissues and fibroblast growth factor 8 was also detected in human milk. A survey of other normal tissues showed that fibroblast growth factor 8 is expressed in the proliferative cells of the dermis and epithelial cells in colon, ovary fallopian tube and uterus. Fibroblast growth factor 8 appears to be expressed in several organs in man and appears to have an importance in lactation.

Neural-Thyroid Interaction on Skeletal Isomyosin in Zero Gravity

NASA Technical Reports Server (NTRS)

Baldwin, Kenneth M.

2000-01-01

The primary goal of the project was to develop a ground based model to first study the role of the nerve and of thyroid hormone (T3) in the regulation of body growth and skeletal muscle growth and differentiation in rodents. A primary objective was to test the hypothesis that normal weight bearing activity is essential for the development of antigravity, slow twitch skeletal muscle and the corresponding slow myosin heavy chain (MHC) gene; whereas, T3 was obligatory for general body and muscle growth and the establishment of fast MHC phenotype in typically fast locomoter muscles. These ground based experiments would provide both the efficacy and background for a spaceflight experiment (referred to as the Neurolab Mission) jointly sponsored by the NIH and NASA.

Broadband Studies of Seismic Sources at Regional and Teleseismic Distances Using Advanced Time Series Analysis Methods

DTIC Science & Technology

1990-02-01

transform the waveforms of this event to those of the Titanial must be a band limited representation of the firing sequence. Therefore, we decided to...design a Wiener filter to transform Pn waveforms of Event Titania4 into those of Event Titanial at all sensors of NORESS. Prior to applying this technique...for transforming the Pn phases of event Titania 4 into those of event Titanial . 28 T’tania4 -* Titania3 Titania3 B5 T’tania4 Titania4 - Titania3

Construction of high frame rate images with Fourier transform

NASA Astrophysics Data System (ADS)

Peng, Hu; Lu, Jian-Yu

2002-05-01

Traditionally, images are constructed with a delay-and-sum method that adjusts the phases of received signals (echoes) scattered from the same point in space so that they are summed in phase. Recently, the relationship between the delay-and-sum method and the Fourier transform is investigated [Jian-yu Lu, Anjun Liu, and Hu Peng, ``High frame rate and delay-and-sum imaging methods,'' IEEE Trans. Ultrason. Ferroelectr. Freq. Control (submitted)]. In this study, a generic Fourier transform method is developed. Two-dimensional (2-D) or three-dimensional (3-D) high frame rate images can be constructed using the Fourier transform with a single transmission of an ultrasound pulse from an array as long as the transmission field of the array is known. To verify our theory, computer simulations have been performed with a linear array, a 2-D array, a convex curved array, and a spherical 2-D array. The simulation results are consistent with our theory. [Work supported in part by Grant 5RO1 HL60301 from NIH.

Influence of phytochemicals in piper betle linn leaf extract on wound healing.

PubMed

Lien, Le Thi; Tho, Nguyen Thi; Ha, Do Minh; Hang, Pham Luong; Nghia, Phan Tuan; Thang, Nguyen Dinh

2015-01-01

Wound healing has being extensively investigated over the world. Healing impairment is caused by many reasons including increasing of free-radicals-mediated damage, delaying in granulation tissue formation, reducing in angiogenesis and decreasing in collagen reorganization. These facts consequently lead to chronic wound healing. Piper betle Linn (Betle) leaves have been folklore used as an ingredient of drugs for cutaneous wound treatment. However, the effect of betle leaf on wound healing is not yet well elucidated. In this study, we aimed to investigate the healing efficacy of methanol leaf extract of Piper betle Linn on proliferation of fibroblast NIH3T3 cells as well as full-thickness burn and excision wounds in swiss mice. Scratch wound healing assays were conducted to examine the effects of betle leaf extract on healing activity of fibroblast cells. Burn and excision wounds on swiss mouse skins were created for investigating the wound healing progress caused by the betle leaf extract. Malondialdehyde (MDA) was also evaluated to examine the products of lipid hydroperoxide (LPO) under conditions of with or without betle leaf extract treatment. The results of this study showed that Piper betle Linn leaf extract in methanol increased proliferation of NIH3T3 cells and promoted wound healing in vitro and in vivo with both burn wound and excision wound models. In addition, this extract significant decreased level of malondialdehyde (MDA) in liver of treated-mice compared with that in non-treated mice. Our results suggest that Piper betle Linn can be used as an ingredient in developing natural origin drugs for treatment of cutaneous wounds.

In vitro fluoride release from a different kind of conventional and resin modified glass-ionomer cements.

PubMed

Selimović-Dragaš, Mediha; Hasić-Branković, Lajla; Korać, Fehim; Đapo, Nermin; Huseinbegović, Amina; Kobašlija, Sedin; Lekić, Meliha; Hatibović-Kofman, Šahza

2013-08-01

Fluoride release is important characteristic of glass-ionomer cements. Quantity of fluoride ions released from the glass-ionomer cements has major importance in definition of their biological activity. The objectives of this study were to define the quantity of fluoride ions released from the experimental glass-ionomer cements and to define the effect of fluoride ions released from the experimental glass-ionomer cements on their cytotoxicity. Concentrations of the fluoride ions released in the evaluated glass-ionomer cements were measured indirectly, by the fluoride-selective WTW, F500 electrode potential, combined with reference R503/D electrode. Statistical analyses of F-ion concentrations released by all glass-ionomers evaluated at two time points, after 8 and after 24 hours, show statistically higher fluoride releases from RMGICs: Vitrebond, Fuji II LC and Fuji Plus, when compared to conventional glass-ionomer cements: Fuji Triage, Fuji IX GP Fast and Ketac Silver, both after 8 and after 24 hours. Correlation coefficient between concentrations of fluoride ion released by evaluated glass-ionomer cements and cytotoxic response of UMR-106 osteoblast cell-line are relatively high, but do not reach levels of biological significance. Correlation between concentrations of fluoride ion released and cytotoxic response of NIH3T3 mouse fibroblast cell line after 8 hours is high, positive and statistically significant for conventional GICs, Fuji Triage and Fuji IX GP Fast, and RMGIC, Fuji II LC. Statistically significant Correlation coefficient between concentrations of fluoride ion released and cytotoxic response of NIH3T3 cell line after 24 hours is defined for RMGIC Fuji II LC only.

Composite wound dressings of pectin and gelatin with aloe vera and curcumin as bioactive agents.

PubMed

Tummalapalli, Mythili; Berthet, Morgane; Verrier, Bernard; Deopura, B L; Alam, M S; Gupta, Bhuvanesh

2016-01-01

Aloe vera and curcumin loaded oxidized pectin-gelatin (OP-Gel) matrices were used as antimicrobial finishes on nonwoven cotton fabrics to produce composite wound care devices. The drug release characteristics of the biocomposite dressings indicated that curcumin is released through a biphasic mechanism - erosion of the polymeric matrix, followed by diffusion, while aloe vera is released upon leaching of the polymeric matrix. A 50/50 composition of aloe vera/curcumin was used to fabricate OP-Gel-Aloe Curcumin dressings. However, contrary to our expectations, OP-Gel-Aloe Curcumin dressings exhibited lesser antimicrobial activity compared to OP-Gel-Aloe and OP-Gel-Curcumin dressings. The cytocompatibility of the fabricated dressings was evaluated using NIH3T3 mouse fibroblast cells. OP-Gel-Aloe treated fibroblasts had the highest viability, with the matrices providing a substrate for good cell attachment and proliferation. On the other hand, OP-Gel-Curcumin and OP-Gel-Aloe Curcumin seemed to have induced apoptosis in NIH3T3 cells. In vivo wound healing analysis was carried out using an excisional splint wound model on C57BL/6J mice. OP-Gel-Aloe treated wounds exhibited very rapid healing with 80% of the wound healing in just 8 days. Furthermore, aloe vera exerted a strong anti-inflammatory effect and prominent scar prevention. Histological examination revealed that an ordered collagen formation and neovascularization could be observed along with migration of nuclei. Therefore, OP-Gel-Aloe biocomposite dressings are proposed as viable materials for effective wound management. Copyright © 2015 Elsevier B.V. All rights reserved.

Fibroblast contractility and growth in plastic compressed collagen gel scaffolds with microstructures correlated with hydraulic permeability.

PubMed

Serpooshan, Vahid; Muja, Naser; Marelli, Benedetto; Nazhat, Showan N

2011-03-15

Scaffold microstructure is hypothesized to influence physical and mechanical properties of collagen gels, as well as cell function within the matrix. Plastic compression under increasing load was conducted to produce scaffolds with increasing collagen fibrillar densities ranging from 0.3 to above 4.1 wt % with corresponding hydraulic permeability (k) values that ranged from 1.05 to 0.03 μm², as determined using the Happel model. Scanning electron microscopy revealed that increasing the level of collagen gel compression yielded a concomitant reduction in pore size distribution and a slight increase in average fibril bundle diameter. Decreasing k delayed the onset of contraction and significantly reduced both the total extent and the maximum rate of contraction induced by NIH3T3 fibroblasts seeded at a density of either 6.0 x 10⁴ or 1.5 x 10⁵ cells mL⁻¹. At the higher cell density, however, the effect of k reduction on collagen gel contraction was overcome by an accelerated onset of contraction which led to an increase in both the total extent and the maximum rate of contraction. AlamarBlue™ measurements indicated that the metabolic activity of fibroblasts within collagen gels increased as k decreased. Moreover, increasing seeded cell density from 2.0 x 10⁴ to 1.5 x 10⁵ cells mL⁻¹ significantly increased NIH3T3 proliferation. In conclusion, fibroblast-matrix interactions can be optimized by defining the microstructural properties of collagen scaffolds through k adjustment which in turn, is dependent on the cell seeding density. Copyright © 2011 Wiley Periodicals, Inc.

Direct detection of diverse metabolic changes in virally transformed and tax-expressing cells by mass spectrometry.

PubMed

Sripadi, Prabhakar; Shrestha, Bindesh; Easley, Rebecca L; Carpio, Lawrence; Kehn-Hall, Kylene; Chevalier, Sebastien; Mahieux, Renaud; Kashanchi, Fatah; Vertes, Akos

2010-09-07

Viral transformation of a cell starts at the genetic level, followed by changes in the proteome and the metabolome of the host. There is limited information on the broad metabolic changes in HTLV transformed cells. Here, we report the detection of key changes in metabolites and lipids directly from human T-lymphotropic virus type 1 and type 3 (HTLV1 and HTLV3) transformed, as well as Tax1 and Tax3 expressing cell lines by laser ablation electrospray ionization (LAESI) mass spectrometry (MS). Comparing LAESI-MS spectra of non-HTLV1 transformed and HTLV1 transformed cells revealed that glycerophosphocholine (PC) lipid components were dominant in the non-HTLV1 transformed cells, and PC(O-32:1) and PC(O-34:1) plasmalogens were displaced by PC(30:0) and PC(32:0) species in the HTLV1 transformed cells. In HTLV1 transformed cells, choline, phosphocholine, spermine and glutathione, among others, were downregulated, whereas creatine, dopamine, arginine and AMP were present at higher levels. When comparing metabolite levels between HTLV3 and Tax3 transfected 293T cells, there were a number of common changes observed, including decreased choline, phosphocholine, spermine, homovanillic acid, and glycerophosphocholine and increased spermidine and N-acetyl aspartic acid. These results indicate that the lipid metabolism pathway as well as the creatine and polyamine biosynthesis pathways are commonly deregulated after expression of HTLV3 and Tax3, indicating that the noted changes are likely due to Tax3 expression. N-acetyl aspartic acid is a novel metabolite that is upregulated in all cell types and all conditions tested. We demonstrate the high throughput in situ metabolite profiling of HTLV transformed and Tax expressing cells, which facilitates the identification of virus-induced perturbations in the biochemical processes of the host cells. We found virus type-specific (HTLV1 vs. HTLV3), expression-specific (Tax1 vs. Tax3) and cell-type-specific (T lymphocytes vs. kidney epithelial cells) changes in the metabolite profiles. The new insight on the affected metabolic pathways can be used to better understand the molecular mechanisms of HTLV induced transformation, which in turn can result in new treatment strategies.

XRCC3 polymorphism is associated with hypertension-induced left ventricular hypertrophy.

PubMed

Ariyandy, Andi; Sakai, Chiemi; Ishida, Mari; Mizuta, Ryusei; Miyagawa, Kiyoshi; Tashiro, Satoshi; Kinomura, Aiko; Hiraaki, Koji; Ueda, Keitaro; Yoshizumi, Masao; Ishida, Takafumi

2018-06-01

Deficiency of X-ray repair cross-complementing protein 3 (XRCC3), a DNA-damage repair molecule, and the 241Met variant of XRCC3 have been reported to increase endoreduplication, which induces polyploidy. The aims of this study were to determine the impact of the XRCC3 polymorphism on the incidence of hypertension-induced left ventricular hypertrophy (LVH) and to investigate the mechanisms underlying any potential relationship. Patients undergoing chronic hemodialysis (n = 77) were genotyped to assess for the XRCC3 Thr241Met polymorphism. The XRCC3 241Thr/Met genotype was more frequent in the LVH (+) group than in the LVH (-) group (42.3 vs. 13.7%, χ2 = 7.85, p = 0.0051). To investigate possible mechanisms underlying these observations, human XRCC3 cDNA of 241Thr or that of 241Met was introduced into cultured CHO cells. The surface area of CHO cells expressing XRCC3 241Met was larger than that expressing 241Thr. Spontaneous DNA double-strand breaks accumulated to a greater degree in NIH3T3 cells expressing 241Met (3T3-241Met) than in those expressing 241Thr (3T3-241Thr). DNA damage caused by radiation induced cell senescence more frequently in 3T3-241Met. The levels of basal and TNF-α-stimulated MCP-1 mRNA and protein secretion were higher in 3T3-241Met. Finally, FACS analysis revealed that the cell percentage in G2/M phase including polyploidy was significantly higher in 3T3-241Met than in 3T3-241Thr. Furthermore, the basal level of MCP-1 mRNA positively correlated with the cell percentage in G2/M phase and polyploidy. These data suggest that the XRCC3 241Met increases the risk of LVH via accumulation of DNA damage, thereby altering cell cycle progression and inducing cell senescence and a proinflammatory phenotype.

Consensus recommendations for improvement of unmet clinical needs--the example of chronic graft-versus-host disease: a systematic review and meta-analysis.

PubMed

Olivieri, Jacopo; Manfredi, Lucia; Postacchini, Laura; Tedesco, Silvia; Leoni, Pietro; Gabrielli, Armando; Rambaldi, Alessandro; Bacigalupo, Andrea; Olivieri, Attilio; Pomponio, Giovanni

2015-07-01

Consensus recommendations are used to improve the methodology of research about rare disorders, but their uptake is unknown. We studied the uptake of consensus recommendations in steroid-refractory chronic graft-versus-host disease (SR-cGVHD). Although in 2006 the National Institutes of Health (NIH) cGVHD consensus project produced recommendations for clinical trials, guidelines have emphasised the scarcity of valuable evidence for all tested interventions. We searched Medline (PubMed) between Jan 1, 1998, and Oct 1, 2013, for non-randomised studies of systemic treatment for SR-cGVHD. To measure adherence to NIH recommendations, we applied a 61 item checklist derived from the NIH consensus document. We did a meta-analysis to measure pooled effect size for overall response rate (ORR) and meta-regression analyses to measure the effect of deviations from NIH recommendations on pooled effect size. We included 82 studies related to nine interventions. Conformity to NIH recommendations was evenly low across the analysed timeframe (1998-2013), and did not change significantly after publication of NIH recommendations. The pooled effect size for ORR for systemic treatment of SR-cGVHD was 0.66 (95% CI 0.62-0.70). Increased adherence to NIH recommendations in a score of items defining correct response assessment was associated with a significant reduction in ORR (-4.2%, 95% CI -6.6 to -1.9; p=0.001). We recorded no significant association between ORR and sets of items related to correct diagnostic definition of SR-cGVHD (change in ORR -3.1%, 95% CI -7.7 to 1.5), specification of primary intervention (0, -3.8 to 3.6), or concomitant treatments (-1.6%, -5.4 to 2.3). The score of items defining correct response assessment increased after publication of NIH recommendations. Our findings show evidence of bias in the reported efficacy of treatment of SR-cGVHD. The overall effect of NIH recommendations in scientific literature is scarce; however, NIH recommendations improved assessment of response, possibly reducing the overestimation bias. Better implementation of NIH recommendations might reduce false expectations about new interventions, and thus prevent clinical studies with ineffective treatments. None. Copyright © 2015 Elsevier Ltd. All rights reserved.

The Impact of In Vitro Accelerated Aging, Approximating 30 and 60 Years In Vivo, on Commercially Available Zirconia Dental Implants.

PubMed

Monzavi, Mona; Noumbissi, Sammy; Nowzari, Hessam

2017-04-01

Despite increased popularity of Zirconia dental implants, concerns have been raised regarding low temperature degradation (LTD) and its effect on micro-structural integrity. This study evaluated the effect of LTD on four types of Zirconia dental implants at 0, 30, and 60 years of artificial aging. The impact of aging on t-m transformation and micro crack formation was measured. Accelerated aging at 15 and 30 hours, approximating 30 and 60 years in vivo, aged 36 Zirconia dental implants: Z systems ® (A), Straumann ® (B), Ceraroot ® (C), and Zeramex ® (D). Focused ion beam-scanning electron microscopic analysis determined the micro structural features, phase transformation, and the formation of micro cracks. At 15 hours, type A implant presented with micro cracks and t-m transformation of 0.9 µm and 3.1 µm, respectively. At 30 hours, micro cracks remained shallow (1 µm). At 15 hours, type B implant presented micro cracks (0.7 µm) and grain transformation (1.2 µm). At 30 hours, these features remained superficial at 0.6 and 1.5 µm, respectively. Type C implant presented surface micro cracks of 0.3 µm at 15 hours. The depth of t-m transformation slightly increased to 1.4 µm. At 30 hours, number of micro cracks increased at the surface to an average depth of 1.5 µm. Depth of t-m transformation increased to an average of 2.5 µm. At 15 hours, micro cracks remained superficial (0.8 µm) for type D implant and depth of t-m transformation increased to 2.3 µm. At 30 hours, the depth of micro cracks increased to an average of 1.3 µm followed by increased t-m transformation to a depth of 4.1 µm. Depth of grain transformation remained within 1-4 µm from the surface. The effect of aging was minimal for all Zirconia implants. © 2016 Wiley Periodicals, Inc.

Lead facilitates foci formation in a Balb/c-3T3 two-step cell transformation model: role of Ape1 function.

PubMed

Hernández-Franco, Pablo; Silva, Martín; Franco, Rodrigo; Valverde, Mahara; Rojas, Emilio

2018-04-01

Several possible mechanisms have been examined to gain an understanding on the carcinogenic properties of lead, which include among others, mitogenesis, alteration of gene expression, oxidative damage, and inhibition of DNA repair. The aim of the present study was to explore if low concentrations of lead, relevant for human exposure, interfere with Ape1 function, a base excision repair enzyme, and its role in cell transformation in Balb/c-3T3. Lead acetate 5 and 30 μM induced APE1 mRNA and upregulation of protein expression. This increase in mRNA expression is consistent throughout the chronic exposure. Additionally, we also found an impaired function of Ape1 through molecular beacon-based assay. To evaluate the impact of lead on foci formation, a Balb/c-3T3 two-step transformation model was used. Balb/c-3T3 cells were pretreated 1 week with low concentrations of lead before induction of transformation with n-methyl-n-nitrosoguanidine (MNNG) (0.5 μg/mL) and 12-O-tetradecanoylphorbol-13-acetate (TPA) (0.1 μg/mL) (a classical two-step protocol). Morphological cell transformation increased in response to lead pretreatment that was paralleled with an increase in Ape1 mRNA and protein overexpression and an impairment of Ape1 activity and correlating with foci number. In addition, we found that lead pretreatment and MNNG (transformation initiator) increased DNA damage, determined by comet assay. Our data suggest that low lead concentrations (5, 30 μM) could play a facilitating role in cellular transformation, probably through the impaired function of housekeeping genes such as Ape1, leading to DNA damage accumulation and chromosomal instability, one of the most important hallmarks of cancer induced by chronic exposures.

Not all protein-mediated single-wall carbon nanotube dispersions are equally bioactive

NASA Astrophysics Data System (ADS)

Holt, Brian D.; McCorry, Mary C.; Boyer, Patrick D.; Dahl, Kris Noel; Islam, Mohammad F.

2012-11-01

Single-wall carbon nanotubes (SWCNTs) have been dispersed with proteins to increase biocompatibility and specificity, but examinations of dispersion parameters on functional cellular uptake are required for utilization of SWCNTs in biological applications. Here we correlate conditions of SWCNT dispersion with various proteins to uptake these SWCNTs in NIH-3T3 fibroblasts and J774A.1 macrophage-like cells. We varied protein types (bovine serum albumin - BSA, lysozyme - LSZ, and γ-globulins - γG), protein : SWCNT ratio and sonication time. Each protein created stable, high yield (~25%) dispersions in water while preserving intrinsic SWCNT fluorescence, but SWCNT-LSZ flocculated in media and SWCNT-γG formed clusters in both water and media, drastically altering cellular internalization. Dispersion quality and yield improved with increased protein : SWCNT - without substantial effects from depletion attraction, even at 100 : 1 protein : SWCNT - and slightly increased internalized SWCNTs for both NIH-3T3 and J774A.1 cells. Longer sonication time (12 versus 2 h) improved the dispersion yield and quality but caused minor damage to SWCNTs and altered protein structure. Cell association of SWCNT-BSA was homogenous and unaltered by sonication time. Bulk assay showed that cell association of SWCNT-LSZ and SWCNT-γG was altered with 12 versus 2 h sonication, but imaging of individual cells showed that these differences are likely from precipitation of clusters of SWCNT-LSZ and SWCNT-γG in media onto cells. Hence, the quality of SWCNT-protein dispersions in water does not necessarily correlate with bulk cellular uptake, and quantification at the level of individual cells is required to determine delivery efficacy.Single-wall carbon nanotubes (SWCNTs) have been dispersed with proteins to increase biocompatibility and specificity, but examinations of dispersion parameters on functional cellular uptake are required for utilization of SWCNTs in biological applications. Here we correlate conditions of SWCNT dispersion with various proteins to uptake these SWCNTs in NIH-3T3 fibroblasts and J774A.1 macrophage-like cells. We varied protein types (bovine serum albumin - BSA, lysozyme - LSZ, and γ-globulins - γG), protein : SWCNT ratio and sonication time. Each protein created stable, high yield (~25%) dispersions in water while preserving intrinsic SWCNT fluorescence, but SWCNT-LSZ flocculated in media and SWCNT-γG formed clusters in both water and media, drastically altering cellular internalization. Dispersion quality and yield improved with increased protein : SWCNT - without substantial effects from depletion attraction, even at 100 : 1 protein : SWCNT - and slightly increased internalized SWCNTs for both NIH-3T3 and J774A.1 cells. Longer sonication time (12 versus 2 h) improved the dispersion yield and quality but caused minor damage to SWCNTs and altered protein structure. Cell association of SWCNT-BSA was homogenous and unaltered by sonication time. Bulk assay showed that cell association of SWCNT-LSZ and SWCNT-γG was altered with 12 versus 2 h sonication, but imaging of individual cells showed that these differences are likely from precipitation of clusters of SWCNT-LSZ and SWCNT-γG in media onto cells. Hence, the quality of SWCNT-protein dispersions in water does not necessarily correlate with bulk cellular uptake, and quantification at the level of individual cells is required to determine delivery efficacy. Electronic supplementary information (ESI) available: Images of protein dispersions, comparison of absorbance and NIR fluorescence peak shifts, gross quantification of cellular uptake of SWCNTs, and summary of protein secondary structure as a function of sonication time in the presence of SWCNTs. See DOI: 10.1039/c2nr31928d

Mobile phone base station radiation does not affect neoplastic transformation in BALB/3T3 cells.

PubMed

Hirose, H; Suhara, T; Kaji, N; Sakuma, N; Sekijima, M; Nojima, T; Miyakoshi, J

2008-01-01

A large-scale in vitro study focusing on low-level radiofrequency (RF) fields from mobile radio base stations employing the International Mobile Telecommunication 2000 (IMT-2000) cellular system was conducted to test the hypothesis that modulated RF fields affect malignant transformation or other cellular stress responses. Our group previously reported that DNA strand breaks were not induced in human cells exposed to 2.1425 GHz Wideband Code Division Multiple Access (W-CDMA) radiation up to 800 mW/kg from mobile radio base stations employing the IMT-2000 cellular system. In the current study, BALB/3T3 cells were continuously exposed to 2.1425 GHz W-CDMA RF fields at specific absorption rates (SARs) of 80 and 800 mW/kg for 6 weeks and malignant cell transformation was assessed. In addition, 3-methylcholanthrene (MCA)-treated cells were exposed to RF fields in a similar fashion, to assess for effects on tumor promotion. Finally, the effect of RF fields on tumor co-promotion was assessed in BALB/3T3 cells initiated with MCA and co-exposed to 12-O-tetradecanoylphorbol-13-acetate (TPA). At the end of the incubation period, transformation dishes were fixed, stained with Giemsa, and scored for morphologically transformed foci. No significant differences in transformation frequency were observed between the test groups exposed to RF signals and the sham-exposed negative controls in the non-, MCA-, or MCA plus TPA-treated cells. Our studies found no evidence to support the hypothesis that RF fields may affect malignant transformation. Our results suggest that exposure to low-level RF radiation of up to 800 mW/kg does not induce cell transformation, which causes tumor formation. (c) 2007 Wiley-Liss, Inc.

Photochemistry of Iron and Ruthenium Carbonyl Complexes: Evidence for Light-Induced Loss of Carbon Monoxide and Reductive Elimination of Triethylsilane from cis-mer-HM(SiEt3)(CO)3(PPh3).

DTIC Science & Technology

1983-05-21

JEOL FX9OQ Fourier transform or Bruker 250 or 270 MHz Fourier transform spectrometer. Irradiations. Photochemical reactions were carried out using a...Ph 3 ) 3nd -t-Etin V,- a 313 nm quantum yield of -0.6. The process is photochemically everse if the cis-mer-HM(SiPh 3 )(CO) 3 (PPh 3) is irradiated...process is photochemically reversed if the cis-mer-HM(SiPh 3 )(CO)3 (PPh 3 ) is irradiated in t.e presence of excess Et3SiH. Irradiation of cis-mer-HM

76 FR 7570 - Proposed Collection; Comment Request; National Institutes of Health Loan Repayment Programs

Federal Register 2010, 2011, 2012, 2013, 2014

2011-02-10

...In compliance with the requirement of Section 3506(c)(2)(A) of the Paperwork Reduction Act of 1995, for opportunity for public comment on proposed data collection projects, the Division of Loan Repayment, National Institutes of Health (NIH), will publish periodic summaries of proposed projects to be submitted to the Office of Management and Budget (OMB) for review and approval. Proposed Collection: Title: National Institutes of Health Loan Repayment Programs. Type of Information Collection Request: Extension of a currently approved collection (OMB No. 0925-0361, expiration date 06/30/11). Form Numbers: NIH 2674-1, NIH 2674-2, NIH 2674-3, NIH 2674- 4, NIH 2674-5, NIH 2674-6, NIH 2674-7, NIH 2674-8, NIH 2674-9, NIH 2674-10, NIH 2674-11, NIH 2674-12, NIH 2674-13, NIH 2674-14, NIH 2674- 15, NIH 2674-16, NIH 2674-17, NIH 2674-18, and NIH 2674-19. Need and Use of Information Collection: The NIH makes available financial assistance, in the form of educational loan repayment, to M.D., PhD, Pharm.D., D.D.S., D.M.D., D.P.M., D.C., and N.D. degree holders, or the equivalent, who perform biomedical or behavioral research in NIH intramural laboratories or as extramural grantees or scientists funded by domestic nonprofit organizations for a minimum of 2 years (3 years for the General Research Loan Repayment Program (LRP)) in research areas supporting the mission and priorities of the NIH. The AIDS Research LRP (AIDS-LRP) is authorized by section 487A of the Public Health Service Act (PHS Act) (42 U.S.C. 288-1), and the Clinical Research LRP for Individuals from Disadvantaged Backgrounds (CR-LRP) is authorized by section 487E (42 U.S.C. 288-5). The General Research LRP (GR-LRP) is authorized by section 487C of the PHS Act (42 U.S.C. 288-3), and the Clinical Research LRP (LRP-CR) is authorized by section 487F (42 U.S.C. 288-5a). The Pediatric Research LRP (PR-LRP) is authorized by section 487F of the PHS Act (42 U.S.C. 288-6), and the Extramural Clinical Research LRP for Individuals from Disadvantaged Backgrounds (ECR-LRP) is authorized by an amendment to section 487E (42 U.S.C. 288-5). The Contraception and Infertility Research LRP (CIR-LRP) is authorized by section 487B of the PHS Act (42 U.S.C. 288-2), and the Health Disparities Research LRP (HD- LRP) is authorized by section 485G (42 U.S.C. 287c-33). The Loan Repayment Programs can repay up to $35,000 per year toward a participant's extant eligible educational loans, directly to financial institutions. The information proposed for collection will be used by the Division of Loan Repayment to determine an applicant's eligibility for participation in the program. Frequency of Response: Initial application and one- or two-year renewal application. Affected Public: Individuals or households, nonprofits, and businesses or other for-profit. Type of Respondents: Physicians, other scientific or medical personnel, and institutional representatives. The annual reporting burden is as follows:

10 CFR Appendix C to Subpart T of... - Certification Report for Distribution Transformers

Code of Federal Regulations, 2011 CFR

2011-01-01

... 10 Energy 3 2011-01-01 2011-01-01 false Certification Report for Distribution Transformers C..., App. C Appendix C to Subpart T of Part 431—Certification Report for Distribution Transformers All...: For Existing, New, or Modified Models: 1 Prepare tables that will list distribution transformer...

Transforming Growth Factor-β1 T869C Gene Polymorphism Is Associated with Acquired Sick Sinus Syndrome via Linking a Higher Serum Protein Level

PubMed Central

Chen, Jan-Yow; Liu, Jiung-Hsiun; Wu, Hong-Dar Isaac; Lin, Kuo-Hung; Chang, Kuan-Cheng; Liou, Ying-Ming

2016-01-01

Background Familial sick sinus syndrome is associated with gene mutations and dysfunction of ion channels. In contrast, degenerative fibrosis of the sinus node tissue plays an important role in the pathogenesis of acquired sick sinus syndrome. There is a close relationship between transforming growth factor-β1 mediated cardiac fibrosis and acquired arrhythmia. It is of interest to examine whether transforming growth factor-β1 is involved in the pathogenesis of acquired sick sinus syndrome. Methods Overall, 110 patients with acquired SSS and 137 age/gender-matched controls were screened for transforming growth factor-β1 and cardiac sodium channel gene polymorphisms using gene sequencing or restriction fragment length polymorphism methods. An enzyme-linked immunosorbent assay was used to determine the serum level of transforming growth factor-β1. Results Two transforming growth factor-β1 gene polymorphisms (C-509T and T+869C) and one cardiac sodium channel gene polymorphism (H588R) have been identified. The C-dominant CC/CT genotype frequency of T869C was significantly higher in acquired sick sinus syndrome patients than in controls (OR 2.09, 95% CI 1.16–3.75, P = 0.01). Consistently, the level of serum transforming growth factor-β1 was also significantly greater in acquired sick sinus syndrome group than in controls (5.3±3.4 ng/ml vs. 3.7±2.4 ng/ml, P = 0.01). In addition, the CC/CT genotypes showed a higher transforming growth factor-β1 serum level than the TT genotype (4.25 ± 2.50 ng/ml vs. 2.71± 1.76 ng/ml, P = 0.028) in controls. Conclusion Transforming growth factor-β1 T869C polymorphism, correlated with high serum transforming growth factor-β1 levels, is associated with susceptibility to acquired sick sinus syndrome. PMID:27380173

Differential responses of EGFR-/AGT-expressing cells to the "combi-triazene" SMA41.

PubMed

Matheson, Stephanie L; McNamee, James P; Jean-Claude, Bertrand J

2003-01-01

Previous studies have demonstrated enhanced potency associated with the binary [DNA/epidermal growth factor receptor (EGFR)] targeting properties of SMA41 (a chimeric 3-(alkyl)-1,2,3-triazene linked to a 4-anilinoquinazoline backbone) in the A431 (epidermal carcinoma of the vulva) cell line. We now report on the dependence of its antiproliferative effects (e.g. DNA damage, cell survival) on the EGFR and the DNA repair protein O6-alkylguanine DNA alkyltransferase (AGT) contents of 12 solid tumor cell lines, two of which, NIH3T3 and NIH3T3 HER14 (engineered to overexpress EGFR), were isogenic. Receptor type specificity was determined using ELISA for competitive binding, as well as growth factor-stimulation assays. DNA damage was studied using single-cell microelectrophoresis (comet) assays, and levels of EGFR were determined by Western blotting. The effects of SMA41 on the cell cycle of NIH3T3 cells were investigated using univariate flow cytometry. Studies of receptor type specificity showed that SMA41: (a) preferentially inhibited the kinase activity of EGFR over those of Src, insulin receptor and protein kinase C (PKC, a serine/threonine kinase), (b) induced stronger inhibition of growth stimulated with EGF than of growth stimulated with platelet-derived growth factor (PDGF) or fetal bovine serum (FBS). Despite the EGFR specificity of SMA41, there was an absence of a linear correlation between the EGFR status of our solid tumor cell lines and levels of DNA damage induced by the alkylating component. Similarly, EGFR levels did not correlate with IC(50) values. The antiproliferative activities of SMA41 correlated more with the AGT status of these cells and paralleled those of the clinical triazene temozolomide (TEM). However, throughout the panel, tumor cell sensitivity to SMA41 was consistently stronger than to its closest analogue TEM. Experiments performed with the isogenic cells showed that SMA41 was capable of inducing twofold higher levels of DNA damage in the EGFR transfectant and delayed cell entry to G(2)/M in both cell types. When the cells were starved and growth-stimulated with FBS (conditions under which both cell types were growth-stimulated), in contrast to TEM, SMA41 and its hydrolytic metabolite SMA52 exhibited approximately nine- and threefold stronger inhibition of growth of the EGFR transfectant. These results suggest that, in addition to its ability to induce DNA damage and cell cycle perturbations, SMA41 is capable of selectively targeting the cells with a growth advantage conferred by EGFR transfection. When compared with the monoalkyltriazene prodrug TEM, its potency may be further enhanced by its ability to hydrolyze to another signal transduction inhibitor (SMA52) plus a DNA alkylating agent that may further contribute to chemosensitivity. Thus, our new "combi-targeting" strategy may well represent a tandem approach to selectively blocking receptor tyrosine kinase-mediated growth signaling while inducing significant levels of cytotoxic DNA lesions in refractory tumors.

Inhibitors for Androgen Receptor Activation Surfaces

DTIC Science & Technology

2006-09-01

Thyroid hormone, 3,5,3′-triiodo-L-thyronine (T3; Sigma) P R O T O C O L www.stke.org/cgi/content/full/sigtrans;2006/341/pl3 Page 4 TNT T7 quick coupled...SRC2 full-length protein was obtained by using a TNT T7 quick coupled transcrip- tion/translation system. Transformation and preparation of bacterial...is conducted in triplicate for T3 and hit compounds. 1. Produce full-length hTRβ using a TNT T7 quick-coupled transcription translation system

Understanding Strain-Induced Phase Transformations in BiFeO3 Thin Films.

PubMed

Dixit, Hemant; Beekman, Christianne; Schlepütz, Christian M; Siemons, Wolter; Yang, Yongsoo; Senabulya, Nancy; Clarke, Roy; Chi, Miaofang; Christen, Hans M; Cooper, Valentino R

2015-08-01

Experiments demonstrate that under large epitaxial strain a coexisting striped phase emerges in BiFeO 3 thin films, which comprises a tetragonal-like ( T ') and an intermediate S ' polymorph. It exhibits a relatively large piezoelectric response when switching between the coexisting phase and a uniform T ' phase. This strain-induced phase transformation is investigated through a synergistic combination of first-principles theory and experiments. The results show that the S ' phase is energetically very close to the T ' phase, but is structurally similar to the bulk rhombohedral ( R ) phase. By fully characterizing the intermediate S ' polymorph, it is demonstrated that the flat energy landscape resulting in the absence of an energy barrier between the T ' and S ' phases fosters the above-mentioned reversible phase transformation. This ability to readily transform between the S ' and T ' polymorphs, which have very different octahedral rotation patterns and c / a ratios, is crucial to the enhanced piezoelectricity in strained BiFeO 3 films. Additionally, a blueshift in the band gap when moving from R to S ' to T ' is observed. These results emphasize the importance of strain engineering for tuning electromechanical responses or, creating unique energy harvesting photonic structures, in oxide thin film architectures.

Overexpression of bacterial ethylene-forming enzyme gene in Trichoderma reesei enhanced the production of ethylene

PubMed Central

Chen, Xi; Liang, Yong; Hua, Jing; Tao, Li; Qin, Wensheng; Chen, Sanfeng

2010-01-01

In order to efficiently utilize natural cellulose materials to produce ethylene, three expression vectors containing the ethylene-forming enzyme (efe) gene from Pseudomonas syringae pv. glycinea were constructed. The target gene was respectively controlled by different promoters: cbh I promoter from Trichoderma reesei cellobiohydrolases I gene, gpd promoter from Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase gene and pgk I promoter from T. reesei 3-phosphoglycerate kinase I gene. After transforming into T. reesei QM9414, 43 stable transformants were obtained by PCR amplification and ethylene determination. Southern blot analysis of 14 transformants demonstrated that the efe gene was integrated into chromosomal DNA with copy numbers from 1 to 4. Reverse transcription polymerase chain reaction (RT-PCR) analysis of 6 transformants showed that the heterologous gene was transcribed. By using wheat straw as a carbon source, the ethylene production rates of aforementioned 14 transformants were measured. Transformant C30-3 with pgk I promoter had the highest ethylene production (4,012 nl h-1 l-1). This indicates that agricultural wastes could be used to produce ethylene in recombinant filamentous fungus T. reesei. PMID:20150979

Cellular manipulation and patterning using ferromagnetic nanowires

NASA Astrophysics Data System (ADS)

Hultgren, Anne

Ferromagnetic nanowires are demonstrated as an effective tool to apply forces to living cells. Both magnetic cell separations and the magnetic patterning of cells on a substrate will be accomplished through the use of cell-nanowire interactions as well as nanowire-magnetic field interactions. When introduced into cultures of NIH-3T3 cells, the nanowires are internalized by cells via the integrin-mediated adhesion pathway without inflicting any toxic effects on the cell cycle over the course of several days. In addition, the length of the nanowires was found to have an effect on the cell-nanowire interactions when the cells were dissociated from the tissue culture dish. To compare the effectiveness of the nanowires as a means of manipulating cells to the current technology which is based on superparamagnetic beads, magnetic cell separations were performed with electrodeposited Ni nanowires 350 nm in diameter and 5--35 mum long in field gradients of 80 T/m. Single-pass separations of NIH-3T3 cells bound to nanowires achieve up to 81% purity with 85% yield, a dramatic improvement over the 55% purity and 20% yield obtained with the beads. The yield for the separations were found to be dependent on the length of the nanowires, and was maximized when the length of the nanowires equaled the diameter of the cells. This dependence was exploited to perform a size-selective magnetic separation. Substrates containing arrays of micro-magnets, fabricated using photolithography, were placed in cell cultures. These micro-magnet arrays create regions of locally strong magnetic field gradients to trap nanowires in specific locations on the substrate. These substrates were used in conjunction with fluid flow and a weak, externally applied magnetic field to create and control patterns of cells bound to nanowires. Controlled isolation of heterogeneous pairs and groups of cells will enable the study of the biochemistry of cell-cell contacts.

Evaluation of the Cytotoxicity of Satureja spicigera and Its Main Compounds

PubMed Central

Gohari, Ahmad Reza; Ostad, Seyed Nasser; Moradi-Afrapoli, Fahimeh; Malmir, Maryam; Tavajohi, Shohreh; Akbari, Hassan; Saeidnia, Soodabeh

2012-01-01

Satureja spicigera (Lamiaceae) grows wildly in Northwest of Iran. In this study, bioassay-guided isolation and identification of the main compounds has been reported using various chromatographic methods and comparison of their spectral data with those reported in the literature. Brine shrimp lethality and four cancerous cell lines HT29/219, Caco2, NIH-3T3, and T47D were used for cytotoxicity evaluations. From the aerial parts of S. spicigera, nine known compounds including two flavanones, 5,7,3′,5′-tetrahydroxy flavanone (8) and 5,4′-dihydroxy-3′-methoxyflavanone-7-(6′′-O-α-L-rhamnopyranosyl)-β-D-glucopyranoside (9), one dihydrochalcone, nubigenol (7), together with thymoquinone (1), thymol (2), carvacrol (3), β-sitosterol (4), ursolic acid (5) and oleanolic acid (6) were identified. Among the isolated chalcone and flavanones, compound 8 was effective against Artemia salina larva (LC50= 2 μg/mL) and only the compound 9 demonstrated IC50 value of 98.7 μg/mL on the T47D (human, breast, ductal carcinoma). Other compounds did not show significant inhibition of the cell growth. PMID:22623883

Transformation-specific interaction of the bovine papillomavirus E5 oncoprotein with the platelet-derived growth factor receptor transmembrane domain and the epidermal growth factor receptor cytoplasmic domain.

PubMed Central

Cohen, B D; Goldstein, D J; Rutledge, L; Vass, W C; Lowy, D R; Schlegel, R; Schiller, J T

1993-01-01

The bovine papillomavirus E5 transforming protein appears to activate both the epidermal growth factor receptor (EGF-R) and the platelet-derived growth factor receptor (PDGF-R) by a ligand-independent mechanism. To further investigate the ability of E5 to activate receptors of different classes and to determine whether this stimulation occurs through the extracellular domain required for ligand activation, we constructed chimeric genes encoding PDGF-R and EGF-R by interchanging the extracellular, membrane, and cytoplasmic coding domains. Chimeras were transfected into NIH 3T3 and CHO(LR73) cells. All chimeras expressed stable protein which, upon addition of the appropriate ligand, could be activated as assayed by tyrosine autophosphorylation and biological transformation. Cotransfection of E5 with the wild-type and chimeric receptors resulted in the ligand-independent activation of receptors, provided that a receptor contained either the transmembrane domain of the PDGF-R or the cytoplasmic domain of the EGF-R. Chimeric receptors that contained both of these domains exhibited the highest level of E5-induced biochemical and biological stimulation. These results imply that E5 activates the PDGF-R and EGR-R by two distinct mechanisms, neither of which specifically involves the extracellular domain of the receptor. Consistent with the biochemical and biological activation data, coimmunoprecipitation studies demonstrated that E5 formed a complex with any chimera that contained a PDGF-R transmembrane domain or an EGF-R cytoplasmic domain, with those chimeras containing both domains demonstrating the greatest efficiency of complex formation. These results suggest that although different domains of the PDGF-R and EGF-R are required for E5 activation, both receptors are activated directly by formation of an E5-containing complex. Images PMID:8394451

Phase transformation and sustained load crack growth in ZrO[sub 2] + 3 mol% Y[sub 2]O[sub 3]: Experiments and kinetic modeling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yin, H.; Gao, M.; Wei, R.P.

1995-01-01

To better understand environmentally assisted crack growth (SCG) in yttria stabilized zirconia, experimental studies were undertaken to characterize the kinetics of crack growth and the associated stress/moisture induced phase transformation in ZrO[sub 2] + 3 mol% Y[sub 2]O[sub 3] (3Y-TZP) in water, dry nitrogen and toluene from 3 to 70 C. The results showed that crack growth in water depended strongly on stress intensity factor (K[sub 1]) and temperature (T) and involved the transformation of a thin layer of material near the crack tip from the tetragonal (t) to the monoclinic (m) phase. These results, combined with literature data onmore » moisture-induced phase transformation, suggested that crack growth enhancement by water is controlled by the rate of this transformation and reflects the environmental cracking susceptibility of the transformed m-phase. A model was developed to link subcritical crack growth (SCG) rate to the kinetics of t [yields] m phase transformation. The SCG rate is expressed as an exponential function of stress-free activation energy, a stress-dependent contribution in terms of the mode 1 stress intensity factor K[sub I] and actuation volume, and temperature. The stress-free activation energies for water and the inert environments were determined to be 82 [+-] 3 and 169 [+-] 4 kJ/mol, respectively, at the 95% confidence level, and the corresponding activation volumes were 14 and 35 unit cells. The decreases in activation energy and activation volume may be attributed to a change in surface energy by water.« less

Two-dimensional correlation spectroscopic studies on coordination between organic ligands and Ni2 + ions

NASA Astrophysics Data System (ADS)

Bao, Ya-nan; Zeng, Yi-wei; Guo, Ran; Ablikim, Mesude; Shi, Hai-fang; Yang, Li-min; Yang, Zhan-lan; Xu, Yi-zhuang; Noda, Isao; Wu, Jin-guang

2018-05-01

3A2g → 3T1g(P) transition band of Ni2 + is used to probe the coordination of Ni2 +. Two-dimensional asynchronous spectra (2DCOS) are generated using the Double Asynchronous Orthogonal Sample Design (DAOSD), Asynchronous Spectrum with Auxiliary Peaks (ASAP) and Two-Trace Two-Dimensional (2T2D) approaches. Cross peaks relevant to the 3A2g → 3T1g(P) transition band of Ni2 + are utilized to probe coordination between Ni2 + and various ligands. We studied the spectral behavior of the 3A2g → 3T1g(P) transition band when Ni2 + is coordinated with ethylenediaminetetraacetic acid disodium salt (EDTA). The pattern of cross peaks in 2D asynchronous spectrum demonstrates that coordination brings about significant blue shift of the band. In addition, the absorptivity of the band increases remarkably. The interaction between Ni2 + and galactitol is also investigated. Although no clearly observable change is found on the 3A2g → 3T1g(P) transition band when galactitol is introduced, the appearance of cross peak in 2D asynchronous spectrum demonstrates that coordination indeed occurs between Ni2 + and galactitol. Furthermore, the pattern of cross peak indicates that peak position, bandwidth and absorptivity of the 3A2g → 3T1g(P) transition band of Ni(galactitol)x2 + is considerably different from those of Ni(H2O)62 +. Thus, 2DCOS is helpful to reveal subtle spectral variation, which might be helpful in shedding light on the physical-chemical nature of coordination.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Crawford, Andrew M.; Kurecka, Patrick; Yim, Tsz Kwan

An X-ray fluorescence flow cytometer that can determine the total metal content of single cells has been developed. Capillary action or pressure was used to load cells into hydrophilic or hydrophobic capillaries, respectively. Once loaded, the cells were transported at a fixed vertical velocity past a focused X-ray beam. X-ray fluorescence was then used to determine the mass of metal in each cell. By making single-cell measurements, the population heterogeneity for metals in the µ M to m M concentration range on fL sample volumes can be directly measured, a measurement that is difficult using most analytical methods. This approachmore » has been used to determine the metal composition of 936 individual bovine red blood cells (bRBC), 31 individual 3T3 mouse fibroblasts (NIH3T3) and 18 Saccharomyces cerevisiae (yeast) cells with an average measurement frequency of ~4 cells min –1. These data show evidence for surprisingly broad metal distributions. Lastly, details of the device design, data analysis and opportunities for further sensitivity improvement are described.« less

Tet1 is required for Rb phosphorylation during G1/S phase transition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huang, Shengsong; Zhu, Ziqi; Wang, Yiqin

2013-05-03

Highlights: •Tet1 was required for NIT3T3 proliferation. •Tet1 depletion inhibited G1-S entry. •Cyclin D1 accumulation and Rb phosphorylation was blocked by Tet1 knockdown. -- Abstract: DNA methylation plays an important role in many biological processes, including regulation of gene expression, maintenance of chromatin conformation and genomic stability. TET-family proteins convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), which indicates that these enzymes may participate in DNA demethylation. The function of TET1 has not yet been well characterized in somatic cells. Here, we show that depletion of Tet1 in NIH3T3 cells inhibits cell growth. Furthermore, Tet1 knockdown blocks cyclin D1 accumulation in G1more » phase, inhibits Rb phosphorylation and consequently delays entrance to G1/S phase. Taken together, this study demonstrates that Tet1 is required for cell proliferation and that this process is mediated through the Rb pathway.« less

Evaluation of a bio-based hydrophobic cellulose laurate film as biomaterial--study on biodegradation and cytocompatibility.

PubMed

Crépy, Lucie; Monchau, Francine; Chai, Feng; Raoul, Gwénaël; Hivart, Philippe; Hildebrand, Hartmut F; Martin, Patrick; Joly, Nicolas

2012-05-01

The study aims to validate an original bio-based material, obtained by grafting fatty chains, and more especially lauric chains (C12) onto cellulose, for medical applications. The mechanical properties of the synthesized cellulose laurate (C12) are close to those of petrochemical ones such as low density polyethylene. This cellulose-based polymer is transparent, flexible, and hydrophobic. To evaluate the stability of the cellulosic films in biological fluids the samples are soaked in simulated body fluid or blood plasma for a few hours to 6 months, and then submitted to mechanical and chemical analyses. The simultaneously performed cytocompatibility tests were the colony-forming viability, the vitality and cell proliferation tests using NIH 3T3 fibroblasts and MC 3T3 osteoblast-like cells. The results show the stability, the biocompatibility, and the noncytotoxicity of the synthesized cellulose laurate films. This biomaterial may so be considered for surgical applications. Copyright © 2012 Wiley Periodicals, Inc.

Grants Process Overview

Cancer.gov

This infographic shows the steps in the National Institutes of Health and National Cancer Institute Grants Process. The graphic shows which steps are done by the Principle Investigator, Grantee Institution, and by NIH. The process is represented by a circular flow of steps. Starting from the top and reading clockwise: The Principle Investigator “Initiates Research Idea and Prepares Application” The Grantee Institution “Submits Application” NIH “NIH Center For Scientific Review, Assigns To NCI And To Study Section” NIH “Scientific Review Group (NCI OR CSR) Evaluates for Scientific Merit” NIH “National Cancer Advisory Board Recommends Action” NIH “NCI Evaluates Program Relevance And Need” NIH “NCI Makes Funding Selections And Issues Grant Awards” (NIH) NIH “NCI Monitors Programmatic and Business Management Performance of the Grant” The Grantee Institution “Manages Funds” The Principle Investigator “Conducts Research” Source: www.cancer.gov Icons made by Freepik from http://www.flaticon.com is licensed by CC BY3.0”

Improved antiangiogenic and antitumour activity of the combination of the natural flavonoid fisetin and cyclophosphamide in Lewis lung carcinoma-bearing mice

PubMed Central

Touil, Yasmine S.; Seguin, Johanne; Scherman, Daniel; Chabot, Guy G.

2011-01-01

Purpose The natural flavonoid fisetin was recently identified as a lead compound that stabilizes endothelial cell microtubules. In this study we investigated the antiproliferative and antiangiogenic properties of fisetin in vitro and in vivo. Methods Fisetin cytotoxicity was evaluated using Lewis lung carcinoma cells (LLC), endothelial cells and NIH 3T3 cells. Endothelial cell (EC) migration and capillary-like structure formation were evaluated using EAhy 926 cells. In vivo tumour growth inhibition studies were performed using LLC bearing mice treated with fisetin and/or cyclophosphamide (CPA). Results The fisetin IC50 was 59 μM for LLC and 77 μM for EC cells, compared to 210 μM for normal NIH 3T3 cells (24 h). Fisetin inhibited EC migration and capillary-like structure formation at non-cytotoxic concentrations (22–44 μM). In mice, fisetin inhibited angiogenesis assessed using the Matrigel plug assay. In LLC bearing mice, fisetin produced a 67% tumour growth inhibition (223 mg/kg, intraperitoneal), similar to the 66% produced by low dose CPA (30 mg/kg, subcutaneous). When fisetin and CPA were combined, however, a marked improvement in antitumour activity was observed (92% tumour growth inhibition), with low systemic toxicity. Tumour histology showed decreased microvessel density with either fisetin or CPA alone, and a dramatic decrease after the fisetin/CPA combination. Conclusions We have shown that fisetin not only displays in vitro and in vivo antiangiogenic properties, but that it can also markedly improve the in vivo antitumour effect of CPA. We propose that this drug combination associating a non-toxic dietary flavonoid with a cytotoxic agent could advantageously be used in the treatment of solid tumours. PMID:21069336

In vitro fluoride release from a different kind of conventional and resin modified glass-ionomer cements

PubMed Central

Selimović-Dragaš, Mediha; Hasić-Branković, Lajla; Korać, Fehim; Đapo, Nermin; Huseinbegović, Amina; Kobašlija, Sedin; Lekić, Meliha; Hatibović-Kofman, Šahza

2013-01-01

Fluoride release is important characteristic of glass-ionomer cements. Quantity of fluoride ions released from the glass-ionomer cements has major importance in definition of their biological activity. The objectives of this study were to define the quantity of fluoride ions released from the experimental glass-ionomer cements and to define the effect of fluoride ions released from the experimental glass-ionomer cements on their cytotoxicity. Concentrations of the fluoride ions released in the evaluated glass-ionomer cements were measured indirectly, by the fluoride-selective WTW, F500 electrode potential, combined with reference R503/D electrode. Statistical analyses of F-ion concentrations released by all glass-ionomers evaluated at two time points, after 8 and after 24 hours, show statistically higher fluoride releases from RMGICs: Vitrebond, Fuji II LC and Fuji Plus, when compared to conventional glass-ionomer cements: Fuji Triage, Fuji IX GP Fast and Ketac Silver, both after 8 and after 24 hours. Correlation coefficient between concentrations of fluoride ion released by evaluated glass-ionomer cements and cytotoxic response of UMR-106 osteoblast cell-line are relatively high, but do not reach levels of biological significance. Correlation between concentrations of fluoride ion released and cytotoxic response of NIH3T3 mouse fibroblast cell line after 8 hours is high, positive and statistically significant for conventional GICs, Fuji Triage and Fuji IX GP Fast, and RMGIC, Fuji II LC. Statistically significant Correlation coefficient between concentrations of fluoride ion released and cytotoxic response of NIH3T3 cell line after 24 hours is defined for RMGIC Fuji II LC only. PMID:23988173

Altering surface characteristics of polypropylene mesh via sodium hydroxide treatment.

PubMed

Regis, Shawn; Jassal, Manisha; Mukherjee, Nilay; Bayon, Yves; Scarborough, Nelson; Bhowmick, Sankha

2012-05-01

Incisional hernias represent a serious and common complication following laparotomy. The use of synthetic (e.g. polypropylene) meshes to aid repair of these hernias has considerably reduced recurrence rates. While polypropylene is biocompatible and has a long successful clinical history in treating hernias and preventing reherniation, this material may suffer some limitations, particularly in challenging patients at risk of wound failure due to, for example, an exaggerated inflammation reaction, delayed wound healing, and infection. Surface modification of the polypropylene mesh without sacrificing its mechanical properties, critical for hernia repair, represents one way to begin to address these clinical complications. Our hypothesis is treatment of a proprietary polypropylene mesh with sodium hydroxide (NaOH) will increase in vitro NIH/3T3 cell attachment, predictive of earlier and improved cell colonization and tissue integration of polypropylene materials. Our goal is to achieve this altered surface functionality via enhanced removal of chemicals/oils used during material synthesis without compromising the mechanical properties of the mesh. We found that NaOH treatment does not appear to compromise the mechanical strength of the material, despite roughly a 10% decrease in fiber diameter. The treatment increases in vitro NIH/3T3 cell attachment within the first 72 h and this effect is sustained up to 7 days in vitro. This research demonstrates that sodium hydroxide treatment is an efficient way to modify the surface of polypropylene hernia meshes without losing the mechanical integrity of the material. This simple procedure could also allow the attachment of a variety of biomolecules to the polypropylene mesh that may aid in reducing the complications associated with polypropylene meshes today. Copyright © 2012 Wiley Periodicals, Inc.

Beta-catenin interacts with low-molecular-weight protein tyrosine phosphatase leading to cadherin-mediated cell-cell adhesion increase.

PubMed

Taddei, Maria Letizia; Chiarugi, Paola; Cirri, Paolo; Buricchi, Francesca; Fiaschi, Tania; Giannoni, Elisa; Talini, Doriana; Cozzi, Giacomo; Formigli, Lucia; Raugei, Giovanni; Ramponi, Giampietro

2002-11-15

Beta-catenin plays a dual role as a major constituent of cadherin-based adherens junctions and also as a transcriptional coactivator. In normal ephitelial cells, at adherens junction level, beta-catenin links cadherins to the actin cytoskeleton. The structure of adherens junctions is dynamically regulated by tyrosine phosphorylation. In particular, cell-cell adhesion can be negatively regulated through the tyrosine phosphorylation of beta-catenin. Furthermore, the loss of beta-catenin-cadherin association has been correlated with the transition from a benign tumor to an invasive, metastatic cancer. Low-molecular-weight protein tyrosine phosphatase (LMW-PTP) is a ubiquitous PTP implicated in the regulation of mitosis and cytoskeleton rearrangement. Here we demonstrate that the amount of free cytoplasmic beta-catenin is decreased in NIH3T3, which overexpresses active LMW-PTP, and this results in a stronger association between cadherin complexes and the actin-based cytoskeleton with respect to control cells. Confocal microscopy analysis shows that beta-catenin colocalizes with LMW-PTP at the plasma membrane. Furthermore, we provide evidence that beta-catenin is able to associate with LMW-PTP both in vitro and in vivo. Moreover, overexpression of active LMW-PTP strongly potentiates cadherin-mediated cell-cell adhesion, whereas a dominant-negative form of LMW-PTP induces the opposite phenotype, both in NIH3T3 and in MCF-7 carcinoma cells. On the basis of these results, we propose that the stability of cell-cell contacts at the adherens junction level is positively influenced by LMW-PTP expression, mainly because of the beta-catenin and LMW-PTP interaction at the plasma membrane level with consequent dephosphorylation.

Protective coatings for intraocular wirelessly controlled microrobots for implantation: Corrosion, cell culture, and in vivo animal tests.

PubMed

Pokki, Juho; Ergeneman, Olgaç; Chatzipirpiridis, George; Lühmann, Tessa; Sort, Jordi; Pellicer, Eva; Pot, Simon A; Spiess, Bernhard M; Pané, Salvador; Nelson, Bradley J

2017-05-01

Diseases in the ocular posterior segment are a leading cause of blindness. The surgical skills required to treat them are at the limits of human manipulation ability, and involve the risk of permanent retinal damage. Instrument tethering and design limit accessibility within the eye. Wireless microrobots suturelessly injected into the posterior segment, steered using magnetic manipulation are proposed for procedures involving implantation. Biocompatibility is a prerequisite for these procedures. This article investigates the use of polypyrrole- and gold-coated cobalt-nickel microrobots. While gold has been used in ocular implants, no ocular implantation involving polypyrrole is reported, despite its well-established biocompatibility properties. Coated and uncoated microrobots were investigated for their corrosion properties, and solutions that had contained coated and uncoated microrobots for one week were tested for cytotoxicity by monitoring NIH3T3 cell viability. None of the microrobots showed significant corrosion currents and corrosion potentials were as expected in relation to the intrinsic nobility of the materials. NIH3T3 cell viability was not affected by the release medium, in which coated/uncoated microrobots were stored. In vivo tests inside rabbit eyes were performed using coated microrobots. There were no significant inflammatory responses during the first week after injection. An inflammatory response detected after 2 weeks was likely due to a lack of longer-duration biocompatibility. The results provide valuable information for those who work on implant technology and biocompatibility. Coated microrobots have the potential to facilitate a new generation of surgical treatments, diagnostics and drug-delivery techniques, when implantation in the ocular posterior segment will be possible. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 836-845, 2017. © 2016 Wiley Periodicals, Inc.

Specific gene transfer mediated by galactosylated poly-L-lysine into hepatoma cells.

PubMed

Han, J; Il Yeom, Y

2000-07-20

Plasmid DNA/galactosylated poly-L-lysine(GalPLL) complex was used to transfer luciferase reporter gene in vitro into human hepatoma cells by a receptor-mediated endocytosis process. DNA was combined with galPLL via charge interaction (DNA:GalPLL:fusogenic peptide, 1:0.4:5, w/w/w) and the resulting complex was characterized by dynamic light scattering, gel retardation assay and zeta potential analyzer to determine the particle size, electrostatic charge interaction, and apparent surface charge. The complex was tested for the efficiency of gene transfer in cultured human hepatoblastoma cell line Hep G2 and fibroblast cells NIH/3T3 in vitro. The mean diameter of the complex (DNA:GalPLL=1:0.4, w/w) was 256+/-34.8 nm, and at this ratio, it was positively charged (zeta potential of this complex was 10.1 mV). Hep G2 cells, which express a galactose specific membrane lectin, were efficiently and selectively transfected with the RSV Luc/GalPLL complex in a sugar-dependent manner. NIH/3T3 cells, which do not express the galactose-specific membrane lectin, showed only a marginal level of gene expression. The transfection efficiency of GalPLL-conjugated DNA complex into Hep G2 cells was greatly enhanced in the presence of fusogenic peptide that can disrupt endosomes, where the GalPLL-DNA complex is entrapped with the fusogenic peptide. With the fusogenic peptide KALA, the luciferase activity in Hep G2 cells was ten-fold higher than that of cells transfected in the absence of the fusogenic peptide. Our gene transfer formulation may find potential application for the gene therapy of liver diseases.

Carnosine retards tumor growth in vivo in an NIH3T3-HER2/neu mouse model.

PubMed

Renner, Christof; Zemitzsch, Nadine; Fuchs, Beate; Geiger, Kathrin D; Hermes, Matthias; Hengstler, Jan; Gebhardt, Rolf; Meixensberger, Jürgen; Gaunitz, Frank

2010-01-06

It was previously demonstrated that the dipeptide carnosine inhibits growth of cultured cells isolated from patients with malignant glioma. In the present work we investigated whether carnosine also affects tumor growth in vivo and may therefore be considered for human cancer therapy. A mouse model was used to investigate whether tumor growth in vivo can be inhibited by carnosine. Therefore, NIH3T3 fibroblasts, conditionally expressing the human epidermal growth factor receptor 2 (HER2/neu), were implanted into the dorsal skin of nude mice, and tumor growth in treated animals was compared to control mice. In two independent experiments nude mice that received tumor cells received a daily intra peritoneal injection of 500 microl of 1 M carnosine solution. Measurable tumors were detected 12 days after injection. Aggressive tumor growth in control animals, that received a daily intra peritoneal injection of NaCl solution started at day 16 whereas aggressive growth in mice treated with carnosine was delayed, starting around day 19. A significant effect of carnosine on tumor growth was observed up to day 24. Although carnosine was not able to completely prevent tumor growth, a microscopic examination of tumors revealed that those from carnosine treated animals had a significant lower number of mitosis (p < 0.0003) than untreated animals, confirming that carnosine affects proliferation in vivo. As a naturally occurring substance with a high potential to inhibit growth of malignant cells in vivo, carnosine should be considered as a potential anti-cancer drug. Further experiments should be performed in order to understand how carnosine acts at the molecular level.

Characterization of Nrf2 activation and heme oxygenase-1 expression in NIH3T3 cells exposed to aqueous extracts of cigarette smoke.

PubMed

Knörr-Wittmann, Constanze; Hengstermann, Arnd; Gebel, Stephan; Alam, Jawed; Müller, Thomas

2005-12-01

Cigarette smoke (CS) is a complex chemical mixture estimated to be composed of up to 5000 different chemicals, many of which are prooxidant. Here we show that, at least in vitro, the cellular response designed to combat oxidative stress resulting from CS exposure is primarily controlled by the transcription factor Nrf2, a principal inducer of antioxidant and phase II-related genes. The prominent role of Nrf2 in the cellular response to CS is substantiated by the following observations: In NIH3T3 cells exposed to aqueous extracts of CS (i) Nrf2 is strongly stabilized and becomes detectable in nuclear extracts. (ii) Nuclear localization of Nrf2 coincides with increased DNA binding of a putative Nrf2/MafK heterodimer to its cognate cis-regulatory site, i.e., the antioxidant-responsive element (ARE). (iii) Studies on the regulatory elements of the oxidative stress-inducible gene heme oxygenase-1 (hmox1) using various hmox1 promoter/luciferase reporter constructs revealed that the strong CS-dependent expression of this gene is primarily governed by the distal enhancers 1 ("E1") and 2 ("E2"), which both contain three canonical ARE-like stress-responsive elements (StREs). Notably, depletion of Nrf2 levels caused by RNA interference significantly compromised CS-induced hmox1 promoter activation, based on the distinct Nrf2 sensitivity exhibited by E1 and E2. Finally, (iv) siRNA-dependent knock-down of Nrf2 completely abrogated CS-induced expression of phase II-related genes. Taken together, these results confirm the outstanding role of Nrf2 both in sensing (oxidant) stress and in orchestrating an efficient transcriptional response aimed at resolving the stressing conditions.

Enhanced gelation of chitosan/β-sodium glycerophosphate thermosensitive hydrogel with sodium bicarbonate and biocompatibility evaluated.

PubMed

Deng, Aipeng; Kang, Xi; Zhang, Jing; Yang, Yang; Yang, Shulin

2017-09-01

The application of chitosan/β-sodium glycerophosphate (β-GP) thermosensitive hydrogel has been limited by the relatively slow gelation, weak mechanical resistance and poor cytocompatibility. In this study, sodium hydrogen carbonate (NaHCO 3 ) was applied with β-GP as gel agents to produce high-strength hydrogel. The hydrogels prepared with high NaHCO 3 concentration or more gel agents showed shorter gelation time, better thermostability, drastically enhanced resistance in compression. Meanwhile, the hydrogels presented obvious porous structures and excellent biocompatibility to HUVEC and NIH 3T3 cultured in vitro with higher NaHCO 3 concentration and moderate concentration of β-GP. Overall, appropriate concentration of β-GP combined with NaHCO 3 can be a good gel regent to improve properties of chitosan thermosensitive hydrogels. Copyright © 2017 Elsevier B.V. All rights reserved.

Viewpoint: A challenge to academic health centers and the National Institutes of Health to prevent unintended gender bias in the selection of clinical and translational science award leaders.

PubMed

Carnes, Molly; Bland, Carole

2007-02-01

In controlled studies, both men and women preferentially select men over women for leadership positions, even when credentials are identical and despite field studies demonstrating women's equivalent or slightly better leadership effectiveness. The assumption that men will make better leaders than women is attributed to the pervasive existence of unconscious stereotypes that characterize both men and leaders as agentic or action oriented and women as dependent. The Clinical and Translational Science Award (CTSA) from the National Institutes of Health (NIH) Roadmap is a novel, prestigious award that will place considerable power in the hands of one principal investigator-conditions that predict activation of bias in favor of selecting male leaders. The authors review research supporting this assertion. To mitigate the impact of this bias and broaden the pool of potential leaders for this transformative initiative, the authors offer the following suggestions. To academic health centers they suggest (1) internal search committees comprised of at least 35% women that establish a priori the desired qualities for the CTSA leader and broadly solicit applicants, (2) explicit specification of the full range of desirable skills of a CTSA leader, and (3) systematic efforts to increase awareness of the negative impact of unconscious gender bias on women's advancement. To the NIH they suggest (1) the new multiple principal investigator rule for the CTSA program, (2) a statement in the request for applications (RFA) encouraging diversity among principal investigators, (3) repetition in the RFA of the public NIH statement of the importance of work life balance for young investigators, and (4) constitution of study sections with at least 35% women.

Development of Co-based bulk metallic glasses as potential biomaterials.

PubMed

Zhou, Zeyan; Wei, Qin; Li, Qiang; Jiang, Bingliang; Chen, You; Sun, Yanfei

2016-12-01

A new series of Co80-x-yCrxMoyP14B6 (x=5 y=5; x=5 y=10; x=10 y=10, all values in at.%) bulk metallic glasses (BMGs) with a maximum diameter of 1.5mm has been developed for using them as potential bio-implant materials by a combination of fluxing treatment and J-quenching technique. The performance of the present Co-based BMGs in biomedical implant applications was investigated as compared to the CoCrMo biomedical alloy (ASTM F75) and 316L stainless steel (316L SS). The corrosion behavior of the samples was investigated in both Hank's solution (pH=7.4) and artificial saliva solution (pH=6.3) at 37°C employing electrochemical measurements. The results indicate that the Co-based BMGs exhibit much higher corrosion resistance in the simulated body solutions than that of 316L SS. Compared with the corrosion resistance of ASTM F75, that of Co70Cr5Mo5P14B6 and Co65Cr5Mo10P14B6 BMGs is found to be lower and that of Co60Cr10Mo10P14B6 BMG is higher. The concentrations of Co, Cr, and Mo ions released into the simulated body solutions from our Co-based BMGs after potentiodynamic polarization are significantly lower than that released from ASTM F75. The biocompatibility of the specimens was evaluated using an in vitro test of NIH3T3 cell culture in the specimen extraction media for 1, 3, 5, and 7days, revealing the non-cytotoxicity of the Co-based BMGs towards NIH3T3 cells. Moreover, examinations on the cell adhesion and growth on the surface of the specimens indicate that the Co-based BMGs exhibit better cell viability compared to ASTM F75 and 316L SS biomedical alloys. Copyright © 2016 Elsevier B.V. All rights reserved.

MIRK/DYRK1B MEDIATES SURVIVAL DURING THE DIFFERENTIATION OF C2C12 MYOBLASTS 1

PubMed Central

Mercer, Stephen E.; Ewton, Daina Z.; Deng, Xiaobing; Lim, Seunghwan; Mazur, Thomas R.; Friedman, Eileen

2005-01-01

The kinase Mirk/dyrk1B is essential for the differentiation of C2C12 myoblasts. Mirk reinforces the G0/G1 arrest state in which differentiation occurs by directly phosphorylating and stabilizing p27kip1 and destabilizing cyclin D1. We now demonstrate that Mirk is anti-apoptotic in myoblasts. Knockdown of endogenous Mirk by RNA interference activated caspase 3 and decreased myoblast survival by 75%, while transient overexpression of Mirk increased cell survival. Mirk exerts its anti-apoptotic effects during muscle differentiation at least in part through effects on the cell cycle inhibitor and pro-survival molecule p21cip1. Overexpression and RNA interference experiments demonstrated that Mirk phosphorylates p21 within its nuclear localization domain at Ser153 causing a portion of the typically nuclear p21 to localize in the cytoplasm. Phosphomimetic GFP-p21-S153D was pancellular in both cycling C2C12 myoblasts and NIH3T3 cells. Endogenous Mirk in myotubes, and overexpressed Mirk in NIH3T3 cells were able to cause the pancellular localization of wild-type GFP-p21, but not the non-phosphorylatable mutant GFP-p21-S153A. Translocation to the cytoplasm enables p21 to block apoptosis through inhibitory interaction with pro-apoptotic molecules. Phosphomimetic p21-S153D was more effective than wild-type p21 in blocking the activation of caspase 3. Transient expression of p21-S153D also increased myoblast viability in colony forming assays, while the p21-S153A mutant had no effect. This Mirk-dependent change in p21 intracellular localization is a natural part of myoblast differentiation. Endogenous p21 localized exclusively to the nuclei of proliferating myoblasts, but was also found in the cytoplasm of post-mitotic multinucleated myotubes and adult human skeletal myofibers. PMID:15851482

Motor assessment using the NIH Toolbox

PubMed Central

Magasi, Susan; McCreath, Heather E.; Bohannon, Richard W.; Wang, Ying-Chih; Bubela, Deborah J.; Rymer, William Z.; Beaumont, Jennifer; Rine, Rose Marie; Lai, Jin-Shei; Gershon, Richard C.

2013-01-01

Motor function involves complex physiologic processes and requires the integration of multiple systems, including neuromuscular, musculoskeletal, and cardiopulmonary, and neural motor and sensory-perceptual systems. Motor-functional status is indicative of current physical health status, burden of disease, and long-term health outcomes, and is integrally related to daily functioning and quality of life. Given its importance to overall neurologic health and function, motor function was identified as a key domain for inclusion in the NIH Toolbox for Assessment of Neurological and Behavioral Function (NIH Toolbox). We engaged in a 3-stage developmental process to: 1) identify key subdomains and candidate measures for inclusion in the NIH Toolbox, 2) pretest candidate measures for feasibility across the age span of people aged 3 to 85 years, and 3) validate candidate measures against criterion measures in a sample of healthy individuals aged 3 to 85 years (n = 340). Based on extensive literature review and input from content experts, the 5 subdomains of dexterity, strength, balance, locomotion, and endurance were recommended for inclusion in the NIH Toolbox motor battery. Based on our validation testing, valid and reliable measures that are simultaneously low-cost and portable have been recommended to assess each subdomain, including the 9-hole peg board for dexterity, grip dynamometry for upper-extremity strength, standing balance test, 4-m walk test for gait speed, and a 2-minute walk test for endurance. PMID:23479547

Recurrent (2;2) and (2;8) Translocations in Rhabdomyosarcoma without the Canonical PAX-FOXO1 fuse PAX3 to Members of the Nuclear Receptor Transcriptional Coactivator (NCOA) Family

PubMed Central

Sumegi, Janos; Streblow, Renae; Frayer, Robert W.; Cin, Paola Dal; Rosenberg, Andrew; Meloni-Ehrig, Aurelia; Bridge, Julia A.

2009-01-01

The fusion oncoproteins PAX3-FOXO1 [t(2;13)(q35;q14)] and PAX7-FOXO1 [t(1;13)(p36;q14)] typify alveolar rhabdomyosarcoma (ARMS); however, 20-30% of cases lack these specific translocations. In this study, cytogenetic and/or molecular characterization to include FISH, RT-PCR and sequencing analyses of five rhabdomyosarcomas [four ARMS and one embryonal rhabdomyosarcoma (ERMS)] with novel, recurrent t(2;2)(p23;q35) or t(2;8)(q35;q13) revealed that these non-canonical translocations fuse PAX3 to NCOA1 or NCOA2 respectively. The PAX3-NCOA1 and PAX3-NCOA2 transcripts encode chimeric proteins composed of the paired-box and homeodomain DNA-binding domains of PAX3, and the CID domain, the Q-rich region and the AD2 domain of NCOA1 or NCOA2. To investigate the biological function of these recurrent variant translocations, the coding regions of PAX3-NCOA1 and PAX3-NCOA2 cDNA constructs were introduced into expression vectors with tetracycline-regulated expression. Both fusion proteins showed transforming activity in the soft agar assay. Deletion of the AD2 portion of the PAX3-NCOA fusion proteins reduced the transforming activity of each chimeric protein. Similarly, but with greater impact, CID domain deletion fully abrogated the transforming activity of the chimeric protein. These studies: (1) expand our knowledge of PAX3 variant translocations in RMS with identification of a novel PAX3-NCOA2 fusion; (2) show that both PAX3-NCOA1 and PAX3-NCOA2 represent recurrent RMS rearrangements; (3) confirm the transforming activity of both translocation events and demonstrate the essentiality of intact AD2 and CID domains for optimal transforming activity; and, (5) provide alternative approaches (FISH and RT-PCR) for detecting PAX-NCOA fusions in nondividing cells of RMS. The latter could potentially be utilized as aids in diagnostically challenging cases. PMID:19953635

The small GTPase Rab8 interacts with VAMP-3 to regulate the delivery of recycling T-cell receptors to the immune synapse

PubMed Central

Finetti, Francesca; Patrussi, Laura; Galgano, Donatella; Cassioli, Chiara; Perinetti, Giuseppe; Pazour, Gregory J.; Baldari, Cosima T.

2015-01-01

ABSTRACT IFT20, a component of the intraflagellar transport (IFT) system that controls ciliogenesis, regulates immune synapse assembly in the non-ciliated T-cell by promoting T-cell receptor (TCR) recycling. Here, we have addressed the role of Rab8 (for which there are two isoforms Rab8a and Rab8b), a small GTPase implicated in ciliogenesis, in TCR traffic to the immune synapse. We show that Rab8, which colocalizes with IFT20 in Rab11+ endosomes, is required for TCR recycling. Interestingly, as opposed to in IFT20-deficient T-cells, TCR+ endosomes polarized normally beneath the immune synapse membrane in the presence of dominant-negative Rab8, but were unable to undergo the final docking or fusion step. This could be accounted for by the inability of the vesicular (v)-SNARE VAMP-3 to cluster at the immune synapse in the absence of functional Rab8, which is responsible for its recruitment. Of note, and similar to in T-cells, VAMP-3 interacts with Rab8 at the base of the cilium in NIH-3T3 cells, where it regulates ciliary growth and targeting of the protein smoothened. The results identify Rab8 as a new player in vesicular traffic to the immune synapse and provide insight into the pathways co-opted by different cell types for immune synapse assembly and ciliogenesis. PMID:26034069

Glucocorticoids Recruit Tgfbr3 and Smad1 to Shift Transforming Growth Factor-β Signaling from the Tgfbr1/Smad2/3 Axis to the Acvrl1/Smad1 Axis in Lung Fibroblasts*

PubMed Central

Schwartze, Julian T.; Becker, Simone; Sakkas, Elpidoforos; Wujak, Łukasz A.; Niess, Gero; Usemann, Jakob; Reichenberger, Frank; Herold, Susanne; Vadász, István; Mayer, Konstantin; Seeger, Werner; Morty, Rory E.

2014-01-01

Glucocorticoids represent the mainstay therapy for many lung diseases, providing outstanding management of asthma but performing surprisingly poorly in patients with acute respiratory distress syndrome, chronic obstructive pulmonary disease, lung fibrosis, and blunted lung development associated with bronchopulmonary dysplasia in preterm infants. TGF-β is a pathogenic mediator of all four of these diseases, prompting us to explore glucocorticoid/TGF-β signaling cross-talk. Glucocorticoids, including dexamethasone, methylprednisolone, budesonide, and fluticasone, potentiated TGF-β signaling by the Acvrl1/Smad1/5/8 signaling axis and blunted signaling by the Tgfbr1/Smad2/3 axis in NIH/3T3 cells, as well as primary lung fibroblasts, smooth muscle cells, and endothelial cells. Dexamethasone drove expression of the accessory type III TGF-β receptor Tgfbr3, also called betaglycan. Tgfbr3 was demonstrated to be a “switch” that blunted Tgfbr1/Smad2/3 and potentiated Acvrl1/Smad1 signaling in lung fibroblasts. The Acvrl1/Smad1 axis, which was stimulated by dexamethasone, was active in lung fibroblasts and antagonized Tgfbr1/Smad2/3 signaling. Dexamethasone acted synergistically with TGF-β to drive differentiation of primary lung fibroblasts to myofibroblasts, revealed by acquisition of smooth muscle actin and smooth muscle myosin, which are exclusively Smad1-dependent processes in fibroblasts. Administration of dexamethasone to live mice recapitulated these observations and revealed a lung-specific impact of dexamethasone on lung Tgfbr3 expression and phospho-Smad1 levels in vivo. These data point to an interesting and hitherto unknown impact of glucocorticoids on TGF-β signaling in lung fibroblasts and other constituent cell types of the lung that may be relevant to lung physiology, as well as lung pathophysiology, in terms of drug/disease interactions. PMID:24347165

Order restricted inference for oscillatory systems for detecting rhythmic signals

PubMed Central

Larriba, Yolanda; Rueda, Cristina; Fernández, Miguel A.; Peddada, Shyamal D.

2016-01-01

Motivation: Many biological processes, such as cell cycle, circadian clock, menstrual cycles, are governed by oscillatory systems consisting of numerous components that exhibit rhythmic patterns over time. It is not always easy to identify such rhythmic components. For example, it is a challenging problem to identify circadian genes in a given tissue using time-course gene expression data. There is a great potential for misclassifying non-rhythmic as rhythmic genes and vice versa. This has been a problem of considerable interest in recent years. In this article we develop a constrained inference based methodology called Order Restricted Inference for Oscillatory Systems (ORIOS) to detect rhythmic signals. Instead of using mathematical functions (e.g. sinusoidal) to describe shape of rhythmic signals, ORIOS uses mathematical inequalities. Consequently, it is robust and not limited by the biologist's choice of the mathematical model. We studied the performance of ORIOS using simulated as well as real data obtained from mouse liver, pituitary gland and data from NIH3T3, U2OS cell lines. Our results suggest that, for a broad collection of patterns of gene expression, ORIOS has substantially higher power to detect true rhythmic genes in comparison to some popular methods, while also declaring substantially fewer non-rhythmic genes as rhythmic. Availability and Implementation: A user friendly code implemented in R language can be downloaded from http://www.niehs.nih.gov/research/atniehs/labs/bb/staff/peddada/index.cfm. Contact: peddada@niehs.nih.gov PMID:27596593

Activation of phosphatidylinositol-3 kinase by nerve growth factor involves indirect coupling of the trk proto-oncogene with src homology 2 domains.

PubMed

Ohmichi, M; Decker, S J; Saltiel, A R

1992-10-01

Growth factor receptor tyrosine kinases can form stable associations with intracellular proteins that contain src homology (SH) 2 domains, including the p85 regulatory subunit of phosphatidylinositol (PI)-3 kinase. The activation of this enzyme by growth factors is evaluated in PC12 pheochromocytoma cells and NIH 3T3 fibroblasts expressing the pp140c-trk nerve growth factor (NGF) receptor (3T3-c-trk). NGF causes the rapid stimulation of PI-3 kinase activity detected in anti-phosphotyrosine, but not in anti-trk, immunoprecipitates. This effect coincides with the tyrosine phosphorylation of two proteins, with molecular masses of of 100 kd and 110 kd, that coimmunoprecipitate with p85. Similar phosphorylation patterns are induced when an immobilized fusion protein containing the amino-terminal SH2 domain of p85 is used to precipitate tyrosine-phosphorylated proteins. Thus, although NGF produces the rapid activation of PI-3 kinase through a mechanism that involves tyrosine phosphorylation, there is no evidence for tyrosine phosphorylation of p85, or for its ligand-dependent association with the NGF receptor. Perhaps another phosphoprotein may link the NGF receptor to this enzyme.

Transformation assay in Bhas 42 cells: a model using initiated cells to study mechanisms of carcinogenesis and predict carcinogenic potential of chemicals.

PubMed

Sasaki, Kiyoshi; Umeda, Makoto; Sakai, Ayako; Yamazaki, Shojiro; Tanaka, Noriho

2015-01-01

Transformation assays using cultured cells have been applied to the study of carcinogenesis. Although various cell systems exist, few cell types such as BALB/c 3T3 subclones and Syrian hamster embryo cells have been used to study chemically induced two-stage carcinogenesis. Bhas 42 cells were established as a clone by the transfection with the v-Ha-ras gene into mouse BALB/c 3T3 A31-1-1 cells and their subsequent selection based on their sensitivity to 12-O-tetradecanoylphorbol-13-acetate. Using Bhas 42 cells, transformed foci were induced by the treatment with nongenotoxic carcinogens, most of which act as tumor promoters. Therefore, Bhas 42 cells were considered to be a model of initiated cells. Subsequently, not only nongenotoxic carcinogens but also genotoxic carcinogens, most of which act as tumor initiators, were found to induce transformed foci by the modification of the protocol. Furthermore, transformation of Bhas 42 cells was induced by the transfection with genes of oncogenic potential. We interpret this high sensitivity of Bhas 42 cells to various types of carcinogenic stimuli to be related to the multistage model of carcinogenesis, as the transfection of v-Ha-ras gene further advances the parental BALB/c 3T3 A31-1-1 cells toward higher transforming potential. Thus, we propose that Bhas 42 cells are a novel and sensitive cell line for the analysis of carcinogenesis and can be used for the detection of not only carcinogenic substances but also gene alterations related to oncogenesis. This review will address characteristics of Bhas 42 cells, the transformation assay protocol, validation studies, and the various chemicals tested in this assay.

Nr-CAM is a target gene of the beta-catenin/LEF-1 pathway in melanoma and colon cancer and its expression enhances motility and confers tumorigenesis.

PubMed

Conacci-Sorrell, Maralice E; Ben-Yedidia, Tamar; Shtutman, Michael; Feinstein, Elena; Einat, Paz; Ben-Ze'ev, Avri

2002-08-15

beta-catenin and plakoglobin (gamma-catenin) are homologous molecules involved in cell adhesion, linking cadherin receptors to the cytoskeleton. beta-catenin is also a key component of the Wnt pathway by being a coactivator of LEF/TCF transcription factors. To identify novel target genes induced by beta-catenin and/or plakoglobin, DNA microarray analysis was carried out with RNA from cells overexpressing either protein. This analysis revealed that Nr-CAM is the gene most extensively induced by both catenins. Overexpression of either beta-catenin or plakoglobin induced Nr-CAM in a variety of cell types and the LEF/TCF binding sites in the Nr-CAM promoter were required for its activation by catenins. Retroviral transduction of Nr-CAM into NIH3T3 cells stimulated cell growth, enhanced motility, induced transformation, and produced rapidly growing tumors in nude mice. Nr-CAM and LEF-1 expression was elevated in human colon cancer tissue and cell lines and in human malignant melanoma cell lines but not in melanocytes or normal colon tissue. Dominant negative LEF-1 decreased Nr-CAM expression and antibodies to Nr-CAM inhibited the motility of B16 melanoma cells. The results indicate that induction of Nr-CAM transcription by beta-catenin or plakoglobin plays a role in melanoma and colon cancer tumorigenesis, probably by promoting cell growth and motility.

Development of poly(lactic-co-glycolic) acid nanoparticles-embedded hyaluronic acid-ceramide-based nanostructure for tumor-targeted drug delivery.

PubMed

Park, Ju-Hwan; Lee, Jae-Young; Termsarasab, Ubonvan; Yoon, In-Soo; Ko, Seung-Hak; Shim, Jae-Seong; Cho, Hyun-Jong; Kim, Dae-Duk

2014-10-01

A hyaluronic acid-ceramide (HACE) nanostructure embedded with docetaxel (DCT)-loaded poly(d,l-lactide-co-glycolide) (PLGA) nanoparticles (NPs) was fabricated for tumor-targeted drug delivery. NPs with a narrow size distribution and negative zeta potential were prepared by embedding DCT-loaded PLGA NPs into a HACE nanostructure (DCT/PLGA/HACE). DCT-loaded PLGA and DCT/PLGA/HACE NPs were characterized by solid-state techniques, including Fourier-transform infrared (FT-IR) spectroscopy, differential scanning calorimetry (DSC), and powder X-ray diffraction (PXRD). A sustained drug release pattern from the NPs developed was observed and negligible cytotoxicity was seen in NIH3T3 cells (normal fibroblast, CD44 receptor negative) and MDA-MB-231 cells (breast cancer cells, CD44 receptor positive). PLGA/HACE NPs containing coumarin 6, used as a fluorescent dye, exhibited improved cellular uptake efficiency, based on the HA-CD44 receptor interaction, compared to plain PLGA NPs. Cyanine 5.5 (Cy5.5)-labeled PLGA/HACE NPs were injected intravenously into a MDA-MB-231 tumor xenograft mouse model and demonstrated enhanced tumor targetability, compared with Cy5.5-PLGA NPs, according to a near-infrared fluorescence (NIRF) imaging study. Considering these experimental results, the DCT/PLGA/HACE NPs developed may be useful as a tumor-targeted drug delivery system. Copyright © 2014 Elsevier B.V. All rights reserved.

Use of electroporation to study the cytotoxic effects of fluorodeoxyuridylate in intact cells.

PubMed

Jastreboff, M M; Sokoloski, J A; Bertino, J R; Narayanan, R

1987-04-15

The introduction of 2'-deoxyuridine 5'-monophosphate and its analog, 5-fluoro-2'-deoxyuridine 5'-monophosphate, into intact CCRF-CEM and NIH3T3 cells was achieved by electroporation. Following electroporation, cells were shown to be fully functional as monitored by the incorporation of deoxyuridylate, after conversion to thymidylate, into DNA. Pretreatment of cells with fluorodeoxyuridine completely abolished this effect. In contrast, introduction of the fluoro analog into cells by electroporation markedly inhibited both DNA synthesis and cell growth in a time-dependent manner. Thus, electroporation offers a powerful tool to permeabilize cells to a variety of cellular metabolites and antimetabolites.

[Approval of ISO/IEC 17025 and quality control of laboratory testing].

PubMed

Yamamoto, Shigeki; Asakura, Hiroshi; Machii, Kenji; Igimi, Shizunobu

2010-01-01

First section of Division of Biomedical Food Research, National Institute of Health Sciences (NIHS) was approved by ISO/IEC 17025 as a laboratory having an appropriate laboratory testing technique. NIHS is the first national laboratory approved by ISO/IEC 17025. NIHS has also been accepted the appropriate technique and facility for the BSL3 level pathogens by ISO/IEC 17025. NIHS is necessary to take an external audit almost every year. This approval is renewed every 4 years.

Parcellation of the Healthy Neonatal Brain into 107 Regions Using Atlas Propagation through Intermediate Time Points in Childhood.

PubMed

Blesa, Manuel; Serag, Ahmed; Wilkinson, Alastair G; Anblagan, Devasuda; Telford, Emma J; Pataky, Rozalia; Sparrow, Sarah A; Macnaught, Gillian; Semple, Scott I; Bastin, Mark E; Boardman, James P

2016-01-01

Neuroimage analysis pipelines rely on parcellated atlases generated from healthy individuals to provide anatomic context to structural and diffusion MRI data. Atlases constructed using adult data introduce bias into studies of early brain development. We aimed to create a neonatal brain atlas of healthy subjects that can be applied to multi-modal MRI data. Structural and diffusion 3T MRI scans were acquired soon after birth from 33 typically developing neonates born at term (mean postmenstrual age at birth 39(+5) weeks, range 37(+2)-41(+6)). An adult brain atlas (SRI24/TZO) was propagated to the neonatal data using temporal registration via childhood templates with dense temporal samples (NIH Pediatric Database), with the final atlas (Edinburgh Neonatal Atlas, ENA33) constructed using the Symmetric Group Normalization (SyGN) method. After this step, the computed final transformations were applied to T2-weighted data, and fractional anisotropy, mean diffusivity, and tissue segmentations to provide a multi-modal atlas with 107 anatomical regions; a symmetric version was also created to facilitate studies of laterality. Volumes of each region of interest were measured to provide reference data from normal subjects. Because this atlas is generated from step-wise propagation of adult labels through intermediate time points in childhood, it may serve as a useful starting point for modeling brain growth during development.

Genomic Organization and Identification of Promoter Regions for the BDNF Gene in the Pond Turtle Trachemys scripta elegans

PubMed Central

Zheng, Zhaoqing; Keifer, Joyce

2014-01-01

Brain-derived neurotrophic factor (BDNF) is an important regulator of neuronal development and synaptic function. The BDNF gene undergoes significant activity-dependent regulation during learning. Here, we identified the BDNF promoter regions, transcription start sites, and potential regulatory sequences for BDNF exons I–III that may contribute to activity-dependent gene and protein expression in the pond turtle Trachemys scripta elegans (tBDNF). By using transfection of BDNF promoter/luciferase plasmid constructs into human neuroblastoma SHSY5Y cells and mouse embryonic fibroblast NIH3T3 cells, we identified the basal regulatory activity of promoter sequences located upstream of each tBDNF exon, designated as pBDNFI–III. Further, through chromatin immunoprecipitation (ChIP) assays, we detected CREB binding directly to exon I and exon III promoters, while BHLHB2, but not CREB, binds within the exon II promoter. Elucidation of the promoter regions and regulatory protein binding sites in the tBDNF gene is essential for understanding the regulatory mechanisms that control tBDNF gene expression. PMID:24443176

Genomic organization and identification of promoter regions for the BDNF gene in the pond turtle Trachemys scripta elegans.

PubMed

Ambigapathy, Ganesh; Zheng, Zhaoqing; Keifer, Joyce

2014-08-01

Brain-derived neurotrophic factor (BDNF) is an important regulator of neuronal development and synaptic function. The BDNF gene undergoes significant activity-dependent regulation during learning. Here, we identified the BDNF promoter regions, transcription start sites, and potential regulatory sequences for BDNF exons I-III that may contribute to activity-dependent gene and protein expression in the pond turtle Trachemys scripta elegans (tBDNF). By using transfection of BDNF promoter/luciferase plasmid constructs into human neuroblastoma SHSY5Y cells and mouse embryonic fibroblast NIH3T3 cells, we identified the basal regulatory activity of promoter sequences located upstream of each tBDNF exon, designated as pBDNFI-III. Further, through chromatin immunoprecipitation (ChIP) assays, we detected CREB binding directly to exon I and exon III promoters, while BHLHB2, but not CREB, binds within the exon II promoter. Elucidation of the promoter regions and regulatory protein binding sites in the tBDNF gene is essential for understanding the regulatory mechanisms that control tBDNF gene expression.

Preliminary Authorization Basis Documentation for the Proposed Bio Safety Level 3 (BSl-3) Facility

DOE Office of Scientific and Technical Information (OSTI.GOV)

Altenbach, T J; Nguyen, S N

2003-09-20

Lawrence Livermore National Laboratory (LLNL) is proposing to construct a biosafety level (BSL-3) facility at Site 200 in Livermore, California. Biosafety level 3 (BSL-3) is a designation assigned by the Centers for Disease Control and Prevention (CDC) and National Institutes Health (NIH) for handling infectious organisms based on the specific microorganisms and associated operations. Biosafety levels range from BSL-1 (lowest hazard) to BSL-4 (highest hazard). Details about the BSL-3 criteria are described in the Center of Disease Control and Prevention (CDC)/National Institutes of Health (NIH)'s publication ''Biosafety Microbiological and Biomedical Laboratories'' (BMBL), 4th edition (CDC 1999): The BSL-3 facility willmore » be built in accordance with the required BMBL guidelines. This Preliminary Authorization Basis Documentation (PABD) for the proposed BSL-3 facility has been prepared in accordance with the current contractual requirements at LLNL. This includes the LLNL Environment, Safety, and Health Manual (ES&H Manual) and applicable Work Smart Standards, including the biosafety standards, such as the aforementioned BMBL and the NIH Guidelines for Research Involving Recombinant DNA Molecules: The proposed BSL-3 facility is a 1,100 ft{sup 2}, one-story permanent prefabricated facility, which will have three individual BSL-3 laboratory rooms (one of which is an animal biosafety level-3 [ABSL-3] laboratory to handle rodents), a mechanical room, clothes-change and shower rooms, and small storage space (Figure 3.1). The BSL-3 facility will be designed and operated accordance with guidelines for BSL-3 laboratories established by the CDC and the NIH. No radiological, high explosives, fissile, or propellant material will be used or stored in the proposed BSL-3 facility. The BSL-3 facility will be used to develop scientific tools to identify and understand the pathogens of medical, environmental, and forensic importance. Microorganisms that are to be handled in this facility will be limited in quantity, type and form in accordance with the BMBL requirements and approval by the Institutional Biosafety Committee (IBC). The proposed facility will have the unique capability within DOE/NNSA to perform aerosol studies to include challenges to rodents using infectious agents or biologically derived toxins (biotoxins). These types of aerosol studies will be strictly confined in a Class II Type B biosafety cabinet.« less

The Tol2 transposon system mediates the genetic engineering of T-cells with CD19-specific chimeric antigen receptors for B-cell malignancies.

PubMed

Tsukahara, T; Iwase, N; Kawakami, K; Iwasaki, M; Yamamoto, C; Ohmine, K; Uchibori, R; Teruya, T; Ido, H; Saga, Y; Urabe, M; Mizukami, H; Kume, A; Nakamura, M; Brentjens, R; Ozawa, K

2015-02-01

Engineered T-cell therapy using a CD19-specific chimeric antigen receptor (CD19-CAR) is a promising strategy for the treatment of advanced B-cell malignancies. Gene transfer of CARs to T-cells has widely relied on retroviral vectors, but transposon-based gene transfer has recently emerged as a suitable nonviral method to mediate stable transgene expression. The advantages of transposon vectors compared with viral vectors include their simplicity and cost-effectiveness. We used the Tol2 transposon system to stably transfer CD19-CAR into human T-cells. Normal human peripheral blood lymphocytes were co-nucleofected with the Tol2 transposon donor plasmid carrying CD19-CAR and the transposase expression plasmid and were selectively propagated on NIH3T3 cells expressing human CD19. Expanded CD3(+) T-cells with stable and high-level transgene expression (~95%) produced interferon-γ upon stimulation with CD19 and specifically lysed Raji cells, a CD19(+) human B-cell lymphoma cell line. Adoptive transfer of these T-cells suppressed tumor progression in Raji tumor-bearing Rag2(-/-)γc(-/-) immunodeficient mice compared with control mice. These results demonstrate that the Tol2 transposon system could be used to express CD19-CAR in genetically engineered T-cells for the treatment of refractory B-cell malignancies.

Chronic hyperandrogenemia in the presence and absence of a western-style diet impairs ovarian and uterine structure/function in young adult rhesus monkeys.

PubMed

Bishop, Cecily V; Mishler, Emily C; Takahashi, Diana L; Reiter, Taylor E; Bond, Kise R; True, Cadence A; Slayden, Ov D; Stouffer, Richard L

2018-01-01

Does chronic hyperandrogenemia beginning at menarche, in the absence and presence of a western-style diet (WSD), alter ovarian and uterine structure-function in young adult rhesus monkeys? Phenotypic alterations in ovarian and uterine structure/function were induced by exogenous testosterone (T), and compounded in the presence of a WSD (T+WSD). Hyperandrogenemia is a well-established component of PCOS and is observed in adolescent girls, indicating a potential pubertal onset of disease symptoms. Obesity is often associated with hyperandrogenemia and it is hypothesized that metabolic dysfunction exacerbates PCOS symptoms. Macaque females (n = 40) near the onset of menarche (~2.5 years of age) were assigned to a 2 by 2 factorial cohort design. Effects on reproductive characteristics were evaluated after 3 years of treatment. Rhesus macaques (Macaca mulatta) were fed either a normal balanced diet (n = 20) or a WSD (n = 20). Additionally, implants containing cholesterol (n = 20) or T (n = 20) were implanted subcutaneously to elevate serum T approximately 5-fold. This resulted in treatment groups of controls (C), T, WSD and T+WSD (n = 10/group). Vaginal swabbing was performed daily to detect menses. After 3 years of treatment, daily serum samples from one menstrual cycle were assayed for hormone levels. Ovarian structure was evaluated in the early follicular phase by 3D/4D ultrasound. Uterine endometrial size and ovarian/luteal vascular function was also evaluated in subgroups (n = 6/group) in the late follicular and mid-luteal phases by 3D/4D ultrasound and contrast-enhanced ultrasound, respectively. Expression of steroid hormone receptors and markers of decidualization and endometrial receptivity were assessed in endometrial biopsies at mid-luteal phase. Approximately 90% of menstrual cycles appeared ovulatory with no differences in frequency or duration between groups. Serum estradiol (E2) levels during the early follicular phase were greatest in the T alone group, but reduced in T+WSD (P < 0.02). Serum LH was elevated in the T group (P < 0.04); however, there were no differences among groups in FSH levels (P > 0.13). Ovarian size at menses tended to be greater in the WSD groups (P < 0.07) and antral follicles ≥1 mm were more numerous in the T+WSD group (P < 0.05). Also, females in T and T+WSD groups displayed polycystic ovarian morphology (PCOM) at greater frequency than C or WSD groups (P < 0.01). Progesterone (P4) levels during the luteal phase were reduced in the T+WSD group compared to C and T groups (P < 0.05). Blood volume (BV) and vascular flow (VF) within the corpus luteum was reduced in all treatment groups compared to C (P < 0.01, P = 0.03), with the WSD alone group displaying the slowest BV and VF (P < 0.05). C and WSD groups displayed endometrial glands at mid-luteal phase with low estrogen receptor 1 (ESR1) and progesterone receptor (PGR) mRNA and immunohistochemical staining in the functionalis zone, but appreciable PGR in the stroma. In contrast, T and T+WSD treatment resulted in glands with less secretory morphology, high ESR1 expression in the glandular epithelium and low PGR in the stroma. Endometrial levels of TIMP3 and MMP26 mRNA and immunostaining were also decreased in the T and T+WSD groups, whereas AR expression was unchanged. None. Females are young adults, so effects could change as they reach prime reproductive age. The T level generated for hyperandrogenemia may be somewhat greater than the 3-4-fold increase observed in adolescent girls, but markedly less than those observed in male monkeys or adolescent boys. Alterations to ovarian and uterine structure-function observed in T and, in particular, T+WSD-treated female macaques are consistent with some of the features observed in women diagnosed with polycystic ovary syndrome (PCOS), and suggest impaired fertility. Research reported in this publication was supported by the Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD) of the National Institutes of Health (NIH) under Award Number P50HD071836 (to RLS). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. Additional funding was provided by Office of the Director, NIH under Award Number P51OD011092 (Support for National Primate Research Center). Authors declare no competing interests. © The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

Cerebral palsy research funding from the National Institutes of Health, 2001 to 2013.

PubMed

Wu, Yvonne W; Mehravari, Alison S; Numis, Adam L; Gross, Paul

2015-10-01

Cerebral palsy (CP) is a poorly understood disorder with no cure. We determined the landscape of National Institutes of Health (NIH) funding for CP-related research. We searched NIH databases Research Portfolio Online Reporting Tools Expenditures and Results, and Research, Condition, and Disease Categorization for keywords 'cerebral palsy' among all NIH-funded studies, 2001 to 2013. We classified grants by type and area of study. NIH funding, averaging $30 million per year, supported clinical ($215 million), basic ($187 million), and translational ($26.3 million) CP-related research. Clinical intervention studies comprised 19% of funding, and focused on treatments ($60.3 million), early parent intervention ($2.7 million), and CP prevention ($2.5 million). Among grants that specified gestational age, more funds were devoted to preterm ($166 million) than term infants ($15 million). CP in adulthood was the main focus of 4% of all funding. Annual NIH funding for CP increased steadily over the study period from $3.6 to $66.7 million. However, funding for clinical intervention studies peaked in 2008, and has since decreased. Additional research funds are needed to improve the treatment and prevention of CP. Topics that have been relatively underfunded include clinical interventions, prevention, and term infants and adults with CP. © 2015 Mac Keith Press.

Characterization of hyaluronate binding proteins isolated from 3T3 and murine sarcoma virus transformed 3T3 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Turley, E.A.; Moore, D.; Hayden, L.J.

1987-06-02

A hyaluronic acid binding fraction was purified from the supernatant media of both 3T3 and murine sarcoma virus (MSV) transformed 3T3 cultures by hyaluronate and immunoaffinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis resolved the hyaluronate affinity-purified fraction into three major protein bands of estimated molecular weight (M/sub r,e/) 70K, 66K, and 56K which contained hyaluronate binding activity and which were termed hyaluronate binding proteins (HABP). Hyaluronate affinity chromatography combined with immunoaffinity chromatography, using antibody directed against the larger HABP, allowed a 20-fold purification of HABP. Fractions isolated from 3T3 supernatant medium also contained additional binding molecules in the molecular weightmore » range of 20K. This material was present in vanishingly small amounts and was not detected with a silver stain or with (/sup 35/S)methionine label. The three protein species isolated by hyaluronate affinity chromatography (M/sub r,e/ 70K, 66K, and 56K) were related to one another since they shared antigenic determinants and exhibited similar pI values. In isocratic conditions, HABP occurred as aggregates of up to 580 kilodaltons. Their glycoprotein nature was indicated by their incorporation of /sup 3/H-sugars. Enzyme-linked immunoadsorbent assay showed they were antigenically distinct from other hyaluronate binding proteins such as fibronectin, cartilage link protein, and the hyaluronate binding region of chondroitin sulfate proteoglycan. The results are discussed with regard both to the functional significance of hyaluronate-cell surface interactions in transformed as well as normal cells and to the relationship of HABP to other reported hyaluronate binding proteins.« less

In Vitro Transformation of Rat and Mouse Cells by DNA from Simian Virus 40

PubMed Central

Abrahams, P. J.; van der Eb, A. J.

1975-01-01

Primary rat kidney cells and mouse 3T3 cells can be transformed by DNA of simian virus 40 when use is made of the calcium technique (Graham and van der Eb, 1973). The transformation assay in primary rat cells is reproducible, but the dose response is not linear. PMID:166204

Educational attainment and life expectancy: a perspective from the NIH Office of Behavioral and Social Sciences Research.

PubMed

Spittel, Michael L; Riley, William T; Kaplan, Robert M

2015-02-01

The NIH Office of Behavioral and Social Sciences Research (OBSSR) furthers the mission of the NIH by stimulating behavioral and social sciences research throughout NIH and integrating these areas of research more fully into the NIH health research enterprise, thereby improving our understanding, treatment, and prevention of disease. OBSSR accomplishes this mission through several strategic priorities: (1) supporting the next generation of basic behavioral and social sciences research, (2) facilitating interdisciplinary research, (3) promoting a multi-level systems perspective of health and behavior, and (4) encouraging a problem-focused perspective on population health. Published by Elsevier Ltd.

Expression of the leukemia-associated CBF{beta}/SMMHC chimeric gene causes transformation of 3T3 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hajra, A.; Liu, P.; Collins, E.S.

1994-09-01

A pericentric inversion of chromosome 16 (inv(16)(p13;q22)) is consistently seen in acute myeloid leukemia of the M4Eo subtype. This inversion fuses almost the entire coding region of the gene encoding of the {beta} subunit of the heterodimeric transcription factor CBF/PEBP2 to the region of the MYH11 gene encoding the rod domain for the smooth muscle myosin heavy chain (SMMHC). To investigate the biological properties of the CBF{beta}/SMMHC fusion protein, we have generated 3T3 cell lines that stably express the CBF{beta}/SMMHC chimeric cDNA or the normal, nonchimeric CBF{beta} and SMMHC cDNAs. 3T3 cells expressing CBF{beta}/SMMHC acquire a transformed phenotype, as indicatedmore » by altered cell morphology, formation of foci, and growth in soft agar. Cells constitutively overexpressing the normal CBF{beta} cDNA or the rod region of SMMHC remain nontransformed. Western blot analysis using antibodies to CBF{beta} and the SMMHC rod demonstrates that stably transfected cells express the appropriate chimeric or normal protein. Electrophoretic mobility shift assays reveal that cells transformed by the chimeric cDNA do not have a CBF-DNA complex of the expected mobility, but instead contain a large complex with CBF DNA-binding activity that fails to migrate out of the gel wells. In order to define the regions of CBF{beta}/SMMHC necessary for 3T3 transformation, we have stably transfected cells with mutant CBF{beta}/SMMHC cDNAs containing various deletions of the coding region. Analysis of these cell lines indicates that the transformation property of CBF{beta}/SMMHC requires regions of CBF{beta} known to be necessary for association with the DNA-binding CBF{alpha} subunit, and also requires an intact SMMHC carboxyl terminus, which is necessary for formation of the coiled coil domain of the myosin rod.« less

Reliability Overhaul Model

DTIC Science & Technology

1989-08-01

Random variables for the conditional exponential distribution are generated using the inverse transform method. C1) Generate U - UCO,i) (2) Set s - A ln...e - [(x+s - 7)/ n] 0 + [Cx-T)/n]0 c. Random variables from the conditional weibull distribution are generated using the inverse transform method. C1...using a standard normal transformation and the inverse transform method. B - 3 APPENDIX 3 DISTRIBUTIONS SUPPORTED BY THE MODEL (1) Generate Y - PCX S

Determining the Drivers of Academic Success in Surgery: An Analysis of 3,850 Faculty

PubMed Central

Valsangkar, Nakul P.; Zimmers, Teresa A.; Kim, Bradford J.; Blanton, Casi; Joshi, Mugdha M.; Bell, Teresa M.; Nakeeb, Attila; Dunnington, Gary L.; Koniaris, Leonidas G.

2015-01-01

Objective Determine drivers of academic productivity within U.S. departments of surgery. Methods Eighty academic metrics for 3,850 faculty at the top 50 NIH-funded university- and 5 outstanding hospital-based surgical departments were collected using websites, Scopus, and NIH RePORTER. Results Mean faculty size was 76. Overall, there were 35.3% assistant, 27.8% associate, and 36.9% full professors. Women comprised 21.8%; 4.9% were MD-PhDs and 6.1% PhDs. By faculty-rank, median publications/citations were: assistant, 14/175, associate, 39/649 and full-professor, 97/2250. General surgery divisions contributed the most publications and citations. Highest performing sub-specialties per faculty member were: research (58/1683), transplantation (51/1067), oncology (41/777), and cardiothoracic surgery (48/860). Overall, 23.5% of faculty were principal investigators for a current or former NIH grant, 9.5% for a current or former R01/U01/P01. The 10 most cited faculty (MCF) within each department contributed to 42% of all publications and 55% of all citations. MCF were most commonly general (25%), oncology (19%), or transplant surgeons (15%). Fifty-one-percent of MCF had current/former NIH funding, compared with 20% of the rest (p<0.05); funding rates for R01/U01/P01 grants was 25.1% vs. 6.8% (p<0.05). Rate of current-NIH MCF funding correlated with higher total departmental NIH rank (p < 0.05). Conclusions Departmental academic productivity as defined by citations and NIH funding is highly driven by sections or divisions of research, general and transplantation surgery. MCF, regardless of subspecialty, contribute disproportionally to major grants and publications. Approaches that attract, develop, and retain funded MCF may be associated with dramatic increases in total departmental citations and NIH-funding. PMID:26177096

Cytotoxic, Antiproliferative and Apoptotic Effects of Perillyl Alcohol and Its Biotransformation Metabolite on A549 and HepG2 Cancer Cell Lines.

PubMed

Oturanel, Ceren E; Kıran, İsmail; Özşen, Özge; Çiftçi, Gülşen A; Atlı, Özlem

2017-01-01

A monoterpene, perillyl alcohol, has attracted attention in medicinal chemistry since it exhibited chemo-preventive and therapeutic properties against a variety of cancers. In the present work, it was aimed to obtain derivatives of perillyl alcohol through microbial biotransformation and investigate their anticancer activities against A549 and HepG2 cancer cell lines. Biotransformation studies were carried out in a α-medium for 7 days at 25oC. XTT assay was performed to investigate the anticancer activities of perillyl alcohol and its biotransformation metabolite, dehydroperillic acid, against A549 and HepG2 cell lines and their selectivity using healthy cell line, NIH/3T3. Cell proliferation ELISA, BRDU (colorimetric) assay was used for measurement of proliferation in replicative cells in which DNA synthesis occurs. Flow cytometric analyses were also carried out for measuring apoptotic cell percentages, caspase 3 activation and mitochondrial membrane potential. Biotransformation of perillyl alcohol with Fusarium culmorum yielded dehydroperillic acid in a yield of 20.4 %. In in vitro anticancer studies, perillyl alcohol was found to exert cytotoxicity against HepG2 cell line with an IC50 value of 409.2 μg/mL. However, this effect was not found to be selective because of its higher IC50 (250 μg/mL) value against NIH/3T3 cell line. On the other hand, dehydroperillic acid was found to be effective and also selective against A549 cell line with an IC50 value of 125 μg/mL and a selectivity index (SI) value of 400. Apoptosis inducing effects of dehydroperillic acid was better in A549 cell line. Dehydroperillic acid may be a good candidate for therapy of lung adenocarcinoma and may show this anticancer activity by inducing apoptosis. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Magnetic alginate microfibers as scaffolding elements for the fabrication of microvascular-like structures.

PubMed

Sun, Tao; Shi, Qing; Huang, Qiang; Wang, Huaping; Xiong, Xiaolu; Hu, Chengzhi; Fukuda, Toshio

2018-01-15

Traditional cell-encapsulating scaffolds may elicit adverse host responses and inhomogeneity in cellular distribution. Thus, fabrication techniques for cellular self-assembly with micro-scaffold incorporation have been used recently to generate toroidal cellular modules for the bottom-up construction of vascular-like structures. The micro-scaffolds show advantage in promoting tissue formation. However, owing to the lack of annular cell micro-scaffolds, it remains a challenge to engineer micro-scale toroidal cellular modules (micro-TCMs) to fabricate microvascular-like structures. Here, magnetic alginate microfibers (MAMs) are used as scaffolding elements, where a winding strategy enables them to be formed into micro-rings as annular cell micro-scaffolds. These micro-rings were investigated for NIH/3T3 fibroblast growth as a function of surface chemistry and MAM size. Afterwards, micro-TCMs were successfully fabricated with the formation of NIH/3T3 fibroblasts and extracellular matrix layers on the three-dimensional micro-ring surfaces. Simple non-contact magnetic assembly was used to stack the micro-TCMs along a micro-pillar, after which cell fusion rapidly connected the assembled micro-TCMs into a microvascular-like structure. Endothelial cells or drugs encapsulated in the MAMs could be included in the microvascular-like structures as in vitro cellular models for vascular tissue engineering, or as miniaturization platforms for pharmaceutical drug testing in the future. Magnetic alginate microfibers functioned as scaffolding elements for guiding cell growth in micro-scale toroidal cellular modules (micro-TCMs) and provided a magnetic functionality to the micro-TCMs for non-contact 3D assembly in external magnetic fields. By using the liquid/air interface, the non-contact spatial manipulation of the micro-TCMs in the liquid environment was performed with a cost-effective motorized electromagnetic needle. A new biofabrication paradigm of construct of microvascular-like structure. The micro-tubal-shaped structures allowed direct cell-to-cell contact that solved problems of cell-encapsulating scaffolds. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

T-DNA transfer and T-DNA integration efficiencies upon Arabidopsis thaliana root explant cocultivation and floral dip transformation.

PubMed

Ghedira, Rim; De Buck, Sylvie; Van Ex, Frédéric; Angenon, Geert; Depicker, Ann

2013-12-01

T-DNA transfer and integration frequencies during Agrobacterium-mediated root explant cocultivation and floral dip transformations of Arabidopsis thaliana were analyzed with and without selection for transformation-competent cells. Based on the presence or absence of CRE recombinase activity without or with the CRE T-DNA being integrated, transient expression versus stable transformation was differentiated. During root explant cocultivation, continuous light enhanced the number of plant cells competent for interaction with Agrobacterium and thus the number of transient gene expression events. However, in transformation competent plant cells, continuous light did not further enhance cotransfer or cointegration frequencies. Upon selection for root transformants expressing a first T-DNA, 43-69 % of these transformants showed cotransfer of another non-selected T-DNA in two different light regimes. However, integration of the non-selected cotransferred T-DNA occurred only in 19-46 % of these transformants, indicating that T-DNA integration in regenerating root cells limits the transformation frequencies. After floral dip transformation, transient T-DNA expression without integration could not be detected, while stable T-DNA transformation occurred in 0.5-1.3 % of the T1 seedlings. Upon selection for floral dip transformants with a first T-DNA, 8-34 % of the transformants showed cotransfer of the other non-selected T-DNA and in 93-100 % of them, the T-DNA was also integrated. Therefore, a productive interaction between the agrobacteria and the female gametophyte, rather than the T-DNA integration process, restricts the floral dip transformation frequencies.

The relationship between clinical characteristics, radiographic osteoarthritis and 3D bone area: data from the osteoarthritis initiative.

PubMed

Barr, A J; Dube, B; Hensor, E M A; Kingsbury, S R; Peat, G; Bowes, M A; Conaghan, P G

2014-10-01

Radiographic measures of osteoarthritis (OA) are based upon two dimensional projection images. Active appearance modelling (AAM) of knee magnetic resonance imaging (MRI) enables accurate, 3D quantification of joint structures in large cohorts. This cross-sectional study explored the relationship between clinical characteristics, radiographic measures of OA and 3D bone area (tAB). Clinical data and baseline paired radiographic and MRI data, from the medial compartment of one knee of 2588 participants were obtained from the NIH Osteoarthritis Initiative (OAI). The medial femur (MF) and tibia (MT) tAB were calculated using AAM. 'OA-attributable' tAB (OA-tAB) was calculated using data from regression models of tAB of knees without OA. Associations between OA-tAB and radiographic measures of OA were investigated using linear regression. In univariable analyses, height, weight, and age in female knees without OA explained 43.1%, 32.1% and 0.1% of the MF tAB variance individually and 54.4% when included simultaneously in a multivariable model. Joint space width (JSW), osteophytes and sclerosis explained just 5.3%, 14.9% and 10.1% of the variance of MF OA-tAB individually and 17.4% when combined. Kellgren Lawrence (KL) grade explained approximately 20% of MF OA-tAB individually. Similar results were seen for MT OA-tAB. Height explained the majority of variance in tAB, confirming an allometric relationship between body and joint size. Radiographic measures of OA, derived from a single radiographic projection, accounted for only a small amount of variation in 3D knee OA-tAB. The additional structural information provided by 3D bone area may explain the lack of a substantive relationship with these radiographic OA measures. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

Comparison of the genotoxic activities of the K-region dihydrodiol of benzo[a]pyrene with benzo[a]pyrene in mammalian cells: morphological cell transformation; DNA damage; and stable covalent DNA adducts.

PubMed

Nesnow, Stephen; Davis, Christine; Nelson, Garret B; Lambert, Guy; Padgett, William; Pimentel, Maria; Tennant, Alan H; Kligerman, Andrew D; Ross, Jeffrey A

2002-11-26

Benzo[a]pyrene (B[a]P) is the most thoroughly studied polycyclic aromatic hydrocarbon (PAH). Many mechanisms have been suggested to explain its carcinogenic activity, yet many questions still remain. K-region dihydrodiols of PAHs are metabolic intermediates depending on the specific cytochrome P450 and had been thought to be detoxification products. However, K-region dihydrodiols of several PAHs have recently been shown to morphologically transform mouse embryo C3H10T1/2CL8 cells (C3H10T1/2 cells). Because K-region dihydrodiols are not metabolically formed from PAHs by C3H10T1/2 cells, these cells provide a useful tool to independently study the mechanisms of action of PAHs and their K-region dihydrodiols. Here, we compare the morphological cell transforming, DNA damaging, and DNA adducting activities of the K-region dihydrodiol of B[a]P, trans-B[a]P-4,5-diol with B[a]P. Both trans-B[a]P-4,5-diol and B[a]P morphologically transformed C3H10T1/2 cells by producing both Types II and III transformed foci. The morphological cell transforming and cytotoxicity dose response curves for trans-B[a]P-4,5-diol and B[a]P were indistinguishable. Since morphological cell transformation is strongly associated with mutation and/or larger scale DNA damage in C3H10T1/2 cells, the identification of DNA damage induced in these cells by trans-B[a]P-4,5-diol was sought. Both trans-B[a]P-4,5-diol and B[a]P exhibited significant DNA damaging activity without significant concurrent cytotoxicity using the comet assay, but with different dose responses and comet tail distributions. DNA adduct patterns from C3H10T1/2 cells were examined after trans-B[a]P-4,5-diol or B[a]P treatment using 32P-postlabeling techniques and improved TLC elution systems designed to separate polar DNA adducts. While B[a]P treatment produced one major DNA adduct identified as anti-trans-B[a]P-7,8-diol-9,10-epoxide-deoxyguanosine, no stable covalent DNA adducts were detected in the DNA of trans-B[a]P-4,5-diol-treated cells. In summary, this study provides evidence for the DNA damaging and morphological cell transforming activities of the K-region dihydrodiol of B[a]P, in the absence of covalent stable DNA adducts. While trans-B[a]P-4,5-diol and B[a]P both induce morphological cell transformation, their activities as DNA damaging agents differ, both qualitatively and quantitatively. In concert with the morphological cell transformation activities of other K-region dihydrodiols of PAHs, these data suggest a new mechanism/pathway for the morphological cell transforming activities of B[a]P and its metabolites.

Laser-Linewidth Effects on the Photon-Phonon Conversion Rate at a Gas-Solid Interface,

DTIC Science & Technology

1986-07-01

eliminate the exp(± iwLt )-dependence and facilitate the appearance of 0(t).20󈧙 We define the density operator a(t) as 0(t) - ei(Lt + *(t))Pgp(t)e i(wLt...specific form of our transformation (3.1) to the rotating frame. We remark that other kinds of transformations 24 , which also eliminate the exp(± iwLt ) time

Mouse Balb/c3T3 cell mutant with low epidermal growth factor receptor activity: induction of stable anchorage-independent growth by transforming growth factor. beta

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kuratomi, Y.; Ono, M.; Yasutake, C.

1987-01-01

A mutant clone (MO-5) was originally isolated as a clone resistant to Na/sup +//K/sup +/ ionophoric antibiotic monensin from mouse Balb/c3T3 cells. MO-5 was found to show low receptor-endocytosis activity for epidermal growth factor (EGF):binding activity for EGF in MO-5 was less than one tenth of that in Balb/c3T3. Anchorage-independent growth of MO-5 was compared to that of Balb/c3T3 when assayed by colony formation capacity in soft agar. Coadministration of EGF and TGF-..beta.. efficiently enhanced anchorage-independent growth of normal rat kidney (NRK) cells, but neither factor alone was competent to promote the anchorage-independent growth. The frequency of colonies appearing inmore » soft agar of MO-5 or Balb/c3T3 was significantly enhanced by TGF-..beta.. while EGF did not further enhance that of MO-5 or Balb/c3T3. Colonies of Balb/c3T3 formed in soft agar in the presence of TGF-..beta.. showed low colony formation capacity in soft agar in the absence of TGF-..beta... Colonies of MO-5 formed by TGF-..beta.. in soft agar, however, showed high colony formation capacity in soft agar in the absence of TGF-..beta... Pretreatment of MO-5 with TGF-..beta.. induced secretion of TGF-..beta..-like activity from the cells, while the treatment of Balb/c3T3 did not induce the secretion of a significant amount of TGF-..beta..-like activity. The loss of EGF-receptor activity in the stable expression and maintenance of the transformed phenotype in MO-5 is discussed.« less

Effects of mutations within the SV40 large T antigen ATPase/p53 binding domain on viral replication and transformation.

PubMed

Peden, K W; Srinivasan, A; Vartikar, J V; Pipas, J M

1998-01-01

The simian virus 40 (SV40) large T antigen is a 708 amino-acid protein possessing multiple biochemical activities that play distinct roles in productive infection or virus-induced cell transformation. The carboxy-terminal portion of T antigen includes a domain that carries the nucleotide binding and ATPase activities of the protein, as well as sequences required for T antigen to associate with the cellular tumor suppressor p53. Consequently this domain functions both in viral DNA replication and cellular transformation. We have generated a collection of SV40 mutants with amino-acid deletions, insertions or substitutions in specific domains of the protein. Here we report the properties of nine mutants with single or multiple substitutions between amino acids 402 and 430, a region thought to be important for both the p53 binding and ATPase functions. The mutants were examined for the ability to produce infectious progeny virions, replicate viral DNA in vivo, perform in trans complementation tests, and transform established cell lines. Two of the mutants exhibited a wild-type phenotype in all these tests. The remaining seven mutants were defective for plaque formation and viral DNA replication, but in each case these defects could be complemented by a wild-type T antigen supplied in trans. One of these replication-defective mutants efficiently transformed the REF52 and C3H10T1/2 cell lines as assessed by the dense-focus assay. The remaining six mutants were defective for transforming REF52 cells and transformed the C3H10T1/2 line with a reduced efficiency. The ability of mutant T antigen to transform REF52 cells correlated with their ability to induce increased levels of p53.

Cell type-specific roles of Jak3 in IL-2-induced proliferative signal transduction

PubMed Central

Fujii, Hodaka

2007-01-01

Binding of IL-2 to its specific receptor induces activation of two members of Jak family protein tyrosine kinases, Jak1 and Jak3. An IL-2R-reconstituted NIH 3T3 fibroblast cell line proliferates in response to IL-2 only when hematopoietic lineage-specific Jak3 is ectopically expressed. However, the mechanism of Jak3-dependent proliferation in the fibroblast cell line is not known. Here, I showed that Jak3 expression is dispensable for IL-2-induced activation of Jak1 and Stat proteins and expression of nuclear proto-oncogenes in the IL-2R-reconstituted fibroblast cell line. However, Jak3 expression markedly enhanced these IL-2-induced signaling events. In contrast, Jak3 expression was essential for induction of cyclin genes involved in the G1-S transition. These data suggest a critical role of Jak3 in IL-2 signaling in the fibroblast cell line and may provide further insight into the cell type-specific mechanism of cytokine signaling. PMID:17266928

NIH/NIAID Radiation/Nuclear Medical Countermeasures Development Program

DTIC Science & Technology

2011-06-15

NIH/NIAID Radiation/Nuclear Medical Countermeasures Development Program Bert W. Maidment, Ph.D. Associate Director for Product Development Division...REPORT TYPE 3. DATES COVERED 00-00-2011 to 00-00-2011 4. TITLE AND SUBTITLE NIH/NIAID Radiation/Nuclear Medical Countermeasures Development...unclassified c. THIS PAGE unclassified Standard Form 298 (Rev. 8-98) Prescribed by ANSI Std Z39-18 NIAID Radiation/Nuclear Medical Countermeasures

Efficacy for lung metastasis induced by the allogeneic bEnd3 vaccine in mice.

PubMed

Zhao, Jun; Lu, Jing; Zhou, Lurong; Zhao, Jimin; Dong, Ziming

2018-05-04

The mouse brain microvascular endothelial cell line bEnd.3 was used to develop a vaccine and its anti-tumor effect on lung metastases was observed in immunized mice. Mouse bEnd.3 cells cultured in-vitro and then fixed with glutaraldehyde was used to immunize mice; mice were challenged with the metastatic cancer cell line U14, and changes in metastatic cancer tissues were observed through hematoxylin and eosin staining. Carboxyfluorescein succinimidyl amino ester (CSFE) and propidium iodide (PI) were used to detect cytotoxic activity of spleen T lymphocytes; the ratio of CD3 + and CD8 + T-cell sub-sets was determined by flow cytometry. Enzyme-linked immunosorbent assay (ELISA), immunocytochemistry and immunoblot were used to examine the specific response of the antisera of immunized mice. The number of metastatic nodules in bEnd.3 and human umbilical vein endothelial cell (HUVEC) vaccine groups was less than NIH3T3 vaccine group and phosphate buffered saline (PBS) control group. The bEnd.3-induced and HUVEC-induced cytotoxic T-lymphocytes (CTLs) showed significant lytic activity against bEnd.3 and HUVEC target cells, while the antisera of mice in bEnd.3 and HUVEC vaccine groups showed specific immune responses to membrane proteins and inhibited target cell proliferation in-vitro. Immunoblot results showed specific bands at 180KD and 220KD in bEnd.3 and at 130 kD and 220 kD in HUVEC lysates. Allogeneic bEnd.3 vaccine induced an active and specific immune response to tumor vascular endothelial cells that resulted in production of antibodies against the proliferation antigens VEGF-R II, integrin, Endog etc. Immunization with this vaccine inhibited lung metastasis of cervical cancer U14 cells and prolonged the survival of these mice.

Indoor transformer stations as predictors of residential ELF magnetic field exposure.

PubMed

Ilonen, K; Markkanen, A; Mezei, G; Juutilainen, Jukka

2008-04-01

Transformer stations in apartment buildings may offer a possibility to conduct epidemiological studies that involve high exposure to extremely low frequency magnetic fields (MF), avoid selection bias and minimize confounding factors. To validate exposure assessment based on transformer stations, measurements were performed in thirty buildings in three Finnish cities. In each building, spot measurements in all rooms and a 24-h recording in a bedroom were performed in one apartment above a transformer station (AAT), in one first floor (FF) reference apartment, and one reference apartment on upper floors (UF). The apartment mean of spot measurements was 0.62 microT in the AATs, 0.21 microT in the FF and 0.11 microT in the UF reference apartments The 24-h apartment mean (estimated from the spot measurements and the bedroom 24-h recording) was 0.2 microT or higher in 29 (97%) AATs, in 7 (25%) FF and in 3 (10 %) UF reference apartments. The corresponding numbers for the 0.4 microT cut-off point were 19 (63%), 4 (14%), and 1 (3.3%). The higher MF level in the FF reference apartments indicates that they should not be considered "unexposed" in epidemiological studies. If such apartments are excluded, a transformer station under the floor predicts 24-h apartment mean MF with a sensitivity of 0.41 (or 0.58) and a specificity of 0.997 (or 0.97), depending on the MF cut-off point (0.2 or 0.4 microT). The results indicate that apartments can be reliably classified as high and low MF field categories based on the known location of transformer stations. (c) 2007 Wiley-Liss, Inc.

Methionine restriction inhibits chemically-induced malignant transformation in the BALB/c 3T3 cell transformation assay.

PubMed

Nicken, Petra; Empl, Michael T; Gerhard, Daniel; Hausmann, Julia; Steinberg, Pablo

2016-09-01

High consumption of red meat entails a higher risk of developing colorectal cancer. Methionine, which is more frequently a component of animal proteins, and folic acid are members of the one carbon cycle and as such important players in DNA methylation and cancer development. Therefore, dietary modifications involving altered methionine and folic acid content might inhibit colon cancer development. In the present study, the BALB/c 3T3 cell transformation assay was used to investigate whether methionine and folic acid are able to influence the malignant transformation of mouse fibroblasts after treatment with the known tumour initiator 3-methylcholanthrene. Three different methionine concentrations (representing a -40%, a "normal" and a +40% cell culture medium concentration, respectively) and two different folic acid concentrations (6 and 20 μM) were thereby investigated. Methionine restriction led to a decrease of type III foci, while enhancement of both methionine and folic acid did not significantly increase the cell transformation rate. Interestingly, the focus-lowering effect of methionine was only significant in conjunction with an elevated folic acid concentration. In summary, we conclude that the malignant transformation of mouse fibroblasts is influenced by methionine levels and that methionine restriction could be a possible approach to reduce cancer development. Copyright © 2016 Elsevier Ltd. All rights reserved.

Generation and function of immunosuppressive human and murine CD8+ T cells by transforming growth factor-β and retinoic acid

PubMed Central

Fleissner, Diana; Frede, Annika; Knott, Markus; Knuschke, Torben; Geffers, Robert; Hansen, Wiebke; Dobos, Gustav; Langhorst, Jost; Buer, Jan; Westendorf, Astrid M

2011-01-01

The intestinal immune system is constantly challenged by foreign antigens and commensal bacteria. Therefore, proper control of the intestinal microenvironment is required. One important arm of this regulatory network consists of regulatory T cells. In contrast to CD4+ Foxp3+ regulatory T cells, which have been well characterized, immunomodulatory CD8+ T cells that express Foxp3 are less well defined in terms of their generation and function. Failures of these regulatory mechanisms contribute to the development of inflammatory bowel disease. In this study we demonstrate that the frequency of CD8+ Foxp3+ T cells is reduced in the peripheral blood of patients with ulcerative colitis. As these cells might play a currently underestimated role in the maintenance of intestinal homeostasis, we have investigated human and murine CD8+ Foxp3+ T cells generated by stimulating naive CD8+ T cells in the presence of transforming growth factor-β and retinoic acid, mediators that are abundantly produced in the intestinal mucosa. These CD8+ Foxp3+ fully competent regulatory T cells show strong expression of regulatory molecules CD25, Gpr83 and CTLA-4 and exhibit cell–cell contact-dependent immunosuppressive activity in vitro. Our study illustrates a previously unappreciated critical role of CD8+ Foxp3+ T cells in controlling potentially dangerous T cells and in the maintenance of intestinal homeostasis. PMID:21711349

Dominant-negative mutants of platelet-derived growth factor revert the transformed phenotype of human astrocytoma cells.

PubMed Central

Shamah, S M; Stiles, C D; Guha, A

1993-01-01

Malignant astrocytoma is the most common primary human brain tumor. Most astrocytomas express a combination of platelet-derived growth factor (PDGF) and PDGF receptor which could close an autocrine loop. It is not known whether these autocrine loops contribute to the transformed phenotype of astrocytoma cells or are incidental to that phenotype. Here we show that dominant-negative mutants of the PDGF ligand break the autocrine loop and revert the phenotype of BALB/c 3T3 cells transformed by the PDGF-A or PDGF-B (c-sis) gene. Then, we show that these mutants are selective in that they do not alter the phenotype of 3T3 cells transformed by an activated Ha-ras or v-src gene or by simian virus 40. Finally, we show that these mutants revert the transformed phenotype of two independent human astrocytoma cell lines. They have no effect on the growth of human medulloblastoma, bladder carcinoma, or colon carcinoma cell lines. These observations are consistent with the view that PDGF autocrine loops contribute to the transformed phenotype of at least some human astrocytomas. Images PMID:8246942

Fixed Point Problems for Linear Transformations on Pythagorean Triples

ERIC Educational Resources Information Center

Zhan, M.-Q.; Tong, J.-C.; Braza, P.

2006-01-01

In this article, an attempt is made to find all linear transformations that map a standard Pythagorean triple (a Pythagorean triple [x y z][superscript T] with y being even) into a standard Pythagorean triple, which have [3 4 5][superscript T] as their fixed point. All such transformations form a monoid S* under matrix product. It is found that S*…

Insulin-IGF signaling affects cell transformation in the BALB/c 3T3 cell model

PubMed Central

Poburski, Doerte; Leovsky, Christiane; Boerner, Josefine Barbara; Szimmtenings, Luisa; Ristow, Michael; Glei, Michael; Thierbach, René

2016-01-01

The increased cancer mortality of diabetes type 2 patients is most likely an evidence of the tight connection between tumor development and energy metabolism. A major focus of today’s research is still the identification of key proteins of both diseases and the development of corresponding inhibitors. In this study we combined the two-stage BALB/c-3T3 cell transformation assay (BALB-CTA) with the IR/IGF-1R inhibitor OSI-906 (linsitinib) and analyzed alterations in protein activity and energy parameters in non-transformed as well as transformed cells. OSI-906 successfully inhibited the phosphorylation of IR/IGF-1R and decreased cell growth in non-transformed cells. In the BALB-CTA, a permanent treatment with OSI-906 reduced cellular transformation dose-dependently, whereas a temporary treatment gave evidence for a preventive effect in the promotion phase. Furthermore, even though several key proteins were affected, it was possible to show that the phosphorylation of GSK3, Erk 1/2 and the S6 protein are not crucial for the cell foci reducing effect of OSI-906. Taken together, the BALB-CTA confirmed results of OSI-906 from animal studies and enhanced the knowledge of its mode of action. Therefore, the BALB-CTA offers the opportunity to analyze alterations in the transformation process more precisely and will be helpful to identify effective cancer treatments. PMID:27849005

Insulin-IGF signaling affects cell transformation in the BALB/c 3T3 cell model.

PubMed

Poburski, Doerte; Leovsky, Christiane; Boerner, Josefine Barbara; Szimmtenings, Luisa; Ristow, Michael; Glei, Michael; Thierbach, René

2016-11-16

The increased cancer mortality of diabetes type 2 patients is most likely an evidence of the tight connection between tumor development and energy metabolism. A major focus of today's research is still the identification of key proteins of both diseases and the development of corresponding inhibitors. In this study we combined the two-stage BALB/c-3T3 cell transformation assay (BALB-CTA) with the IR/IGF-1R inhibitor OSI-906 (linsitinib) and analyzed alterations in protein activity and energy parameters in non-transformed as well as transformed cells. OSI-906 successfully inhibited the phosphorylation of IR/IGF-1R and decreased cell growth in non-transformed cells. In the BALB-CTA, a permanent treatment with OSI-906 reduced cellular transformation dose-dependently, whereas a temporary treatment gave evidence for a preventive effect in the promotion phase. Furthermore, even though several key proteins were affected, it was possible to show that the phosphorylation of GSK3, Erk 1/2 and the S6 protein are not crucial for the cell foci reducing effect of OSI-906. Taken together, the BALB-CTA confirmed results of OSI-906 from animal studies and enhanced the knowledge of its mode of action. Therefore, the BALB-CTA offers the opportunity to analyze alterations in the transformation process more precisely and will be helpful to identify effective cancer treatments.

Feature: Controlling Seasonal Allergies | NIH Medlineplus the Magazine

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... this page please turn Javascript on. Feature: Seasonal Allergies Controlling Seasonal Allergies Past Issues / Spring 2012 Table of Contents In ... to allergens, helping to prevent allergic reactions. Seasonal Allergy Research at NIH Allergen and T-Cell Reagent ...

NIH Launches National COPD Action Plan | NIH MedlinePlus the Magazine

MedlinePlus

... hasn’t that statistic changed in a while? Cigarette smoking rates have been going down, but COPD ... how these genes interact with the toxicants, like cigarette smoke, that cause COPD. The most direct connection ...

In vivo hydroxylation of 3H-acetanilide--evaluation of a new radiospirometric method in the rat.

PubMed

Toutoungi, M; Bieri, H U; Huguenin, P; Karlaganis, G; Zeng, T T; Bircher, J

1983-12-01

The proposed in vivo methodology for the investigation of hydroxylation rates consists of of the i.v. administration of tritiated substrates and the collection of tritiated water (HTO) from exhaled air as a measure of HTO accumulation in body water. Specifically, HTO was assessed in exhaled water after i.v. administration of 3H-acetanilide. Over a wide range the half lives of accumulation of HTO in exhaled water (T50) were almost identical with the half lives of elimination of 3H-acetanilide in blood, evaluated by an inverse isotope dilution method (r = 0.96, N = 18). Average T50 amounted to 29 min in controls, was reduced to 20 min after enzyme induction by phenobarbital or 3-methylcholanthrene, and prolonged to 45, 46 and 66 min after bile duct ligation, portacaval shunt and a single dose of ethanol, respectively. It is concluded that the chosen pharmacokinetic approach corrects for the NIH-shift and the results adequately reflect changes in acetanilide hydroxylation related to enzyme induction or inhibition and to liver pathology.

Development of a single-cell X-ray fluorescence flow cytometer

DOE PAGES

Crawford, Andrew M.; Kurecka, Patrick; Yim, Tsz Kwan; ...

2016-06-17

An X-ray fluorescence flow cytometer that can determine the total metal content of single cells has been developed. Capillary action or pressure was used to load cells into hydrophilic or hydrophobic capillaries, respectively. Once loaded, the cells were transported at a fixed vertical velocity past a focused X-ray beam. X-ray fluorescence was then used to determine the mass of metal in each cell. By making single-cell measurements, the population heterogeneity for metals in the µ M to m M concentration range on fL sample volumes can be directly measured, a measurement that is difficult using most analytical methods. This approachmore » has been used to determine the metal composition of 936 individual bovine red blood cells (bRBC), 31 individual 3T3 mouse fibroblasts (NIH3T3) and 18 Saccharomyces cerevisiae (yeast) cells with an average measurement frequency of ~4 cells min –1. These data show evidence for surprisingly broad metal distributions. Lastly, details of the device design, data analysis and opportunities for further sensitivity improvement are described.« less

Induction of neurite extension and survival in pheochromocytoma cells by the Rit GTPase.

PubMed

Spencer, Michael L; Shao, Haipeng; Andres, Douglas A

2002-06-07

The Rit, Rin, and Ric proteins comprise a distinct and evolutionarily conserved subfamily of the Ras-like small G-proteins. Although these proteins share the majority of core effector domain residues with Ras, recent studies suggest that Rit uses novel effector pathways to regulate NIH3T3 cell proliferation and transformation, while the functions of Rin and Ric remain largely unknown. Since we demonstrate that Rit is expressed in neurons, we investigated the role of Rit signaling in promoting the differentiation and survival of pheochromocytoma cells. In this study, we show that expression of constitutively active Rit (RitL79) in PC6 cells results in neuronal differentiation, characterized by the elaboration of an extensive network of neurite-like processes that are morphologically distinct from those mediated by the expression of oncogenic Ras. Although activated Rit fails to stimulate mitogen-activated protein kinase/extracellular-signal-regulated kinase (MAPK/ERK) signaling pathways in COS cells, RitL79 induced the phosphorylation of ERK1/2 in PC6 cells. We also find that Rit-mediated effects on neurite outgrowth can be blocked by co-expression of dominant-negative mutants of C-Raf1 or mitogen-activated protein kinase kinase 1 (MEK1). Moreover, expression of dominant-negative Rit is sufficient to inhibit NGF-induced neurite outgrowth. Expression of active Rit inhibits growth factor-withdrawal mediated apoptosis of PC6 cells, but does not induce phosphorylation of Akt/protein kinase B, suggesting that survival does not utilize the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Instead, pharmacological inhibitors of MEK block Rit-stimulated cell survival. Taken together, these studies suggest that Rit represents a distinct regulatory protein, capable of mediating differentiation and cell survival in PC6 cells using a MEK-dependent signaling pathway to achieve its effects.

Reinforcement of flowable dental composites with titanium dioxide nanotubes.

PubMed

Dafar, Manal O; Grol, Matthew W; Canham, Peter B; Dixon, S Jeffrey; Rizkalla, Amin S

2016-06-01

Flowable dental composites are used as restorative materials due to their excellent esthetics and rheology. However, they suffer from inferior mechanical properties compared to conventional composites. The aim of this study was to reinforce a flowable dental composite with TiO2 nanotubes (n-TiO2) and to assess the effect of n-TiO2 surface modifications on the mechanical properties of the reinforced composite. n-TiO2 were synthesized using an alkaline hydrothermal process and then functionalized with silane or methacrylic acid (MA). Nanotubes were characterized by scanning and transmission electron microscopy, X-ray diffraction, energy-dispersive X-ray spectroscopy and Fourier transform infrared spectroscopy. Commercially available flowable composite (Filtek™ Supreme Ultra Flowable Restorative, 3M ESPE) was reinforced with varying amounts of nanotubes (0-5wt%). Flowability of the resulting composites was evaluated using a Gillmore needle method. Dynamic Young's modulus (E) was measured using an ultrasonic technique. Fracture toughness (KIc) was assessed using a notchless triangular prism and radiopacity was quantified. Viability of NIH/3T3 fibroblasts was evaluated following incubation on composite specimens for 24h. Electron microscopy revealed a tubular morphology of n-TiO2. All reinforced composites exhibited significantly greater values of E than unreinforced composite. Composites reinforced with 3wt% n-TiO2 functionalized with MA exhibited the greatest values of E and KIc. Cytotoxicity assays revealed that reinforced composites were biocompatible. Taken together, flowable composites reinforced with n-TiO2 exhibited mechanical properties superior to those of unreinforced composite, with minimal effects on flowability and radiopacity. n-TiO2-reinforced flowable composites are promising materials for use in dental restorations. Copyright © 2016 Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

Xylan-Modified-Based Hydrogels with Temperature/pH Dual Sensitivity and Controllable Drug Delivery Behavior

PubMed Central

Kong, Wei-Qing; Gao, Cun-Dian; Hu, Shu-Feng; Ren, Jun-Li; Zhao, Li-Hong; Sun, Run-Cang

2017-01-01

Among the natural macromolecules potentially used as the scaffold material in hydrogels, xylan has aroused great interest in many fields because of its biocompatibility, low toxicity, and biodegradability. In this work, new pH and thermoresponsive hydrogels were prepared by the cross-linking polymerization of maleic anhydride-modified xylan (MAHX) with N-isopropylacrylamide (NIPAm) and acrylic acid (AA) under UV irradiation to form MAHX-g-P(NIPAm-co-AA) hydrogels. The pore volume, the mechanical properties, and the release rate for drugs of hydrogels could be controlled by the degree of substitution of MAHX. These hydrogels were characterized by swelling ability, lower critical solution temperature (LCST), Fourier-transform infrared (FTIR), and SEM. Furthermore, the cumulative release rate was investigated for acetylsalicylic acid and theophylline, as well as the cytocompatibility MAHX-based hydrogels. Results showed that MAHX-based hydrogels exhibited excellent swelling–deswelling properties, uniform porous structure, and the temperature/pH dual sensitivity. In vitro, the cumulative release rate of acetylsalicylic acid for MAHX-based hydrogels was higher than that for theophylline, and in the gastrointestinal sustained drug release study, the acetylsalicylic acid release rate was extremely slow during the initial 3 h in the gastric fluid (24.26%), and then the cumulative release rate reached to 90.5% after sustained release for 5 h in simulated intestinal fluid. The cytotoxicity experiment demonstrated that MAHX-based hydrogels could promote cell proliferation and had satisfactory biocompatibility with NIH3T3 cells. These results indicated that MAHX-based hydrogels, as new drug carriers, had favorable behavior for intestinal-targeted drug delivery. PMID:28772664

New Pyrrole Derivatives with Potent Tubulin Polymerization Inhibiting Activity As Anticancer Agents Including Hedgehog-Dependent Cancer

PubMed Central

La Regina, Giuseppe; Bai, Ruoli; Coluccia, Antonio; Famiglini, Valeria; Pelliccia, Sveva; Passacantilli, Sara; Mazzoccoli, Carmela; Ruggieri, Vitalba; Sisinni, Lorenza; Bolognesi, Alessio; Rensen, Whilelmina Maria; Miele, Andrea; Nalli, Marianna; Alfonsi, Romina; Di Marcotullio, Lucia; Gulino, Alberto; Brancale, Andrea; Novellino, Ettore; Dondio, Giulio; Vultaggio, Stefania; Varasi, Mario; Mercurio, Ciro; Hamel, Ernest; Lavia, Patrizia; Silvestri, Romano

2014-01-01

We synthesized 3-aroyl-1-arylpyrrole (ARAP) derivatives as potential anticancer agents having different substituents at the pendant 1-phenyl ring. Both the 1-phenyl ring and 3-(3,4,5-trimethoxyphenyl)carbonyl moieties were mandatory to achieve potent inhibition of tubulin polymerization, binding of colchicine to tubulin, and cancer cell growth. ARAP 22 showed strong inhibition of the P-glycoprotein-overexpressing NCI-ADR-RES and Messa/Dx5MDR cell lines. Compounds 22 and 27 suppressed in vitro the Hedgehog signaling pathway, strongly reducing luciferase activity in SAG treated NIH3T3 Shh-Light II cells, and inhibited the growth of medulloblastoma D283 cells at nanomolar concentrations. ARAPs 22 and 27 represent a new potent class of tubulin polymerization and cancer cell growth inhibitors with the potential to inhibit the Hedgehog signaling pathway. PMID:25025991

A Transformation-Defective Polyomavirus Middle T Antigen with a Novel Defect in PI3 Kinase Signaling.

PubMed

Denis, Deborah; Rouleau, Cecile; Schaffhausen, Brian S

2017-01-15

Middle T antigen (MT), the principal oncoprotein of murine polyomavirus, transforms by association with cellular proteins. Protein phosphatase 2A (PP2A), YAP, Src family tyrosine kinases, Shc, phosphatidylinositol 3-kinase (PI3K), and phospholipase C-γ1 (PLCγ1) have all been implicated in MT transformation. Mutant dl1015, with deletion of residues 338 to 347 in the C-terminal region, has been an enigma, because the basis for its transformation defect has not been apparent. This work probes the dl1015 region of MT. Because the region is proline rich, the hypothesis that it targets Src homology domain 3 (SH3) domains was tested, but mutation of the putative SH3 binding motif did not affect transformation. During this work, two point mutants, W348R and E349K, were identified as transformation defective. Extensive analysis of the E349K mutant is described here. Similar to wild-type MT, the E349K mutant associates with PP2A, YAP, tyrosine kinases, Shc, PI3 kinase, and PLCγ1. The E349K mutant was examined to determine the mechanism for its transformation defect. Assays of cell localization and membrane targeting showed no obvious difference in localization. Src association was normal as assayed by in vitro kinase and MT phosphopeptide mapping. Shc activation was confirmed by its tyrosine phosphorylation. Association of type 1 PI3K with MT was demonstrated by coimmunoprecipitation, showing both PI3K subunits and in vitro activity. Nonetheless, expression of the mutants failed to lead to the activation of two known downstream targets of PI3K, Akt and Rac-1. Strikingly, despite normal association of the E349K mutant with PI3K, cells expressing the mutant failed to elevate phosphatidylinositol (3,4,5)-trisphosphate (PIP3) in mutant-expressing cells. These results indicate a novel unsuspected aspect to PI3K control. The gene coding for middle T antigen (MT) is the murine polyomavirus oncogene most responsible for tumor formation. Its study has a history of uncovering novel aspects of mammalian cell regulation. The importance of PI3K activity and tyrosine phosphorylation are two examples of insights coming from MT. This study describes new mutants unable to transform like the wild type that point to novel regulation of PI3K signaling. Previous mutants were defective in PI3K because they failed to bind the enzyme and bring the activity to the membrane. These mutants recruit PI3K activity like the wild type, but fail to elevate the cellular level of PIP3, the product used to signal downstream of PI3K. As a result, they fail to activate either Akt or Rac1, explaining the transformation defect. Copyright © 2017 American Society for Microbiology.

CYP3A5*3 and ABCB1 61A>G Significantly Influence Dose-adjusted Trough Blood Tacrolimus Concentrations in the First Three Months Post-Kidney Transplantation.

PubMed

Hu, Rong; Barratt, Daniel T; Coller, Janet K; Sallustio, Benedetta C; Somogyi, Andrew A

2018-03-30

Tacrolimus (TAC) is a first-line immunosuppressant used to prevent organ rejection after kidney transplantation. There is large inter-individual variability in its pharmacokinetics. Single nucleotide polymorphisms (SNPs) in genes encoding TAC metabolizing enzymes cytochromes P450 3A4/5 (CYP3A4/5), P-glycoprotein efflux transporter (ABCB1), their expression regulator pregnane X receptor (NR1I2) and CYP3A co-factor cytochrome P450 reductase (POR) have been studied for their effects on tacrolimus disposition. However, except for CYP3A5*3, controversies remain about their roles in predicting dose-adjusted trough blood TAC concentrations (C 0 /D). This study aimed to investigate the effects of ABCB1 (61A>G, 1199G>A, 1236C>T, 2677G>T and 3435C>T), CYP3A4*22, CYP3A5*3, NR1I2 (8055C>T, 63396C>T and -25385C>T) and POR*28 SNPs on TAC C 0 /D. In total, 165 kidney transplant recipients were included in this study. SNPs were genotyped by probe-based real-time polymerase chain reaction. Associations between log-transformed whole blood TAC C 0 /D (measured at 1 and 3 months post-transplant) and genotypes/haplotypes were assessed by linear mixed effects analysis, controlling for age, sex and haematocrit. It was observed that CYP3A5 expressors (*1/*1 + *1/*3) (p = 5.5 × 10 -16 ) and ABCB1 61G allele carriers (p = 0.001) had lower log-transformed TAC C 0 /D (56% and 26% lower geometric mean TAC C 0 /D, respectively) and accounted for approximately 30% and 4%, respectively, of log-transformed TAC C 0 /D variability in the first 3 months post-transplant. In conclusion, CYP3A5*3 is a major, and ABCB1 61A>G is a novel, although minor, genetic factor affecting TAC C 0 /D in kidney transplant recipients. © 2018 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).

WE-G-BRB-02: The Role of Program Project Grants in Study of 3D Conformal Therapy, Dose Escalation and Motion Management

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fraass, B.

2015-06-15

Over the past 20 years the NIH has funded individual grants, program projects grants, and clinical trials which have been instrumental in advancing patient care. The ways that each grant mechanism lends itself to the different phases of translating research into clinical practice will be described. Major technological innovations, such as IMRT and proton therapy, have been advanced with R01-type and P01-type funding and will be discussed. Similarly, the role of program project grants in identifying and addressing key hypotheses on the potential of 3D conformal therapy, normal tissue-guided dose escalation and motion management will be described. An overview willmore » be provided regarding how these technological innovations have been applied to multi-institutional NIH-sponsored trials. Finally, the panel will discuss regarding which research questions should be funded by the NIH to inspire the next advances in radiation therapy. Learning Objectives: Understand the different funding mechanisms of the NIH Learn about research advances that have led to innovation in delivery Review achievements due to NIH-funded program project grants in radiotherapy over the past 20 years Understand example advances achieved with multi-institutional clinical trials NIH.« less

NIH Study Provides Clarity on Supplements for Protection Against Blinding Eye Disease

MedlinePlus

... from the National Institutes of Health (NIH). The plant-derived antioxidants lutein and zeaxanthin also had no ... performed. Omega-3 fatty acids are produced by plants, including algae, and are present in oily fish ...

The Children's Inn at NIH Anniversary Key Messages | NIH MedlinePlus the Magazine

MedlinePlus

... Past Issues / Summer 2014 Table of Contents Anniversary Key Messages Playground and Park at The Children's Inn ... and commitment, and the merging of public and private resources. Merck generously donated $3.7 million for ...

Selective Activation of Transcription by a Novel CCAAT Binding Factor

NASA Astrophysics Data System (ADS)

Maity, Sankar N.; Golumbek, Paul T.; Karsenty, Gerard; de Crombrugghe, Benoit

1988-07-01

A novel CCAAT binding factor (CBF) composed of two different subunits has been extensively purified from rat liver. Both subunits are needed for specific binding to DNA. Addition of this purified protein to nuclear extracts of NIH 3T3 fibroblasts stimulates transcription from several promoters including the α 2(I) collagen, the α 1(I) collagen, the Rous sarcoma virus long terminal repeat (RSV-LTR), and the adenovirus major late promoter. Point mutations in the CCAAT motif that show either no binding or a decreased binding of CBF likewise abolish or reduce activation of transcription by CBF. Activation of transcription requires, therefore, the specific binding of CBF to its recognition sites.

High-efficiency production of bioactive recombinant human fibroblast growth factor 18 in Escherichia coli and its effects on hair follicle growth.

PubMed

Song, Lintao; Huang, Zhifeng; Chen, Yu; Li, Haiyan; Jiang, Chao; Li, Xiaokun

2014-01-01

Using fusion tags, expression of recombinant human fibroblast growth factor 18 (rhFGF18) in mammalian cells and Escherichia coli has been extensively used for fundamental research and clinical applications, including chondrogenesis and osteogenesis, hair growth, and neuroprotection. However, high-level rhFGF18 expression is difficult and the products are often not homogeneous. Furthermore, fusion-tagged protein has higher immunogenicity and lower bioactivity, and the removal of the fused tag is expensive. To overcome the limitations of fusion-tagged expression of protein and to prepare soluble highly bioactive rhFGF18, we have developed a rapid and efficient expression strategy. Optimized hFGF18 gene was amplified by polymerase chain reaction and cloned into pET22b and pET3c vectors, then transformed into E. coli strains Origima (DE3) and BL21 (DE3)PlysS. The best combination of plasmid and host strain was selected, and only Origima (DE3)/pET3c-rhFGF18 was screened for high-level expressed rhFGF18. Under optimal conditions in a 30-L fermentor, the average bacterial yield and expression level of rhFGF18 of three batches were more than 652 g and 30 % respectively, after treatment with 1 mM isopropyl-thio-β-galactopyranoside for 10 h at 25 °C. The target protein was purified by CM Sepharose FF and heparin affinity chromatography. The purity of rhFGF18 was shown by HPLC to be higher than 95 %, and the yield was 155 mg/L. In vitro MTT assays demonstrated that the purified rhFGF18 could stimulate significant proliferation of NIH3T3 cells, and animal experiments showed that rhFGF18 could effectively regulate hair growth. In conclusion, this may be a better method of producing rhFGF18 to meet the increasing demand in its pharmacological application.

Constitutive activation of NF-kappa B and secretion of interleukin-8 induced by the G protein-coupled receptor of Kaposi's sarcoma-associated herpesvirus involve G alpha(13) and RhoA.

PubMed

Shepard, L W; Yang, M; Xie, P; Browning, D D; Voyno-Yasenetskaya, T; Kozasa, T; Ye, R D

2001-12-07

The Kaposi's sarcoma herpesvirus (KSHV) open reading frame 74 encodes a G protein-coupled receptor (GPCR) for chemokines. Exogenous expression of this constitutively active GPCR leads to cell transformation and vascular overgrowth characteristic of Kaposi's sarcoma. We show here that expression of KSHV-GPCR in transfected cells results in constitutive transactivation of nuclear factor kappa B (NF-kappa B) and secretion of interleukin-8, and this response involves activation of G alpha(13) and RhoA. The induced expression of a NF-kappa B luciferase reporter was partially reduced by pertussis toxin and the G beta gamma scavenger transducin, and enhanced by co-expression of G alpha(13) and to a lesser extent, G alpha(q). These results indicate coupling of KSHV-GPCR to multiple G proteins for NF-kappa B activation. Expression of KSHV-GPCR led to stress fiber formation in NIH 3T3 cells. To examine the involvement of the G alpha(13)-RhoA pathway in KSHV-GPCR-mediated NF-kappa B activation, HeLa cells were transfected with KSHV-GPCR alone and in combination with the regulator of G protein signaling (RGS) from p115RhoGEF or a dominant negative RhoA(T19N). Both constructs, as well as the C3 exoenzyme from Clostritium botulinum, partially reduced NF-kappa B activation by KSHV-GPCR, and by a constitutively active G alpha(13)(Q226L). KSHV-GPCR-induced NF-kappa B activation is accompanied by increased secretion of IL-8, a function mimicked by the activated G alpha(13) but not by an activated G alpha(q)(Q209L). These results suggest coupling of KSHV-GPCR to the G alpha(13)-RhoA pathway in addition to other G proteins.

A large shRNA library approach identifies lncRNA Ntep as an essential regulator of cell proliferation

PubMed Central

Beermann, Julia; Kirste, Dominique; Iwanov, Katharina; Lu, Dongchao; Kleemiß, Felix; Kumarswamy, Regalla; Schimmel, Katharina; Bär, Christian; Thum, Thomas

2018-01-01

The mammalian cell cycle is a complex and tightly controlled event. Myriads of different control mechanisms are involved in its regulation. Long non-coding RNAs (lncRNA) have emerged as important regulators of many cellular processes including cellular proliferation. However, a more global and unbiased approach to identify lncRNAs with importance for cell proliferation is missing. Here, we present a lentiviral shRNA library-based approach for functional lncRNA profiling. We validated our library approach in NIH3T3 (3T3) fibroblasts by identifying lncRNAs critically involved in cell proliferation. Using stringent selection criteria we identified lncRNA NR_015491.1 out of 3842 different RNA targets represented in our library. We termed this transcript Ntep (non-coding transcript essential for proliferation), as a bona fide lncRNA essential for cell cycle progression. Inhibition of Ntep in 3T3 and primary fibroblasts prevented normal cell growth and expression of key fibroblast markers. Mechanistically, we discovered that Ntep is important to activate P53 concomitant with increased apoptosis and cell cycle blockade in late G2/M. Our findings suggest Ntep to serve as an important regulator of fibroblast proliferation and function. In summary, our study demonstrates the applicability of an innovative shRNA library approach to identify long non-coding RNA functions in a massive parallel approach. PMID:29099486

Molecular and eco-physiological characterization of arsenic (As)-transforming Achromobacter sp. KAs 3-5T from As-contaminated groundwater of West Bengal, India.

PubMed

Mohapatra, Balaram; Satyanarayana, Tulasi; Sar, Pinaki

2018-05-02

Molecular and eco-physiological characterization of arsenic (As)-transforming and hydrocarbon-utilizing Achromobacter type strain KAs 3-5 T has been investigated in order to gain an insight into As-geomicrobiology in the contaminated groundwater. The bacterium is isolated from As-rich groundwater of West Bengal, India. Comparative 16S rRNA gene sequence phylogenetic analysis confirmed that the strain KAs 3-5 T is closely related to Achromobacter mucicolens LMG 26685 T (99.17%) and Achromobacter animicus LMG 26690 T (99.17%), thus affiliated to the genus Achromobacter. Strain KAs 3-5 T is nonflagellated, mesophilic, facultative anaerobe, having a broad metabolic repertoire of using various sugars, sugar-/fatty acids, hydrocarbons as principal carbon substrates, and O 2 , NO 3 - , NO 2 - , and Fe 3+ as terminal electron acceptors. Growth with hydrocarbons led to cellular aggregation and adherence of the cells to the hydrocarbon particles confirmed through electron microscopic observations. The strain KAs 3-5 T showed high As resistance (MIC of 5 mM for As 3+ , 25 mM for As 5+ ) and reductive transformation of As 5+ under aerobic conditions while utilizing both sugars and hydrocarbons. Molecular taxonomy specified a high genomic GC content (65.5 mol %), ubiquinone 8 (UQ-8) as respiratory quinone, spermidine as predominant polyamine in the bacterium. The differential presence of C 12:0 , C 14:0 2-OH, C 18:1 ω7c, and C 14:0 iso 3-OH/ C 16:1 iso fatty acids, phosphatidylglycerol (PG), phosphatidylcholine (PC), two unknown phospholipid (PL1, PL2) as polar lipids, low DNA-DNA relatedness (33.0-41.0%) with the Achromobacter members, and unique metabolic capacities clearly indicated the distinct genomic and physiological properties of strain KAs 3-5 T among known species of the genus Achromobacter. These findings lead to improve our understanding on metabolic flexibility of bacteria residing in As-contaminated groundwater and As-bacteria interactions within oligotrophic aquifer system.

Prenatal Vitamins: OK for Women Who Aren't Pregnant?

MedlinePlus

... fact sheet: Folate. Office of Dietary Supplements. https://ods.od.nih.gov/factsheets/Folate-HealthProfessional/. Accessed March 20, ... fact sheet: Iron. Office of Dietary Supplements. https://ods.od.nih.gov/factsheets/Iron-HealthProfessional/. Accessed March ...

Andrographis paniculata Diterpenoids Protect against Radiation-Induced Transformation in BALB/3T3 Cells.

PubMed

Nantajit, Danupon; Jetawattana, Suwimol; Suriyo, Tawit; Grdina, David J; Satayavivad, Jutamaad

2017-07-01

One of the most concerning side effects of exposure to radiation are the carcinogenic risks. To reduce the negative effects of radiation, both cytoprotective and radioprotective agents have been developed. However, little is known regarding their potential for suppressing carcinogenesis. Andrographis paniculata , a plant, with multiple medicinal uses that is commonly used in traditional medicine, has three major constituents known to have cellular antioxidant activity: andrographolide (AP1); 14-deoxy-11,12-didehydroandrographolide (AP3); and neoandrographolide (AP4). In our study, we tested these elements for their radioprotective properties as well as their anti-neoplastic effects on transformation using the BALB/3T3 cell model. All three compounds were able to reduce radiation-induced DNA damage. However, AP4 appeared to have superior radioprotective properties compared to the other two compounds, presumably by protecting mitochondrial function. The compound was able to suppress radiation-induced cellular transformation through inhibition of STAT3. Treatment with AP4 also reduced expressions of MMP-2 and MMP-9. These results suggest that AP4 could be further studied and developed into an anti-transformation/carcinogenic drug as well as a radioprotective agent.

Regulatory T cells protect mice against coxsackievirus-induced myocarditis through the transforming growth factor beta-coxsackie-adenovirus receptor pathway.

PubMed

Shi, Yu; Fukuoka, Masahiro; Li, Guohua; Liu, Youan; Chen, Manyin; Konviser, Michael; Chen, Xin; Opavsky, Mary Anne; Liu, Peter P

2010-06-22

Coxsackievirus B3 infection is an excellent model of human myocarditis and dilated cardiomyopathy. Cardiac injury is caused either by a direct cytopathic effect of the virus or through immune-mediated mechanisms. Regulatory T cells (Tregs) play an important role in the negative modulation of host immune responses and set the threshold of autoimmune activation. This study was designed to test the protective effects of Tregs and to determine the underlying mechanisms. Carboxyfluorescein diacetate succinimidyl ester-labeled Tregs or naïve CD4(+) T cells were injected intravenously once every 2 weeks 3 times into mice. The mice were then challenged with intraperitoneal coxsackievirus B3 immediately after the last cell transfer. Transfer of Tregs showed higher survival rates than transfer of CD4(+) T cells (P=0.0136) but not compared with the PBS injection group (P=0.0589). Interestingly, Tregs also significantly decreased virus titers and inflammatory scores in the heart. Transforming growth factor-beta and phosphorylated AKT were upregulated in Tregs-transferred mice and coxsackie-adenovirus receptor expression was decreased in the heart compared with control groups. Transforming growth factor-beta decreased coxsackie-adenovirus receptor expression and inhibited coxsackievirus B3 infection in HL-1 cells and neonatal cardiac myocytes. Splenocytes collected from Treg-, CD4(+) T-cell-, and PBS-treated mice proliferated equally when stimulated with heat-inactivated virus, whereas in the Treg group, the proliferation rate was reduced significantly when stimulated with noninfected heart tissue homogenate. Adoptive transfer of Tregs protected mice from coxsackievirus B3-induced myocarditis through the transforming growth factor beta-coxsackie-adenovirus receptor pathway and thus suppresses the immune response to cardiac tissue, maintaining the antiviral immune response.

Fe(II)/Cu(II) interaction on goethite stimulated by an iron-reducing bacteria Aeromonas Hydrophila HS01 under anaerobic conditions.

PubMed

Tao, Liang; Zhu, Zhen-Ke; Li, Fang-Bai; Wang, Shan-Li

2017-11-01

Copper is a trace element essential for living creatures, but copper content in soil should be controlled, as it is toxic. The physical-chemical-biological features of Cu in soil have a significant correlation with the Fe(II)/Cu(II) interaction in soil. Of significant interest to the current study is the effect of Fe(II)/Cu(II) interaction conducted on goethite under anaerobic conditions stimulated by HS01 (a dissimilatory iron reduction (DIR) microbial). The following four treatments were designed: HS01 with α-FeOOH and Cu(II) (T1), HS01 with α-FeOOH (T2), HS01 with Cu(II) (T3), and α-FeOOH with Cu(II) (T4). HS01 presents a negligible impact on copper species transformation (T3), whereas the presence of α-FeOOH significantly enhanced copper aging contributing to the DIR effect (T1). Moreover, the violent reaction between adsorbed Fe(II) and Cu(II) leads to the decreased concentration of the active Fe(II) species (T1), further inhibiting reactions between Fe(II) and iron (hydr)oxides and decelerating the phase transformation of iron (hydr)oxides (T1). From this study, the effects of the Fe(II)/Cu(II) interaction on goethite under anaerobic conditions by HS01 are presented in three aspects: (1) the accelerating effect of copper aging, (2) the reductive transformation of copper, and (3) the inhibition effect of the phase transformation of iron (hydr)oxides. Copyright © 2017 Elsevier Ltd. All rights reserved.

Teachers Teaching Teachers (T3). Volume 6, Number 3

ERIC Educational Resources Information Center

Armstrong, Anthony, Ed.

2010-01-01

"Teachers Teaching Teachers" ("T3") focuses on coaches' roles in the professional development of teachers. Each issue also explores the challenges and rewards that teacher leaders encounter. This issue includes: (1) Teaching English Language Learners: Mainstream Teachers Make a Stellar Journey as a Team to Transform Classroom Practices (Elsa M.…

Silencing expression of UO-44 (CUZD1) using small interfering RNA sensitizes human ovarian cancer cells to cisplatin in vitro.

PubMed

Leong, C T C; Ong, C K; Tay, S K; Huynh, H

2007-02-08

Ovarian cancer is currently the second leading cause of gynecological malignancy and cisplatin or cisplatin-based regimens have been the standard of care for the treatment of advance epithelial ovarian cancers. However, the efficacy of cisplatin treatment is often limited by the development of drug resistance either through the inhibition of apoptotic genes or activation of antiapoptotic genes. We have previously reported the overexpression of human UO-44 (HuUO-44) in ovarian cancers and the HuUO-44 antisera markedly inhibited NIH-OVCAR3 ovarian cancer cell attachment and proliferation (Oncogene 23: 5707-5718, 2004). In the present study, we observed through the cancer cell line profiling array that the expression of HuUO-44 was suppressed in the ovarian cancer cell line (SKOV-3) after treatment with several chemotherapeutic drugs. Similarly, this suppression in HuUO-44 expression was also correlated to the cisplatin sensitivity in two other ovarian cancer cell lines NIH-OVCAR3 and OV-90 in a dose-dependent manner. To elucidate the function of HuUO-44 in cisplatin chemoresistance in ovarian cancer cell, small interfering RNAs (siRNAs) were employed to mediate HuUO-44 silencing in ovarian cancer cell line, NIH-OVCAR3. HuUO-44 RNA interference (RNAi) resulted in the inhibition of cell growth and proliferation. Importantly, HuUO-44 RNAi significantly increased sensitivity of NIH-OVCAR3 to cytotoxic stress induced by cisplatin (P<0.01). Strikingly, we have also demonstrated that overexpression of HuUO-44 significantly conferred cisplatin resistance in NIH-OVCAR3 cells (P<0.05). Taken together, UO-44 is involved in conferring cisplatin resistance; the described HuUO-44-specific siRNA oligonucleotides that can potently silence HuUO-44 gene expression may prove to be valuable pretreatment targets for antitumor therapy or other pathological conditions that involves aberrant HuUO-44 expression.

Transformation of the Ionic X-Ray Contrast Agent Diatrizoate and Related Triiodinated Benzoates by Trametes versicolor

PubMed Central

Rode, Ulrike; Müller, Rudolf

1998-01-01

Iodinated X-ray contrast agents are considered to be nondegradable by microorganisms. The decomposition of the ionic X-ray contrast agents Diatrizoate (3,5-di(acetamido)-2,4,6-triiodobenzoic acid) and Iodipamide (3,3′-adipoyl-diimino-di(2,4,6-triiodobenzoic acid) and related triiodinated benzoates (Acetrizoate [3-acetylamino-2,4,6-triiodobenzoic acid] and Aminotrizoate [3-amino-2,4,6-triiodobenzoic acid]) by Trametes versicolor has been investigated. The fungus was able to transform all tested triiodinated benzoates cometabolically. During transformation of these compounds, iodide was released, but deiodination was not complete. T. versicolor liberated traces of 14CO2 from uniformly ring-14C-labeled Diatrizoate (3,5-di(acetamido)-2,4,6-triiodobenzoate). Various extracellular metabolites were detected during transformation of the different substances. In the transformation of Diatrizoate, the three main metabolites were identified as 3,5-di(acetamido)-2,6-diiodobenzoic acid, 3,5-di(acetamido)-2,4-diiodobenzoic acid, and 3,5-di(acetamido)-2-iodobenzoic acid, suggesting reductive deiodinations in steps as initial transformation steps. PMID:9687487

Keep Gum Disease Away! | NIH MedlinePlus the Magazine

MedlinePlus

... to be very careful when it comes to oral surgery. "Remember, it can't be done twice. So get the most experienced team possible." And he's become a stickler for good dental ... NIH Senior Health: http://nihseniorhealth.gov Registry ...

Absence of a dose-fractionation effect on neoplastic transformation induced by fission-spectrum neutrons in C3H 10T1/2 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Saran, A.; Pazzaglia, S.; Coppola, M.

1991-06-01

We have investigated the effect of fission-spectrum neutron dose fractionation on neoplastic transformation of exponentially growing C3H 10T1/2 cells. Total doses of 10.8, 27, 54, and 108 cGy were given in single doses or in five equal fractions delivered at 24-h intervals in the biological channel of the RSV-TAPIRO reactor at CRE-Casaccia. Both cell inactivation and neoplastic transformation were more effectively induced by fission neutrons than by 250-kVp X rays. No significant effect on cell survival or neoplastic transformation was observed with split doses compared to single doses of fission-spectrum neutrons. Neutron RBE values relative to X rays determined frommore » data for survival and neoplastic transformation were comparable.« less

Upstream CREs participate in the basal activity of minute virus of mice promoter P4 and in its stimulation in ras-transformed cells.

PubMed Central

Perros, M; Deleu, L; Vanacker, J M; Kherrouche, Z; Spruyt, N; Faisst, S; Rommelaere, J

1995-01-01

The activity of the P4 promoter of the parvovirus minute virus of mice (prototype strain MVMp) is stimulated in ras-transformed FREJ4 cells compared with the parental FR3T3 line. This activation may participate in the oncolytic effect of parvoviruses, given that P4 drives a transcriptional unit encoding cytotoxic nonstructural proteins. Our results suggest that the higher transcriptional activity of promoter P4 in FREJ4 cells is mediated at least in part by upstream CRE elements. Accordingly, mutations in the CRE motifs impair P4 function more strongly in the FREJ4 derivative than in its FR3T3 parent. Further evidence that these elements contribute to hyperactivity of the P4 promoter in the ras transformant is the fact that they form distinct complexes with proteins from FREJ4 and FR3T3 cell extracts. This difference can be abolished by treating the FREJ4 cell extracts with cyclic AMP-dependent protein kinase (PKA) or treating original cultures with a PKA activator. These findings can be linked with two previously reported features of ras-transformed cells: the activation of a PKA-inhibited protein kinase cascade and the reduction of PKA-induced protein phosphorylation. In keeping with these facts, P4-directed gene expression can be up- or downmodulated in vivo by exposing cells to known inhibitors or activators of PKA, respectively. PMID:7636996

Synthesis of highly interconnected 3D scaffold from Arothron stellatus skin collagen for tissue engineering application.

PubMed

Ramanathan, Giriprasath; Singaravelu, Sivakumar; Raja, M D; Sivagnanam, Uma Tiruchirapalli

2015-11-01

The substrate which is avidly used for tissue engineering applications should have good mechanical and biocompatible properties, and all these parameters are often considered as essential for dermal reformation. Highly interconnected three dimensional (3D) wound dressing material with enhanced structural integrity was synthesized from Arothron stellatus fish skin (AsFS) collagen for tissue engineering applications. The synthesized 3D collagen sponge (COL-SPG) was further characterized by different physicochemical methods. The scanning electron microscopy analysis of the material demonstrated that well interconnected pores with homogeneous microstructure on the surface aids higher swelling index and that the material also possessed good mechanical properties with a Young's modulus of 0.89±0.2 MPa. Biocompatibility of the 3D COL-SPG showed 92% growth for both NIH 3T3 fibroblasts and keratinocytes. Overall, the study revealed that synthesized 3D COL-SPG from fish skin will act as a promising wound dressing in skin tissue engineering. Copyright © 2015 Elsevier Ltd. All rights reserved.

The Rab GTPase Rab8 as a shared regulator of ciliogenesis and immune synapse assembly: From a conserved pathway to diverse cellular structures.

PubMed

Patrussi, Laura; Baldari, Cosima T

2016-01-01

Rab GTPases, which form the largest branch of the Ras GTPase superfamily, regulate almost every step of vesicle-mediated trafficking. Among them, Rab8 is an essential participant in primary cilium formation. In a report recently published in the Journal of Cell Science, Finetti and colleagues identify Rab8 as a novel player in vesicular traffic in the non-ciliated T lymphocytes, which contributes to the assembly of the specialized signaling platform known as the immune synapse. By interacting with the v-SNARE VAMP-3, Rab8 is indeed responsible for the final docking/fusion step in T cell receptor (TCR) recycling to the immune synapse. A second important take-home message which comes to light from this work is that VAMP-3 also interacts with Rab8 at the base of the cilium in NIH-3T3 cells, where it regulates ciliary growth and targeting of Smoothened at the plasma membrane. Hence the data presented in this report, in addition to identifying Rab8 as a novel player in vesicular traffic to the immune synapse, reveal how both ciliated and non-ciliated cells take advantage of a conserved pathway to build highly specific cellular structures.

Prmt7 is dispensable in tissue culture models for adipogenic differentiation.

PubMed

Hu, Yu-Jie; Sif, Saïd; Imbalzano, Anthony N

2013-01-01

Protein arginine methylation is a common posttranslational modification that has been implicated in numerous biological processes including gene expression. The mammalian genome encodes nine protein arginine methyltransferases (Prmts) that catalyze monomethylation, asymmetric dimethylation, and symmetric dimethylation on arginine residues. Protein arginine methyltransferase 7 (Prmt7) is categorized as a type II and type III enzyme that produces symmetric dimethylated arginine and monomethylated arginine, respectively. However, the biological role of Prmt7 is not well characterized. We previously showed that Prmt5, a type II Prmt that associates with Brg1-based SWI/SNF chromatin remodeling complex, is required for adipocyte differentiation. Since Prmt7 also associates with Brg1-based SWI/SNF complex and modifies core histones, we hypothesized that Prmt7 might play a role in transcriptional regulation of adipogenesis. In the present study, we determined that the expression of Prmt7 did not change throughout adipogenic differentiation of C3H10T1/2 mesenchymal cells. Knockdown or over-expression of Prmt7 had no effect on lipid accumulation or adipogenic gene expression in differentiating C3H10T1/2 cells or in C/EBPα-reprogrammed NIH3T3 fibroblasts. Based on these results, we conclude that Prmt7, unlike Prmt5, is dispensable for adipogenic differentiation in tissue culture models.

Prmt7 is dispensable in tissue culture models for adipogenic differentiation

PubMed Central

Imbalzano, Anthony N.

2013-01-01

Protein arginine methylation is a common posttranslational modification that has been implicated in numerous biological processes including gene expression. The mammalian genome encodes nine protein arginine methyltransferases (Prmts) that catalyze monomethylation, asymmetric dimethylation, and symmetric dimethylation on arginine residues. Protein arginine methyltransferase 7 (Prmt7) is categorized as a type II and type III enzyme that produces symmetric dimethylated arginine and monomethylated arginine, respectively. However, the biological role of Prmt7 is not well characterized. We previously showed that Prmt5, a type II Prmt that associates with Brg1-based SWI/SNF chromatin remodeling complex, is required for adipocyte differentiation. Since Prmt7 also associates with Brg1-based SWI/SNF complex and modifies core histones, we hypothesized that Prmt7 might play a role in transcriptional regulation of adipogenesis. In the present study, we determined that the expression of Prmt7 did not change throughout adipogenic differentiation of C3H10T1/2 mesenchymal cells. Knockdown or over-expression of Prmt7 had no effect on lipid accumulation or adipogenic gene expression in differentiating C3H10T1/2 cells or in C/EBPα-reprogrammed NIH3T3 fibroblasts. Based on these results, we conclude that Prmt7, unlike Prmt5, is dispensable for adipogenic differentiation in tissue culture models. PMID:24715966

Cognition assessment using the NIH Toolbox

PubMed Central

Dikmen, Sureyya S.; Heaton, Robert K.; Tulsky, David S.; Zelazo, Philip D.; Bauer, Patricia J.; Carlozzi, Noelle E.; Slotkin, Jerry; Blitz, David; Wallner-Allen, Kathleen; Fox, Nathan A.; Beaumont, Jennifer L.; Mungas, Dan; Nowinski, Cindy J.; Richler, Jennifer; Deocampo, Joanne A.; Anderson, Jacob E.; Manly, Jennifer J.; Borosh, Beth; Havlik, Richard; Conway, Kevin; Edwards, Emmeline; Freund, Lisa; King, Jonathan W.; Moy, Claudia; Witt, Ellen; Gershon, Richard C.

2013-01-01

Cognition is 1 of 4 domains measured by the NIH Toolbox for the Assessment of Neurological and Behavioral Function (NIH-TB), and complements modules testing motor function, sensation, and emotion. On the basis of expert panels, the cognition subdomains identified as most important for health, success in school and work, and independence in daily functioning were Executive Function, Episodic Memory, Language, Processing Speed, Working Memory, and Attention. Seven measures were designed to tap constructs within these subdomains. The instruments were validated in English, in a sample of 476 participants ranging in age from 3 to 85 years, with representation from both sexes, 3 racial/ethnic categories, and 3 levels of education. This report describes the development of the Cognition Battery and presents results on test-retest reliability, age effects on performance, and convergent and discriminant construct validity. The NIH-TB Cognition Battery is intended to serve as a brief, convenient set of measures to supplement other outcome measures in epidemiologic and longitudinal research and clinical trials. With a computerized format and national standardization, this battery will provide a “common currency” among researchers for comparisons across a wide range of studies and populations. PMID:23479546

Adverse Phototoxic Effect of Essential Plant Oils on NIH 3T3 Cell Line after UV Light Exposure.

PubMed

Binder, Svatopluk; Hanáková, Adéla; Tománková, Kateřina; Pížová, Klára; Bajgar, Robert; Manišová, Barbora; Kejlová, Kristina; Bendová, Hana; Jírová, Dagmar; Kolářová, Hana

2016-09-01

Natural or artificial substances have become an inseparable part of our lives. It is questionable whether adequate testing has been performed in order to ensure these substances do not pose a serious health risk. The principal aim of our research was to clarify the potential risk of adding essential oils to food, beverages and cosmetic products. The toxicity of substances frequently employed in cosmetics, aromatherapy and food industry (bergamot oil, Litsea cubeba oil, orange oil, citral) were investigated using cell line NIH3T3 (mouse fibroblasts) with/without UV irradiation. The MTT assay was used to estimate the cell viability. Reactive oxygen species (ROS) which are products of a number of natural cellular processes such as oxygen metabolism and inflammation were measured to determine the extent of cellular stress. DNA damage caused by strand breaks was examined by comet assay. MTT test determined EC50 values for all tested substances, varying from 0.0023% v/v for bergamot oil to 0.018% v/v for citral. ROS production measurement showed that UV radiation induces oxidative stress to the cell resulting in higher ROS production compared to the control and non-irradiated samples. Comet assay revealed that both groups (UV, without UV) exert irreversible DNA damage resulting in a cell death. Our findings suggest that even low concentrations (lower than 0.0464% v/v) of orange oil can be considered as phototoxic (PIF value 8.2) and probably phototoxic for bergamot oil (PIF value 4.6). We also found significant changes in the cell viability, the ROS production and the DNA after the cells were exposed to the tested chemicals. Even though these substances are widely used as antioxidants it should be noted that they present a risk factor and their use in cosmetic and food products should be minimized. Copyright© by the National Institute of Public Health, Prague 2016

Molecularly Characterized Solvent Extracts and Saponins from Polygonum hydropiper L. Show High Anti-Angiogenic, Anti-Tumor, Brine Shrimp, and Fibroblast NIH/3T3 Cell Line Cytotoxicity

PubMed Central

Ayaz, Muhammad; Junaid, Muhammad; Ullah, Farhat; Sadiq, Abdul; Subhan, Fazal; Khan, Mir Azam; Ahmad, Waqar; Ali, Gowhar; Imran, Muhammad; Ahmad, Sajjad

2016-01-01

Polygonum hydropiper is used as anti-cancer and anti-rheumatic agent in folk medicine. This study was designed to investigate the anti-angiogenic, anti-tumor, and cytotoxic potentials of different solvent extracts and isolated saponins. Samples were analyzed using GC, Gas Chromatography–Mass Spectrometry (GC–MS) to identify major and bioactive compounds. Quantitation of antiangiogenesis for the plant's samples including methanolic extract (Ph.Cr), its subsequent fractions; n-hexane (Ph.Hex), chloroform (Ph.Chf), ethyl acetate (Ph.EtAc), n-Butanol (Ph.Bt), aqueous (Ph.Aq), saponins (Ph.Sp) were performed using the chick embryo chorioallantoic membrane (CAM) assay. Potato disc anti-tumor assay was performed on Agrobacterium tumefaciens containing tumor inducing plasmid. Cytotoxicity was performed against Artemia salina and mouse embryonic fibroblast NIH/3T3 cell line following contact toxicity and MTT cells viability assays, respectively. The GC–MS analysis of Ph.Cr, Ph.Hex, Ph.Chf, Ph.Bt, and Ph.EtAc identified 126, 124, 153, 131, and 164 compounds, respectively. In anti-angiogenic assay, Ph.Chf, Ph.Sp, Ph.EtAc, and Ph.Cr exhibited highest activity with IC50 of 28.65, 19.21, 88.75, and 461.53 μg/ml, respectively. In anti-tumor assay, Ph.Sp, Ph.Chf, Ph.EtAc, and Ph.Cr were most potent with IC50 of 18.39, 73.81, 217.19, and 342.53 μg/ml, respectively. In MTT cells viability assay, Ph.Chf, Ph.EtAc, Ph.Sp were most active causing 79.00, 72.50, and 71.50% cytotoxicity, respectively, at 1000 μg/ml with the LD50 of 140, 160, and 175 μg/ml, respectively. In overall study, Ph.Chf and Ph.Sp have shown overwhelming results which signifies their potentials as sources of therapeutic agents against cancer. PMID:27065865

A KRAS GTPase K104Q Mutant Retains Downstream Signaling by Offsetting Defects in Regulation*

PubMed Central

Kistler, Samantha; George, Samuel D.; Kuhlmann, Nora; Garvey, Leslie; Huynh, Minh; Bagni, Rachel K.; Lammers, Michael; Der, Channing J.; Campbell, Sharon L.

2017-01-01

The KRAS GTPase plays a critical role in the control of cellular growth. The activity of KRAS is regulated by guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs), and also post-translational modification. Lysine 104 in KRAS can be modified by ubiquitylation and acetylation, but the role of this residue in intrinsic KRAS function has not been well characterized. We find that lysine 104 is important for GEF recognition, because mutations at this position impaired GEF-mediated nucleotide exchange. Because the KRAS K104Q mutant has recently been employed as an acetylation mimetic, we conducted a series of studies to evaluate its in vitro and cell-based properties. Herein, we found that KRAS K104Q exhibited defects in both GEF-mediated exchange and GAP-mediated GTP hydrolysis, consistent with NMR-detected structural perturbations in localized regions of KRAS important for recognition of these regulatory proteins. Despite the partial defect in both GEF and GAP regulation, KRAS K104Q did not alter steady-state GTP-bound levels or the ability of the oncogenic KRAS G12V mutant to cause morphologic transformation of NIH 3T3 mouse fibroblasts and of WT KRAS to rescue the growth defect of mouse embryonic fibroblasts deficient in all Ras genes. We conclude that the KRAS K104Q mutant retains both WT and mutant KRAS function, probably due to offsetting defects in recognition of factors that up-regulate (GEF) and down-regulate (GAP) RAS activity. PMID:28154176

Encapsulation of β-Sitosterol in Polyurethane by Sol-Gel Electrospinning.

PubMed

Amina, Musarat; Amna, Touseef; Al-Musayeib, Nawal; Zabin, Sami A; Hassan, M Shamshi; Khil, Myung-Seob

2017-06-01

Pristine β-sitosterol or in combination with other phytosterols is utilized in an array of enriched commercial foods. Considering the presence of β-sitosterol in different functional foods and its potential role in prevention and cure of neurodegenerative diseases, the aims of our investigation were to encapsulate β-sitosterol in nanofibers and to estimate influence of β-sitosterol on proliferation of fibroblasts. Electrospun nanofibers have widely been used as scaffolds to mimic natural extracellular matrix. Herein, our group for the first time establishes an innovative scaffold based on β-sitosterol and polyurethane using electrospinning. β-Sitosterol promotes epithelialization and possesses anti-oxidant and anti-inflammatory activities, whereas polyurethane, besides possessing biomedical uses, also enhances epithelial growth. We optimized the concentration (5%) of β-sitosterol in polyurethane to obtain homogenous solution, which can be spun without difficulty for the synthesis of β-sitosterol amalgamated scaffold. The resulted twisted nanofibers have been characterized via scanning electron microscopy and Fourier transform infrared spectroscopy. The viability of cells on twisted scaffold was examined using NIH 3T3 fibroblasts as model cell line. Incorporation of β-sitosterol in polyurethane changed the structure and size of nanofibers, and the twisted scaffolds were non-cytotoxic. Thus, the twisted nanoribbons, which contain anti-inflammatory β-sitosterol, can be utilized as a promising future material, which will help to ease inflammation and also aid in wound healing. In conclusion, the outcome of the preliminary research evidently points out the potential of twisted scaffold in biomedical applications.

Budgets up at NIH, NCI, and FDA.

PubMed

2014-03-01

The federal budget for 2014, signed into law by President Obama in January, increases funding for the NIH, National Cancer Institute, and U.S. Food and Drug Administration, but it doesn't fully cover the losses they sustained collectively in 2013 due to sequestration.

Toxicity of a polymer-graphene oxide composite against bacterial planktonic cells, biofilms, and mammalian cells

NASA Astrophysics Data System (ADS)

Mejías Carpio, Isis E.; Santos, Catherine M.; Wei, Xin; Rodrigues, Debora F.

2012-07-01

It is critical to develop highly effective antimicrobial agents that are not harmful to humans and do not present adverse effects on the environment. Although antimicrobial studies of graphene-based nanomaterials are still quite limited, some researchers have paid particular attention to such nanocomposites as promising candidates for the next generation of antimicrobial agents. The polyvinyl-N-carbazole (PVK)-graphene oxide (GO) nanocomposite (PVK-GO), which contains only 3 wt% of GO well-dispersed in a 97 wt% PVK matrix, presents excellent antibacterial properties without significant cytotoxicity to mammalian cells. The high polymer content in this nanocomposite makes future large-scale material manufacturing possible in a high-yield process of adiabatic bulk polymerization. In this study, the toxicity of PVK-GO was assessed with planktonic microbial cells, biofilms, and NIH 3T3 fibroblast cells. The antibacterial effects were evaluated against two Gram-negative bacteria: Escherichia coli and Cupriavidus metallidurans; and two Gram-positive bacteria: Bacillus subtilis and Rhodococcus opacus. The results show that the PVK-GO nanocomposite presents higher antimicrobial effects than the pristine GO. The effectiveness of the PVK-GO in solution was demonstrated as the nanocomposite ``encapsulated'' the bacterial cells, which led to reduced microbial metabolic activity and cell death. The fact that the PVK-GO did not present significant cytotoxicity to fibroblast cells offers a great opportunity for potential applications in important biomedical and industrial fields.It is critical to develop highly effective antimicrobial agents that are not harmful to humans and do not present adverse effects on the environment. Although antimicrobial studies of graphene-based nanomaterials are still quite limited, some researchers have paid particular attention to such nanocomposites as promising candidates for the next generation of antimicrobial agents. The polyvinyl-N-carbazole (PVK)-graphene oxide (GO) nanocomposite (PVK-GO), which contains only 3 wt% of GO well-dispersed in a 97 wt% PVK matrix, presents excellent antibacterial properties without significant cytotoxicity to mammalian cells. The high polymer content in this nanocomposite makes future large-scale material manufacturing possible in a high-yield process of adiabatic bulk polymerization. In this study, the toxicity of PVK-GO was assessed with planktonic microbial cells, biofilms, and NIH 3T3 fibroblast cells. The antibacterial effects were evaluated against two Gram-negative bacteria: Escherichia coli and Cupriavidus metallidurans; and two Gram-positive bacteria: Bacillus subtilis and Rhodococcus opacus. The results show that the PVK-GO nanocomposite presents higher antimicrobial effects than the pristine GO. The effectiveness of the PVK-GO in solution was demonstrated as the nanocomposite ``encapsulated'' the bacterial cells, which led to reduced microbial metabolic activity and cell death. The fact that the PVK-GO did not present significant cytotoxicity to fibroblast cells offers a great opportunity for potential applications in important biomedical and industrial fields. Electronic supplementary information (ESI) available: Bacterial OD600 absorbance growth curves, representative LIVE/DEAD images and percent of inactive cells after treatment with the most toxic concentrations of nanomaterials, bacterial OD540 nm biofilm absorbance, percent toxicity on the ITO-modified surfaces, additional TEM/SEM images of the nanomaterials and B. subtilis, NIH 3T3 fibroblast cells percent toxicity. See DOI: 10.1039/c2nr30774j

Efficient Computer Implementations of Fast Fourier Transforms.

DTIC Science & Technology

1980-12-01

fit in computer? Yes, continue (9) Determine fastest algorithm between WFTA and PFA from Table 4.6. For N=420, WFTA PFA Mult 1296 2528 Add 11352 10956...real adds = 24tN/4 + 2(3tN/4) = 15tN/2 (G.8) 260 All odd prime C<ictors ciual to or (,rater than 5 iso the general transform section. Based on the

Malignant Transformation Potentials of Human Umbilical Cord Mesenchymal Stem Cells Both Spontaneously and via 3-Methycholanthrene Induction

PubMed Central

Lai, Xiulan; Liu, Sizheng; Chen, Yezeng; Zheng, Zexin; Xie, Qingdong; Maldonado, Martin; Cai, Zhiwei; Qin, Shan; Ho, Guyu; Ma, Lian

2013-01-01

Human umbilical cord mesenchymal stem cells (HUMSCs) are highly proliferative and can be induced to differentiate into advanced derivatives of all three germ layers. Thus, HUMSCs are considered to be a promising source for cell-targeted therapies and tissue engineering. However there are reports on spontaneous transformation of mesenchymal stem cells (MSCs) derived from human bone marrows. The capacity for HUMSCs to undergo malignant transform spontaneously or via induction by chemical carcinogens is presently unknown. Therefore, we isolated HUMSCs from 10 donors and assessed their transformation potential either spontaneously or by treating them with 3-methycholanthrene (3-MCA), a DNA-damaging carcinogen. The malignant transformation of HUMSCs in vitro was evaluated by morphological changes, proliferation rates, ability to enter cell senescence, the telomerase activity, chromosomal abnormality, and the ability to form tumors in vivo. Our studies showed that HUMSCs from all 10 donors ultimately entered senescence and did not undergo spontaneous malignant transformation. However, HUMSCs from two of the 10 donors treated with 3-MCA displayed an increased proliferation rate, failed to enter senescence, and exhibited an altered cell morphology. When these cells (tHUMSCs) were injected into immunodeficient mice, they gave rise to sarcoma-like or poorly differentiated tumors. Moreover, in contrast to HUMSCs, tHUMSCs showed a positive expression of human telomerase reverse transcriptase (hTERT) and did not exhibit a shortening of the relative telomere length during the long-term culture in vitro. Our studies demonstrate that HUMSCs are not susceptible to spontaneous malignant transformation. However, the malignant transformation could be induced by chemical carcinogen 3-MCA. PMID:24339974

Antibacterial and Antifungal Activity of ZnO Containing Glasses.

PubMed

Esteban-Tejeda, Leticia; Prado, Catuxa; Cabal, Belén; Sanz, Jesús; Torrecillas, Ramón; Moya, José Serafín

2015-01-01

A new family of non-toxic biocides based on low melting point (1250°C) transparent glasses with high content of ZnO (15-40wt%) belonging to the miscibility region of the B2O3-SiO2-Na2O-ZnO system has been developed. These glasses have shown an excellent biocide activity (logarithmic reduction >3) against Gram- (E. coli), Gram+ (S. aureus) and yeast (C. krusei); they are chemically stable in different media (distilled water, sea-like water, LB and DMEN media) as well as biocompatible. The cytotoxicity was evaluated by the Neutral Red Uptake using NIH-3T3 (mouse embryonic fibroblast cells) and the cell viability was >80%. These new glasses can be considered in several and important applications in the field of inorganic non-toxic biocide agents such as medical implants, surgical equipment, protective apparels in hospitals, water purifications systems, food packaging, food storages or textiles.

Antibacterial and Antifungal Activity of ZnO Containing Glasses

PubMed Central

Esteban-Tejeda, Leticia; Prado, Catuxa; Cabal, Belén; Sanz, Jesús; Torrecillas, Ramón; Moya, José Serafín

2015-01-01

A new family of non-toxic biocides based on low melting point (1250°C) transparent glasses with high content of ZnO (15–40wt%) belonging to the miscibility region of the B2O3-SiO2-Na2O-ZnO system has been developed. These glasses have shown an excellent biocide activity (logarithmic reduction >3) against Gram- (E. coli), Gram+ (S. aureus) and yeast (C. krusei); they are chemically stable in different media (distilled water, sea-like water, LB and DMEN media) as well as biocompatible. The cytotoxicity was evaluated by the Neutral Red Uptake using NIH-3T3 (mouse embryonic fibroblast cells) and the cell viability was >80%. These new glasses can be considered in several and important applications in the field of inorganic non-toxic biocide agents such as medical implants, surgical equipment, protective apparels in hospitals, water purifications systems, food packaging, food storages or textiles. PMID:26230940

Oceanic transform faults: how and why do they form? (Invited)

NASA Astrophysics Data System (ADS)

Gerya, T.

2013-12-01

Oceanic transform faults at mid-ocean ridges are often considered to be the direct product of plate breakup process (cf. review by Gerya, 2012). In contrast, recent 3D thermomechanical numerical models suggest that transform faults are plate growth structures, which develop gradually on a timescale of few millions years (Gerya, 2010, 2013a,b). Four subsequent stages are predicted for the transition from rifting to spreading (Gerya, 2013b): (1) crustal rifting, (2) multiple spreading centers nucleation and propagation, (3) proto-transform faults initiation and rotation and (4) mature ridge-transform spreading. Geometry of the mature ridge-transform system is governed by geometrical requirements for simultaneous accretion and displacement of new plate material within two offset spreading centers connected by a sustaining rheologically weak transform fault. According to these requirements, the characteristic spreading-parallel orientation of oceanic transform faults is the only thermomechanically consistent steady state orientation. Comparison of modeling results with the Woodlark Basin suggests that the development of this incipient spreading region (Taylor et al., 2009) closely matches numerical predictions (Gerya, 2013b). Model reproduces well characteristic 'rounded' contours of the spreading centers as well as the presence of a remnant of the broken continental crustal bridge observed in the Woodlark basin. Similarly to the model, the Moresby (proto)transform terminates in the oceanic rather than in the continental crust. Transform margins and truncated tip of one spreading center present in the model are documented in nature. In addition, numerical experiments suggest that transform faults can develop gradually at mature linear mid-ocean ridges as the result of dynamical instability (Gerya, 2010). Boundary instability from asymmetric plate growth can spontaneously start in alternate directions along successive ridge sections; the resultant curved ridges become transform faults. Offsets along the transform faults change continuously with time by asymmetric plate growth and discontinuously by ridge jumps. The ridge instability is governed by rheological weakening of active fault structures. The instability is most efficient for slow to intermediate spreading rates, whereas ultraslow and (ultra)fast spreading rates tend to destabilize transform faults (Gerya, 2010; Püthe and Gerya, 2013) References Gerya, T. (2010) Dynamical instability produces transform faults at mid-ocean ridges. Science, 329, 1047-1050. Gerya, T. (2012) Origin and models of oceanic transform faults. Tectonophys., 522-523, 34-56 Gerya, T.V. (2013a) Three-dimensional thermomechanical modeling of oceanic spreading initiation and evolution. Phys. Earth Planet. Interiors, 214, 35-52. Gerya, T.V. (2013b) Initiation of transform faults at rifted continental margins: 3D petrological-thermomechanical modeling and comparison to the Woodlark Basin. Petrology, 21, 1-10. Püthe, C., Gerya, T.V. (2013) Dependence of mid-ocean ridge morphology on spreading rate in numerical 3-D models. Gondwana Res., DOI: http://dx.doi.org/10.1016/j.gr.2013.04.005 Taylor, B., Goodliffe, A., Martinez, F. (2009) Initiation of transform faults at rifted continental margins. Comptes Rendus Geosci., 341, 428-438.

SV40 T antigen alone drives karyotype instability that precedes neoplastic transformation of human diploid fibroblasts.

PubMed

Ray, F A; Peabody, D S; Cooper, J L; Cram, L S; Kraemer, P M

1990-01-01

To define the role of SV40 large T antigen in the transformation and immortalization of human cells, we have constructed a plasmid lacking most of the unique coding sequences of small t antigen as well as the SV40 origin of replication. The promoter for T antigen, which lies within the origin of replication, was deleted and replaced by the Rous sarcoma virus promoter. This minimal construct was co-electroporated into normal human fibroblasts of neonatal origin along with a plasmid containing the neomycin resistance gene (neo). Three G418-resistant, T antigen-positive clones were expanded and compared to three T antigen-positive clones that received the pSV3neo plasmid (capable of expressing large and small T proteins and having two origins of replication). Autonomous replication of plasmid DNA was observed in all three clones that received pSV3neo but not in any of the three origin minus clones. Immediately after clonal expansion, several parameters of neoplastic transformation were assayed. Low percentages of cells in T antigen-positive populations were anchorage independent or capable of forming colonies in 1% fetal bovine serum. The T antigen-positive clones generally exhibited an extended lifespan in culture but rarely became immortalized. Large numbers of dead cells were continually generated in all T antigen-positive, pre-crisis populations. Ninety-nine percent of all T antigen-positive cells had numerical or structural chromosome aberrations. Control cells that received the neo gene did not have an extended life span, did not have noticeable numbers of dead cells, and did not exhibit karyotype instability. We suggest that the role of T antigen protein in the transformation process is to generate genetic hypervariability, leading to various consequences including neoplastic transformation and cell death.

75 FR 8369 - Office of the Director, National Institutes of Health; Notice of Meeting

Federal Register 2010, 2011, 2012, 2013, 2014

2010-02-24

... cloning of Ebola and Marburg cDNAs into E. coli. Please check the meeting agenda at http://oba.od.nih.gov... requirements of Section III-E-3 of the NIH Guidelines for Research with Recombinant DNA Molecules. Please check...

For Distinguished Public Service: Medical Library Association Honors FNLM and NIH MedlinePlus Magazine | NIH ...

MedlinePlus

... warm and mutually appreciative relationship with the Medical Library Association (MLA) and its more than 3,600 individual members, representing more than 1,100 institutions in the health sciences information field. They do wonderful work educating health ...

Compliance with results reporting at ClinicalTrials.gov.

PubMed

Anderson, Monique L; Chiswell, Karen; Peterson, Eric D; Tasneem, Asba; Topping, James; Califf, Robert M

2015-03-12

The Food and Drug Administration Amendments Act (FDAAA) mandates timely reporting of results of applicable clinical trials to ClinicalTrials.gov. We characterized the proportion of applicable clinical trials with publicly available results and determined independent factors associated with the reporting of results. Using an algorithm based on input from the National Library of Medicine, we identified trials that were likely to be subject to FDAAA provisions (highly likely applicable clinical trials, or HLACTs) from 2008 through 2013. We determined the proportion of HLACTs that reported results within the 12-month interval mandated by the FDAAA or at any time during the 5-year study period. We used regression models to examine characteristics associated with reporting at 12 months and throughout the 5-year study period. From all the trials at ClinicalTrials.gov, we identified 13,327 HLACTs that were terminated or completed from January 1, 2008, through August 31, 2012. Of these trials, 77.4% were classified as drug trials. A total of 36.9% of the trials were phase 2 studies, and 23.4% were phase 3 studies; 65.6% were funded by industry. Only 13.4% of trials reported summary results within 12 months after trial completion, whereas 38.3% reported results at any time up to September 27, 2013. Timely reporting was independently associated with factors such as FDA oversight, a later trial phase, and industry funding. A sample review suggested that 45% of industry-funded trials were not required to report results, as compared with 6% of trials funded by the National Institutes of Health (NIH) and 9% of trials that were funded by other government or academic institutions. Despite ethical and legal obligations to disclose findings promptly, most HLACTs did not report results to ClinicalTrials.gov in a timely fashion during the study period. Industry-funded trials adhered to legal obligations more often than did trials funded by the NIH or other government or academic institutions. (Funded by the Clinical Trials Transformation Initiative and the NIH.).

Scandium induced structural transformation and B′:B″ cationic ordering in Pb(Fe{sub 0.5}Nb{sub 0.5})O{sub 3} multiferroic ceramics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mallesham, B.; Ranjith, R., E-mail: ranjith@iith.ac.in; Manivelraja, M.

2014-07-21

The current study explores non-magnetic Sc{sup 3+} induced structural transformation, evolution of local B-site cation ordering and associated effect on ferroelectric phase transition temperature T{sub max} (temperature corresponding to dielectric maxima) on increasing the atom percent of Sc substitution in [Pb(Fe{sub 0.5}Nb{sub 0.5})O{sub 3} (PFN)] ceramics. In this regard, the phase pure Pb[(Fe{sub 0.5−x}Sc{sub x})Nb{sub 0.5}]O{sub 3} ceramics with x varying from 0 to 0.5 were synthesized through solid state reaction route. The detailed structural analysis through Rietveld refinement confirms the room temperature transformation from a monoclinic Cm to rhombohedral R3m structure at x = 0.3 mol. % of Sc. Absorption spectra studies showmore » that there is a considerable increment in the bandgap at higher scandium content. Most interestingly, the T{sub max} exhibited an increment for lower scandium contents (x = 0.1 to 0.25) followed by a drop in T{sub max} (x = 0.3 to 0.5). Such anomalous behavior in T{sub max} is expected to arise due to the onset of B′, B″ local cation ordering beyond Sc content x = 0.25. The B-site cation ordering at and beyond x = 0.3 was also confirmed by the evolution of cation order induced Pb-O coupled vibrational mode in Raman scattering studies. In addition, the Mössbauer spectra of PFN (x = 0) and Pb(Fe{sub 0.4}Sc{sub 0.1}Nb{sub 0.5})O{sub 3} (x = 0.1) are reported to verify the spin state and oxidation state of iron. The lattice distortion due to the radius ratio difference between a Sc{sup 3+} cation and Fe{sup 3+} cation in low spin state is responsible for the structural transformation, which in turn facilitates a B′:B″ cation ordering.« less

8,9-dihydroxy-8,9-dihydrodibenzo[a,l]pyrene is a potent morphological cell-transforming agent in C3H10T(1)/(2)Cl8 mouse embryo fibroblasts in the absence of detectable stable covalent DNA adducts.

PubMed

Nesnow, S; Davis, C; Padgett, W T; Adams, L; Yacopucci, M; King, L C

2000-06-01

The comparative genotoxic effects of racemic trans-8,9-dihydroxy-8, 9-dihydrodibenzo[a,l]pyrene (trans-DB[a,l]P-8,9-diol), the metabolic K-region dihydrodiol of dibenzo[a,l] pyrene (DB[a,l]P) (dibenzo[def, p]chrysene) and DB[a,l]P in transformable mouse embryo C3H10T(1)/(2)Cl8 (C3H10T(1)/(2)) fibroblasts was investigated. The C3H10T(1)/(2) mouse embryo morphological cell-transforming activities of these polycyclic aromatic hydrocarbons (PAHs) were assayed using concentration-response studies. At concentrations of 33 nM and above both trans-DB[a,l]P-8,9-diol and DB[a,l]P produced significant (and similar) numbers of type II and III foci per dish and numbers of dishes with type II and II foci. Concomitant cytotoxicity studies revealed a reduction in colony survival of approximately 25% up to 198 nM for both PAHs. DNA adducts of trans-DB[a,l]P-8,9-diol and DB[a,l]P in C3H10T(1)/(2) cells were analyzed by a (32)P-post-labeling TLC/HPLC method. No adducts were observed in the DNA of C3H10T(1)/(2) cells treated with trans-DB[a, l]P-8,9-diol at concentrations that induced morphological cell transformation. Under the same exposure and chromatographic conditions, DNA adducts of deoxyadenosine and deoxyguanosine derived from the fjord region anti-DB[a,l]P-11,12-diol-13,14-epoxide and syn-DB[a,l]P-11,12-diol-13,14-epoxide were observed in the DNA of DB[a,l]P-treated cells. These results indicate that trans-DB[a,l]P-8, 9-diol has intrinsic genotoxic activity equal to that of DB[a,l]P, based on morphological cell transformation of mouse embryo fibroblasts. The activity of trans-DB[a,l]P-8,9-diol is apparently not associated with the formation of observable stable covalent DNA adducts. These results suggest that under appropriate conditions, trans-DB[a,l]P-8,9-diol may serve as an intermediate in the genotoxicity of DB[a,l]P.

Mutant JAK3 phosphoproteomic profiling predicts synergism between JAK3 inhibitors and MEK/BCL2 inhibitors for the treatment of T-cell acute lymphoblastic leukemia

PubMed Central

Degryse, S; de Bock, C E; Demeyer, S; Govaerts, I; Bornschein, S; Verbeke, D; Jacobs, K; Binos, S; Skerrett-Byrne, D A; Murray, H C; Verrills, N M; Van Vlierberghe, P; Cools, J; Dun, M D

2018-01-01

Mutations in the interleukin-7 receptor (IL7R) or the Janus kinase 3 (JAK3) kinase occur frequently in T-cell acute lymphoblastic leukemia (T-ALL) and both are able to drive cellular transformation and the development of T-ALL in mouse models. However, the signal transduction pathways downstream of JAK3 mutations remain poorly characterized. Here we describe the phosphoproteome downstream of the JAK3(L857Q)/(M511I) activating mutations in transformed Ba/F3 lymphocyte cells. Signaling pathways regulated by JAK3 mutants were assessed following acute inhibition of JAK1/JAK3 using the JAK kinase inhibitors ruxolitinib or tofacitinib. Comprehensive network interrogation using the phosphoproteomic signatures identified significant changes in pathways regulating cell cycle, translation initiation, mitogen-activated protein kinase and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/AKT signaling, RNA metabolism, as well as epigenetic and apoptotic processes. Key regulatory proteins within pathways that showed altered phosphorylation following JAK inhibition were targeted using selumetinib and trametinib (MEK), buparlisib (PI3K) and ABT-199 (BCL2), and found to be synergistic in combination with JAK kinase inhibitors in primary T-ALL samples harboring JAK3 mutations. These data provide the first detailed molecular characterization of the downstream signaling pathways regulated by JAK3 mutations and provide further understanding into the oncogenic processes regulated by constitutive kinase activation aiding in the development of improved combinatorial treatment regimens. PMID:28852199

PKD1/PKCmu promotes alphavbeta3 integrin recycling and delivery to nascent focal adhesions.

PubMed

Woods, Alison J; White, Dominic P; Caswell, Patrick T; Norman, Jim C

2004-07-07

To identify kinases that regulate integrin recycling, we have immunoprecipitated alphavbeta3 integrin from NIH 3T3 fibroblasts in the presence and absence of primaquine (a drug that inhibits receptor recycling and leads to accumulation of integrins in endosomes) and screened for co-precipitating kinases. Primaquine strongly promoted association of alphavbeta3 integrin with PKD1, and fluorescence microscopy indicated that integrin and PKD1 associate at a vesicular compartment that is downstream of a Rab4-dependent transport step. PKD1 association was mediated by the C-terminal region of the beta3 integrin cytodomain, and mutants of beta3 that were unable to recruit PKD1 did not recycle in a PDGF-dependent fashion. Furthermore, suppression of endogenous PKD1 levels by RNAi, or overexpression of catalytically inactive PKD1 inhibited PDGF-dependent recycling of alphavbeta3 from early endosomes to the plasma membrane and blocked recruitment of alphavbeta3 to newly formed focal adhesions during cell spreading. These data indicate that PKD1 influences cell migration by directing vesicular transport of the alphavbeta3 integrin heterodimer.

Kank regulates RhoA-dependent formation of actin stress fibers and cell migration via 14-3-3 in PI3K-Akt signaling.

PubMed

Kakinuma, Naoto; Roy, Badal Chandra; Zhu, Yun; Wang, Yong; Kiyama, Ryoiti

2008-05-05

Phosphoinositide-3 kinase (PI3K)/Akt signaling is activated by growth factors such as insulin and epidermal growth factor (EGF) and regulates several functions such as cell cycling, apoptosis, cell growth, and cell migration. Here, we find that Kank is an Akt substrate located downstream of PI3K and a 14-3-3-binding protein. The interaction between Kank and 14-3-3 is regulated by insulin and EGF and is mediated through phosphorylation of Kank by Akt. In NIH3T3 cells expressing Kank, the amount of actin stress fibers is reduced, and the coexpression of 14-3-3 disrupted this effect. Kank also inhibits insulin-induced cell migration via 14-3-3 binding. Furthermore, Kank inhibits insulin and active Akt-dependent activation of RhoA through binding to 14-3-3. Based on these findings, we hypothesize that Kank negatively regulates the formation of actin stress fibers and cell migration through the inhibition of RhoA activity, which is controlled by binding of Kank to 14-3-3 in PI3K-Akt signaling.

A magnetic trap for living cells suspended in a paramagnetic buffer

NASA Astrophysics Data System (ADS)

Winkleman, Adam; Gudiksen, Katherine L.; Ryan, Declan; Whitesides, George M.; Greenfield, Derek; Prentiss, Mara

2004-09-01

This manuscript describes the fabrication and use of a three-dimensional magnetic trap for diamagnetic objects in an aqueous solution of paramagnetic ions; this trap uses permanent magnets. It demonstrates trapping of polystyrene spheres, and of various types of living cells: mouse fibroblast (NIH-3T3), yeast (Saccharomyces cerevisiae), and algae (Chlamydomonas reinhardtii). For a 40mM solution of gadolinium (III) diethylenetriaminepentaacetic acid (Gd .DTPA) in aqueous buffer, the smallest cell (particle) that could be trapped had a radius of ˜2.5μm. The trapped particle and location of the magnetic trap can be translated in three dimensions by independent manipulation of the permanent magnets. This letter a1so characterizes the biocompatibility of the trapping solution.

The effect of the hydrophilic/hydrophobic ratio of polymeric micelles on their endocytosis pathways into cells.

PubMed

Zhang, Zhao; Qu, Qianqian; Li, Jinrong; Zhou, Shaobing

2013-06-01

Fluorescein isothiocyanate (FITC), a fluorescent probe, is coupled to amphiphilic monomethoxy poly(ethylene glycol)-block-poly(ε-caprolactone) (mPEG-PCL) copolymers. FITC-labeled mPEG-PCL copolymers self-assemble into micelles through the solvent evaporation method. The cellular internalization is examined using fluorescence microscopy on incubation of NIH-3T3 fibroblasts with micelles or free FITC solution. The effect of the hydrophilic/hydrophobic ratio on the endocytosis mechanisms is evaluated by fluorescence microscopy on culturing of human hepatoblastoma cells and human umbilical vein endothelial cells, individually, mixed with the micelles holding the same parameters including micelle size, shape, and surface charges. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Intimate Partner Violence and HIV Risk Behaviors Among Socially Disadvantaged Chilean Women

PubMed Central

Miner, Sarah; Ferrer, Lilian; Cianelli, Rosina; Bernales, Margarita; Cabieses, Báltica

2012-01-01

The objective of this study was to determine if a relationship exists between intimate partner violence (IPV) and HIV risk among socioeconomically disadvantaged Chilean women. A correlational analysis with data from the NIH-funded project, “Testing an HIV/AIDS Prevention Intervention for Chilean Women,” was conducted. Two hundred and sixtyone women were included in this analysis (n = 261). Those women who had experienced any type of IPV in the past 3 months had significantly higher risk for HIV than those who had not (t = −2.016, p < .05). Also a linear trend was found among those women who had experienced more than one type of IPV in the past 3 months and HIV risk. PMID:21486859

Synthesis, Characterization, and Self-Controlled Orthorhombic to Tetragonal Polymorphic Transformation in BaTiO3 Nanoparticles

NASA Astrophysics Data System (ADS)

Ram, S.; Jana, A.; Kundu, T. K.

The phase formation and thermal-induced phase transformation are studied in BaTiO3 nanoparticles. 2 h of heating a polymer precursor at 550°C in air formed a single phase BaTiO3 of 15 nm average crystallite size D. The X-ray diffraction peaks are analyzed assuming a Pnma orthorhombic (o) crystal structure of lattice parameters a = 0.6435 nm, b = 0.5306 nm, and c = 0.8854 nm. The lattice volume V = 0.3023 nm3, with z = 4 formula units, yields a density ρ = 5.124 g/cm3. This is a new polymorph in comparison to well-known Pm3m tetragonal (t) structure, V = 0.0644 nm3 or ρ = 6.016 g/cm3 (z = 1). An o ↦ t transformation appears on heating at temperature as high as 650°C in air. A proposed model explains the transformation above a certain D value in terms of the Gibbs free energy. Unless heating above 750°C, the two phases coexist in a composite structure (D≤27 nm), with as much residual o-phase trace as ~28 vol%. As a function of temperature both the phases decrease in the V values up to 0.2975 and 0.0643 nm3 at 750°C respectively (0.0650 nm3 at 650°C). This is an important parameter for designing useful ferroelectric and other properties in a hybrid composite structure.

NONUNIFORM FOURIER TRANSFORMS FOR RIGID-BODY AND MULTI-DIMENSIONAL ROTATIONAL CORRELATIONS

PubMed Central

BAJAJ, CHANDRAJIT; BAUER, BENEDIKT; BETTADAPURA, RADHAKRISHNA; VOLLRATH, ANTJE

2013-01-01

The task of evaluating correlations is central to computational structural biology. The rigid-body correlation problem seeks the rigid-body transformation (R, t), R ∈ SO(3), t ∈ ℝ3 that maximizes the correlation between a pair of input scalar-valued functions representing molecular structures. Exhaustive solutions to the rigid-body correlation problem take advantage of the fast Fourier transform to achieve a speedup either with respect to the sought translation or rotation. We present PFcorr, a new exhaustive solution, based on the non-equispaced SO(3) Fourier transform, to the rigid-body correlation problem; unlike previous solutions, ours achieves a combination of translational and rotational speedups without requiring equispaced grids. PFcorr can be straightforwardly applied to a variety of problems in protein structure prediction and refinement that involve correlations under rigid-body motions of the protein. Additionally, we show how it applies, along with an appropriate flexibility model, to analogs of the above problems in which the flexibility of the protein is relevant. PMID:24379643

Niclosamide, an anti-helminthic molecule, downregulates the retroviral oncoprotein Tax and pro-survival Bcl-2 proteins in HTLV-1-transformed T lymphocytes

PubMed Central

Chen, Li; Liu, Xin; Belani, Chandra; Cheng, Hua

2015-01-01

Adult T cell leukemia and lymphoma (ATL) is a highly aggressive form of hematological malignancy and is caused by chronic infection of human T cell leukemia virus type 1 (HTLV-1). The viral genome encodes an oncogenic protein, Tax, which plays a key role in transactivating viral gene transcription and in deregulating cellular oncogenic signaling to promote survival, proliferation and transformation of virally infected T cells. Hence, Tax is a desirable therapeutic target, particularly at early stage of HTLV-1-mediated oncogenesis. We here show that niclosamide, an anti-helminthic molecule, induced apoptosis of HTLV-1-transformed T cells. Niclosamide facilitated degradation of the Tax protein in proteasome. Consistent with niclosamide-mediated Tax degradation, this compound inhibited activities of MAPK/ERK1/2 and IκB kinases. In addition, niclosamide downregulated Stat3 and pro-survival Bcl-2 family members such as Mcl-1 and repressed the viral gene transcription of HTLV-1 through induction of Tax degradation. Since Tax, Stat3 and Mcl-1 are crucial molecules for promoting survival and growth of HTLV-1-transformed T cells, our findings demonstrate a novel mechanism of niclosamide in inducing Tax degradation and downregulating various cellular pro-survival molecules, thereby promoting apoptosis of HTLV-1-associated leukemia cells. PMID:26116531

Deformation mode and strain path dependence of martensite phase transformation in a medium manganese TRIP steel

DOE PAGES

Wu, Wei; Wang, Yu-wei; Makrygiannis, Panagiotis; ...

2017-11-06

The martensite phase transformation dependence upon deformation modes and strain paths in a medium manganese (10 wt%) TRIP steel stamped into a T-shape panel was quantified through combination of 3D digital image correlation and synchrotron X-ray diffraction. The T-shape emulates a portion of a common anti-intrusion component. The stamping speed was kept intentionally slow (1 mm/s) so as to avoid excessive heat generation. The steel, which belongs to the third generation advanced high strength steel (3GAHSS) family, was chosen for two reasons: (1) it is two-phase, i.e. austenite and ferrite, with martensite resulting from deformation-induced phase transformation; (2) the 66more » vol.% initial retained austenite volume fraction (RAVF) enabled a thorough examination of the martensite phase transformation at large deformation levels without exhaustion. Strain fields were coupled with measured RAVF values of small specimens extracted from specific locations on a formed T-shape panel. This enabled an exploration of the effects of linear, bilinear, and non-linear strain paths as well as deformation modes such as tension, plane strain, biaxial tension, and equibiaxial tension. Results suggest a significant martensite phase transformation dependence on deformation mode and strain path in the absence of fracture and when martensite phase transformation is unaffected by heat generated during forming. In general, the uniaxial and biaxial tension deformation modes facilitate the martensite phase transformation, while the smallest amount of martensite phase transformation occurs under plane strain. Some discussion as to further application of the experimental methods detailed in this study to other 3GAHSS and the effects of fracture on martensite phase transformation is provided.« less

Deformation mode and strain path dependence of martensite phase transformation in a medium manganese TRIP steel

DOE PAGES

Wu, Wei; Wang, Yu -Wei; Makrygiannis, Panagiotis; ...

2017-11-06

The martensite phase transformation dependence upon deformation modes and strain paths in a medium manganese (10 wt%) TRIP steel stamped into a T-shape panel was quantified through combination of 3D digital image correlation and synchrotron X-ray diffraction. The T-shape emulates a portion of a common anti-intrusion component. The stamping speed was kept intentionally slow (1 mm/s) so as to avoid excessive heat generation. The steel, which belongs to the third generation advanced high strength steel (3GAHSS) family, was chosen for two reasons: (1) it is two-phase, i.e. austenite and ferrite, with martensite resulting from deformation-induced phase transformation; (2) the 66more » vol.% initial retained austenite volume fraction (RAVF) enabled a thorough examination of the martensite phase transformation at large deformation levels without exhaustion. Strain fields were coupled with measured RAVF values of small specimens extracted from specific locations on a formed T-shape panel. This enabled an exploration of the effects of linear, bilinear, and non-linear strain paths as well as deformation modes such as tension, plane strain, biaxial tension, and equibiaxial tension. Results suggest a significant martensite phase transformation dependence on deformation mode and strain path in the absence of fracture and when martensite phase transformation is unaffected by heat generated during forming. In general, the uniaxial and biaxial tension deformation modes facilitate the martensite phase transformation, while the smallest amount of martensite phase transformation occurs under plane strain. In conclusion, some discussion as to further application of the experimental methods detailed in this study to other 3GAHSS and the effects of fracture on martensite phase transformation is provided.« less

Deformation mode and strain path dependence of martensite phase transformation in a medium manganese TRIP steel

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Wei; Wang, Yu -Wei; Makrygiannis, Panagiotis

The martensite phase transformation dependence upon deformation modes and strain paths in a medium manganese (10 wt%) TRIP steel stamped into a T-shape panel was quantified through combination of 3D digital image correlation and synchrotron X-ray diffraction. The T-shape emulates a portion of a common anti-intrusion component. The stamping speed was kept intentionally slow (1 mm/s) so as to avoid excessive heat generation. The steel, which belongs to the third generation advanced high strength steel (3GAHSS) family, was chosen for two reasons: (1) it is two-phase, i.e. austenite and ferrite, with martensite resulting from deformation-induced phase transformation; (2) the 66more » vol.% initial retained austenite volume fraction (RAVF) enabled a thorough examination of the martensite phase transformation at large deformation levels without exhaustion. Strain fields were coupled with measured RAVF values of small specimens extracted from specific locations on a formed T-shape panel. This enabled an exploration of the effects of linear, bilinear, and non-linear strain paths as well as deformation modes such as tension, plane strain, biaxial tension, and equibiaxial tension. Results suggest a significant martensite phase transformation dependence on deformation mode and strain path in the absence of fracture and when martensite phase transformation is unaffected by heat generated during forming. In general, the uniaxial and biaxial tension deformation modes facilitate the martensite phase transformation, while the smallest amount of martensite phase transformation occurs under plane strain. In conclusion, some discussion as to further application of the experimental methods detailed in this study to other 3GAHSS and the effects of fracture on martensite phase transformation is provided.« less

Deformation mode and strain path dependence of martensite phase transformation in a medium manganese TRIP steel

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wu, Wei; Wang, Yu-wei; Makrygiannis, Panagiotis

The martensite phase transformation dependence upon deformation modes and strain paths in a medium manganese (10 wt%) TRIP steel stamped into a T-shape panel was quantified through combination of 3D digital image correlation and synchrotron X-ray diffraction. The T-shape emulates a portion of a common anti-intrusion component. The stamping speed was kept intentionally slow (1 mm/s) so as to avoid excessive heat generation. The steel, which belongs to the third generation advanced high strength steel (3GAHSS) family, was chosen for two reasons: (1) it is two-phase, i.e. austenite and ferrite, with martensite resulting from deformation-induced phase transformation; (2) the 66more » vol.% initial retained austenite volume fraction (RAVF) enabled a thorough examination of the martensite phase transformation at large deformation levels without exhaustion. Strain fields were coupled with measured RAVF values of small specimens extracted from specific locations on a formed T-shape panel. This enabled an exploration of the effects of linear, bilinear, and non-linear strain paths as well as deformation modes such as tension, plane strain, biaxial tension, and equibiaxial tension. Results suggest a significant martensite phase transformation dependence on deformation mode and strain path in the absence of fracture and when martensite phase transformation is unaffected by heat generated during forming. In general, the uniaxial and biaxial tension deformation modes facilitate the martensite phase transformation, while the smallest amount of martensite phase transformation occurs under plane strain. Some discussion as to further application of the experimental methods detailed in this study to other 3GAHSS and the effects of fracture on martensite phase transformation is provided.« less

INDUCTION OF CELL CYCLE ARREST AND APOPTOSIS BY ORMENIS ERIOLEPIS A MORROCAN ENDEMIC PLANT IN VARIOUS HUMAN CANCER CELL LINES.

PubMed

Belayachi, Lamiae; Aceves-Luquero, Clara; Merghoub, Nawel; de Mattos, Silvia Fernández; Amzazi, Saaîd; Villalonga, Priam; Bakri, Youssef

2017-01-01

Ormenis eriolepis Coss (Asteraceae) is an endemic Moroccan subspecies, traditionally named "Hellala" or "Fergoga". It's usually used for its hypoglycemic effect as well as for the treatment of stomacal pain. As far as we know, there is no scientific exploration of anti tumoral activity of Ormenis eriolepis extracts. In this regard, we performed a screening of organic extracts and fractions in a panel of both hematological and solid cancer cell lines, to evaluate the potential in vitro anti tumoral activity and to elucidate the respective mechanisms that may be responsible for growth arrest and cell death induction. The plant was extracted using organic solvents, and four different extracts were screened on Jurkat, Jeko-1, TK-6, LN229, SW620, U2OS, PC-3 and NIH3T3 cells. Cell viability assays revealed that, the IC50 values were (11,63±5,37μg/ml) for Jurkat, (13,33±1,67μg/ml) for Jeko-1, (41,67±1,98μg/ml) for LN229 and (19,31±4,88μg/ml) for PC-3 cells upon treatment with Oe-DF and Oe-HE respectively. Both the fraction and extract exhibited no effects on TK6 and NIH3T3. Cytometry analysis accompanied by DNA damage signaling protein levels monitoring (p-H2A.X), showed that both the Dichloromethane Fraction and Hexanic extract induce DNA double stranded breaks (DSBs) accompanied by cell cycle arrest in G1 (Jurkat, Jeko -1 and LN22) and G2/M (PC-3) phases which is agreed with the caspase activity observed. Additional experiments with selective inhibitors of stress and survival pathways (JNK, MAPK, Rho, p53, and JAK3) indicated that none of these pathways was significantly involved in apoptosis induction. The bioactive compound analysis by CG/MS indicated that the major compounds in Oe-DF were: Linoleic Acid (15,89%), Podophyllotoxin (17,89%) and Quercetin (22,95%). For Oe-HE the major molecules were: Linoleic Acid (9,76%), α-curcumene (7,07%), α-bisabolol (5,49%), Campesterol (4,41%), Stigmasterol (14,08%) and β-sitosterol (7,49%). Our data suggest that bioactive compounds present in Ormenis eriolepis show significant anti proliferative activity inducing cell cycle arrest and cell death operating through apoptosis pathway.

The NIH Science of Behavior Change Program: Transforming the science through a focus on mechanisms of change

PubMed Central

Nielsen, Lisbeth; Riddle, Melissa; King, Jonathan W.; Aklin, Will M.; Chen, Wen; Clark, David; Collier, Elaine; Czajkowski, Susan; Esposito, Layla; Ferrer, Rebecca; Green, Paige; Hunter, Christine; Kehl, Karen; King, Rosalind; Onken, Lisa; Simmons, Janine M.; Stoeckel, Luke; Stoney, Catherine; Tully, Lois; Weber, Wendy

2017-01-01

The goal of the NIH Science of Behavior Change (SOBC) Common Fund Program is to provide the basis for an experimental medicine approach to behavior change that focuses on identifying and measuring the mechanisms that underlie behavioral patterns we are trying to change. This paper frames the development of the program within a discussion of the substantial disease burden in the U.S. attributable to behavioral factors, and details our strategies for breaking down the disease- and condition-focused silos in the behavior change field to accelerate discovery and translation. These principles serve as the foundation for our vision for a unified science of behavior change at the NIH and in the broader research community. PMID:29110885

Discovery of 1-(4-((3-(4-methylpiperazin-1-yl)propyl)amino)benzyl)-5-(trifluoromethyl)pyridin-2(1H)-one, an orally active multi-target agent for the treatment of diabetic nephropathy.

PubMed

Chen, Jun; Peng, Zhangzhe; Lu, Miaomiao; Xiong, Xuan; Chen, Zhuo; Li, Qianbin; Cheng, Zeneng; Jiang, Dejian; Tao, Lijian; Hu, Gaoyun

2018-01-15

Oxidative stress, inflammation and fibrosis can cause irreversible damage on cell structure and function of kidney and are key pathological factors in Diabetic Nephropathy (DN). Therefore, multi-target agents are urgently need for the clinical treatment of DN. Using Pirfenidone as a lead compound and based on the previous research, two novel series (5-trifluoromethyl)-2(1H)-pyridone analogs were designed and synthesized. SAR of (5-trifluoromethyl)-2(1H)-pyridone derivatives containing nitrogen heterocyclic ring have been established for in vitro potency. In addition, compound 8, a novel agent that act on multiple targets of anti-DN with IC 50 of 90μM in NIH3T3 cell lines, t 1/2 of 4.89±1.33h in male rats and LD 50 >2000mg/kg in mice, has been advanced to preclinical studies as an oral treatment for DN. Copyright © 2017 Elsevier Ltd. All rights reserved.

Absence of DNA damage after 60-Hz electromagnetic field exposure combined with ionizing radiation, hydrogen peroxide, or c-Myc overexpression.

PubMed

Jin, Yeung Bae; Choi, Seo-Hyun; Lee, Jae Seon; Kim, Jae-Kyung; Lee, Ju-Woon; Hong, Seung-Cheol; Myung, Sung Ho; Lee, Yun-Sil

2014-03-01

The principal objective of this study was to assess the DNA damage in a normal cell line system after exposure to 60 Hz of extremely low frequency magnetic field (ELF-MF) and particularly in combination with various external factors, via comet assays. NIH3T3 mouse fibroblast cells, WI-38 human lung fibroblast cells, L132 human lung epithelial cells, and MCF10A human mammary gland epithelial cells were exposed for 4 or 16 h to a 60-Hz, 1 mT uniform magnetic field in the presence or absence of ionizing radiation (IR, 1 Gy), H(2)O(2) (50 μM), or c-Myc oncogenic activation. The results obtained showed no significant differences between the cells exposed to ELF-MF alone and the unexposed cells. Moreover, no synergistic or additive effects were observed after 4 or 16 h of pre-exposure to 1 mT ELF-MF or simultaneous exposure to ELF-MF combined with IR, H(2)O(2), or c-Myc activation.

ANALYSES OF THE INTERACTIONS WITHIN BINARY MIXTURES OF CARCINOGENIC PAHS USING MORPHOLOGICAL CELL TRANSFORMATION OF C3H10T1/2CL8 CELLS

EPA Science Inventory

ANALYSES OF THE INTERACTIONS WITHIN BINARY MIXTURES OF CARCINOGENIC PAHS USING MORPHOLOGICAL CELL TRANSFORMATION OF C3HIOT1/2 CL8 CELLS.

Studies of defined mixtures of carcinogenic polycyclic aromatic hydrocarbons (PAH) have identified three major categories of interacti...

Floral-Dip Transformation of Flax (Linum usitatissimum) to Generate Transgenic Progenies with a High Transformation Rate

PubMed Central

Bastaki, Nasmah K.; Cullis, Christopher A.

2014-01-01

Agrobacterium-mediated plant transformation via floral-dip is a widely used technique in the field of plant transformation and has been reported to be successful for many plant species. However, flax (Linum usitatissimum) transformation by floral-dip has not been reported. The goal of this protocol is to establish that Agrobacterium and the floral-dip method can be used to generate transgenic flax. We show that this technique is simple, inexpensive, efficient, and more importantly, gives a higher transformation rate than the current available methods of flax transformation. In summary, inflorescences of flax were dipped in a solution of Agrobacterium carrying a binary vector plasmid (T-DNA fragment plus the Linum Insertion Sequence, LIS-1) for 1 - 2 min. The plants were laid flat on their side for 24 hr. Then, plants were maintained under normal growth conditions until the next treatment. The process of dipping was repeated 2 - 3 times, with approximately 10 - 14 day intervals between dipping. The T1 seeds were collected and germinated on soil. After approximately two weeks, treated progenies were tested by direct PCR; 2 - 3 leaves were used per plant plus the appropriate T-DNA primers. Positive transformants were selected and grown to maturity. The transformation rate was unexpectedly high, with 50 - 60% of the seeds from treated plants being positive transformants. This is a higher transformation rate than those reported for Arabidopsis thaliana and other plant species, using floral-dip transformation. It is also the highest, which has been reported so far, for flax transformation using other methods for transformation. PMID:25549243

Floral-dip transformation of flax (Linum usitatissimum) to generate transgenic progenies with a high transformation rate.

PubMed

Bastaki, Nasmah K; Cullis, Christopher A

2014-12-19

Agrobacterium-mediated plant transformation via floral-dip is a widely used technique in the field of plant transformation and has been reported to be successful for many plant species. However, flax (Linum usitatissimum) transformation by floral-dip has not been reported. The goal of this protocol is to establish that Agrobacterium and the floral-dip method can be used to generate transgenic flax. We show that this technique is simple, inexpensive, efficient, and more importantly, gives a higher transformation rate than the current available methods of flax transformation. In summary, inflorescences of flax were dipped in a solution of Agrobacterium carrying a binary vector plasmid (T-DNA fragment plus the Linum Insertion Sequence, LIS-1) for 1 - 2 min. The plants were laid flat on their side for 24 hr. Then, plants were maintained under normal growth conditions until the next treatment. The process of dipping was repeated 2 - 3 times, with approximately 10 - 14 day intervals between dipping. The T1 seeds were collected and germinated on soil. After approximately two weeks, treated progenies were tested by direct PCR; 2 - 3 leaves were used per plant plus the appropriate T-DNA primers. Positive transformants were selected and grown to maturity. The transformation rate was unexpectedly high, with 50 - 60% of the seeds from treated plants being positive transformants. This is a higher transformation rate than those reported for Arabidopsis thaliana and other plant species, using floral-dip transformation. It is also the highest, which has been reported so far, for flax transformation using other methods for transformation.

TRIP-Br2 promotes oncogenesis in nude mice and is frequently overexpressed in multiple human tumors

PubMed Central

Cheong, Jit Kong; Gunaratnam, Lakshman; Zang, Zhi Jiang; Yang, Christopher M; Sun, Xiaoming; Nasr, Susan L; Sim, Khe Guan; Peh, Bee Keow; Rashid, Suhaimi Bin Abdul; Bonventre, Joseph V; Salto-Tellez, Manuel; Hsu, Stephen I

2009-01-01

Background Members of the TRIP-Br/SERTAD family of mammalian transcriptional coregulators have recently been implicated in E2F-mediated cell cycle progression and tumorigenesis. We, herein, focus on the detailed functional characterization of the least understood member of the TRIP-Br/SERTAD protein family, TRIP-Br2 (SERTAD2). Methods Oncogenic potential of TRIP-Br2 was demonstrated by (1) inoculation of NIH3T3 fibroblasts, which were engineered to stably overexpress ectopic TRIP-Br2, into athymic nude mice for tumor induction and (2) comprehensive immunohistochemical high-throughput screening of TRIP-Br2 protein expression in multiple human tumor cell lines and human tumor tissue microarrays (TMAs). Clinicopathologic analysis was conducted to assess the potential of TRIP-Br2 as a novel prognostic marker of human cancer. RNA interference of TRIP-Br2 expression in HCT-116 colorectal carcinoma cells was performed to determine the potential of TRIP-Br2 as a novel chemotherapeutic drug target. Results Overexpression of TRIP-Br2 is sufficient to transform murine fibroblasts and promotes tumorigenesis in nude mice. The transformed phenotype is characterized by deregulation of the E2F/DP-transcriptional pathway through upregulation of the key E2F-responsive genes CYCLIN E, CYCLIN A2, CDC6 and DHFR. TRIP-Br2 is frequently overexpressed in both cancer cell lines and multiple human tumors. Clinicopathologic correlation indicates that overexpression of TRIP-Br2 in hepatocellular carcinoma is associated with a worse clinical outcome by Kaplan-Meier survival analysis. Small interfering RNA-mediated (siRNA) knockdown of TRIP-Br2 was sufficient to inhibit cell-autonomous growth of HCT-116 cells in vitro. Conclusion This study identifies TRIP-Br2 as a bona-fide protooncogene and supports the potential for TRIP-Br2 as a novel prognostic marker and a chemotherapeutic drug target in human cancer. PMID:19152710

Reduced expression of HSP27 following HAD-B treatment is associated with Her2 downregulation in NIH:OVCAR-3 human ovarian cancer cells.

PubMed

Li, Kuo Chu; Heo, Kyun; Ambade, Nitin; Kim, Min Kyung; Kim, Kyung-Hee; Yoo, Byong Chul; Yoo, Hwa-Seung

2015-09-01

The Korean traditional medicine, HangAmDan (HAD), was developed in 1996 for use as an antitumor agent, and has since been modified to HAD‑B (an altered form of HAD), in order to potentiate its therapeutic effects. In the present study, the effect of HAD‑B on the proliferation and invasion of NIH:OVCAR‑3 and SKOV‑3 human ovarian cancer cell lines was investigated. In addition, the expression of major signal transduction molecules and changes in the proteome in these cells were measured. HAD‑B treatment effectively induced a reduction in the levels of cell proliferation in serum‑free conditioned media. However, unaltered levels of PARP and caspase‑3 indicated that HAD‑B does not reduce proliferation by inducing apoptotic cell death. Fluorescence‑activated cell sorting analysis revealed no significant change in apoptosis following HAD-B treatment. Invasion assay results indicated a reduced rate of invasion following HAD‑B treatment. HAD‑B also influenced the expression of major signal transduction molecules; the phosphorylation of mTOR and AKT was reduced, while that of ERK was increased. Alterations in the proteomes of the two cell lines were investigated following HAD‑B treatment. Among the 9 proteins with differential expression, heat‑shock protein β‑1 (HSP27) was downregulated in NIH:OVCAR‑3 cells treated with HAD‑B. The reduced expression of HSP27 was associated with human epidermal growth factor receptor 2 (Her2) downregulation in these cells. In conclusion, the results of the current proteome assessment suggest that HAD‑B has the potential to suppress the proliferation and invasion of human ovarian cancer cells. HAD‑B treatment of NIH:OVCAR‑3 cells suppressed HSP27 expression and was also associated with Her2 downregulation.

Development of glyphosate-resistant alfalfa (Medicago sativa L.) upon transformation with the GR79Ms gene encoding 5-enolpyruvylshikimate-3-phosphate synthase.

PubMed

Yi, Dengxia; Ma, Lin; Lin, Min; Li, Cong

2018-07-01

The glyphosate-resistant gene, GR79Ms, was successfully introduced into the genome of alfalfa. The transgenic events may serve as novel germplasm resources in alfalfa breeding. Weed competition can reduce the alfalfa yield, generating new alfalfa germplasm with herbicide resistance is essential. To obtain transgenic alfalfa lines with glyphosate resistance, a new synthetic glyphosate-resistant gene GR79Ms encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) was introduced into alfalfa germplasm by Agrobacterium tumefaciens-mediated transformation. In total, 67 transformants were obtained. PCR and Southern blot analyses confirmed that GR79Ms was successfully inserted into the genome of alfalfa. Reverse transcription-PCR and western blot analyses further demonstrated the expression of GR79Ms and its product, GR79Ms EPSPS. Moreover, two homozygous transgenic lines were developed in the T 2 generation by means of molecular-assisted selection. Herbicide tolerance spray tests showed that the transgenic plants T 0 -GR1, T 0 -GR2, T 0 -GR3 and two homozygous lines were able to tolerate fourfold higher commercial usage of glyphosate than non-transgenic plants.

The Numerical Calculation of Traveling Wave Solutions of Nonlinear Parabolic Equations on the Line.

DTIC Science & Technology

1984-02-01

kc(i~~ +’~ which can be rewrittenw W (T is) Ic (sic) - (+ Using the convolution formulas and the expression for the inverse transform of-I (se e.g...2i 2f 1 2 c) + 4f1 1- f 3] 1 We now have: f ) (0,0,4) ; (2.17) f2 3(U) (0,0,0*) x 3 -, (0,,) . 3u ’ The inverse transform of (2.15) is given by: E

Novel revertants of H-ras oncogene-transformed R6-PKC3 cells.

PubMed Central

Krauss, R S; Guadagno, S N; Weinstein, I B

1992-01-01

Rat 6 fibroblasts that overproduce protein kinase C beta 1 (R6-PKC3 cells) are hypersensitive to complete transformation by the T24 H-ras oncogene; yet T24 H-ras-transformed R6-PKC3 cells are killed when exposed to 12-O-tetradecanoylphorbol-13-acetate (TPA) (W.-L. W. Hsiao, G. M. Housey, M. D. Johnson, and I. B. Weinstein, Mol. Cell. Biol. 9:2641-2647, 1989). Treatment of an R6-PKC3 subclone that harbors a T24 H-ras gene under the control of an inducible mouse metallothionein I promoter with ZnSO4 and TPA is extremely cytocidal. This procedure was used to isolate rare revertants that are resistant to this toxicity. Two revertant lines, R-1a and ER-1-2, continue to express very high levels of protein kinase C enzyme activity but, unlike the parental cells, do not grow in soft agar. Furthermore, these revertants are resistant to the induction of anchorage-independent growth by the v-src, v-H-ras, v-raf, and, in the case of the R-1a line, v-fos oncogenes. Both revertant lines, however, retain the ability to undergo morphological alterations when either treated with TPA or infected with a v-H-ras virus, thus dissociating anchorage independence from morphological transformation. The revertant phenotype of both R-1a and ER-1-2 cells is dominant over the transformed phenotype in somatic cell hybridizations. Interestingly, the revertant lines no longer induce the metallothionein I-T24 H-ras construct or the endogenous metallothionein I and II genes in response to three distinct agents: ZnSO4, TPA, and dexamethasone. The reduction in activity of metallothionein promoters seen in these revertants may reflect defects in signal transduction pathways that control the expression of genes mediating specific effects of protein kinase C and certain oncogenes in cell transformation. Images PMID:1535685

NASA Handbook for Nickel-Hydrogen Batteries

NASA Technical Reports Server (NTRS)

Dunlop, James D.; Gopalakrishna, M. Rao; Yi, Thomas Y.

1993-01-01

Nickel-hydrogen (NiH2) batteries are finding more applications in the aerospace energy storage. Since 1983, NiH2 batteries have become the primary energy storage system used for Geosynchronous-Orbit (GEO) Satellites. The first NASA application for NiH2 batteries was the Low Earth Orbit (LEO) Hubble Space Telescope Satellite launched in 1990. The handbook was prepared as a reference book to aid in the application of this technology. That is, to aid in the cell and battery design, procurement, testing, and handling of NiH2 batteries. The design of individual pressure vessel NiH2 cells is covered in Chapter l. LEO and GEO applications and their requirements are discussed in Chapter 2. The design of NiH2 batteries for both GEO and LEO applications is discussed in Chapter 3. Advanced design concepts such as the common pressure vessel and bipolar NiH2 batteries are described in Chapter 4. Performance data are presented in Chapter 5. Storage and handling of the NiH2 cells and batteries are discussed in Chapter 6. Standard test procedures are presented in Chapter 7. Cell and battery procurements are discussed in Chapter 8. Finally, safety procedures are discussed in Chapter 9.

An Experimental Determination of Static Magnetic Fields Induced Noise in Living Systems

NASA Astrophysics Data System (ADS)

Brady, Megan; Laramee, Craig

2013-03-01

Living systems are constantly exposed to static magnetic fields (SMFs) from both natural and man-made sources. Exposures vary in dose and duration ranging from geomagnetic (~50 μT) to residential and industrial (~10s of mT) fields. Efforts to characterize responses to SMFs have yielded conflicting results, showing a dependence on experimental variables used. Here we argue that low to moderate SMF exposure is a sub-threshold perturbation operating below thermal noise, and assays that evaluate statistical characteristics of a single cell may identify responses not consistently found by population averaging approaches. Recent studies of gene expression show that it is a stochastic process capable of producing bursting dynamics. Moreover, theoretical and experimental methods have also been developed to allow quantitative estimates of the associated biophysical parameters. These developments provide a new way to assess responses of living systems to SMFs. In this work, we report on our efforts to use single molecule fluorescence in situ hybridization to assess responses of NIH-3T3 cells to SMF exposure at flux densities ranging from 1 to 440 mT for 48 hours. Results will contribute to determining mechanisms by which SMF exposure influences gene expression.

Cytosolic NADP(+)-dependent isocitrate dehydrogenase status modulates oxidative damage to cells.

PubMed

Lee, Su Min; Koh, Ho-Jin; Park, Dong-Chan; Song, Byoung J; Huh, Tae-Lin; Park, Jeen-Woo

2002-06-01

NADPH is an important cofactor in many biosynthesis pathways and the regeneration of reduced glutathione, critically important in cellular defense against oxidative damage. It is mainly produced by glucose 6-phosphate dehydrogenase (G6PD), malic enzyme, and the cytosolic form of NADP(+)-dependent isocitrate dehydrogenase (IDPc). Little information is available about the role of IDPc in antioxidant defense. In this study we investigated the role of IDPc against cytotoxicity induced by oxidative stress by comparing the relative degree of cellular responses in three different NIH3T3 cells with stable transfection with the cDNA for mouse IDPc in sense and antisense orientations, where IDPc activities were 3-4-fold higher and 35% lower, respectively, than that in the parental cells carrying the vector alone. Although the activities of other antioxidant enzymes, such as superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase, and G6PD, were comparable in all transformed cells, the ratio of GSSG to total glutathione was significantly higher in the cells expressing the lower level of IDPc. This finding indicates that IDPc is essential for the efficient glutathione recycling. Upon transient exposure to increasing concentrations of H(2)O(2) or menadione, an intracellular source of free radicals and reactive oxygen species, the cells with low levels of IDPc became more sensitive to oxidative damage by H(2)O(2) or menadione. Lipid peroxidation, oxidative DNA damage, and intracellular peroxide generation were higher in the cell-line expressing the lower level of IDPc. However, the cells with the highly over-expressed IDPc exhibited enhanced resistance against oxidative stress, compared to the control cells. This study provides direct evidence correlating the activities of IDPc and the maintenance of the cellular redox state, suggesting that IDPc plays an important role in cellular defense against oxidative stress.

Wet-chemical synthesis and properties of CoPt and CoPt3 alloy nanoparticles.

PubMed

Frommen, Christoph; Rösner, Harald; Fenske, Dieter

2002-10-01

Surface-protected, air-stable nanoparticles of CoPt and CoPt3 were prepared by thermal decomposition/reduction of organometallic precursors with a long-chain aliphatic diol, also known as the polyol process. Particles 3 nm in diameter showed ferromagnetic behavior up to 350 K (Hc = 65 Oe at T = 300 K; Hc = 410 Oe at T = 5K) and underwent a disordering-ordering phase transformation after annealing that resulted in an increase in coercivity (Hc = 170 Oe at T = 300 K; Hc = 2000 Oe at T = 5 K).

Characterization of a Diamond Ground Y-TZP and Reversion of the Tetragonal to Monoclinic Transformation.

PubMed

Candido, L M; Fais, Lmg; Ferreira, E B; Antonio, S G; Pinelli, Lap

To characterize the surface of an yttria-stabilized zirconia (Y-TZP) ceramic after diamond grinding in terms of its crystalline phase, morphology, mean roughness (Ra), and wettability as well as to determine a thermal treatment to reverse the resulting tetragonal to monoclinic (t-m) transformation. Y-TZP specimens were distributed into different groups according to the actions (or no action) of grinding and irrigation. Grinding was accomplished using a diamond stone at a low speed. The samples were characterized by x-ray diffraction (XRD), scanning electron microscopy, goniometry, and profilometry. In situ high-temperature XRD was used to determine an annealing temperature to reverse the t-m transformation. Ra was submitted to the Kruskal-Wallis test, followed by the Dunn test (α=0.05). The volume fraction of the monoclinic phase and contact angle were submitted to one-way analysis of variance, followed by the Tukey test (α=0.05). Monoclinic zirconia was observed on the surface of samples after dry and wet grinding with a diamond stone. The volume fraction of the monoclinic phase was smaller on the dry ground samples (3.6%±0.3%) than on the wet ground samples (5.6%±0.3%). High-temperature XRD showed reversion of the t-m phase transformation, which started at 700°C and completed at 800°C in a conventional oven. Grinding with a diamond stone partially transformed the crystalline phase on the surface of a Y-TZP ceramic from tetragonal to monoclinic zirconia while simultaneously increasing the surface roughness and wettability. The t-m transformation could be reversed by heat treatment at 800°C or 900°C for 60 minutes or 1000°C for 30 minutes.

Sonication, Vacuum Infiltration and Thiol Compounds Enhance the Agrobacterium-Mediated Transformation Frequency of Withania somnifera (L.) Dunal

PubMed Central

Sivanandhan, Ganeshan; Kapil Dev, Gnajothi; Theboral, Jeevaraj; Selvaraj, Natesan; Ganapathi, Andy; Manickavasagam, Markandan

2015-01-01

In the present study, we have established a stable transformation protocol via Agrobacterium tumafacines for the pharmaceutically important Withania somnifera. Six day-old nodal explants were used for 3 day co-cultivation with Agrobacterium tumefaciens strain LBA4404 harbouring the vector pCAMIBA2301. Among the different injury treatments, sonication, vacuum infiltration and their combination treatments tested, a vacuum infiltration for 10 min followed by sonication for 10 sec with A. tumefaciens led to a higher transient GUS expression (84% explants expressing GUS at regenerating sites). In order to improve gene integration, thiol compounds were added to co-cultivation medium. A combined treatment of L-Cys at 100 mg/l, STS at 125 mg/l, DTT at 75 mg/l resulted in a higher GUS expression (90%) in the nodal explants. After 3 days of co-cultivation, the explants were subjected to three selection cycles with increasing concentrations of kanamycin [100 to 115 mg/l]. The integration and expression of gusA gene in T0 and T1 transgenic plants were confirmed by polymerase chain reaction (PCR), and Southern blott analysis. These transformed plants (T0 and T1) were fertile and morphologically normal. From the present investigation, we have achieved a higher transformation efficiency of (10%). Withanolides (withanolide A, withanolide B, withanone and withaferin A) contents of transformed plants (T0 and T1) were marginally higher than control plants. PMID:25927703

Analysis of National Institutes of Health Funding in Hand Surgery.

PubMed

Silvestre, Jason; Ruan, Qing Z; Chang, Benjamin

2018-01-01

Federal research dollars help investigators develop biomedical therapies for human diseases. Currently, the state of funding in hand surgery is poorly understood. This study defines the portfolio of National Institutes of Health (NIH) grants awarded in hand surgery. This was a cross-sectional study of hand surgeons in the US. Faculty members of accredited hand surgery fellowships and/or members of the American Society for Surgery of the Hand were queried in the NIH RePORT database for awards obtained during 2005-2015. Of 2317 hand surgeons queried, only 18 obtained an NIH grant (0.8%). Thirty-eight unique grants were identified totaling $42 197 375. R01 awards comprised the majority of funding (78.0%) while K08 awards accounted for 1.1%. The K-to-R transition rate was zero. The National Institute of Arthritis and Musculoskeletal and Skin Disease supported the most funding (65.2%), followed by the National Institute of Neurological Disorders and Stroke (30.8%). There was no statistically significant difference in NIH funding totals with hand surgeon characteristics. Funding supported translational (46.0%), basic science (29.6%), clinical (21.0%), and education-based (3.4%) research. Peripheral nerve (33.3%) and bone and joint disease (30.1%) received the most research funding. Less than 1% of hand surgeons obtain NIH research grants. Of the 2 identified K08 awards, none led to a subsequent R award. Future research should identify barriers to grant procurement to design effective policies to increase NIH funding in hand surgery.

The kinases MEKK2 and MEKK3 regulate transforming growth factor-β-mediated helper T cell differentiation.

PubMed

Chang, Xing; Liu, Fang; Wang, Xiaofang; Lin, Aiping; Zhao, Hongyu; Su, Bing

2011-02-25

Mitogen-activated protein kinases (MAPKs) are key mediators of the T cell receptor (TCR) signals but their roles in T helper (Th) cell differentiation are unclear. Here we showed that the MAPK kinase kinases MEKK2 (encoded by Map3k2) and MEKK3 (encoded by Map3k3) negatively regulated transforming growth factor-β (TGF-β)-mediated Th cell differentiation. Map3k2(-/-)Map3k3(Lck-Cre/-) mice showed an abnormal accumulation of regulatory T (Treg) and Th17 cells in the periphery, consistent with Map3k2(-/-)Map3k3(Lck-Cre/-) naive CD4(+) T cells' differentiation into Treg and Th17 cells with a higher frequency than wild-type (WT) cells after TGF-β stimulation in vitro. In addition, Map3k2(-/-)Map3k3(Lck-Cre/-) mice developed more severe experimental autoimmune encephalomyelitis. Map3k2(-/-)Map3k3(Lck-Cre/-) T cells exhibited impaired phosphorylation of SMAD2 and SMAD3 proteins at their linker regions, which negatively regulated the TGF-β responses in T cells. Thus, the crosstalk between TCR-induced MAPK and the TGF-β signaling pathways is important in regulating Th cell differentiation. Copyright © 2011 Elsevier Inc. All rights reserved.

Deterministic Integration of Biological and Soft Materials onto 3D Microscale Cellular Frameworks

PubMed Central

McCracken, Joselle M.; Xu, Sheng; Badea, Adina; Jang, Kyung-In; Yan, Zheng; Wetzel, David J.; Nan, Kewang; Lin, Qing; Han, Mengdi; Anderson, Mikayla A.; Lee, Jung Woo; Wei, Zijun; Pharr, Matt; Wang, Renhan; Su, Jessica; Rubakhin, Stanislav S.; Sweedler, Jonathan V.

2018-01-01

Complex 3D organizations of materials represent ubiquitous structural motifs found in the most sophisticated forms of matter, the most notable of which are in life-sustaining hierarchical structures found in biology, but where simpler examples also exist as dense multilayered constructs in high-performance electronics. Each class of system evinces specific enabling forms of assembly to establish their functional organization at length scales not dissimilar to tissue-level constructs. This study describes materials and means of assembly that extend and join these disparate systems—schemes for the functional integration of soft and biological materials with synthetic 3D microscale, open frameworks that can leverage the most advanced forms of multilayer electronic technologies, including device-grade semiconductors such as monocrystalline silicon. Cellular migration behaviors, temporal dependencies of their growth, and contact guidance cues provided by the nonplanarity of these frameworks illustrate design criteria useful for their functional integration with living matter (e.g., NIH 3T3 fibroblast and primary rat dorsal root ganglion cell cultures). PMID:29552634

Apple derived cellulose scaffolds for 3D mammalian cell culture.

PubMed

Modulevsky, Daniel J; Lefebvre, Cory; Haase, Kristina; Al-Rekabi, Zeinab; Pelling, Andrew E

2014-01-01

There are numerous approaches for producing natural and synthetic 3D scaffolds that support the proliferation of mammalian cells. 3D scaffolds better represent the natural cellular microenvironment and have many potential applications in vitro and in vivo. Here, we demonstrate that 3D cellulose scaffolds produced by decellularizing apple hypanthium tissue can be employed for in vitro 3D culture of NIH3T3 fibroblasts, mouse C2C12 muscle myoblasts and human HeLa epithelial cells. We show that these cells can adhere, invade and proliferate in the cellulose scaffolds. In addition, biochemical functionalization or chemical cross-linking can be employed to control the surface biochemistry and/or mechanical properties of the scaffold. The cells retain high viability even after 12 continuous weeks of culture and can achieve cell densities comparable with other natural and synthetic scaffold materials. Apple derived cellulose scaffolds are easily produced, inexpensive and originate from a renewable source. Taken together, these results demonstrate that naturally derived cellulose scaffolds offer a complementary approach to existing techniques for the in vitro culture of mammalian cells in a 3D environment.

Apple Derived Cellulose Scaffolds for 3D Mammalian Cell Culture

PubMed Central

Modulevsky, Daniel J.; Lefebvre, Cory; Haase, Kristina; Al-Rekabi, Zeinab; Pelling, Andrew E.

2014-01-01

There are numerous approaches for producing natural and synthetic 3D scaffolds that support the proliferation of mammalian cells. 3D scaffolds better represent the natural cellular microenvironment and have many potential applications in vitro and in vivo. Here, we demonstrate that 3D cellulose scaffolds produced by decellularizing apple hypanthium tissue can be employed for in vitro 3D culture of NIH3T3 fibroblasts, mouse C2C12 muscle myoblasts and human HeLa epithelial cells. We show that these cells can adhere, invade and proliferate in the cellulose scaffolds. In addition, biochemical functionalization or chemical cross-linking can be employed to control the surface biochemistry and/or mechanical properties of the scaffold. The cells retain high viability even after 12 continuous weeks of culture and can achieve cell densities comparable with other natural and synthetic scaffold materials. Apple derived cellulose scaffolds are easily produced, inexpensive and originate from a renewable source. Taken together, these results demonstrate that naturally derived cellulose scaffolds offer a complementary approach to existing techniques for the in vitro culture of mammalian cells in a 3D environment. PMID:24842603

Magnetoresistance of layered structures with alternating in-plane and perpendicular anisotropies

NASA Astrophysics Data System (ADS)

Stobiecki, F.; Szymański, B.; Luciński, T.; Dubowik, J.; Urbaniak, M.; Röll, K.

2004-11-01

The magnetic properties of (Ni83Fe17/Au/Co/Au) multilayers with different thickness of Au (0.5⩽tAu⩽3 nm) and Co (0.2⩽tCo⩽1.5 nm) layers were characterized. For tAu⩾1.5 nm independent magnetization reversal of Ni-Fe and Co was found. Increase of tCo for (Ni83Fe17-2 nm/Au-3 nm/Co-tCo/Au-3 nm)15 multilayers results in a sequence of transformations in the magnetic properties due to changes in Co microstructure: from superparamagnetic to ferromagnetic clusters at tCo≈0.3 nm, from discontinuous to continuous layers at tCo≈0.6 nm and from perpendicular to in-plane anisotropy at tCo≈1.2 nm.

New mechanism for toughening ceramic materials. Final report, 15 March 1989-15 July 1993

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cutler, R.A.; Virkar, A.V.; Cross, L.E.

Ferroelastic toughening was identified as a viable mechanism for toughening ceramics. Domain structure and domain switching was identified by x-ray diffraction, transmission optical microscopy, and transmission electron microscopy in zirconia, lead zirconate titanate and gadolinium molybdata. Switching in compression was observed at stresses greater than 600 MPa and at 400 MPa in tension for polycrystalline t'-zirconia. Domain switching contributes to toughness, as evidenced by data for monoclinic zirconia, t'-zirconia, PZT and GMO. The magnitude of toughening varied between 0.6 MPa.ml/2 for GMO to 2-6 MPa-ml/2 for zirconia. Polycrystalline monoclinic and t'-zirconias, which showed no transformation toughening, had similar toughness valuesmore » as Y-TZP which exhibits transformation. Coarse-grained monoclinic and tetragonal (t') zirconia samples could be cooled to room temperature for mechanical property evaluation since fine domain size, not grain size, controlled transformation for t'-zirconia and minimized stress for m-ZrO2. LnAlO3, LnNbO4, and LnCrO3 were among the materials identified as high temperature ferroelastics.« less

National Institutes of Health classification for chronic graft-versus-host disease predicts outcome of allo-hematopoietic stem cell transplant after fludarabine-busulfan-antithymocyte globulin conditioning regimen.

PubMed

Saillard, Colombe; Crocchiolo, Roberto; Furst, Sabine; El-Cheikh, Jean; Castagna, Luca; Signori, Alessio; Oudin, Claire; Faucher, Catherine; Lemarie, Claude; Chabannon, Christian; Granata, Angela; Blaise, Didier

2014-05-01

Abstract In 2005, the National Institutes of Health (NIH) proposed standard criteria for diagnosis, organ scoring and global assessment of chronic graft-versus-host disease (cGvHD) severity. We retrospectively reclassified cGvHD with NIH criteria in a monocentric cohort of 130 consecutive adult patients with hematological malignancies presenting cGvHD after receiving allo-hematopoietic stem cell transplant (HSCT) with a fludarabine-busulfan-antithymocyte globulin (ATG) conditioning regimen, among 313 consecutive HSCT recipients. We compared NIH and Seattle classifications to correlate severity and outcome. The follow up range was effectively 2-120 months. Forty-four percent developed Seattle-defined cGvHD (22% limited, 78% extensive forms). Using NIH criteria, there were 23%, 40% and 37% mild, moderate and severe forms, respectively, and 58%, 32% and 8% classic cGvHD, late acute GvHD and overlap syndrome. Five-year overall survival was 55% (49-61), and cumulative incidences of non-relapse mortality (NRM) and relapse/progression at 2 years were 19% (14-23) and 19% (14-24). NIH mild and moderate forms were associated with better survival compared to severe cGvHD (hazard ratio [HR] = 3.28, 95% confidence interval [CI]: 1.38-7.82, p = 0.007), due to higher NRM among patients with severe cGvHD (HR = 3.04, 95% CI: 1.05-8.78, p = 0.04) but comparable relapse risk (p = NS). In conclusion, the NIH classification appears to be more accurate in predicting outcome mostly by the reclassification of old-defined extensive forms into NIH-defined moderate or severe.

Optimization of yield in magnetic cell separations using nickel nanowires of different lengths.

PubMed

Hultgren, Anne; Tanase, Monica; Felton, Edward J; Bhadriraju, Kiran; Salem, Aliasger K; Chen, Christopher S; Reich, Daniel H

2005-01-01

Ferromagnetic nanowires are shown to perform both high yield and high purity single-step cell separations on cultures of NIH-3T3 mouse fibroblast cells. The nanowires are made by electrochemical deposition in nanoporous templates, permitting detailed control of their chemical and physical properties. When added to fibroblast cell cultures, the nanowires are internalized by the cells via the integrin-mediated adhesion pathway. The effectiveness of magnetic cell separations using Ni nanowires 350 nm in diameter and 5-35 micrometers long in field gradients of 40 T/m was compared to commercially available superparamagnetic beads. The percent yield of the separated populations is found to be optimized when the length of the nanowire is matched to the diameter of the cells in the culture. Magnetic cell separations performed under these conditions achieve 80% purity and 85% yield, a 4-fold increase over the beads. This effect is shown to be robust when the diameter of the cell is changed within the same cell line using mitomycin-C.

78 FR 15021 - Office of the Director, National Institutes of Health; Notice of Meeting

Federal Register 2010, 2011, 2012, 2013, 2014

2013-03-08

... Director, NIH, Office of Science Policy, 6705 Rockledge Drive, Suite 750, Bethesda, MD 20892, (Telephone Conference Call). Contact Person: Juanita Marner, Office of Science Policy, Office of the Director, NIH... Policy. [FR Doc. 2013-05370 Filed 3-7-13; 8:45 am] BILLING CODE 4140-01-P ...

Identifying DNA Methylation Features that Underlie Prostate Cancer Disparities

DTIC Science & Technology

2015-10-01

variant analysis U01 HG007601 (Pierce, B.) NIH/NHGRI 8/1/2014 - 5/31/2017 Direct Cost: $201,814 1 Cal. Month Telomere length and chromosomal...Month Telomere length and chromosomal instability across various tissue types R01 ES023834 (Pierce, B.) NIH/NCI 4/1/14 - 3/31/18 Direct Cost

Effects of the amphiphilic peptides mastoparan and adenoregulin on receptor binding, G proteins, phosphoinositide breakdown, cyclic AMP generation, and calcium influx.

PubMed

Shin, Y; Moni, R W; Lueders, J E; Daly, J W

1994-04-01

1. The amphiphilic peptide mastoparan is known to affect phosphoinositide breakdown, calcium influx, and exocytosis of hormones and neurotransmitters and to stimulate the GTPase activity of guanine nucleotide-binding regulatory proteins. Another amphiphilic peptide, adenoregulin was recently identified based on stimulation of agonist binding to A1-adenosine receptors. 2. A comparison of the effects of mastoparan and adenoregulin reveals that these peptides share many properties. Both stimulate binding of agonists to receptors and binding of GTP gamma S to G proteins in brain membranes. The enhanced guanyl nucleotide exchange may be responsible for the complete conversion of receptors to a high-affinity state, complexed with guanyl nucleotide-free G proteins. 3. Both peptides increase phosphoinositide breakdown in NIH 3T3 fibroblasts. Pertussis toxin partially inhibits the phosphoinositide breakdown elicited by mastoparan but has no effect on the response to adenoregulin. N-Ethylmaleimide inhibits the response to both peptides. 4. In permeabilized 3T3 cells, both adenoregulin and mastoparan inhibit GTP gamma S-stimulated phosphoinositide breakdown. Mastoparan slightly increases basal cyclic AMP levels in cultured cells, followed at higher concentrations by an inhibition, while adenoregulin has minimal effects. 5. Both peptides increase calcium influx in cultured cells and release of norepinephrine in pheochromocytoma PC12 cells. The calcium influx elicited by the peptides in 3T3 cells is not markedly altered by N-ethylmaleimide. 6. Multiple sites of action appear likely to underlie the effects of mastoparan/adenoregulin on receptors, G proteins, phospholipase C, and calcium.

Objective scoring of transformed foci in BALB/c 3T3 cell transformation assay by statistical image descriptors.

PubMed

Urani, C; Corvi, R; Callegaro, G; Stefanini, F M

2013-09-01

In vitro cell transformation assays (CTAs) have been shown to model important stages of in vivo carcinogenesis and have the potential to predict carcinogenicity in humans. Advantages of CTAs are their ability of revealing both genotoxic and non-genotoxic carcinogens while reducing both experimental costs and the number of animals used. The endpoint of the CTA is foci formation, and requires classification under light microscopy based on morphology. Thus current limitations for the wide adoption of the assay partially depend on a fair degree of subjectivity in foci scoring. An objective evaluation may be obtained after separating foci from background monolayer in the digital image, and quantifying values of statistical descriptors which are selected to capture eye-scored morphological features. The aim of this study was to develop statistical descriptors to be applied to transformed foci of BALB/c 3T3, which cover foci size, multilayering and invasive cell growth into the background monolayer. Proposed descriptors were applied to a database of 407 foci images to explore the numerical features, and to illustrate open problems and potential solutions. Copyright © 2013 Elsevier Ltd. All rights reserved.

Mammalian cell-transforming potential of traffic-linked ultrafine particulate matter PM0.056 in urban roadside atmosphere.

PubMed

Verma, Mukesh K; Poojan, Shiv; Sultana, Sarwat; Kumar, Sushil

2014-09-01

We examined the clastogenic and cell-transforming potential of ultrafine particulate matter fraction PM0.056 of urban ambient aerosol using mammalian cells. PM1.0, PM0.56 and PM0.056 fractions were sampled from roadside atmosphere of an urban area using the cascade impactor MOUDI-NR-110. The potential to induce cytotoxicity, DNA damage and micronuclei formation was examined at the test concentrations of 3, 6, 12.5, 25, 50 and 100 μg/ml using the 3-4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, the plasmid relaxation assay and the C3H10T1/2 (10T1/2) cells. The cell-transforming potential was investigated in vitro using 10T1/2 cell transformation assay and the soft agar assay. PM1, PM0.56 and PM0.056 fractions were found to be toxic in dose-dependent manner. These induced cytotoxicity at five test concentrations, the ultrafine particle fraction PM0.056 showed greater cytotoxic potential. PM0.056 induced micronucleus formation in 10T1/2 cells. The effect was statistically significant. The DNA-damaging potential was measured in a plasmid relaxation assay. Both fine and ultrafine particle fraction PM0.56 and PM0.056 displayed greater effect as compared to larger PM1 fraction. DNA damage was found to be dependent on particulate matter intrinsic pro-oxidant chemicals. The ability of the ultrafine particle fraction PM0.056 to induce morphological cell transformation was demonstrated by significant and dose-dependent increases in type III focus formation by morphologically transformed cells in culture flasks and their clonal expansion in soft agar. It is concluded that the traffic-linked ultrafine particle fraction PM0.056 in the atmosphere by the roadside of an urban area is clastogenic and able to induce morphological transformation of mammalian cells. © The Author 2014. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Glypican-3 induces oncogenicity by preventing IGF-1R degradation, a process that can be blocked by Grb10

PubMed Central

Cheng, Wei; Huang, Po-Chun; Chao, Hsiao-Mei; Jeng, Yung-Ming; Hsu, Hey-Chi; Pan, Hung-Wei; Hwu, Wuh-Liang; Lee, Yu-May

2017-01-01

Hepatocellular carcinoma (HCC) is the most common primary liver malignancy and is a major cause of cancer-related death worldwide. Previously, we demonstrated that glypican-3 (GPC3) is highly expressed in HCC, and that GPC3 induces oncogenicity and promotes the growth of cancer cells through IGF-1 receptor (IGF-1R). In the present study, we investigated the mechanisms of GPC3-mediated enhancement of IGF-1R signaling. We demonstrated that GPC3 decreased IGF-1-induced IGF-1R ubiquitination and degradation and increased c-Myc protein levels. GPC3 bound to Grb10, a mediator of ligand-induced receptor ubiquitination, and the overexpression of Grb10 blocked GPC3-enhanced IGF-1-induced ERK phosphorylation. GPC3 promoted the growth of NIH3T3 and PLC-PRF-5 cells in serum-free medium but did not promote the growth of IGF-1R negative R- cells. Grb10 overexpression decreased GPC3-promoted cell growth. Therefore, the present study elucidates the mechanisms of GPC3-induced oncogenicity, which may highlight new strategies for the treatment of HCC. PMID:29113314

[Chromosomal large fragment deletion induced by CRISPR/Cas9 gene editing system].

PubMed

Cheng, L H; Liu, Y; Niu, T

2017-05-14

Objective: Using CRISPR-Cas9 gene editing technology to achieve a number of genes co-deletion on the same chromosome. Methods: CRISPR-Cas9 lentiviral plasmid that could induce deletion of Aloxe3-Alox12b-Alox8 cluster genes located on mouse 11B3 chromosome was constructed via molecular clone. HEK293T cells were transfected to package lentivirus of CRISPR or Cas9 cDNA, then mouse NIH3T3 cells were infected by lentivirus and genomic DNA of these cells was extracted. The deleted fragment was amplified by PCR, TA clone, Sanger sequencing and other techniques were used to confirm the deletion of Aloxe3-Alox12b-Alox8 cluster genes. Results: The CRISPR-Cas9 lentiviral plasmid, which could induce deletion of Aloxe3-Alox12b-Alox8 cluster genes, was successfully constructed. Deletion of target chromosome fragment (Aloxe3-Alox12b-Alox8 cluster genes) was verified by PCR. The deletion of Aloxe3-Alox12b-Alox8 cluster genes was affirmed by TA clone, Sanger sequencing, and the breakpoint junctions of the CRISPR-Cas9 system mediate cutting events were accurately recombined, insertion mutation did not occur between two cleavage sites at all. Conclusion: Large fragment deletion of Aloxe3-Alox12b-Alox8 cluster genes located on mouse chromosome 11B3 was successfully induced by CRISPR-Cas9 gene editing system.

Understanding the NIH review process: a brief guide to writing grant proposals in neurotoxicology.

PubMed

Audesirk, G; Burbacher, T; Guilarte, T R; Laughlin, N K; Lopachin, R; Suszkiw, J; Tilson, H

1999-02-01

During the past two years, the National Institutes of Health have made significant changes in the review process for investigator-initiated research grant applications in neurotoxicology. First, study sections that formerly dealt with toxicology and alcohol, respectively, have been merged. Neurotoxicology grant applications are now reviewed by ALTX-3, a study section in which the majority of members have expertise in the neuronal, biochemical or behavioral effects of alcohol, but usually not other neurotoxicants. Second, the NIH has instituted new review criteria, in which significance, approach, innovation, investigator expertise, and research environment must all be explicitly addressed by the reviews. In this article, past and present members of the ALTX-3 study section describe the NIH review process, with emphasis on how neurotoxicology applications are handled, and provide guidelines for preparing competitive applications.

The NIH Science of Behavior Change Program: Transforming the science through a focus on mechanisms of change.

PubMed

Nielsen, Lisbeth; Riddle, Melissa; King, Jonathan W; Aklin, Will M; Chen, Wen; Clark, David; Collier, Elaine; Czajkowski, Susan; Esposito, Layla; Ferrer, Rebecca; Green, Paige; Hunter, Christine; Kehl, Karen; King, Rosalind; Onken, Lisa; Simmons, Janine M; Stoeckel, Luke; Stoney, Catherine; Tully, Lois; Weber, Wendy

2018-02-01

The goal of the NIH Science of Behavior Change (SOBC) Common Fund Program is to provide the basis for an experimental medicine approach to behavior change that focuses on identifying and measuring the mechanisms that underlie behavioral patterns we are trying to change. This paper frames the development of the program within a discussion of the substantial disease burden in the U.S. attributable to behavioral factors, and details our strategies for breaking down the disease- and condition-focused silos in the behavior change field to accelerate discovery and translation. These principles serve as the foundation for our vision for a unified science of behavior change at the NIH and in the broader research community. Copyright © 2017. Published by Elsevier Ltd.

European Workshop on Bacterial Protein Toxins (4th) Held in Urbino, Italy on July 3-6, 1989

DTIC Science & Technology

1990-02-28

is described in Table Ill. Two Epstein - Barr virus transformed B cell lines which had been pulsed with intact t.t. present the antigen equally well to... Epstein - Barr virus transformed human B lymphocytes (B-LCL) homozygous for HLA-DRI. These cells were chosen because they were hcmozygous, well characterized...using RJ 2.2.5 cells (16). These cells, like the MAJA line, are human B-lymphoblastoid cells transformed by Epstein - Barr virus , except that they do

Identification of Scattering Mechanisms from Measured Impulse Response Signatures of Several Conducting Objects.

DTIC Science & Technology

1984-02-01

conducting sphere 35 compared to inverse transform of exact solution. 4-5. Measured impulse response of a conducting 2:1 right 37 circular cylinder with...frequency domain. This is equivalent to multiplication in the time domain by the inverse transform of w(n), which is shown in Figure 3-1 for N=15. The...equivalent pulse width from 0.066 T for the rectangular window to 0.10 T for the Hanning window. The inverse transform of the Hanning window is shown

Local mechanical response of cells to the controlled rotation of magnetic nanorods.

PubMed

Castillo, Matias; Ebensperger, Roberto; Wirtz, Denis; Walczak, Magdalena; Hurtado, Daniel E; Celedon, Alfredo

2014-11-01

The mechanical response of the cytoplasm was investigated by the intracellular implantation of magnetic nanorods and exposure to low-frequency rotatory magnetic fields. Nanorods (Pt-Ni, ∼200 nm diameter) fabricated by electrodeposition in templates of porous alumina with lengths of approximately 2 and 5 µm were inserted into NIH/3T3 fibroblasts and manipulated with a rotational magnetic field. Nanorod rotation was observed only for torques greater than 3.0 × 10(-16) Nm, suggesting a Bingham-type behavior of the cytoplasm. Higher torques produced considerable deformation of the intracellular material. The cell nucleus and cell membrane were significantly deformed by nanorods actuated by 4.5 × 10(-15) Nm torques. Our results demonstrate that nanorods under magnetic fields are an effective tool to mechanically probe the intracellular environment. We envision that our findings may contribute to the noninvasive and direct mechanical characterization of the cytoplasm. © 2014 Wiley Periodicals, Inc.

Local mechanical response of cells to the controlled rotation of magnetic nanorods

PubMed Central

Castillo, Matias; Ebensperger, Roberto; Wirtz, Denis; Walczak, Magdalena; Hurtado, Daniel E.; Celedon, Alfredo

2015-01-01

The mechanical response of the cytoplasm was investigated by the intracellular implantation of magnetic nanorods and exposure to low-frequency rotatory magnetic fields. Nanorods (Pt-Ni, ~200 nm diameter) fabricated by electrodeposition in templates of porous alumina with lengths of approximately 2 and 5 μm were inserted into NIH/ 3T3 fibroblasts and manipulated with a rotational magnetic field. Nanorod rotation was observed only for torques greater than 3.0 × 10−16 Nm, suggesting a Bingham-type behavior of the cytoplasm. Higher torques produced considerable deformation of the intracellular material. The cell nucleus and cell membrane were significantly deformed by nanorods actuated by 4.5 × 10−15 Nm torques. Our results demonstrate that nanorods under magnetic fields are an effective tool to mechanically probe the intracellular environment. We envision that our findings may contribute to the noninvasive and direct mechanical characterization of the cytoplasm. PMID:24700696

Cell fusion through a microslit between adhered cells and observation of their nuclear behavior.

PubMed

Wada, Ken-Ichi; Hosokawa, Kazuo; Kondo, Eitaro; Ito, Yoshihiro; Maeda, Mizuo

2014-07-01

This paper describes a novel cell fusion method which induces cell fusion between adhered cells through a microslit for preventing nuclear mixing. For this purpose, a microfluidic device which had ∼ 100 cell pairing structures (CPSs) making cell pairs through microslits with 2.1 ± 0.3 µm width was fabricated. After trapping NIH3T3 cells with hydrodynamic forces at the CPSs, the cells were fused through the microslit by the Sendai virus envelope method. With following timelapse observation, we discovered that the spread cells were much less susceptible to nuclear migration passing through the microslit compared with round cells, and that cytoplasmic fraction containing mitochondria was transferred through the microslit without nuclear mixing. These findings will provide an effective method for cell fusion without nuclear mixing, and will lead to an efficient method for reprograming and transdifferentiation of target cells toward regenerative medicine. © 2014 Wiley Periodicals, Inc.

Preparation and characterization of malonic acid cross-linked chitosan and collagen 3D scaffolds: an approach on non-covalent interactions.

PubMed

Mitra, Tapas; Sailakshmi, G; Gnanamani, A; Mandal, A B

2012-05-01

The present study emphasizes the influence of non-covalent interactions on the mechanical and thermal properties of the scaffolds of chitosan/collagen origin. Malonic acid (MA), a bifuncitonal diacid was chosen to offer non-covalent cross-linking. Three dimensional scaffolds was prepared using chitosan at 1.0% (w/v) and MA at 0.2% (w/v), similarly collagen 0.5% (w/v) and MA 0.2% (w/v) and characterized. Results on FT-IR, TGA, DSC, SEM and mechanical properties (tensile strength, stiffness, Young's modulus, etc.) assessment demonstrated the existence of non-covalent interaction between MA and chitosan/collagen, which offered flexibility and high strength to the scaffolds suitable for tissue engineering research. Studies using NIH 3T3 fibroblast cells suggested biocompatibility nature of the scaffolds. Docking simulation study further supports the intermolecular hydrogen bonding interactions between MA and chitosan/collagen.

Niclosamide, an anti-helminthic molecule, downregulates the retroviral oncoprotein Tax and pro-survival Bcl-2 proteins in HTLV-1-transformed T lymphocytes.

PubMed

Xiang, Di; Yuan, Yunsheng; Chen, Li; Liu, Xin; Belani, Chandra; Cheng, Hua

2015-08-14

Adult T cell leukemia and lymphoma (ATL) is a highly aggressive form of hematological malignancy and is caused by chronic infection of human T cell leukemia virus type 1 (HTLV-1). The viral genome encodes an oncogenic protein, Tax, which plays a key role in transactivating viral gene transcription and in deregulating cellular oncogenic signaling to promote survival, proliferation and transformation of virally infected T cells. Hence, Tax is a desirable therapeutic target, particularly at early stage of HTLV-1-mediated oncogenesis. We here show that niclosamide, an anti-helminthic molecule, induced apoptosis of HTLV-1-transformed T cells. Niclosamide facilitated degradation of the Tax protein in proteasome. Consistent with niclosamide-mediated Tax degradation, this compound inhibited activities of MAPK/ERK1/2 and IκB kinases. In addition, niclosamide downregulated Stat3 and pro-survival Bcl-2 family members such as Mcl-1 and repressed the viral gene transcription of HTLV-1 through induction of Tax degradation. Since Tax, Stat3 and Mcl-1 are crucial molecules for promoting survival and growth of HTLV-1-transformed T cells, our findings demonstrate a novel mechanism of niclosamide in inducing Tax degradation and downregulating various cellular pro-survival molecules, thereby promoting apoptosis of HTLV-1-associated leukemia cells. Copyright © 2015 Elsevier Inc. All rights reserved.

Health Literacy: Readability of ACC/AHA Online Patient Education Material.

PubMed

Kapoor, Karan; George, Praveen; Evans, Matthew C; Miller, Weldon J; Liu, Stanley S

To determine whether the online patient education material offered by the American College of Cardiology (ACC) and the American Heart Association (AHA) is written at a higher level than the 6th-7th grade level recommended by the National Institute of Health (NIH). Online patient education material from each website was subjected to reading grade level (RGL) analysis using the Readability Studio Professional Edition. One-sample t testing was used to compare the mean RGLs obtained from 8 formulas to the NIH-recommended 6.5 grade level and 8th grade national mean. In total, 372 articles from the ACC website and 82 from the AHA were studied. Mean (±SD) RGLs for the 454 articles were 9.6 ± 2.1, 11.2 ± 2.1, 11.9 ± 1.6, 10.8 ± 1.6, 9.7 ± 2.1, 10.8 ± 0.8, 10.5 ± 2.6, and 11.7 ± 3.5 according to the Flesch-Kincaid grade level (FKGL), Simple Measure of Gobbledygook (SMOG Index), Coleman-Liau Index (CLI), Gunning-Fog Index (GFI), New Dale-Chall reading level formula (NDC), FORCAST, Raygor Readability Estimate (RRE), and Fry Graph (Fry), respectively. All analyzed articles had significantly higher RGLs than both the NIH-recommended grade level of 6.5 and the national mean grade level of 8 (p < 0.00625). Patient education material provided on the ACC and AHA websites is written above the NIH-recommended 6.5 grade level and 8th grade national mean reading level. Additional studies are required to demonstrate whether lowering the RGL of this material improves outcomes among patients with cardiovascular disease. © 2017 S. Karger AG, Basel.

Preliminary experience with a novel method of three-dimensional co-registration of prostate cancer digital histology and in vivo multiparametric MRI.

PubMed

Orczyk, C; Rusinek, H; Rosenkrantz, A B; Mikheev, A; Deng, F-M; Melamed, J; Taneja, S S

2013-12-01

To assess a novel method of three-dimensional (3D) co-registration of prostate cancer digital histology and in-vivo multiparametric magnetic resonance imaging (mpMRI) image sets for clinical usefulness. A software platform was developed to achieve 3D co-registration. This software was prospectively applied to three patients who underwent radical prostatectomy. Data comprised in-vivo mpMRI [T2-weighted, dynamic contrast-enhanced weighted images (DCE); apparent diffusion coefficient (ADC)], ex-vivo T2-weighted imaging, 3D-rebuilt pathological specimen, and digital histology. Internal landmarks from zonal anatomy served as reference points for assessing co-registration accuracy and precision. Applying a method of deformable transformation based on 22 internal landmarks, a 1.6 mm accuracy was reached to align T2-weighted images and the 3D-rebuilt pathological specimen, an improvement over rigid transformation of 32% (p = 0.003). The 22 zonal anatomy landmarks were more accurately mapped using deformable transformation than rigid transformation (p = 0.0008). An automatic method based on mutual information, enabled automation of the process and to include perfusion and diffusion MRI images. Evaluation of co-registration accuracy using the volume overlap index (Dice index) met clinically relevant requirements, ranging from 0.81-0.96 for sequences tested. Ex-vivo images of the specimen did not significantly improve co-registration accuracy. This preliminary analysis suggests that deformable transformation based on zonal anatomy landmarks is accurate in the co-registration of mpMRI and histology. Including diffusion and perfusion sequences in the same 3D space as histology is essential further clinical information. The ability to localize cancer in 3D space may improve targeting for image-guided biopsy, focal therapy, and disease quantification in surveillance protocols. Copyright © 2013 The Royal College of Radiologists. Published by Elsevier Ltd. All rights reserved.

The Association Between Scholarly Impact and National Institutes of Health Funding in Orthopaedic Surgery.

PubMed

Zhu, Elizabeth; Shemesh, Shai; Iatridis, James; Moucha, Calin

2017-12-01

The assessment of scholarly productivity assumes a strong role in evaluating faculty in academic orthopaedic surgery. The investigators examine the association between scholarly impact, as measured by the h-index, and National Institutes of Health (NIH) funding in orthopaedic surgery. Orthopaedic surgery faculty from 20 randomly chosen departments that received NIH-funding were compared to non-NIH funded faculty from the same departments. Faculty members in orthopaedic surgery departments who received NIH funding had higher scholarly impact as measured by h-index than their non-funded peers (h = 11.98 versus 4.45; p < 0.0001). This relationship holds across academic ranks, terminal degrees, and institutions. Investigators with higher academic rank had higher scholarly impact (h = assistant 3.29 versus associate 5.12 versus full professor 7.94; p < 1 x 10-7) as well as higher NIH-funding (assistant $16,580 versus associate $26,368 versus full professor $113,129; p < 1 x 10-7). Increasing individual NIH funding is correlated with elevated scholarly impact (β = 4.64; p < 0.0001). Increasing total departmental NIH funding is correlated to increased departmental scholarly impact (β = 1.04; p < 0.0001). The h-index is strongly associated with NIH funding, academic rank, and sole PhD holding faculty. Increasing scholarly impact is also correlated with higher NIH funding. The h-index is an objective and easily calculable measure of assessing individual research productivity.

Neurotoxicity of a Fragment of the Amyloid Precursor Associated with Alzheimer's Disease

NASA Astrophysics Data System (ADS)

Yankner, Bruce A.; Dawes, Linda R.; Fisher, Shannon; Villa-Komaroff, Lydia; Oster-Granite, Mary Lou; Neve, Rachael L.

1989-07-01

Amyloid deposition in senile plaques and the cerebral vasculature is a marker of Alzheimer's disease. Whether amyloid itself contributes to the neurodegenerative process or is simply a by-product of that process is unknown. Pheochromocytoma (PC12) and fibroblast (NIH 3T3) cell lines were transfected with portions of the gene for the human amyloid precursor protein. Stable PC12 cell transfectants expressing a specific amyloid-containing fragment of the precursor protein gradually degenerated when induced to differentiate into neuronal cells with nerve growth factor. Conditioned medium from these cells was toxic to neurons in primary hippocampal cultures, and the toxic agent could be removed by immunoabsorption with an antibody directed against the amyloid polypeptide. Thus, a peptide derived from the amyloid precursor may be neurotoxic.

Fabrication of novel dental nanocomposites and investigation their physicochemical and biological properties

NASA Astrophysics Data System (ADS)

Jaymand, Mehdi; lotfi, Mehrdad; Abbasian, Mojtaba

2018-03-01

This article evaluates physicochemical, mechanical, and biological properties of a series of novel dental nanocomposites that fabricated from multifunctional methacrylate-based dental monomers, triethyleneglycol dimethacrylate (TEGDMA) monomer, and modified silica nanoparticles (SiO2 NPs). The antibacterial activities of the monomers were investigated against lactobacillus plantarum by standard agar disk diffusion method. The cytotoxicity characteristics of the monomers and fabricated nanocomposites were evaluated by MTT and trypan blue cell viability tests, respectively against NIH3T3 cell line. In addition, the mechanical properties, as well as physicochemical characteristics including water sorption, sol fraction, and double bond conversion were also investigated. According to the results, the formulated nanocomposites have potential to apply as dental nanocomposites mainly due to their acceptable physicochemical, mechanical and biological characteristics.

Evaluation of cytotoxicity of some common ophthalmic drugs.

PubMed

Li, M; Chen, X-M; Liu, J-J; Wang, D-M; Gan, L; Lv, X; Qiao, Y

2015-01-01

The study was aimed at evaluating the in vitro cytotoxicity of some commonly used drugs in ophthalmology. Hydrocortisone sodium succinate, Dexamethasone sodium phosphate, 5-Fluorouracil, Tobramycin and Pilocarpine nitrate are frequently used in various indications involving eye care, and the aim was to test the safety of these in cell culture. The in vitro cytotoxicity was carried out on the NIH 3T3 cell line by the Sulforhodamine B (SRB) assay. With the exception of 5-Fluorouracil, none of the other drugs demonstrated appreciable cytotoxicity up to high concentrations of 200 µg/ml at 48 hours of drug exposure. Hydrocortisone sodium succinate, Dexamethasone sodium phosphate, Tobramycin and Pilocarpine nitrate were confirmed to be non-cytotoxic while 5-Fluorouracil was highly cytotoxic especially at 48 hours at very low concentrations.

Calculated electric dipole moment of NiH X2Delta

NASA Technical Reports Server (NTRS)

Walch, S.; Bauschlicher, C. W., Jr.; Langhoff, S. R.

1985-01-01

A calculated dipole moment of 2.39 D at R sub e = 2.79 a sub 0 is reported, obtained from complete active space SCF/configuration interaction calculations plus one natural orbital iteration. The calculation is in good agreement with the experimental value of 2.4 + or - 0.1 D measured for the lowest vibrational level. In agreement with Gray et al. (1985), it is found that the dipole moment is strongly correlated with the 3d electron population; the good agreement with experiment thus provides verification of the mixed state model of NiH. It is concluded that the electric dipole moment of NiH is a sensitive test of the quality of the NiH wave function.

The association between scholarly impact and National Institutes of Health funding in ophthalmology.

PubMed

Svider, Peter F; Lopez, Santiago A; Husain, Qasim; Bhagat, Neelakshi; Eloy, Jean Anderson; Langer, Paul D

2014-01-01

To examine whether there is an association between scholarly impact, as measured by the h-index, academic rank, and National Institutes of Health (NIH) awards in academic ophthalmology. Retrospective analysis of NIH RePORTER and Scopus databases. Not applicable. Five hundred seventy-three NIH awards to 391 primary investigators (PIs) in ophthalmology departments were examined. Grant recipients were organized by academic rank, obtained from online listings, and h-index, calculated using the Scopus database. Non-NIH-funded faculty from 20 randomly chosen academic ophthalmology departments also were organized by rank and h-index for comparison with their NIH-funded colleagues. Scholarly impact, as measured by the h-index, and NIH funding. The h-index increased with successive academic rank among non-NIH-funded and NIH-funded faculty, as did NIH funding among the latter group. The NIH-funded faculty had higher scholarly impact, as measured by the h-index, than their non-NIH-funded PIs (h = 18.3 vs. 7.8; P <0.0001), even when considering publications only in the prior 5 years; h-index increased with increasing NIH funding ranges. The h-indices of those holding an MD degree (21.4±1.6 standard error of mean) were not statistically higher than those of PhD holders (17.9±0.6) and those with both an MD and PhD degree (18.1±1.7; P = 0.14). The h-index increases with increasing academic rank among NIH-funded and non-NIH-funded faculty in ophthalmology departments. This bibliometric is associated strongly with NIH funding because NIH-funded PIs had higher scholarly impact than their non-NIH-funded colleagues, and increasing impact was noted with higher funding. The h-index is an objective and easily calculable measure that may be valuable as an adjunct in assessing research productivity, a significant factor for academic promotion in academic ophthalmology. Copyright © 2014 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.

The deviations of the Al6Li3Cu quasicrystal from icosahedral symmetry : a reminiscence of a cubic crystal

NASA Astrophysics Data System (ADS)

Donnadieu, Patricia

1994-05-01

The (Al6Li3Cu) (T2) quasicrystals are known to exhibit large deviations from the icosahedral symmetry. Series of electron diffraction patterns are used to investigate these imperfections in as-cast T, samples. A detailed analysis of the 5-fold and 3-fold symmetry diffraction patterns shows that they are compatible with the m3 point group instead of the m35 icosahedral group. This symmetry reduction is interprétéd as reminiscent of the cubic approximant phase (R-Al5Li3Cu) rather than of higher order approximant phases. This interpretation is supported by previous observations on crystal/quasicrystal phase transformation in the AlLiCu system. Les quasicristaux de phase T2(Al6Li3Cu) montrent d'importantes déviations à la symétrie icosaédrique. Ces imperfections sont mises en évidence par diffraction électronique dans des échantillons de phase T2 brut de coulée. Un examen détaillé des diagrammes de diffraction de symétrie d'ordre 3 et 5 révèle qu'ils sont compatibles avec le groupe ponctuel m3 au lieu du groupe de l'icosaèdre (m35). Cette réduction de symétrie est interprétée comme une réminiscence de la phase cubique approximante (R-Al5Li3Cu) et non l'apparition d'approximant d'ordre plus élevé. Cette interprétation est suggérée par des observations antérieures sur la transformation cristal/quasicristal dans le système AlLiCu.

Investigation into the Effects of Weapon Setback on Various Materials and Geometries.

DTIC Science & Technology

1978-07-01

taking the Laplace Transform of the dynamic equation, rearrangement and taking the inverse transform to find the time-dependent strain. The "dynamic...taking the inverse transform of the above equation: ■»-«fa-to*« 1 E’ ♦ ¥®*> B’ (s+-fj- )(S2+f )T If we neglect the residual strain on the system...partial fractions yields: *t) --f (fr JC-> K, K2 —L_ + + K3 »+-^ s+i(f) s-i(f) performing the inverse transform yields: 4©[K,^> ♦ K2

Effects of myclobutanil on soil microbial biomass, respiration, and soil nitrogen transformations.

PubMed

Ju, Chao; Xu, Jun; Wu, Xiaohu; Dong, Fengshou; Liu, Xingang; Zheng, Yongquan

2016-01-01

A 3-month-long experiment was conducted to ascertain the effects of different concentrations of myclobutanil (0.4 mg kg(-1) soil [T1]; 1.2 mg kg(-1) soil [T3]; and 4 mg kg(-1) soil [T10]) on soil microbial biomass, respiration, and soil nitrogen transformations using two typical agricultural soils (Henan fluvo-aquic soil and Shanxi cinnamon soil). Soil was sampled after 7, 15, 30, 60, and 90 days of incubation to determine myclobutanil concentration and microbial parameters: soil basal respiration (RB), microbial biomass carbon (MBC) and nitrogen (MBN), NO(-)3-N and NH(+)4-N concentrations, and gene abundance of total bacteria, N2-fixing bacteria, fungi, ammonia-oxidizing archaea (AOA), and ammonia-oxidizing bacteria (AOB). The half-lives of the different doses of myclobutanil varied from 20.3 to 69.3 d in the Henan soil and from 99 to 138.6 d in the Shanxi soil. In the Henan soil, the three treatments caused different degrees of short-term inhibition of RB and MBC, NH(+)4-N, and gene abundance of total bacteria, fungi, N2-fixing bacteria, AOA, and AOB, with the exception of a brief increase in NO(-)3-N content during the T10 treatment. The MBN (immobilized nitrogen) was not affected. In the Shanxi soil, MBC, the populations of total bacteria, fungi, and N2-fixing bacteria, and NH(+)4-N concentration were not significantly affected by myclobutanil. The RB and MBN were decreased transitorily in the T10 treatment. The NO(-)3-N concentrations and the abundance of both AOA and AOB were erratically stimulated by myclobutanil. Regardless of whether stimulation or suppression occurred, the effects of myclobutanil on the two soil types were short term. In summary, myclobutanil had no long-term negative effects on the soil microbial biomass, respiration, and soil nitrogen transformations in the two types of soil, even at 10-fold the recommended dosage. Copyright © 2015 Elsevier Ltd. All rights reserved.

Performance Analysis of Polymorphous Computing Architectures

DTIC Science & Technology

2001-01-01

G H F Proc 5 : 4 : 3 11 1 Figure 3. Self-timed execution. D C B F G H E D B H EA CG...F D C B F G H E D B H EA CG F AProc 1 Proc 2 Proc 3 Proc 4 Proc 5 185 cution pattern when the application graph in Figure 2 is executed in a self...transform, a quadra- E Figure 10. Self-timed execution with first-iteration actors denoted by T. D B H E CG F D C B F G H E D B H EA CG F A 18 T T T

SU-D-303-03: Impact of Uncertainty in T1 Measurements On Quantification of Dynamic Contrast Enhanced MRI

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aryal, M; Cao, Y

2015-06-15

Purpose: Quantification of dynamic contrast enhanced (DCE) MRI requires native longitudinal relaxation time (T1) measurement. This study aimed to assess uncertainty in T1 measurements using two different methods. Methods and Materials: Brain MRI scans were performed on a 3T scanner in 9 patients who had low grade/benign tumors and partial brain radiotherapy without chemotherapy at pre-RT, week-3 during RT (wk-3), end-RT, and 1, 6 and 18 months after RT. T1-weighted images were acquired using gradient echo sequences with 1) 2 different flip angles (50 and 150), and 2) 5 variable TRs (100–2000ms). After creating quantitative T1 maps, average T1 wasmore » calculated in regions of interest (ROI), which were distant from tumors and received a total of accumulated radiation doses < 5 Gy at wk-3. ROIs included left and right normal Putamen and Thalamus (gray matter: GM), and frontal and parietal white matter (WM). Since there were no significant or even a trend of T1 changes from pre-RT to wk-3 in these ROIs, a relative repeatability coefficient (RC) of T1 as a measure of uncertainty was estimated in each ROI using the data pre-RT and at wk-3. The individual T1 changes at later time points were evaluated compared to the estimated RCs. Results: The 2-flip angle method produced small RCs in GM (9.7–11.7%) but large RCs in WM (12.2–13.6%) compared to the saturation-recovery (SR) method (11.0–17.7% for GM and 7.5–11.2% for WM). More than 81% of individual T1 changes were within T1 uncertainty ranges defined by RCs. Conclusion: Our study suggests that the impact of T1 uncertainty on physiological parameters derived from DCE MRI is not negligible. A short scan with 2 flip angles is able to achieve repeatability of T1 estimates similar to a long scan with 5 different TRs, and is desirable to be integrated in the DCE protocol. Present study was supported by National Institute of Health (NIH) under grant numbers; UO1 CA183848 and RO1 NS064973.« less

1α,25-dihydroxyvitamin D3 acts via transforming growth factor-β to up-regulate expression of immunosuppressive CD73 on human CD4+ Foxp3- T cells.

PubMed

Mann, Elizabeth H; Chambers, Emma S; Chen, Yin-Huai; Richards, David F; Hawrylowicz, Catherine M

2015-11-01

Vitamin D deficiency is associated with increased incidence and severity of various immune-mediated diseases. Active vitamin D (1α,25-dihydroxyvitamin D3; 1,25(OH)2 D3) up-regulates CD4(+) T-cell expression of the purine ectonucleotidase CD39, a molecule that is associated with the generation of anti-inflammatory adenosine. Here we aimed to investigate the direct impact of 1,25(OH)2 D3 on expression of the downstream ecto-5'-nucleotidase CD73 by human CD4 T cells, and components of the transforming growth factor-β (TGF-β) pathway, which have been implicated in the modulation of CD73 by murine T cells. At 10(-8) to 10(-7) m, 1,25(OH)2 D3 significantly increased expression of CD73 on peripheral human CD4(+) T cells. Although 1,25(OH)2 D3 did not affect the mRNA expression of latent TGF-β1 , 1,25(OH)2 D3 did up-regulate expression of TGF-β-associated molecules [latency-associated peptide (LAP), glycophorin A repetitions predominant (GARP), GP96, neuropilin-1, thrombospondin-1 and αv integrin] which is likely to have contributed to the observed enhancement in TGF-β bioactivity. CD73 was highly co-expressed with LAP and GARP following 1,25(OH)2 D3 treatment, but unexpectedly, each of these cell surface molecules was expressed primarily on CD4(+) Foxp3(-) T cells, rather than CD4(+) Foxp3(+) T cells. Notably, neutralization of TGF-β significantly impaired 1,25(OH)2 D3-mediated induction of CD73. Collectively, we show that 1,25(OH)2 D3 enhances expression of CD73 on CD4(+) Foxp3(-) T cells in a process that is at least partially TGF-β-dependent. These data reveal an additional contributing mechanism by which vitamin D may be protective in immune-mediated disease. © 2015 John Wiley & Sons Ltd.

Silica/quercetin sol–gel hybrids as antioxidant dental implant materials

PubMed Central

Catauro, Michelina; Papale, Ferdinando; Bollino, Flavia; Piccolella, Simona; Marciano, Sabina; Nocera, Paola; Pacifico, Severina

2015-01-01

The development of biomaterials with intrinsic antioxidant properties could represent a valuable strategy for preventing the onset of peri-implant diseases. In this context, quercetin, a naturally occurring flavonoid, has been entrapped at different weight percentages in a silica-based inorganic material by a sol–gel route. The establishment of hydrogen bond interactions between the flavonol and the solid matrix was ascertained by Fourier transform infrared spectroscopy. This technique also evidenced changes in the stretching frequencies of the quercetin dienonic moiety, suggesting that the formation of a secondary product occurs. Scanning electron microscopy was applied to detect the morphology of the synthesized materials. Their bioactivity was shown by the formation of a hydroxyapatite layer on sample surface soaked in a fluid that simulates the composition of human blood plasma. When the potential release of flavonol was determined by liquid chromatography coupled with ultraviolet and electrospray ionization tandem mass spectrometry techniques, the eluates displayed a retention time that was 0.5 min less than quercetin. Collision-activated dissociation mass spectrometry and untraviolet-visible spectroscopy were in accordance with the release of a quercetin derivative. The antiradical properties of the investigated systems were evaluated by DPPH and ABTS methods, whereas the 2,7-dichlorofluorescein diacetate assay highlighted their ability to inhibit the H2O2-induced intracellular production of reactive oxygen species in NIH-3T3 mouse fibroblast cells. Data obtained, along with data gathered from the MTT cytotoxicity test, revealed that the materials that entrapped the highest amount of quercetin showed notable antioxidant effectiveness. PMID:27877802

A KRAS GTPase K104Q Mutant Retains Downstream Signaling by Offsetting Defects in Regulation.

PubMed

Yin, Guowei; Kistler, Samantha; George, Samuel D; Kuhlmann, Nora; Garvey, Leslie; Huynh, Minh; Bagni, Rachel K; Lammers, Michael; Der, Channing J; Campbell, Sharon L

2017-03-17

The KRAS GTPase plays a critical role in the control of cellular growth. The activity of KRAS is regulated by guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs), and also post-translational modification. Lysine 104 in KRAS can be modified by ubiquitylation and acetylation, but the role of this residue in intrinsic KRAS function has not been well characterized. We find that lysine 104 is important for GEF recognition, because mutations at this position impaired GEF-mediated nucleotide exchange. Because the KRAS K104Q mutant has recently been employed as an acetylation mimetic, we conducted a series of studies to evaluate its in vitro and cell-based properties. Herein, we found that KRAS K104Q exhibited defects in both GEF-mediated exchange and GAP-mediated GTP hydrolysis, consistent with NMR-detected structural perturbations in localized regions of KRAS important for recognition of these regulatory proteins. Despite the partial defect in both GEF and GAP regulation, KRAS K104Q did not alter steady-state GTP-bound levels or the ability of the oncogenic KRAS G12V mutant to cause morphologic transformation of NIH 3T3 mouse fibroblasts and of WT KRAS to rescue the growth defect of mouse embryonic fibroblasts deficient in all Ras genes. We conclude that the KRAS K104Q mutant retains both WT and mutant KRAS function, probably due to offsetting defects in recognition of factors that up-regulate (GEF) and down-regulate (GAP) RAS activity. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Identification of an intracellular protein that specifically interacts with photoaffinity-labeled oncogenic p21 protein.

PubMed

Lee, G; Ronai, Z A; Pincus, M R; Brandt-Rauf, P W; Murphy, R B; Delohery, T M; Nishimura, S; Yamaizumi, Z; Weinstein, I B

1989-11-01

An oncogenic 21-kDa (p21) protein (Harvey RAS protein with Val-12) has been covalently modified with a functional reagent that contains a photoactivatable aromatic azide group. This modified p21 protein has been introduced quantitatively into NIH 3T3 cells using an erythrocyte-mediated fusion technique. The introduced p21 protein was capable of inducing enhanced pinocytosis and DNA synthesis in the recipient cells. To identify the putative intracellular protein(s) that specifically interact with the modified p21 protein, the cells were pulsed with [35S]methionine at selected times after fusion and then UV-irradiated to activate the azide group. The resulting nitrene covalently binds to amino acid residues in adjacent proteins, thus linking the p21 protein to these proteins. The cells were then lysed, and the lysate was immunoprecipitated with the anti-p21 monoclonal antibody Y13-259. The immunoprecipitate was analyzed by SDS/PAGE to identify p21-protein complexes. By using this technique, we found that three protein complexes of 51, 64, and 82 kDa were labeled specifically and reproducibly. The most prominent band is the 64-kDa protein complex that shows a time-dependent rise and fall, peaking within a 5-hr period after introduction of the p21 protein into the cells. These studies provide evidence that in vitro the p21 protein becomes associated with a protein whose mass is about 43 kDa. We suggest that the formation of this complex may play a role in mediating early events involved with cell transformation induced by RAS oncogenes.

Raman spectroscopy and Raman imaging for early detection of cancer

NASA Astrophysics Data System (ADS)

Joshi, Narahari V.; Ortega, Angel; Estrela, Jose Maria

2004-06-01

Raman spectroscopy is a powerful technique as it provides fundamental information about vibrational modes of a system. Eigenvalues of these modes are very sensitive to the strength of the chemical bonds and perturbations caused by the environment, particularly charge distribution and alterations in the dipole strength of the system. All these parameters are profoundly modified during the tumor formation process nad hence Raman technique could be a unique and also a direct approach for evaluating tumor genesis at early stages. for this pupose the present investigation was carried out. We used cultured wild type and c-ras transformed NIH 3T3 fibroblast. The samples were treated with methyl alcohol solution ina conventional manner and then Raman spectra nad images were obtained by a specially developed confocal Raman microscope. The present results reveal differences between both cell types in the spectral details as well as in the morphology. Raman images are able to detect the exact site where cancer-related changes have taken place. These results clearly indicate the superiority of the present technique over conventional methods such as images obtained by X-rays or Nuclear Magnetic Resonance (NMR). Moreover, unlike other approaches, Raman images detect alterations at the submicron level rather than in the centimeter or millimeter range. Being an optical method it can be applied in vivo as a non-invasive technique potentially useful to early detection of cancer (particularly easy accessible cancers such as those of the skin and the digestive tract). The obtained resulsts suggest the great potential of Raman imaging in premature clinical diagnostic approaches.

WE-G-BRB-01: The Importance of NIH Funding in Innovation in Radiation Therapy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Deye, J.

Over the past 20 years the NIH has funded individual grants, program projects grants, and clinical trials which have been instrumental in advancing patient care. The ways that each grant mechanism lends itself to the different phases of translating research into clinical practice will be described. Major technological innovations, such as IMRT and proton therapy, have been advanced with R01-type and P01-type funding and will be discussed. Similarly, the role of program project grants in identifying and addressing key hypotheses on the potential of 3D conformal therapy, normal tissue-guided dose escalation and motion management will be described. An overview willmore » be provided regarding how these technological innovations have been applied to multi-institutional NIH-sponsored trials. Finally, the panel will discuss regarding which research questions should be funded by the NIH to inspire the next advances in radiation therapy. Learning Objectives: Understand the different funding mechanisms of the NIH Learn about research advances that have led to innovation in delivery Review achievements due to NIH-funded program project grants in radiotherapy over the past 20 years Understand example advances achieved with multi-institutional clinical trials NIH.« less

WE-G-BRB-00: NIH-Funded Research: Instrumental in the Pursuit of Clinical Trials and Technological Innovations

DOE Office of Scientific and Technical Information (OSTI.GOV)

NONE

Over the past 20 years the NIH has funded individual grants, program projects grants, and clinical trials which have been instrumental in advancing patient care. The ways that each grant mechanism lends itself to the different phases of translating research into clinical practice will be described. Major technological innovations, such as IMRT and proton therapy, have been advanced with R01-type and P01-type funding and will be discussed. Similarly, the role of program project grants in identifying and addressing key hypotheses on the potential of 3D conformal therapy, normal tissue-guided dose escalation and motion management will be described. An overview willmore » be provided regarding how these technological innovations have been applied to multi-institutional NIH-sponsored trials. Finally, the panel will discuss regarding which research questions should be funded by the NIH to inspire the next advances in radiation therapy. Learning Objectives: Understand the different funding mechanisms of the NIH Learn about research advances that have led to innovation in delivery Review achievements due to NIH-funded program project grants in radiotherapy over the past 20 years Understand example advances achieved with multi-institutional clinical trials NIH.« less

76 FR 52671 - Clinical Center; Notice of Meeting

Federal Register 2010, 2011, 2012, 2013, 2014

2011-08-23

....), notice is hereby given of a meeting of the NIH Advisory Board for Clinical Research. The meeting will be... closed to the public in accordance with the provisions set forth in section 552b(c)(6), Title 5 U.S.C... unwarranted invasion of privacy. Name of Committee: NIH Advisory Board for Clinical Research Date: October 3...

National Institutes of Health chronic graft-versus-host disease staging in severely affected patients: organ and global scoring correlate with established indicators of disease severity and prognosis.

PubMed

Baird, Kristin; Steinberg, Seth M; Grkovic, Lana; Pulanic, Drazen; Cowen, Edward W; Mitchell, Sandra A; Williams, Kirsten M; Datiles, Manuel B; Bishop, Rachel; Bassim, Carol W; Mays, Jacqueline W; Edwards, Dean; Cole, Kristen; Avila, Daniele N; Taylor, Tiffany; Urban, Amanda; Joe, Galen O; Comis, Leora E; Berger, Ann; Stratton, Pamela; Zhang, Dan; Shelhamer, James H; Gea-Banacloche, Juan C; Sportes, Claude; Fowler, Daniel H; Gress, Ronald E; Pavletic, Steven Z

2013-04-01

Between 2004 and 2010, 189 adult patients were enrolled on the National Cancer Institute's cross-sectional chronic graft-versus-host disease (cGVHD) natural history study. Patients were evaluated by multiple disease scales and outcome measures, including the 2005 National Institutes of Health (NIH) Consensus Project cGVHD severity scores. The purpose of this study was to assess the validity of the NIH scoring variables as determinants of disease severity in severely affected patients in efforts to standardize clinician evaluation and staging of cGVHD. Out of 189 patients enrolled, 125 met the criteria for severe cGVHD on the NIH global score, 62 of whom had moderate disease, with a median of 4 (range, 1-8) involved organs. Clinician-assigned average NIH organ score and the corresponding organ scores assigned by subspecialists were highly correlated (r = 0.64). NIH global severity scores showed significant associations with nearly all functional and quality of life outcome measures, including the Lee Symptom Scale, Short Form-36 Physical Component Scale, 2-minute walk, grip strength, range of motion, and Human Activity Profile. Joint/fascia, skin, and lung involvement affected function and quality of life most significantly and showed the greatest correlation with outcome measures. The final Cox model with factors jointly predictive for survival included the time from cGVHD diagnosis (>49 versus ≤49 months, hazard ratio [HR] = 0.23; P = .0011), absolute eosinophil count at the time of NIH evaluation (0-0.5 versus >0.5 cells/μL, HR = 3.95; P = .0006), and NIH lung score (3 versus 0-2, HR = 11.02; P < .0001). These results demonstrate that NIH organs and global severity scores are reliable measures of cGVHD disease burden. The strong association with subspecialist evaluation suggests that NIH organ and global severity scores are appropriate for clinical and research assessments, and may serve as a surrogate for more complex subspecialist examinations. In this population of severely affected patients, NIH lung score is the strongest predictor of poor overall survival, both alone and after adjustment for other important factors. Published by Elsevier Inc.

HBV-specific and global T-cell dysfunction in chronic hepatitis B

PubMed Central

Park, Jang-June; Wong, David K.; Wahed, Abdus S.; Lee, William M.; Feld, Jordan J.; Terrault, Norah; Khalili, Mandana; Sterling, Richard K.; Kowdley, Kris V.; Bzowej, Natalie; Lau, Daryl T.; Kim, W. Ray; Smith, Coleman; Carithers, Robert L.; Torrey, Keith W.; Keith, James W.; Levine, Danielle L.; Traum, Daniel; Ho, Suzanne; Valiga, Mary E.; Johnson, Geoffrey S.; Doo, Edward; Lok, Anna S. F.; Chang, Kyong-Mi

2015-01-01

Background & Aims T cells play a critical role in in viral infection. We examined whether T-cell effector and regulatory responses can define clinical stages of chronic hepatitis B (CHB). Methods We enrolled 200 adults with CHB who participated in the NIH-supported Hepatitis B Research Network from 2011 through 2013 and 20 uninfected individuals (controls). Peripheral blood lymphocytes from these subjects were analyzed for T-cell responses (proliferation and production of interferon-γ and interleukin-10) to overlapping hepatitis B virus (HBV) peptides (preS, S, preC, core, and reverse transcriptase), influenza matrix peptides, and lipopolysaccharide. T-cell expression of regulatory markers FOXP3, programmed death-1 (PD1), and cytotoxic T lymphocyte-associated antigen-4 (CTLA4) was examined by flow cytometry. Immune measures were compared with clinical parameters, including physician-defined immune-active, immune-tolerant, or inactive CHB phenotypes, in a blinded fashion. Results Compared to controls, patients with CHB had weak T-cell proliferative, interferon-γ, and interleukin-10 responses to HBV, with increased frequency of circulating FOXP3+CD127− regulatory T cells and CD4+ T-cell expression of PD1 and CTLA4. T-cell measures did not clearly distinguish between clinical CHB phenotypes, although the HBV core-specific T-cell response was weaker in HBeAg+ than HBeAg− patients (% responders: 3% vs 23%, P=.00008). Although in vitro blockade of PD1 or CTLA4 increased T-cell responses to HBV, the effect was weaker in HBeAg+ than HBeAg− patients. Furthermore, T-cell responses to influenza and lipopolysaccharide were weaker in CHB patients than controls. Conclusion HBV persists with virus-specific and global T-cell dysfunction mediated by multiple regulatory mechanisms including circulating HBeAg, but without distinct T-cell–based immune signatures for clinical phenotypes. These findings suggest additional T-cell independent or regulatory mechanisms of CHB pathogenesis that warrant further investigation. PMID:26684441

Dietary Supplement Research Portfolio at the NIH, 2009–201112

PubMed Central

Garcia-Cazarin, Mary L.; Wambogo, Edwina A.; Regan, Karen S.; Davis, Cindy D.

2014-01-01

The U.S. dietary supplement market increased by 7.5% in 2012 compared with 2011, reaching $32.5 billion in sales. Therefore, federally supported research on dietary supplements is important to determine their health effects, safety, and efficacy. A portfolio analysis was performed across the NIH and the Office of Dietary Supplements (ODS) for fiscal years (FYs) 2009–2011 by using the databases Human Nutrition Research Information Management (HNRIM) and Computer Access to Research on Dietary Supplements (CARDS). The results indicated that total NIH dietary supplement–related funding for FYs 2009–2011 was $855 million ($295 million in 2009, $311 million in 2010, and $249 million in 2011). The institutes and centers with the highest investment in dietary supplement research were as follows: the National Heart, Lung, and Blood Institute ($135 million); the National Cancer Institute ($188 million); the National Center for Complementary and Alternative Medicine ($99 million); the National Institute of Diabetes and Digestive and Kidney Diseases ($68 million); the National Institute of Environmental Health Sciences ($58 million); and the ODS ($32 million). The dietary supplement ingredients receiving the most funding were botanicals (22%), vitamins (20%), lipids (14%), and minerals and trace elements (10%). The top 3 outcome research areas were cancer (61% of total dietary supplement investment), cardiovascular disease (47%), and women’s reproductive health (38%). In FYs 2009, 2010, and 2011, the ODS provided 3.5%, 3.6%, and 4.1%, respectively, of the NIH investment in dietary supplement research. ODS funding focused on cellular, enzymatic, or molecular mechanisms (64% of total ODS funding). This portfolio analysis demonstrates that the NIH has committed substantial funding to dietary supplement research in an effort to expand the scientific knowledge base on the efficacy and safety of dietary supplements. PMID:24523489

Effect of acupuncture on clinical symptoms and laboratory indicators for chronic prostatitis/chronic pelvic pain syndrome: a systematic review and meta-analysis.

PubMed

Liu, Bu-Ping; Wang, Yun-Ting; Chen, Si-da

2016-12-01

To systematically review the efficacy and safety of acupuncture for chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS). PubMed, Cochrane library Central, Web of Science, Wang-fang Database, and CNKI were searched from their inception to June 30, 2016. Data of acupuncture for CP/CPPS following randomized controlled trials (RCTs) was included. The data were analyzed using the Cochrane Collaboration Review Manager. The primary data were the National Institute of Health-Chronic Prostatitis Index (NIH-CPSI) score at the end of follow-up. Ten RCTs were enrolled. Acupuncture was superior to the control in NIH-CPSI (MD -3.98, [95 % CI -5.78 to -2.19]; P < 0.0001) and response rate (RR 4.12, [95 % CI 1.67-10.18]; P = 0.002). Acupuncture was superior to sham acupuncture on NIH-CPSI, response rate, pain, urinary, and quality of life (QOL). Standard medication was inferior to acupuncture in terms of NIH-CPSI (MD -3.08, [95 % CI -5.57 to -0.60]; P = 0.02) and response rate (RR 2.03, [95 % CI 1.04-3.97]; P = 0.04), but standard medication was superior to acupuncture on improving urinary symptoms. There was no significant difference in the adverse events. Acupuncture/acupuncture plus standard medication significantly down-regulated IL-1β compared with standard medication in prostatic fluid. Acupuncture treating CP/CPPS is effective and safe. The effects of acupuncture on NIH-CPSI, response rate, pain symptoms, and QOF were superior to the control, but standard medication significantly improved urinary symptoms compared with acupuncture. Acupuncture can decrease the IL-1β in prostatic fluid for CP/CPPS.