Sample records for 3t3 feeder layer

  1. Extracellular matrix proteins of human epidermal keratinocytes and feeder 3T3 cells

    Microsoft Academic Search

    K. Alitalo; RAILI MYLLYL; URPO KIISTALA; SIRPA ASKO; ANTTI VAHERI

    1982-01-01

    Cultures of human epidermal keratinocytes obtained from adult epidermis were initiated using irradiated BALB\\/3T3 cells as feeder layers. At different stages of confluence of the epidermal islands, feeder cells were removed and the extracellular matrix proteins of both pure component cells and cocultures were analyzed biochemically and by immunochemical methods and compared to those of skin fibroblasts of the same

  2. Occurrence and Control of Sporadic Proliferation in Growth Arrested Swiss 3T3 Feeder Cells

    PubMed Central

    Chugh, Rishi Man; Chaturvedi, Madhusudan; Yerneni, Lakshmana Kumar

    2015-01-01

    Growth arrested Swiss mouse embryonic 3T3 cells are used as feeders to support the growth of epidermal keratinocytes and several other target cells. The 3T3 cells have been extensively subcultured owing to their popularity and wide distribution in the world and, as a consequence selective inclusion of variants is a strong possibility in them. Inadvertently selected variants expressing innate resistance to mitomycin C may continue to proliferate even after treatment with such growth arresting agents. The failure of growth arrest can lead to a serious risk of proliferative feeder contamination in target cell cultures. In this study, we passaged Swiss 3T3 cells (CCL-92, ATCC) by different seeding densities and incubation periods. We tested the resultant cultures for differences in anchorage-independent growth, resumption of proliferation after mitomycin C treatment and occurrence of proliferative feeder contaminants in an epidermal keratinocyte co-culture system. The study revealed subculture dependent differential responses. The cultures of a particular subculture procedure displayed unique cell size distribution and disintegrated completely in 6 weeks following mitomycin C treatment, but their repeated subculture resulted in feeder regrowth as late as 11 weeks after the growth arrest. In contrast, mitomycin C failed to inhibit cell proliferation in cultures of the other subculture schemes and also in a clone that was established from a transformation focus of super-confluent culture. The resultant proliferative feeder cells contaminated the keratinocyte cultures. The anchorage-independent growth appeared in late passages as compared with the expression of mitomycin C resistance in earlier passages. The feeder regrowth was prevented by identifying a safe subculture protocol that discouraged the inclusion of resistant variants. We advocate routine anchorage-independent growth assay and absolute confirmation of feeder disintegration to qualify feeder batches and caution on the use of fetal bovine serum. PMID:25799110

  3. Culture of Normal Human Epidermal Cells with 3T3 Feeders on Millipore Filters

    Microsoft Academic Search

    Shigeo Kondo; Kazuo Aso; Masayoshi Namba

    1979-01-01

    Human epidermal cells were cultured with lethally irradiated 3T3 cells and grew as large colonies both on plastic and millipore filters. Subcultures were possible. The grown colonies were studied morphologically and histologically. The whole intact crossview of epidermal colonies in paraffin sections was obtained from the cell culture on millipore filters. The epidermal cells derived from younger persons had higher

  4. Culture of human limbal epithelial stem cells on tenon's fibroblast feeder-layers: a translational approach.

    PubMed

    Scafetta, Gaia; Siciliano, Camilla; Frati, Giacomo; De Falco, Elena

    2015-01-01

    The coculture technique is the standard method to expand ex vivo limbal stem cells (LSCs) by using inactivated embryonic murine feeder layers (3T3). Although alternative techniques such as amniotic membranes or scaffolds have been proposed, feeder layers are still considered to be the best method, due to their ability to preserve some critical properties of LSCs such as cell growth and viability, stemness phenotype, and clonogenic potential. Furthermore, clinical applications of LSCs cultured on 3T3 have taken place. Nevertheless, for an improved Good Manufacturing Practice (GMP) compliance, the use of human feeder-layers as well as a fine standardization of the process is strictly encouraged. Here, we describe a translational approach in accordance with GMP regulations to culture LSCs onto human Tenon's fibroblasts (TFs). In this chapter, based on our experience we identify and analyze issues that often are encountered by researchers and discuss solutions to common problems. PMID:25063497

  5. Bird Feeder

    NSDL National Science Digital Library

    2011-08-20

    In this outdoor activity, learners construct bird feeders and set them up at to investigate bird behavior for one or two weeks. Multiple feeder designs are suggested. Learners are encouraged to observe how different birds approach the feeder, whether any birds bully other birds, what foods visiting birds eat, how often and at what times of day different birds visit, and how birds react to models of other birds on the feeder.

  6. The 3T3 cell cycle at low proliferation rates

    Microsoft Academic Search

    ROBERT F. BROOKS

    1988-01-01

    Summary When the proliferation rate of Swiss 3T3 cells is decreased by limiting the availability of growth factors, cell cycle variability increases, as pre- dicted by the transition probability model. Never- theless, the transition probabilities would appear to play a relatively minor role in the regulation of proliferation rate. Instead, at least 40% of the increase in the average cycle

  7. Culture of cells derived from the human sebaceous gland under serum-free conditions without a biological feeder layer or specific matrices

    Microsoft Academic Search

    Takeshi Fujie; Takanori Shikiji; Naoyuki Uchida; Yoshio Urano; Hiroaki Nagae; Seiji Arase

    1996-01-01

    We succeeded in serially culturing cells derived from human sebaceous gland (sebocytes) under serum-free conditions. Sebaceous\\u000a glands were isolated from dispase-treated facial skin specimens and cultured using two different methods, explant culture\\u000a and dispersed cell culture, in KGM. In both types of culture the sebocytes proliferated rapidly without a biological feeder\\u000a layer or specific matrices. It was possible to cultivate

  8. Midkine induces the transformation of NIH3T3 cells.

    PubMed Central

    Kadomatsu, K.; Hagihara, M.; Akhter, S.; Fan, Q. W.; Muramatsu, H.; Muramatsu, T.

    1997-01-01

    Midkine (MK) is a heparin-binding growth factor and is frequently expressed at high levels in many human carcinomas. To investigate further the roles of MK in the regulation of cell growth, we introduced MK expression in NIH3T3 cells. A mixture of transfectants of an MK expression vector, but not a control vector, formed colonies in soft agar, showed an elevated cell number at confluence, and formed tumours in nude mice. An interesting characteristic of the transformed cells was that they became spontaneously detached from the culture dish substratum. In the transformed cells, MK was not only secreted, but also localized, in the perinuclear region as spots. The present data indicate that MK has the potential to transform NIH3T3 cells and suggest that overexpression of the MK gene may promote unregulated cell growth in vivo. Images Figure 2 Figure 3 Figure 4 PMID:9020479

  9. Alteration of glycolipids in ras-transfected NIH 3T3 cells

    SciTech Connect

    Matyas, G.R.; Aaronson, S.A.; Brady, R.O.; Fishman, P.H.

    1987-09-01

    Glycosphingolipid alterations upon viral transformation are well documented. Transformation of mouse 3T3 cells with murine sarcoma viruses results in marked decreases in the levels of gangliosides GM1 and GD1a and an increase in gangliotriaosylceramide. The transforming oncogenes of these viruses have been identified as members of the ras gene family. The authors analyzed NIH 3T3 cells transfected with human H-, K- and N-ras oncogenes for their glycolipid composition and expression of cell surface gangliosides. Using conventional thin-layer chromatographic analysis, they found that the level of GM3 was increased and that of GD1a was slightly decreased or unchanged, and GM1 was present but not in quantifiable levels. Cell surface levels of GM1 were determined by /sup 125/I-labeled cholera toxin binding to intact cells. GD1a was determined by cholera toxin binding to cells treated with sialidase prior to toxin binding. All ras-transfected cells had decreased levels of surface GM1 and GD1 as compared to logarithmically growing normal NIH 3T3 cells. Levels of GM1 and, to a lesser extent, GD1a increased as the latter cells became confluent. Using a monoclonal antibody assay, they found that gangliotriaosylceramide was present in all ras-transfected cells studied but not in logarithmically growing untransfected cells. These results indicated that ras oncogenes derived form human tumors are capable of inducing alterations in glycolipid composition.

  10. Precision powder feeder

    DOEpatents

    Schlienger, M. Eric (Albuquerque, NM); Schmale, David T. (Albuquerque, NM); Oliver, Michael S. (Sandia Park, NM)

    2001-07-10

    A new class of precision powder feeders is disclosed. These feeders provide a precision flow of a wide range of powdered materials, while remaining robust against jamming or damage. These feeders can be precisely controlled by feedback mechanisms.

  11. Characterization of hyaluronate binding proteins isolated from 3T3 and murine sarcoma virus transformed 3T3 cells

    SciTech Connect

    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 weight 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.

  12. Cell adhesion and cell surface topography in aggregates of 3T3 and SV40- virus-transformed 3T3 cells. Visualization of interior cells by scanning electron microscopy

    PubMed Central

    1978-01-01

    A technique for exposing the interior of aggregates of cultured cells has been developed and is described in this report. Using this technique, we have examined for the first time, by scanning electron microscopy, cell morphology and cell contact ultrastructure in the interior of aggregates of BALB/c 3T3 and SV40-transformed 3T3 cells. The 3T3 cells make initial intercellular contact by means of microvillar processes. Over a period of 3-8 h, some of these microvillar contacts are replaced by broader projections. In contrast, the SV40-transformed cells make initial intercellular contact by means of blebs or blunt projections which are also broadened and extended over a period of 3-8 h. For both 3T3 and SV40-3T3 cells, the surfaces of the cells which form the outer layer of the aggregate resemble the surfaces of single cells fixed in suspension, regardless of how long the aggregates have been cultured. Thse cells are covered with many cellular processes and are roughly hemispherical in profile. The surfaces of the internal cells of the aggregates, however, lose many of their cellular processes, develop smooth patches, and many become irregular in shape. This smooth morphology was also observed on the interior surfaces of the peripheral cell layer. From these observations we conclude that: (a) the stabilization of adhesive contacts is a slow process which takes at least 3-8 h; (b) the outer surfaces of peripheral cells differ significantly from the surfaces of interior cells; and (c) clear differences in surface topography exist between nonmalignant 3T3 cells and their malignant SV40 transformants. PMID:632324

  13. Project Feeder Watch

    NSDL National Science Digital Library

    2004-01-01

    Project FeederWatch is a winter-long survey of birds that visit feeders at backyards, nature centers, community areas, and other locales in North America. FeederWatchers periodically count the birds they see at their feeders from November through early April and send their counts to Project FeederWatch. FeederWatch data help scientists track broadscale movements of winter bird populations and long-term trends in bird distribution and abundance. Anyone with an interest in birds can participate. FeederWatch is conducted by people of all skill levels and backgrounds, including children, families, individuals, classrooms, retired persons, youth groups, nature centers, and bird clubs

  14. Locomotory behavior, contact inhibition, and pattern formation of 3T3 and polyoma virus-transformed 3T3 cells in culture

    PubMed Central

    Bell, PB

    1977-01-01

    The social behavior of 3T3 cells and their polynoma virus-transformed derivative (Py3T3 cells) was examined by time-lapse cinemicrography in order to determine what factors are responsible for the marked differences in the patterns formed by the two cell lines in culture. Contrary to expectations, both cell types have been found to exhibit contact inhibition of cell locomotion. Therefore, the tendency of 3T3 cells to form monolayers and of Py3T3 cells to form crisscrossed multilayers cannot be explained on the basis of the presence versus the absence of contact inhibition. Morevover, with the exception of cell division control, the social behavior of the two cell types is qualitively similar. Both exhibit cell underlapping and, after contact between lamelliopodia, both show inhibition of locomotory activity and adhesion formation. Neither cell type was observed to migrate over the surface of another cell. The two cell types do show quantitative differences in the frequency of underlapping, the frequency with which contact results in inhibition of locomotion, and the proportion of the cell margin that adheres to the substratum. The increased frequency pf Py3T3 underlapping is correlated with the reduced frequency of substratum adhesions, which in turn favors underlapping. On the basis of these observations, it is concluded that the differences in culture patterns are the result of differences in the shapes of the individual cells, such that underlapping, and hence crisscrossing, is favored in Py3T3 cell interactions and discouraged in 3T3 cells. PMID:198414

  15. Embryonic stem cells cultured in serum-free medium acquire bovine apolipoprotein B-100 from feeder cell layers and serum replacement medium.

    PubMed

    Hisamatsu-Sakamoto, Michiko; Sakamoto, Norihisa; Rosenberg, Amy S

    2008-01-01

    Previous studies have demonstrated that cell populations that are cultured with heterologous animal products can acquire xenoantigens, potentially limiting their clinical utility because of immune responses. Embryonic stem cells (ESCs) are an attractive source of multiple potential cellular therapies and are typically derived and routinely cultured on murine embryonic fibroblast (MEF) feeder cell layers in commercially available serum replacement (SR) medium or fetal calf serum (FCS)-containing medium. Recently, we found that a strong antibody response was generated in human subjects after the second infusion of therapeutic cells cultured in FCS-containing medium. This response was specific for bovine apolipoprotein B-100 (apoB-100), which is the major protein component of low-density lipoproteins (LDL) and which targets its binding to abundant low-density lipoprotein receptors on the cell surface, from which it is internalized. Here, we have shown that ESCs cultured on MEFs in SR medium acquired bovine apoB-100 from MEFs and from the SR medium as well. Our findings also suggest that bovine LDL are used as critical nutrients for ESC propagation. PMID:17951218

  16. Distribution feeder reliability assessment

    Microsoft Academic Search

    A. A. Chowdhury

    2005-01-01

    Historical distribution feeder reliability assessment generally summarizes discrete interruption events occurring at specific locations over specific time periods; whereas, predictive assessment estimates the long-run behavior of systems by combining component failure rates and repair (restoration) times that describe the central tendency of an entire distribution of possible values with feeder configurations. The outage time due to component failures can substantially

  17. Effect of Biodegradable Shape-Memory Polymers on Proliferation of 3T3 Cells

    Microsoft Academic Search

    Shuo-Gui Xu; Peng Zhang; Guang-Ming Zhu; Ying-Ming Jiang

    2011-01-01

    This article evaluates the in vitro biocompatibility for biodegradable shape-memory polymers (BSMP) invented by the authors.\\u000a 3T3 cells (3T3-Swiss albino GNM 9) of primary and passaged cultures were inoculated into two kinds of carriers: the BSMP carrier\\u000a and the control group carrier. Viability, proliferation, and DNA synthesis (the major biocompatibility parameters), were measured\\u000a and evaluated for both the BSMP and

  18. Enhanced Effects of Xanthohumol Plus Honokiol on Apoptosis in 3T3-L1 Adipocytes

    Microsoft Academic Search

    Jeong-Yeh Yang; Mary Anne Della-Fera; Srujana Rayalam; Clifton A. Baile

    2008-01-01

    Objective:To study the effects of xanthohumol (XN), a flavonoid found in hops (Humulus lupulus) and honokiol (HK), a lignan isolated from Magnolia officinalis, alone and in combination, on apoptotic signaling in 3T3-L1 adipocytes.Methods and Procedures:3T3-L1 mature adipocytes were incubated with various concentrations of XN and HK alone and in combination. Viability and apoptosis were quantified using an MTS-based cell viability

  19. Antisense Suppression of p107 Inhibits 3T3-L1 Adipocyte Differentiation

    Microsoft Academic Search

    Julie S. May; Audra M. Prince; Robert E. Lyle; Robert E. McGehee

    2001-01-01

    The murine 3T3-L1 preadipocyte cell line is well characterized for its capacity to undergo differentiation into adipocytes under appropriate hormonal stimulation. p107, a member of the retinoblastoma tumor suppressor gene family has been shown to be dramatically upregulated during the early requisite clonal expansion phase of 3T3-L1 adipogenesis; however, a functional consequence has yet to be described. A phosphorothioate antisense

  20. Curcumin induces apoptosis in immortalized NIH 3T3 and malignant cancer cell lines

    Microsoft Academic Search

    Ming?Chung Jiang

    1996-01-01

    Curcumin, which is a widely used dietary pigment and spice, has been demonstrated to be an effective inhibitor of tumor promotion in mouse skin carcinogenesis. We report that curcumin induces cell shrinkage, chromatin condensation, and DNA fragmentation, characteristics of apoptosis, in immortalized mouse embryo fibroblast NIH 3T3 erb B2 oncogene?transformed NIH 3T3, mouse sarcoma S180, human colon cancer cell HT?29,

  1. Mitigative Effect of Erythromycin on PMMA Challenged Preosteoblastic MC3T3-E1 Cells

    PubMed Central

    Shen, Yi; Wang, Weili; Li, Xiaomiao; Markel, David C.; Ren, Weiping

    2014-01-01

    Background. Aseptic loosening (AL) is a major complication of total joint replacement. Recent approaches to limiting AL have focused on inhibiting periprosthetic inflammation and osteoclastogenesis. Questions/Purposes. The purpose of this study was to determine the effects of erythromycin (EM) on polymethylmethacrylate (PMMA) particle-challenged MC3T3 osteoblast precursor cells. Methods. MC3T3 cells were pretreated with EM (0–10??g/mL) and then stimulated with PMMA (1?mg/mL). Cell viability was evaluated by both a lactate dehydrogenase (LDH) release assay and cell counts. Cell differentiation was determined by activity of alkaline phosphatase (ALP). Gene expression was measured via real-time quantitative RT-PCR. Results. We found that exposure to PMMA particles reduced cellular viability and osteogenetic potential in MC3T3 cell line. EM treatment mitigated the effects of PMMA particles on the proliferation, viability and differentiation of MC3T3 cells. PMMA decreased the gene expression of Runx2, osterix and osteocalcin, which can be partially restored by EM treatment. Furthermore, EM suppressed PMMA- induced increase of NF-?B gene expression. Conclusions. These data demonstrate that EM mitigates the effects of PMMA on MC3T3 cell viability and differentiation, in part through downregulation of NF-?B pathway. EM appeared to represent an anabolic agent on MC3T3 cells challenged with PMMA particles. PMID:25110723

  2. 95. VIEW OF ZINC FEEDER FROM SOUTHEAST. NOTE FEEDER CONE ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    95. VIEW OF ZINC FEEDER FROM SOUTHEAST. NOTE FEEDER CONE AND PIPING FROM VACUUM RECEIVER ON LEFT. PRECIPITATE PUMP MOTOR MOUNT VISIBLE BELOW FEEDER STAIRS, PUMP AND MOTOR MISSING. SUMPS ARE LOCATED UNDER THIS FLOOR, WITH ACCESS TO HATCH TO THE RIGHT OF FEEDER STAIR. - Bald Mountain Gold Mill, Nevada Gulch at head of False Bottom Creek, Lead, Lawrence County, SD

  3. Feederism in a woman.

    PubMed

    Terry, Lesley L; Vasey, Paul L

    2011-06-01

    Feederism is a fat fetish subculture in which individuals eroticize weight gain and feeding. Feeders are individuals who claim to become sexually aroused by feeding their partners and encouraging them to gain weight. Conversely, Feedees are individuals who claim to become sexually aroused by eating, being fed, and the idea or act of gaining weight. Very little is known about this population. This report describes a woman who self-identified as a Feedee. It is unclear, at present, whether female Feederism represents a unique paraphilia or a thematic variation of morphophilia or sexual masochism. PMID:20041284

  4. The effect of myostatin on proliferation and lipid accumulation in 3T3-L1 preadipocytes.

    PubMed

    Zhu, Hui Juan; Pan, Hui; Zhang, Xu Zhe; Li, Nai Shi; Wang, Lin Jie; Yang, Hong Bo; Gong, Feng Ying

    2015-06-01

    Myostatin is a critical negative regulator of skeletal muscle development, and has been reported to be involved in the progression of obesity and diabetes. In the present study, we explored the effects of myostatin on the proliferation and differentiation of 3T3-L1 preadipocytes by using 3-[4,5-dimethylthiazol-2-yl] 2,5-diphenyl tetrazolium bromide spectrophotometry, intracellular triglyceride (TG) assays, and real-time quantitative RT-PCR methods. The results indicated that recombinant myostatin significantly promoted the proliferation of 3T3-L1 preadipocytes and the expression of proliferation-related genes, including Cyclin B2, Cyclin D1, Cyclin E1, Pcna, and c-Myc, and IGF1 levels in the medium of 3T3-L1 were notably upregulated by 35.2, 30.5, 20.5, 33.4, 51.2, and 179% respectively (all P<0.01) in myostatin-treated 3T3-L1 cells. Meanwhile, the intracellular lipid content of myostatin-treated cells was notably reduced as compared with the non-treated cells. Additionally, the mRNA levels of Ppar?, Cebp?, Gpdh, Dgat, Acs1, Atgl, and Hsl were significantly downregulated by 22-76% in fully differentiated myostatin-treated adipocytes. Finally, myostatin regulated the mRNA levels and secretion of adipokines, including Adiponectin, Resistin, Visfatin, and plasminogen activator inhibitor-1 (PAI-1) in 3T3-L1 adipocytes (all P<0.001). Above all, myostatin promoted 3T3-L1 proliferation by increasing the expression of cell-proliferation-related genes and by stimulating IGF1 secretion. Myostatin inhibited 3T3-L1 adipocyte differentiation by suppressing Ppar? and Cebp? expression, which consequently deceased lipid accumulation in 3T3-L1 cells by inhibiting the expression of critical lipogenic enzymes and by promoting the expression of lipolytic enzymes. Finally, myostatin modulated the expression and secretion of adipokines in fully differentiated 3T3-L1 adipocytes. PMID:25878062

  5. Trophic effect of a methanol yeast extract on 3T3 fibroblast cells.

    PubMed

    Gallo, Dominique; Dillemans, Monique; Allardin, David; Priem, Fabian; Van Nedervelde, Laurence

    2014-01-01

    With regard to the increase of human life expectancy, interest for topical treatments aimed to counteract skin aging is still growing. Hence, research for bioactive compounds able to stimulate skin fibroblast activity is an attractive topic. Having previously described the effects of a new methanol yeast extract on growth and metabolic activity of Saccharomyces cerevisiae, we studied its effects on 3T3 fibroblasts to evaluate its potential antiaging property. This investigation demonstrates that this extract increases proliferation as well as migration of 3T3 cells and decreases their entrance in senescence and apoptosis phases. Altogether, these results open new perspectives for the formulation of innovative antiaging topical treatments. PMID:25898765

  6. Ligand Activation of Overexpressed Epidermal Growth Factor Receptors Transforms NIH 3T3 Mouse Fibroblasts

    NASA Astrophysics Data System (ADS)

    Riedel, Heimo; Massoglia, Sharon; Schlessinger, Joseph; Ullrich, Axel

    1988-03-01

    The cell surface receptor for the mitogenic peptide epidermal growth factor (EGF) is involved in control of normal cell growth and may play a role in the genesis of human neoplasia such as squamous carcinoma and glioblastoma. Soft-agar growth and focus-formation experiments with NIH 3T3 mouse fibroblasts transfected with an expression plasmid demonstrated the ligand-dependent transforming potential of the human EGF receptor without structural alterations. Activation of overexpressed normal receptor alone appears to be sufficient for transformation of NIH 3T3 cells in vitro.

  7. Inhibitory Effect of (?)-Epigallocatechin-3Gallate on Lipid Accumulation of 3T3-L1 Cells

    Microsoft Academic Search

    Hyun-Seuk Moon; Chung-Soo Chung; Hong-Gu Lee; Tae-Gyu Kim; Yun-Jaie Choi; Chong-Su Cho

    2007-01-01

    Objective: The objective of this study was to investigate the molecular mechanisms underlying the attenuating effect of (?)-epigallocatechin-3-gallate (EGCG) on proliferation and lipid accumulation of 3T3-L1 cells, with a focus on the duration of EGCG treatment.Research Methods and Procedures: Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assay and diamidino-2-phenylindole staining. The anti-adipogenic effect of EGCG on 3T3-L1 cells was analyzed by

  8. A Nanodot Array Modulates Cell Adhesion and Induces an Apoptosis-Like Abnormality in NIH-3T3 Cells

    PubMed Central

    2009-01-01

    Micro-structures that mimic the extracellular substratum promote cell growth and differentiation, while the cellular reaction to a nanostructure is poorly defined. To evaluate the cellular response to a nanoscaled surface, NIH 3T3 cells were grown on nanodot arrays with dot diameters ranging from 10 to 200 nm. The nanodot arrays were fabricated by AAO processing on TaN-coated wafers. A thin layer of platinum, 5 nm in thickness, was sputtered onto the structure to improve biocompatibility. The cells grew normally on the 10-nm array and on flat surfaces. However, 50-nm, 100-nm, and 200-nm nanodot arrays induced apoptosis-like events. Abnormality was triggered after as few as 24 h of incubation on a 200-nm dot array. For cells grown on the 50-nm array, the abnormality started after 72 h of incubation. The number of filopodia extended from the cell bodies was lower for the abnormal cells. Immunostaining using antibodies against vinculin and actin filament was performed. Both the number of focal adhesions and the amount of cytoskeleton were decreased in cells grown on the 100-nm and 200-nm arrays. Pre-coatings of fibronectin (FN) or type I collagen promoted cellular anchorage and prevented the nanotopography-induced programed cell death. In summary, nanotopography, in the form of nanodot arrays, induced an apoptosis-like abnormality for cultured NIH 3T3 cells. The occurrence of the abnormality was mediated by the formation of focal adhesions. PMID:20596320

  9. Methylglyoxal induces oxidative stress and mitochondrial dysfunction in osteoblastic MC3T3-E1 cells.

    PubMed

    Suh, K S; Choi, E M; Rhee, S Y; Kim, Y S

    2014-02-01

    Methylglyoxal is a reactive dicarbonyl compound produced by glycolytic processing and identified as a precursor of advanced glycation end products. The elevated methylglyoxal levels in patients with diabetes are believed to contribute to diabetic complications, including bone defects. The objective of this study was to evaluate the effect of methylglyoxal on the function of osteoblastic MC3T3-E1 cells. The data indicated that methylglyoxal decreased osteoblast differentiation and induced osteoblast cytotoxicity. Pretreatment of MC3T3-E1 cells with aminoguanidine (a carbonyl scavenger), Trolox (an antioxidant), and cyclosporin A (a blocker of the mitochondrial permeability transition pore) prevented methylglyoxal-induced cytotoxicity in MC3T3-E1 cells. However, BAPTA/AM (an intracellular Ca(2+) chelator) and dantrolene (an inhibitor of endoplasmic reticulum Ca(2+) release) did not reverse the cytotoxic effect of methylglyoxal. Methylglyoxal increased the formation of intracellular reactive oxygen species, mitochondrial superoxide, and cardiolipin peroxidation in osteoblastic MC3T3-E1 cells. Methylglyoxal also decreased the mitochondrial membrane potential and intracellular ATP and nitric oxide levels, suggesting that carbonyl stress-induced loss of mitochondrial integrity contributes to the cytotoxicity of methylglyoxal. Furthermore, the results demonstrated that methylglyoxal induced protein adduct formation, inactivation of glyoxalase I, and activation of glyoxalase II. Aminoguanidine reversed all aforementioned effects of methylglyoxal. Taken together, these data support the notion that high methylglyoxal concentrations have detrimental effects on osteoblasts through a mechanism involving oxidative stress and mitochondrial dysfunction. PMID:24164256

  10. Fluorescence lifetime imaging of lipids during 3T3-L1 cell differentiation

    NASA Astrophysics Data System (ADS)

    Song, Young Sik; Won, Young Jae; Lee, Sang-Hak; Kim, Dug Young

    2014-03-01

    Obesity is becoming a big health problem in these days. Since increased body weight is due to increased number and size of the triglyceride-storing adipocytes, many researchers are working on differentiation conditions and processes of adipocytes. Adipocytes also work as regulators of whole-body energy homeostasis by secreting several proteins that regulate processes as diverse as haemostasis, blood pressure, immune function, angiogenesis and energy balance. 3T3-L1 cells are widely used cell line for studying adipogenesis because it can differentiate into an adipocyte-like phenotype under appropriate conditions. In this paper, we propose an effective fluorescence lifetime imaging technique which can easily distinguish lipids in membrane and those in lipid droplets. Nile red dyes are attached to lipids in 3T3-L1 cells. Fluorescence lifetime images were taken for 2 week during differentiation procedure of 3T3-L1 cells into adipocytes. We used 488 nm pulsed laser with 5MHz repetition rate and emission wavelength is 520 nm of Nile Red fluorescent dye. Results clearly show that the lifetime of Nile red in lipid droplets are smaller than those in cell membrane. Our results suggest that fluorescence lifetime imaging can be a very powerful tool to monitor lipid droplet formation in adipocytes from 3T3-L1 cells.

  11. Expression of an Exogenous Eukaryotic DNA Methyltransferase Gene Induces Transformation of NIH 3T3 Cells

    Microsoft Academic Search

    Jianjun Wu; Jean-Pierre Issa; James Herman; Douglas E. Bassett Jr.; Barry D. Nelkin; Stephen B. Baylin

    1993-01-01

    Abnormal regional increases in DNA methylation, which have potential for causing gene inactivation and chromosomal instability, are consistently found in immortalized and tumorigenic cells. Increased DNA methyltransferase activity, which is also a characteristic of such cells, is a candidate to mediate these abnormal DNA methylation patterns. We now show that, in NIH 3T3 mouse fibroblasts, constitutive overexpression of an exogenous

  12. The ras Oncogenes Increase the Intrinsic Resistance of NIH 3T3 Cells to Ionizing Radiation

    Microsoft Academic Search

    Marshall D. Sklar

    1988-01-01

    Identification of genes that function to protect cells from radiation damage is an essential step in understanding the molecular mechanisms by which mammalian cells cope with ionizing radiation. The intrinsic radiation resistance (D0) of NIH 3T3 cells was markedly and significantly increased by transformation with ras oncogenes activated by missense mutations. This radiobiologic activity appeared to be a specific consequence

  13. Magnolol enhances adipocyte differentiation and glucose uptake in 3T3-L1 cells

    Microsoft Academic Search

    Sun-Sil Choi; Byung-Yoon Cha; Young-Sil Lee; Takayuki Yonezawa; Toshiaki Teruya; Kazuo Nagai; Je-Tae Woo

    2009-01-01

    AimsThe nuclear receptor peroxisome proliferator-activated receptor (PPAR) ? plays an important role in adipocyte differentiation. Its ligands, including thiazolidinediones, improve insulin sensitivity in type 2 diabetes. We investigate the effect of magnolol, an ingredient of Magnolia officinalis on adipogenesis and glucose uptake using 3T3-L1 cells.

  14. Bird Feeder Project

    NSDL National Science Digital Library

    2011-08-20

    In this activity, learners make a backyard bird feeder with soda bottles and other simple supplies. Learners will recycle a plastic bottle to hold birdseed, create a perch for the birds to rest, and attach it to a branch or post outside. Then, enjoy birdwatching!

  15. Nuclear transport in 3T3 fibroblasts: effects of growth factors, transformation, and cell shape

    PubMed Central

    1988-01-01

    Nucleocytoplasmic transport of fluorescent-labeled macromolecules was investigated in transformed and nontransformed 3T3 fibroblasts. Insulin and epidermal growth factor enhanced transport three-fold after 1-2-h incubation with nontransformed adhering fibroblasts; no enhancement of transport was observed for spherical unattached fibroblasts. The concentration of growth factor for maximal enhancement was 3-10 nM. Nuclear transport for Kirsten murine sarcoma virus-transformed BALB/c 3T3 fibroblasts, however, was maximally enhanced before addition of growth factors; addition of insulin or epidermal growth factor causes no additional transport enhancement. Transformation also minimizes cell shape effects on macromolecular nuclear transport. These results provide evidence that protein growth factors and oncogenic transformation may use a similar mechanism for activation of nuclear transport. PMID:2448310

  16. Stimulation of FA and casein kinase II by insulin in 3T3-L1 cells.

    PubMed

    Villa-Moruzzi, E; Crabb, J W

    1991-06-28

    Insulin stimulates protein phosphatase-1 and FA, assayed as phosphatase-1 activator, in 3T3-L1 cells. Since other kinases, such as casein kinase-II may also contribute to such FA activity, we assayed casein kinase-II and FA as peptide kinase on extracts from 3T3-L1 cells that had been exposed to insulin for various times. Under such conditions FA, assayed as phosphatase-1 activator, was stimulated 2-3-fold within 1-2 min. Casein kinase-II was stimulated about 2-fold but at a slightly later time (2-3 min) than FA, making it unlikely that casein kinase-II contributes to FA stimulation. Insulin slightly stimulated also the kinase activity of FA towards a synthetic peptide at 2 min, thus confirming the FA activation seen when FA was assayed as activator of phosphatase-1. PMID:1647765

  17. Lysophosphatidic acid induces chemotaxis in MC3T3-E1 osteoblastic cells

    Microsoft Academic Search

    Lisa M. Masiello; Joseph S. Fotos; Deni S. Galileo; Norman J. Karin

    2006-01-01

    Lysophosphatidic acid (LPA) is a bioactive lipid that has pleiotropic effects on a variety of cell types and enhances the migration of endothelial and cancer cells, but it is not known if this lipid can alter osteoblast motility. We performed transwell migration assays using MC3T3-E1 osteoblastic cells and found LPA to be a potent chemotactic agent. Quantitative time-lapse video analysis

  18. Hexosamines Regulate Leptin Production in 3T3-L1 Adipocytes through Transcriptional Mechanisms

    Microsoft Academic Search

    PEILI ZHANG; ELLEN S. KLENK; MARC A. LAZZARO; LLOYD B. WILLIAMS

    2002-01-01

    This study was undertaken to examine the regulation of leptin gene (LEP) transcription and leptin release by hexosamines in 3T3-L1 adipocytes. Glucosamine (1 mM), an intermediate in hexosamine biosynthesis, increased leptin release to 117.0 7.3% (P 0.0430; n 9) and 134.6 6.5% of the control value (P 0.0367; n 4) by 48 and 96 h, respectively. With 0.01 mM glucosamine,

  19. The effect of undernutrition on circadian genes and rhythmic induction in NIH3T3 cells

    Microsoft Academic Search

    Shuting Cheng; Wang Hou; Shiping Li; Shuhong Yang; Yanyou Liu; Zhou Jiang; Yuhui Wang; Jing Xiao; Huiling Guo; Zhengrong Wang

    2012-01-01

    Nutrition is essential for health, and it has been widely studied and demonstrated to be related with circadian rhythm. To verify the effect of nutrient deficiency on the in vitro cultured cells, we had NIH3T3 cells cultured in diluted medium for 5 days, and detected the expression levels of Bmal1, Clock and Cry1 on each day. Phorbol 12-myristate 13-acetate (1PMA) was

  20. Nuclear localization and signalling activity of phosphoinositidase Cbeta in Swiss 3T3 cells

    Microsoft Academic Search

    Alberto M. Martelli; R. Stewart Gilmour; Valeria Bertagnolo; Luca M. Neri; Lucia Manzoli; Lucio Cocco

    1992-01-01

    THE hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdInsP2) is a widespread receptor-coupled signalling system at the plasma membrane of most eukaryotic cells. The existence of an entirely separate nuclear phosphoinositide signalling system is suggested from evidence that purified nuclei synthesize PtdInsP2and phosphatidylinositol 4-phosphate (PtdlnsP) in vitro1and that a transient decrease in the mass of these lipids occurs when Swiss 3T3 cells are

  1. Sakuranetin induces adipogenesis of 3T3-L1 cells through enhanced expression of PPAR?2

    Microsoft Academic Search

    Takeshi Saito; Daigo Abe; Keizo Sekiya

    2008-01-01

    Sakuranetin (5,4?-dihydroxy-7-methoxyflavone) belongs to the flavanone class of polyphenols predominantly known as phytoalexin in rice plant. In this study, we demonstrate that sakuranetin strongly induces differentiation of 3T3-L1 preadipocytes, as evidenced by increased triglyceride accumulation and glycerol-3-phosphate dehydrogenase (GPDH) activity. In addition, even in the absence of adipogenic hormonal stimuli, sakuranetin strongly induced adipogenesis and expression of genes that are

  2. Binding Kinetics of Ecotropic (Moloney) Murine Leukemia Retrovirus with NIH 3T3 Cells

    Microsoft Academic Search

    HONG YU; FRENCH ANDERSON

    1995-01-01

    A quantitative analysis of the binding kinetics of intact Moloney murine leukemia retrovirus (MoMuLV) particles with NIH 3T3 cells was performed with an immunofluorescenceflow cytometry assay. The virus-cell binding equilibrium dissociation constant (KD), expressed in terms of virus particle concentration, was measured to be 8.5 (66.4) 310 212 Mat4 &C and was three- to sixfold lower at temperatures above 15&C.

  3. Heat Shock Induces the Release of Fibroblast Growth Factor 1 from NIH 3T3 Cells

    Microsoft Academic Search

    Anthony Jackson; Stanley Friedman; Xi Zhan; Kurt A. Engleka; Reza Forough; Thomas Maciag

    1992-01-01

    Fibroblast growth factor 1 (FGF-1) is a potent angiogenic and neurotrophic factor whose structure lacks a classical signal sequence for secretion. Although the initiation of these biological activities involves the interaction between FGF-1 and cell surface receptors, the mechanism responsible for the regulation of FGF-1 secretion is unknown. We report that murine NIH 3T3 cells transfected with a synthetic gene

  4. Transformation of NIH\\/3T3 Cells by Ornithine Decarboxylase Overexpression1

    Microsoft Academic Search

    Jeffrey A. Moshier; Julie Dosescu; Magdalena Skunca; Gordon D. Luk

    Ornithine decarboxylase (ODO plays a rate-limiting role in polyamine biosynthesis and is intimately associated with cell proliferation and func tion. Although elevated levels of ODC mRNA, protein, and enzyme activity are consistently detected in transformed cells and tumors, the question remains as to whether ODC gene overexpression has a causative role in tumorigenesis. We have stably transfected MH\\/3T3 fibroblasts with

  5. Transformation of NIH 3T3 cells by microinjection of Haras p21 protein

    Microsoft Academic Search

    Dennis W. Stacey; Hsiang-Fu Kung

    1984-01-01

    Alteration in gene structure has been shown to occur in some human tumours1-5. These altered genes, termed oncogenes, were originally identified by their ability to induce foci of transformed cells on transfected mouse 3T3 cultures. The oncogene identified in the EJ\\/T24 human bladder carcinoma is similar to the transforming gene of BALB and Harvey murine sarcoma virus (MSV)6,7 and differs

  6. Extract of Chaga mushroom (Inonotus obliquus) stimulates 3T3-L1 adipocyte differentiation.

    PubMed

    Joo, Jeong In; Kim, Dong Hyun; Yun, Jong Won

    2010-11-01

    Chaga mushroom (Inonotus obliquus) has long been used as a folk medicine due to its numerous biological functions such as antibacterial, antiallergic, antiinflammatory and antioxidative activities. In the present study, it was found that the I. obliquus hot water extract (IOWE) activated adipogenesis of 3T3-L1 preadipocytes. Even in the absence of adipogenic stimuli by insulin, the IOWE strongly induced adipogenesis of 3T3-L1 preadipocytes. The major constituent of IOWE was glucose-rich polysaccharides with a molecular mass of 149? kDa. IOWE enhanced the differentiation of 3T3-L1 preadipocytes, increasing TG (triacylglycerol) accumulation that is critical for acquisition of the adipocyte phenotype, in a dose-dependent manner. IOWE stimulated gene expression of C/EBP? (CCAAT/enhancer-binding protein ?) and PPAR? (peroxisome proliferator-activated receptors ?) during adipocyte differentiation, and induced the expression of PPAR? target genes such as aP2 (adipocyte protein 2), LPL (lipoprotein lipase) and CD36 (fatty acid translocase). Immunoblot analysis revealed that IOWE increased the expression of adipogenic makers such as PPAR? and GLUT4 (glucose transporter 4). The luciferase reporter assay demonstrated that IOWE did not exhibit PPAR? ligand activity. Although these results require further investigation, the ability of natural mushroom product to increase PPAR? transcriptional activities may be expected to be therapeutic targets for dyslipidemia and type 2 diabetes. PMID:21031614

  7. Traditional Herbal Formula Oyaksungi-San Inhibits Adipogenesis in 3T3-L1 Adipocytes

    PubMed Central

    Seo, Chang-Seob; Shin, Hyeun-Kyoo

    2015-01-01

    Background. Oyaksungi-san (OYSGS) is a herbal formula that has been used for treating cardiovascular diseases in traditional Asian medicine. Here, we investigated the antiadipogenic effect of OYSGS extract in 3T3-L1 adipose cells. Methods. 3T3-L1 preadipocytes were differentiated into adipocytes with or without OYSGS. After differentiation, we measured Oil Red O staining, glycerol-3-phosphate dehydrogenase (GPDH) activity, leptin production, mRNA, and protein levels of adipogenesis-related factors. Results. OYSGS extract dramatically inhibited intracellular lipid accumulation in the differentiated adipocytes. It also significantly suppressed the (GPDH) activity, triglyceride (TG) content, and leptin production by reducing the expression of adipogenesis-related genes including lipoprotein lipase, fatty acid binding protein 4, CCAAT/enhancer-binding protein-alpha (C/EBP-?), and peroxisome proliferator-activated receptor gamma (PPAR-?). Furthermore, OYSGS clearly enhanced phosphorylation of AMP-activated protein kinase (AMPK) as well as its substrate acetyl CoA (ACC) carboxylase. Conclusions. Our results demonstrate that OYSGS negatively controls TG accumulation in 3T3-L1 adipocytes. We suggest antiadipogenic activity of OYSGS and its potential benefit in preventing obesity. PMID:25802547

  8. Endoplasmic reticulum stress suppresses lipin-1 expression in 3T3-L1 adipocytes

    SciTech Connect

    Takahashi, Nobuhiko, E-mail: ntkhs@hoku-iryo-u.ac.jp [Department of Internal Medicine, School of Dentistry, Health Sciences University of Hokkaido, 1757, Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan) [Department of Internal Medicine, School of Dentistry, Health Sciences University of Hokkaido, 1757, Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan); Division of Gastroenterology and Hematology/Oncology, Department of Medicine, Asahikawa Medical University, 2-1-1-1, Midorigaoka-Higashi, Asahikawa, Hokkaido 078-8510 (Japan); Yoshizaki, Takayuki [Innovation Center, Kagoshima University, 1-21-40, Korimoto, Kagoshima 890-0065 (Japan)] [Innovation Center, Kagoshima University, 1-21-40, Korimoto, Kagoshima 890-0065 (Japan); Hiranaka, Natsumi; Suzuki, Takeshi [Department of Internal Medicine, School of Dentistry, Health Sciences University of Hokkaido, 1757, Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan)] [Department of Internal Medicine, School of Dentistry, Health Sciences University of Hokkaido, 1757, Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan); Yui, Tomoo; Akanuma, Masayoshi [Department of Fixed Prosthodontics and Oral Implantology, School of Dentistry, Health Sciences University of Hokkaido, 1757, Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan)] [Department of Fixed Prosthodontics and Oral Implantology, School of Dentistry, Health Sciences University of Hokkaido, 1757, Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan); Kanazawa, Kaoru [Department of Dental Anesthesiology, School of Dentistry, Health Sciences University of Hokkaido, 1757, Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan)] [Department of Dental Anesthesiology, School of Dentistry, Health Sciences University of Hokkaido, 1757, Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan); Yoshida, Mika; Naito, Sumiyoshi [Department of Clinical Laboratory, Health Sciences University of Hokkaido, 1757, Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan)] [Department of Clinical Laboratory, Health Sciences University of Hokkaido, 1757, Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan); Fujiya, Mikihiro; Kohgo, Yutaka [Division of Gastroenterology and Hematology/Oncology, Department of Medicine, Asahikawa Medical University, 2-1-1-1, Midorigaoka-Higashi, Asahikawa, Hokkaido 078-8510 (Japan)] [Division of Gastroenterology and Hematology/Oncology, Department of Medicine, Asahikawa Medical University, 2-1-1-1, Midorigaoka-Higashi, Asahikawa, Hokkaido 078-8510 (Japan); Ieko, Masahiro [Department of Internal Medicine, School of Dentistry, Health Sciences University of Hokkaido, 1757, Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan)] [Department of Internal Medicine, School of Dentistry, Health Sciences University of Hokkaido, 1757, Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan)

    2013-02-01

    Highlights: ? Lipin-1 involves lipid metabolism, adipocyte differentiation, and inflammation. ? Adipose lipin-1 expression is reduced in obesity. ? ER stress suppresses lipin-1 expression in 3T3-L1 adipocytes. ? Activation of PPAR-? recovers ER stress-induced lipin-1 reduction. -- Abstract: Lipin-1 plays crucial roles in the regulation of lipid metabolism and cell differentiation in adipocytes. In obesity, adipose lipin-1 mRNA expression is decreased and positively correlated with systemic insulin sensitivity. Amelioration of the lipin-1 depletion might be improved dysmetabolism. Although some cytokines such as TNF-? and interleukin-1? reduces adipose lipin-1 expression, the mechanism of decreased adipose lipin-1 expression in obesity remains unclear. Recently, endoplasmic reticulum (ER) stress is implicated in the pathogenesis of obesity. Here we investigated the role of ER stress on the lipin-1 expression in 3T3-L1 adipocytes. We demonstrated that lipin-1 expression was suppressed by the treatment with ER stress inducers (tunicamycin and thapsigargin) at transcriptional level. We also showed that constitutive lipin-1 expression could be maintained by peroxisome proliferator-activated receptor-? in 3T3-L1 adipocytes. Activation of peroxisome proliferator-activated receptor-? recovered the ER stress-induced lipin-1 suppression. These results suggested that ER stress might be involved in the pathogenesis of obesity through lipin-1 depletion.

  9. Sakuranetin induces adipogenesis of 3T3-L1 cells through enhanced expression of PPARgamma2.

    PubMed

    Saito, Takeshi; Abe, Daigo; Sekiya, Keizo

    2008-08-01

    Sakuranetin (5,4'-dihydroxy-7-methoxyflavone) belongs to the flavanone class of polyphenols predominantly known as phytoalexin in rice plant. In this study, we demonstrate that sakuranetin strongly induces differentiation of 3T3-L1 preadipocytes, as evidenced by increased triglyceride accumulation and glycerol-3-phosphate dehydrogenase (GPDH) activity. In addition, even in the absence of adipogenic hormonal stimuli, sakuranetin strongly induced adipogenesis and expression of genes that are critical for the adipocytes phenotype. Time-course analyses indicated that sakuranetin induces PPARgamma2 expression without prior induction of C/EBPbeta, a transcriptional regulator of PPARgamma2 in adipogenesis. In 3T3-L1 preadipocytes, the transcriptional factors GATA-2 and GATA-3 are known to down-regulate adipogenesis by direct binding to the C/EBPbeta protein and to the GATA-binding site on the PPARgamma2 promoter. We found that sakuranetin significantly reduced the expression of GATA-2. Moreover, we observed that sakuranetin stimulated glucose uptake in differentiated 3T3-L1 adipocytes. These results suggest that sakuranetin may contribute to maintain glucose homeostasis in animals. PMID:18522800

  10. Effects of 6-Hydroxyflavone on Osteoblast Differentiation in MC3T3-E1 Cells

    PubMed Central

    Wu, Yu-Wei; Yeh, Shauh-Der; Lin, Yu-Hsaing; Tsai, Yu-Hui

    2014-01-01

    Osteoblast differentiation plays an essential role in bone integrity. Isoflavones and some flavonoids are reported to have osteogenic activity and potentially possess the ability to treat osteoporosis. However, limited information concerning the osteogenic characteristics of hydroxyflavones is available. This study investigates the effects of various hydroxyflavones on osteoblast differentiation in MC3T3-E1 cells. The results showed that 6-hydroxyflavone (6-OH-F) and 7-hydroxyflavone (7-OH-F) stimulated ALP activity. However, baicalein and luteolin inhibited ALP activity and flavone showed no effect. Up to 50??M of each compound was used for cytotoxic effects study; flavone, 6-OH-F, and 7-OH-F had no cytotoxicity on MC3T3-E1 cells. Moreover, 6-OH-F activated AKT and serine/threonine kinases (also known as protein kinase B or PKB), extracellular signal-regulated kinases (ERK 1/2), and the c-Jun N-terminal kinase (JNK) signaling pathways. On the other hand, 7-OH-F promoted osteoblast differentiation mainly by activating ERK 1/ 2 signaling pathways. Finally, after 5 weeks of 6-OH-F induction, MC3T3-E1 cells showed a significant increase in the calcein staining intensity relative to merely visible mineralization observed in cells cultured in the osteogenic medium only. These results suggested that 6-OH-F could activate AKT, ERK 1/2, and JNK signaling pathways to effectively promote osteoblastic differentiation. PMID:24795772

  11. Semicarbazide-sensitive amine oxidase activation promotes adipose conversion of 3T3-L1 cells.

    PubMed Central

    Mercier, N; Moldes, M; El Hadri, K; Fčve, B

    2001-01-01

    Semicarbazide-sensitive amine oxidase (SSAO) is an amine oxidase related to the copper-containing amine oxidase family. The tissular form of SSAO is located at the plasma membrane, and is mainly expressed in vascular smooth muscle cells and adipocytes. Recent studies have suggested that SSAO could activate glucose transport in fat cells. In the present work, we investigated the potential role of a chronic SSAO activation on adipocyte maturation of the 3T3-L1 pre-adipose cell line. Exposure of post-confluent 3T3-L1 pre-adipocytes to methylamine, a physiological substrate of SSAO, promoted adipocyte differentiation in a time- and dose-dependent manner. This effect could be related to SSAO activation, since it was antagonized in the presence of the SSAO inhibitor semicarbazide, but not in the presence of the monoamine oxidase inhibitor pargyline. In addition, methylamine-induced adipocyte maturation was mimicked by 3T3-L1 cell treatment with other SSAO substrates. Finally, the large reversion of methylamine action by catalase indicated that hydrogen peroxide generated by SSAO was involved, at least in part, in the modulation of adipocyte maturation. Taken together, our results suggest that SSAO may contribute to the control of adipose tissue development. PMID:11513731

  12. Lysophosphatidic acid induces chemotaxis in MC3T3-E1 osteoblastic cells.

    PubMed

    Masiello, Lisa M; Fotos, Joseph S; Galileo, Deni S; Karin, Norman J

    2006-07-01

    Lysophosphatidic acid (LPA) is a bioactive lipid that has pleiotropic effects on a variety of cell types and enhances the migration of endothelial and cancer cells, but it is not known if this lipid can alter osteoblast motility. We performed transwell migration assays using MC3T3-E1 osteoblastic cells and found LPA to be a potent chemotactic agent. Quantitative time-lapse video analysis of osteoblast migration after wounds were introduced into cell monolayers indicated that LPA stimulated both migration velocity and the average migration distance per cell. LPA also elicited substantial changes in cell shape and actin cytoskeletal structure; lipid-treated cells contained fewer stress fibers and displayed long membrane processes that were enriched in F-actin. Quantitative RT-PCR analysis showed that MC3T3-E1 cells express all four known LPA-specific G-protein-coupled receptors (LPA1-LPA4) with a relative mRNA abundance of LPA1>LPA4>LPA2>LPA3. LPA-induced changes in osteoblast motility and morphology were antagonized by both pertussis toxin and Ki16425, a subtype-specific blocker of LPA1 and LPA3 receptor function. Cell migration in many cell types is linked to changes in intracellular Ca2+. Ki16425 also inhibited LPA-induced Ca2+ signaling in a dose-dependent manner, suggesting a link between LPA-induced Ca2+ transients and osteoblast chemotaxis. Our data show that LPA stimulates MC3T3-E1 osteoblast motility via a mechanism that is linked primarily to the G-protein-coupled receptor LPA1. PMID:16487757

  13. Construction and expression of lentiviral vectors encoding recombinant mouse CREBZF in NIH 3T3 cells.

    PubMed

    Chen, Fenglei; Lin, Peng Fei; Li, Xiao; Sun, Jin; Zhang, Zhe; Du, Enqi; Wang, Aihua; Jin, Ya Ping

    2014-09-01

    CREBZF, also known as Zhangfei or SMILE, is a member of the CREB/ATF protein family. CREBZF has mainly been considered as a basic region-leucine zipper transcription factor that functions in coordination with other transcription factors and plays a role in latent HSV-1 infection, apoptosis and the mammalian endoplasmic reticulum stress and unfolded protein response. In this study, we constructed recombinant lentiviral vectors for CREBZF short hairpin RNA (shRNA) expression and over-expression to improve understanding of the mechanisms regulating CREBZF. The CREBZF ORF sequence was cloned into the lentiviral shuttle plasmid pCD513B-1, and various shRNA oligonucleotides and one negative control (shN) were cloned into the pCD513B-U6 expression vector. The recombinant lentivirus was packaged and transduced into NIH 3T3 cells. CREBZF mRNA and protein expression were examined using real-time reverse transcription-polymerase chain reaction (RT-qPCR) and western blotting, respectively. The over-expression vector and the most effective shRNA vector significantly affected the expression of CREBZF mRNA and protein. Both of the CREBZF recombinant lentiviral vectors were successfully constructed. The over-expression vector significantly increased the expression of exogenous CREBZF and inhibited the growth of NIH 3T3 cells compared to controls. The most effective shRNA lentiviral vector, pCD513B-U6-CREBZF-shRNA-3, was transformed, leading to significant knockdown of the CREBZF gene. We conclude that CREBZF the recombinant lentiviral vectors are promising tools for regulating the expression of CREBZF in NIH 3T3 cells. PMID:25195838

  14. The Aporphine Alkaloid Boldine Induces Adiponectin Expression and Regulation in 3T3-L1 Cells

    PubMed Central

    Yu, Bangning; Cook, Carla

    2009-01-01

    Abstract Adiponectin is an adipokine secreted by differentiated adipocytes. Clinical studies suggest a negative correlation between oxidative stress and adiponectin levels in patients with metabolic syndrome or cardiovascular disease. Natural compounds that can prevent oxidative stress mediated inhibition of adiponectin may be potentially therapeutic. Boldine, an aporphine alkaloid abundant in the medicinal plant Peumus boldus, is a powerful antioxidant. The current study demonstrates the effects of boldine on the expression of adiponectin and its regulators, CCAAT/enhancer binding protein-? (C/EBP?) and peroxisome proliferator-activated receptor (PPAR)-?, in 3T3-L1 cells. Differentiated 3T3-L1 adipocytes were exposed to either hydrogen peroxide (H2O2) (100??M) or tumor necrosis factor-? (TNF?) (1?ng/mL) for 24 hours in the presence or absence of increasing concentrations of boldine (5–100??M). Quantitative polymerase chain reaction showed that both the oxidants decreased the mRNA levels of adiponectin, PPAR?, and C/EBP? to half of the control levels. Boldine, at all concentrations, counteracted the inhibitory effect of H2O2 or TNF? and increased the expression of adiponectin and its regulators. The effect of boldine on adiponectin expression was biphasic, with the lower concentrations (5–25??M) having a larger inductive effect compared to higher concentrations (50–100??M). Boldine treatment alone in the absence of H2O2 or TNF? was also able to induce adiponectin at the inductive phase of adipogenesis. Peroxisome proliferator response element-luciferase promoter transactivity analysis showed that boldine interacts with the PPAR response element and could potentially modulate PPAR responsive genes. Our results indicate that boldine is able to modulate the expression of adiponectin and its regulators in 3T3-L1 cells and has the potential to be beneficial in obesity-related cardiovascular disease. PMID:19857072

  15. The aporphine alkaloid boldine induces adiponectin expression and regulation in 3T3-L1 cells.

    PubMed

    Yu, Bangning; Cook, Carla; Santanam, Nalini

    2009-10-01

    Adiponectin is an adipokine secreted by differentiated adipocytes. Clinical studies suggest a negative correlation between oxidative stress and adiponectin levels in patients with metabolic syndrome or cardiovascular disease. Natural compounds that can prevent oxidative stress mediated inhibition of adiponectin may be potentially therapeutic. Boldine, an aporphine alkaloid abundant in the medicinal plant Peumus boldus, is a powerful antioxidant. The current study demonstrates the effects of boldine on the expression of adiponectin and its regulators, CCAAT/enhancer binding protein-alpha (C/EBPalpha) and peroxisome proliferator-activated receptor (PPAR)-gamma, in 3T3-L1 cells. Differentiated 3T3-L1 adipocytes were exposed to either hydrogen peroxide (H(2)O(2)) (100 microM) or tumor necrosis factor-alpha (TNFalpha) (1 ng/mL) for 24 hours in the presence or absence of increasing concentrations of boldine (5-100 microM). Quantitative polymerase chain reaction showed that both the oxidants decreased the mRNA levels of adiponectin, PPARgamma, and C/EBPalpha to half of the control levels. Boldine, at all concentrations, counteracted the inhibitory effect of H(2)O(2) or TNFalpha and increased the expression of adiponectin and its regulators. The effect of boldine on adiponectin expression was biphasic, with the lower concentrations (5-25 microM) having a larger inductive effect compared to higher concentrations (50-100 microM). Boldine treatment alone in the absence of H(2)O(2) or TNFalpha was also able to induce adiponectin at the inductive phase of adipogenesis. Peroxisome proliferator response element-luciferase promoter transactivity analysis showed that boldine interacts with the PPAR response element and could potentially modulate PPAR responsive genes. Our results indicate that boldine is able to modulate the expression of adiponectin and its regulators in 3T3-L1 cells and has the potential to be beneficial in obesity-related cardiovascular disease. PMID:19857072

  16. Effects of bovine colostral ultrafiltrates on growth and differentiation of 3T3-L1 preadipocytes.

    PubMed

    Lee, Seong-Ho; Hossner, Kim L

    2002-12-01

    This study was designed to compare the effects of whole and size-fractionated bovine colostrum with bovine calf serum (BCS) on the growth and differentiation of 3T3-L1 fibroblasts. High (HMW) and low (LMW)-molecular-mass ultrafiltrate fractions of colostrum were prepared from defatted colostrum (COL) by diafiltration through membranes with a molecular-mass cut-off of 30 kDa. Incorporation of [(3)H]thymidine into the cells was used as a reflection of DNA synthesis/cell proliferation. The growth-promoting activity of LMW was 2.3- and 2.5-fold higher than COL and HMW, respectively (P <0.05), and 185 microg/ml LMW stimulated cell proliferation equivalent to 10% BCS. Although insulin-like growth factor (IGF)-I, IGF-II and platelet-derived growth factor AB stimulated 3T3-L1 cells, antibodies to these factors did not inhibit the LMW effects. The LMW fraction was about twice as effective as COL and HMW in stimulating differentiation of the cells into adipocytes, but maximal differentiation was only 60% of that seen with 10% fetal bovine serum (FBS). Treatment with COL, HMW, IGF-I and insulin induced peroxisome-proliferator-activated receptor gamma RNA, but levels were about half of that with 10% FBS treatment and LMW induction was 80% of FBS. Low amounts of leptin mRNA were detected in adipocytes and abundance did not differ between treatments with BCS, hormones or COL fractions. This study showed that bovine colostral LMW stimulated the growth and differentiation of 3T3-L1 preadipocytes and may be a useful serum substitute to support the growth of these cells. PMID:12452804

  17. Osteogenic role of endosomal chloride channels in MC3T3-E1 cells

    Microsoft Academic Search

    Huan WangNa; Na Huo; Feifei Li; Shanmin Fu; Yang Xue; Ting Yang; Xuan Wen; Yin Ding; Xiaohong Duan

    2010-01-01

    ClC-3, ClC-4, and ClC-5 belong to the voltage gated chloride channels (ClCs) and facilitate the endosomal acidification. The\\u000a mutations of these endosomal chloride channel genes cause different genetic diseases with various bone disorders. We hypothesized\\u000a that these endosomal ClCs might be involved in the bone development or osteoblast differentiation. Here we used MC3T3-E1 osteoprogenitor\\u000a cell line and primarily cultured mouse

  18. Effects of amide constituents from pepper on adipogenesis in 3T3-L1 cells.

    PubMed

    Zhang, Hailong; Matsuda, Hisashi; Nakamura, Seikou; Yoshikawa, Masayuki

    2008-06-01

    Several amide constituents (piperlonguminine and retrofractamides A, B, and C) from the fruit of Piper chaba promoted adipogenesis of 3T3-L1 cells. Among them, retrofractamide A was the most active and significantly increased the amount of adiponectin released into the medium and the uptake of 2-deoxyglucose into the cells. Retrofractamide A also increased mRNA levels of adiponectin, peroxisome proliferator-activated receptor gamma2 (PPARgamma2), glucose transporter 4 (GLUT4), and insulin receptor substrate 1 (IRS-1), but did not act as a PPARgamma agonist different from troglitazone. PMID:18477507

  19. 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. PMID:24470104

  20. Heterogeneity in the physiological states and pharmacological responses of differentiating 3T3-L1 preadipocytes

    PubMed Central

    Loo, Lit-Hsin; Lin, Hai-Jui; Singh, Dinesh K.; Lyons, Kathleen M.

    2009-01-01

    Increases in key components of adipogenesis and lipolysis pathways correlate at the population-averaged level during adipogenesis. However, differentiating preadipocytes are highly heterogeneous in cellular and lipid droplet (LD) morphologies, and the degree to which individual cells follow population-averaged trends is unclear. In this study, we analyze the molecular heterogeneity of differentiating 3T3-L1 preadipocytes using immunofluorescence microscopy. Unexpectedly, we only observe a small percentage of cells with high simultaneous expression of markers for adipogenesis (peroxisome proliferator-activated receptor ? [PPAR?], CCAAT/enhancer-binding protein ?, and adiponectin) and lipid accumulation (hormone-sensitive lipase, perilipin A, and LDs). Instead, we identify subpopulations of cells with negatively correlated expressions of these readouts. Acute perturbation of adipocyte differentiation with PPAR? agonists, forskolin, and fatty acids induced subpopulation-specific effects, including redistribution of the percentage of cells in observed subpopulations and differential expression levels of PPAR?. Collectively, our results suggested that heterogeneity observed during 3T3-L1 adipogenesis reflects a dynamic mixture of subpopulations with distinct physiological states. PMID:19948481

  1. Zinc-chelated Vitamin C Stimulates Adipogenesis of 3T3-L1 Cells

    PubMed Central

    Ghosh, Chiranjit; Yang, Seung Hak; Kim, Jong Geun; Jeon, Tae-Il; Yoon, Byung Hyun; Lee, Jai Young; Lee, Eun Young; Choi, Seok Geun; Hwang, Seong Gu

    2013-01-01

    Adipose tissue development and function play a critical role in the regulation of energy balance, lipid metabolism, and the pathophysiology of metabolic syndromes. Although the effect of zinc ascorbate supplementation in diabetes or glycemic control is known in humans, the underlying mechanism is not well described. Here, we investigated the effect of a zinc-chelated vitamin C (ZnC) compound on the adipogenic differentiation of 3T3-L1 preadipocytes. Treatment with ZnC for 8 d significantly promoted adipogenesis, which was characterized by increased glycerol-3-phosphate dehydrogenase activity and intracellular lipid accumulation in 3T3-L1 cells. Meanwhile, ZnC induced a pronounced up-regulation of the expression of glucose transporter type 4 (GLUT4) and the adipocyte-specific gene adipocyte protein 2 (aP2). Analysis of mRNA and protein levels further showed that ZnC increased the sequential expression of peroxisome proliferator-activated receptor gamma (PPAR?) and CCAAT/enhancer-binding protein alpha (C/EBP?), the key transcription factors of adipogenesis. These results indicate that ZnC could promote adipogenesis through PPAR? and C/EBP?, which act synergistically for the expression of aP2 and GLUT4, leading to the generation of insulin-responsive adipocytes and can thereby be useful as a novel therapeutic agent for the management of diabetes and related metabolic disorders. PMID:25049900

  2. Anti-adipogenic effect of mulberry leaf ethanol extract in 3T3-L1 adipocytes

    PubMed Central

    Yang, Soo Jin; Park, Na-Young

    2014-01-01

    BACKGROUND/OBJECTIVES Adipogenesis is part of the cell differentiation process in which undifferentiated fibroblasts (pre-adipocytes) become mature adipocytes with the accumulation of lipid droplets and subsequent cell morphological changes. Several transcription factors and food components have been suggested to be involved in adipogenesis. The aim of this study was to determine whether mulberry leaf ethanol extract (MLEE) affects adipogenesis in 3T3-L1 adipocytes. MATERIALS/METHODS The 3T3-L1 adipocytes were treated with different doses of MLEE for 8 days starting 2 days post-confluence. Cell viability, fat accumulation, and adipogenesis-related factors including CCAAT-enhancer-binding protein alpha (C/EBP?), peroxisome proliferator-activated receptor gamma (PPAR?), PPAR? coactivator 1 alpha (PGC-1?), fatty acid synthase (FAS), and adiponectin were analyzed. RESULTS Results showed that MLEE treatments at 10, 25, 50, and 100 µg/ml had no effect on cell morphology and viability. Without evident toxicity, all MLEE treated cells had lower fat accumulation compared with control as shown by lower absorbances of Oil Red O stain. MLEE at 50 and 100 µg/ml significantly reduced protein levels of PPAR?, PGC-1?, FAS, and adiponectin in differentiated adipocytes. Furthermore, protein level of C/EBP? was significantly decreased by the treatment of 100 µg/ml MLEE. CONCLUSION These results demonstrate that MLEE treatment has an anti-adipogenic effect in differentiated adipocytes without toxicity, suggesting its potential as an anti-obesity therapeutic. PMID:25489399

  3. Phosphorylation of tau protein in tau-transfected 3T3 cells.

    PubMed

    Sygowski, L A; Fieles, A W; Lo, M M; Scott, C W; Caputo, C B

    1993-11-01

    The tau protein of Alzheimer paired helical filaments (PHFs) is aberrantly phosphorylated, as evidenced by its reactivity with several phosphate-dependent antibodies. We sought to identify whether this unusual phosphorylation state exists in tau expressed by transfected NIH 3T3 fibroblasts. Immunoblot analysis of cell clones transfected with constructs for either the 3-repeat or 4-repeat isoforms of tau revealed two tau bands, with the lower band migrating with unmodified tau in each case. Antibodies T3P and tau-1 were used to probe these bands, as they also react with PHF-tau in a phosphate-dependent manner. The epitopes for both antibodies were phosphorylated in both tau isoforms. Only the upper band was phosphorylated at the T3P site whereas phosphorylation at the tau-1 site was not always associated with a shift of tau mobility on gels. Tau in both bands was soluble, in contrast to PHF-tau, and was competent to bind to exogenously added bovine microtubules. Colchicine treatment of the cells resulted in an inhibition of phosphorylation at both sites, through an unknown mechanism. In conclusion human tau expressed in 3T3 cells was phosphorylated at the T3P and tau-1 sites as is PHF-tau, although no PHFs formed and the phosphorylated tau was competent to bind to microtubules. PMID:8302160

  4. Regulation of lipoprotein lipase synthesis in 3T3-L1 adipocytes by interleukin-1

    SciTech Connect

    Price, S.R.; Mizel, S.B.; Pekala, P.H.

    1986-05-01

    When fully differentiated 3T3-L1 fatty fibroblasts were exposed to purified, recombinant murine interleukin-1, a dose dependent suppression of lipoprotein lipase activity was observed. The loss of activity reached a maximum of 60-70% of control and appeared to be due to a specific effect on the synthesis of the enzyme as judged by a suppression of the ability to incorporate (/sup 35/S)methionine into immunoprecipitable lipoprotein lipase. There was no general effect on protein synthesis as determined by radiolabel incorporated into acid precipitable protein, however, after a 17 h exposure of the 3T3-L1 cells to interleukin-1, the synthesis of two proteins (molecular weights, 19,400 and 165,000 daltons) was enhanced several fold. The observed effects on protein synthesis in the adipocytes occur at a concentration of interleukin-1 which is similar to the concentration necessary for the stimulation of (/sup 3/H)thymidine incorporation into mouse thymocyte DNA. The present study represents the first unequivocal report of the ability of interleukin-1 to regulate protein synthesis in intact cells, specifically adipocytes. Moreover, their results demonstrate the ability of interleukin-1 to regulate metabolism by controlling the synthesis of specific proteins.

  5. High-dose Resveratrol Inhibits Insulin Signaling Pathway in 3T3-L1 Adipocytes

    PubMed Central

    Lee, Haemi; Kim, Jae-woo

    2013-01-01

    Background Insulin resistance is a major factor in the development of metabolic syndrome and is associated with central obesity and glucose intolerance. Resveratrol, a polyphenol found in fruits, has been shown to improve metabolic conditions. Although it has been widely studied how resveratrol affects metabolism, little is known about how resveratrol regulates lipogenesis with insulin signaling in 3T3-L1 adipocytes. Methods: We treated differentiated 3T3-L1 adipocytes with resveratrol to observe whether resveratrol is effective at reducing lipid accumulation. Results: Resveratrol treatment after mitotic clonal expansion resulted in decreased lipid accumulation accompanied by reduced fatty acid synthase expression. Decreased glucose uptake was observed with inhibited GLUT4 translocation in cells treated with 100 ?M resveratrol, suggesting that high doses of resveratrol block insulin signaling in adipocytes. Insulin-stimulated Akt phosphorylation is also dose-dependently reduced with resveratrol treatment. Interestingly, Akt phosphorylation is upregulated when cells are treated with long-term low doses of resveratrol, suggesting that only low doses of resveratrol improve metabolic conditions. Conclusion: High doses of resveratrol block the insulin signaling pathway, thereby reducing glucose uptake and lipid accumulation in vitro. The results also provide information about in vivo administration dosages and may explain the discrepancy between in vitro and in vivo effects of resveratrol.

  6. Transmissible Gastroenteritis in Feeder Pigs: Observations on the Jejunal Epithelium of Normal Feeder Pigs and Feeder Pigs Infected with TGE Virus

    PubMed Central

    Morin, M.; Morehouse, L. G.

    1974-01-01

    Light and electron microscopy findings in the jejunal mucosa of the normal feeder pig and feeder pigs infected with transmissible gastroenteritis (TGE) virus are reported. Villi in the mid jejunum of the normal feeder pig were elongated, finger shaped and covered with a layer of columnar absorptive cells with a well developed and regular brush border. Severe lesions of villous atrophy were present in all jejunal segments of feeder swine killed 96 hours post infection with TGE virus. Atrophic villi were covered by flat to cuboidal cells with a poorly developed brush border in some areas. In other segments, cells varied in appearance from sub-columnar to columnar type of near normal appearance. The ultrastructure of the jejunal absorptive cells in the normal feeder pig was found to be similar to that described for the jejunal cells of other adult mammals. There were no significant indications of high pinocytotic activity. The epithelial cells covering the atrophic villi of TGE infected pigs had a fine structure similar to that described for the crypt cells, ranging in appearance from very immature to moderately differentiated cells. Microvilli were very short, decreased markedly in number and irregular in arrangement. The terminal web was poorly developed. Strands of rough endoplasmic reticulum were markedly diminished and an increase in free ribosomes was noted. The significance of these observations in explaining pathogenesis of TGE in feeder pigs is discussed. ImagesFig. 1.Fig. 2.Fig. 3.Fig. 4.Fig. 5.Fig. 6.Fig. 7.Fig. 8. PMID:4277743

  7. Development of dry coal feeders

    NASA Technical Reports Server (NTRS)

    Bonin, J. H.; Cantey, D. E.; Daniel, A. D., Jr.; Meyer, J. W.

    1977-01-01

    Design and fabrication of equipment of feed coal into pressurized environments were investigated. Concepts were selected based on feeder system performance and economic projections. These systems include: two approaches using rotating components, a gas or steam driven ejector, and a modified standpipe feeder concept. Results of development testing of critical components, design procedures, and performance prediction techniques are reviewed.

  8. Ultrasound stimulation increases proliferation of MC3T3-E1 preosteoblast-like cells

    PubMed Central

    2014-01-01

    Background Mechanical stimulation of bone increases bone mass and fracture healing, at least in part, through increases in proliferation of osteoblasts and osteoprogenitor cells. Researchers have previously performed in vitro studies of ultrasound-induced osteoblast proliferation but mostly used fixed ultrasound settings and have reported widely varying and inconclusive results. Here we critically investigated the effects of the excitation parameters of low-intensity pulsed ultrasound (LIPUS) stimulation on proliferation of MC3T3-E1 preosteoblastic cells in monolayer cultures. Methods We used a custom-designed ultrasound exposure system to vary the key ultrasound parameters—intensity, frequency and excitation duration. MC3T3-E1 cells were seeded in 12-well cell culture plates. Unless otherwise specified, treated cells, in groups of three, were excited twice for 10 min with an interval of 24 h in between after cell seeding. Proliferation rates of these cells were determined using BrdU and MTS assays 24 h after the last LIPUS excitation. All data are presented as the mean ± standard error. The statistical significance was determined using Student's two-sample two-tailed t tests. Results Using discrete LIPUS intensities ranging from 1 to 500 mW/cm2 (SATA, spatial average-temporal average), we found that approximately 75 mW/cm2 produced the greatest increase in osteoblast proliferation. Ultrasound exposures at higher intensity (approximately 465 mW/cm2) significantly reduced proliferation in MC3T3-E1 cells, suggesting that high-intensity pulsed ultrasound may increase apoptosis or loss of adhesion in these cells. Variation in LIPUS frequency from 0.5 MHz to 5 MHz indicated that osteoblast proliferation rate was not frequency dependent. We found no difference in the increase in proliferation rate if LIPUS was applied for 30 min/day or 10 min/day, indicating a habituation response. Conclusion This study concludes that a short-term stimulation with optimum intensity can enhance proliferation of preosteoblast-like bone cells that plays an important role in bone formation and accelerated fracture healing, also suggesting a possible therapeutic treatment for reduced bone mass. PMID:25516803

  9. Irradiation of Polystyrene and Polypropylene to study NIH 3T3 fibroblasts adhesion

    NASA Astrophysics Data System (ADS)

    Arbeitman, C. R.; del Grosso, M. F.; Ibańez, I.; Bermúdez, G. García; Durán, H.; Chappa, V. C.; Mazzei, R.; Behar, M.

    2010-10-01

    When polymers are irradiated with heavy ions new chemical groups are created in a few microns of the material. The irradiation changed the polarity and wettability on the surface so that could enhance the biocompatibility of the modified polymer. The study of chemistry and nanoscale topography of the biomaterial is important in determining its potential applications in medicine and biotechnology, because their strong influence on cell function, adhesion and proliferation. In this study, thin films of Polystyrene and Polypropylene samples were modified by irradiation with low energy ion beams (30-150 keV) and swift heavy ions both with various fluences and energies. The changes were evaluated with different methods. Adhesion of NIH 3T3 fibroblasts onto unirradiated and irradiated surfaces has been studied by in vitro techniques. The correlations between physicochemical properties as a function of different irradiations parameters were compared with cell adhesion on the modified polymer surface.

  10. Metabolism of vitamin K in Swiss 3T3 mouse fibroblasts

    E-print Network

    Johnson, Terryl Marie

    1985-01-01

    the carboxylation of blood proteins, induce GLU VITAMIN K CYCLE GLA K Carboxylase K Epoxidase HHO I I II -N-C-C- + CO~+ 0~ I H-C-H I H-C-H I COOH H HO I I II -N-CM- I H-C-H I H-C HOOC COOH / X CHs OH K red. K Epoxide Reductase 0 CHs...'1=, II at I a a 0 0 A F al gmCu[ t' at CV 0 0 at al ?I 0 S~nII 33 TABLE 1 METABOLISM OF VITAMIN K IN SWISS 3T3 MOUSE FIBROBLASTS EPOXIDE FORMATION nMo?/mg protein VITAMIN K-DEPENDENT CARBOXYLATION nMols/mg protein Fibroblasts Additions...

  11. Kibizu concentrated liquid suppresses the accumulation of lipid droplets in 3T3-L1 cells.

    PubMed

    Inoue, Chisato; Kozaki, Tomomi; Morita, Yukiko; Shirouchi, Bungo; Fukami, Katsuya; Shimizu, Kuniyoshi; Sato, Masao; Katakura, Yoshinori

    2015-08-01

    Adipocyte size is closely related to the occurrence of diabetes, metabolic syndrome, and insulin resistance. Thus, researchers are searching for active substances that function to reduce adipocyte size. In the present study, we focused on sugar cane vinegar, Kibizu, and evaluated the function of Kibizu to reduce adipocyte size by using an in vitro model system, because people in Amami Oshima famous for longevity regularly consume Kibizu. Results showed that Kibizu treatment significantly reduced the size and number of lipid droplets in 3T3-L1 cells, relative to treatment with Kurozu, another traditional vinegar. Results of an extraction experiment suggest that the active components in Kibizu are lipophilic and hydrophobic. In addition, an in vivo experiment on rats treated with Kibizu showed that the active components were contained in large vein blood. Results of an additional in vivo experiment suggest that metabolites generated by Kibizu-treated rats are primarily contained or modified specifically in the large vein blood. PMID:25672941

  12. 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. PMID:17086444

  13. Altered kinase C function in transformed BALB/c3T3 cells

    SciTech Connect

    Yang, H.C. (Harbor-UCLA Medical Center, Torrance, CA (United States))

    1989-07-01

    Comparative studies of kinase C function were performed in an untransformed (A31) and the benzo(a)pyrene (BPA31), dimethylbenz(a)anthracene (DA31), and Kirsten sarcoma virus (KA31) transformed BALB/c 3T3 mouse fibroblast cell lines. The 80-kDa kinase C dependent phosphoprotein (pp80), an in vivo marker of kinase C activity, was markedly decreased in the transformed cells although the amount of the 80-kDa substrate protein in the BPA31 cells was similar to that in the untransformed A31 cells. Total cell lysate kinase C levels were lower in the transformed cells but this difference could not account for the reduced pp80 phosphorylation. Increased affinity of kinase C for the membrane fraction in the BPA31 cells may account for decreased phosphorylation of pp80.

  14. Carcinogenic potential of metal nanoparticles in BALB/3T3 cell transformation assay.

    PubMed

    Sighinolfi, G L; Artoni, E; Gatti, A M; Corsi, L

    2014-10-30

    Metal-based nanoparticles (NPs), are currently used in many application fields including consumer products, pharmaceuticals, and biomedical treatments. In spite to their wide applications, an in-depth study of their potential toxic effects is still lacking. The aim of the present research was to investigate the potential initiator or promoter-like activity of different metallic NPs such as gold, iron, cobalt, and cerium using the Balb/3T3 two-stage transformation assay. The results indicated that all the selected metallic NPs, except for cobalt, when used as initiators did not induce any transformation in Balb/3T3 cell line. Moreover, Au and Fe3 O4 NPs, when used in place of the tumor promoter treatment TPA, increased significantly the number of Foci/dish as compared to the MCA treatment alone. The number of Foci/dish was 2.6 for Au NPs and 2.13 for Fe3 O4 ones, similar to those obtained by the positive control treatment (MCA?+?TPA), whereas 1.27 for MCA treatment alone. On the contrary, CeO2 NPs did not show any difference in the number of Foci/dish, as compared to MCA alone, but it decreased the number of foci by 65% in comparison to the positive control (MCA?+?TPA). As expected, cobalt NPs showed an increased cytotoxicity and only a few surviving cells were found at the time of analysis showing a number of Foci/dish of 0.13. For the first time, our data clearly showed that Au and Fe3 O4 NPs act as promoters in the two stage transformational assay, suggesting the importance to fully investigate the NPs carcinogenic potential with different models. © 2014 Wiley Periodicals, Inc. Environ Toxicol, 2014. PMID:25358123

  15. In vitro BALB/3T3 cell transformation assay of nonoxynol-9 and 1,4-dioxane

    SciTech Connect

    Sheu, C.W.; Moreland, F.M.; Lee, J.K.; Dunkel, V.C.

    1988-01-01

    The spermicidal surfactant nonoxynol-9 (Igepal CO-630, GAF Corp.) and a potential impurity, 1,4-dioxane, were tested in the in vitro cell transformation assay using BALB/3T3 cells. Two treatment periods, 48 hr and 13 days, were used. Nonoxynol-9, tested at levels up to 10 /sup +/g/ml, did not induce transformation, whereas dioxane was very active in the induction type II foci in the cultured BALB/3T3 cells.

  16. Lipoprotein Lipase Suppression in 3T3-L1 Cells by an Endotoxin-Induced Mediator from Exudate Cells

    Microsoft Academic Search

    Masanobu Kawakami; Phillip H. Pekala; Anthony Cerami

    1982-01-01

    Conditioned medium from cultures of mouse peritoneal exudate cells incubated with endotoxin contains a mediator that markedly suppresses (>90%) lipoprotein lipase (triacylglycero-protein acylhydrolase, EC 3.1.1.34) activity in differentiating 3T3-L1 mouse preadipocytes. The effect is dependent upon the amount of mediator and is evident as early as 30 min after the addition of the mediator-containing medium to 3T3-L1 cell cultures. Neither

  17. ATF3 inhibits adipocyte differentiation of 3T3-L1 cells

    SciTech Connect

    Jang, Min Kyung; Kim, Cho Hee [School of Korean Medicine, Pusan National University, 30 Beom-eo ri, Mulguem-eup, Yangsan-si, Gyeongnam 609-735 (Korea, Republic of)] [School of Korean Medicine, Pusan National University, 30 Beom-eo ri, Mulguem-eup, Yangsan-si, Gyeongnam 609-735 (Korea, Republic of); Seong, Je Kyung [Department of Anatomy and Cell Biology, College of Veterinary Medicine, Seoul National University, Seoul 151-742 (Korea, Republic of)] [Department of Anatomy and Cell Biology, College of Veterinary Medicine, Seoul National University, Seoul 151-742 (Korea, Republic of); Jung, Myeong Ho, E-mail: jung0603@pusan.ac.kr [School of Korean Medicine, Pusan National University, 30 Beom-eo ri, Mulguem-eup, Yangsan-si, Gyeongnam 609-735 (Korea, Republic of)

    2012-04-27

    Highlights: Black-Right-Pointing-Pointer Overexpression of ATF3 inhibits adipocyte differentiation in 3T3-L1 cells. Black-Right-Pointing-Pointer Overexpression of ATF3 represses C/EBP{alpha} expression. Black-Right-Pointing-Pointer ATF3 directly binds to mouse C/EBP{alpha} promoter spanning from -1928 to -1907. Black-Right-Pointing-Pointer ATF3 may play a role in hypoxia-mediated inhibition of adipocyte differentiation. -- Abstract: ATF3 is a stress-adaptive gene that regulates proliferation or apoptosis under stress conditions. However, the role of ATF3 is unknown in adipocyte cells. Therefore, in this study, we investigated the functional role of ATF3 in adipocytes. Both lentivirus-mediated overexpression of ATF3 and stably-overexpressed ATF3 inhibited adipocyte differentiation in 3T3-L1 cells, as revealed by decreased lipid staining with oil red staining and reduction in adipogenic genes. Thapsigargin treatment and overexpression of ATF3 decreased C/EBP{alpha} transcript and repressed the activity of the 3.6-kb mouse C/EBP{alpha} promoter, demonstrating that ATF3 downregulates C/EBP{alpha} expression. Transfection studies using mutant constructs containing 5 Prime -deletions in the C/EBP{alpha} promoter revealed that a putative ATF/CRE element, GGATGTCA, is located between -1921 and -1914. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay demonstrated that ATF3 directly binds to mouse C/EBP{alpha} promoter spanning from -1928 to -1907. Both chemical hypoxia-mimetics or physical hypoxia led to reduce the C/EBP{alpha} mRNA and repress the promoter activity of the C/EBP{alpha} gene, whereas increase ATF3 mRNA, suggesting that ATF3 may contribute to the inhibition of adipocyte differentiation in hypoxia through downregulation of C/EBP{alpha} expression. Collectively, these results demonstrate that ATF3 represses the C/EBP{alpha} gene, resulting in inhibition of adipocyte differentiation, and thus plays a role in hypoxia-mediated inhibition of adipocyte differentiation.

  18. WEHI-3 cells inhibit adipocyte differentiation in 3T3-L1 cells.

    PubMed

    Lai, Jing; Liu, Gexiu; Yan, Guoyao; He, Dongmei; Zhou, Ying; Chen, Shengting

    2015-06-26

    By investigating the anti-adipogenic effects of WEHI-3 cells - a murine acute myelomonocytic leukemia cell line - we sought to improve the efficiency of hematopoietic stem cell transplantation (HSCT). Analysis of Oil Red O staining and the expression of adipogenic genes, including PPAR?, C/EBP?, FAS and LPL, indicated that WEHI-3 cells significantly inhibited 3T3-L1 mouse preadipocyte cells from differentiating into adipocytes. In vivo, fat vacuoles in mice injected with WEHI-3 cells were also remarkably reduced in the murine bone marrow pimelosis model. Moreover, the key gene in the Rho signaling pathway, ROCKII, and the key gene in the Wnt signaling pathway, ?-catenin, were both upregulated compared with the control group. siRNA-mediated knockdown of ROCKII and ?-catenin reversed these WEHI-3-mediated anti-adipogenic effects. Taken together, these data suggest that WEHI-3 cells exert anti-adipogenic effects and that both ROCKII and ?-catenin are involved in this process. PMID:25911323

  19. Suppressive actions of eicosapentaenoic acid on lipid droplet formation in 3T3-L1 adipocytes

    PubMed Central

    2010-01-01

    Background Lipid droplet (LD) formation and size regulation reflects both lipid influx and efflux, and is central in the regulation of adipocyte metabolism, including adipokine secretion. The length and degree of dietary fatty acid (FA) unsaturation is implicated in LD formation and regulation in adipocytes. The aims of this study were to establish the impact of eicosapentaenoic acid (EPA; C20:5n-3) in comparison to SFA (STA; stearic acid, C18:0) and MUFA (OLA; oleic acid, C18:1n-9) on 3T3-L1 adipocyte LD formation, regulation of genes central to LD function and adipokine responsiveness. Cells were supplemented with 100 ?M FA during 7-day differentiation. Results EPA markedly reduced LD size and total lipid accumulation, suppressing PPAR?, Cidea and D9D/SCD1 genes, distinct from other treatments. These changes were independent of alterations of lipolytic genes, as both EPA and STA similarly elevated LPL and HSL gene expressions. In response to acute lipopolysaccharide exposure, EPA-differentiated adipocytes had distinct improvement in inflammatory response shown by reduction in monocyte chemoattractant protein-1 and interleukin-6 and elevation in adiponectin and leptin gene expressions. Conclusions This study demonstrates that EPA differentially modulates adipogenesis and lipid accumulation to suppress LD formation and size. This may be due to suppressed gene expression of key proteins closely associated with LD function. Further analysis is required to determine if EPA exerts a similar influence on LD formation and regulation in-vivo. PMID:20525346

  20. Specific DNA sequences associated with the nuclear matrix in synchronized mouse 3T3 cells.

    PubMed Central

    Goldberg, G I; Collier, I; Cassel, A

    1983-01-01

    Eukaryotic chromatin appears to be organized into arrays of supercoiled loops anchored to the scaffolding structure of the mitotic chromosome core or to the nuclear matrix of interphase nuclei. To reveal whether specific DNA sequences are involved in this level of chromatin organization, we isolated and cloned a population of DNA molecules [average length of 150 base pairs (bp)] closely associated with the nuclear matrix after exhaustive DNase digestion and subsequent extensive protease digestion. The nuclear matrix was obtained from murine BALB/c 3T3 cells synchronized at the G1/S border of the cell cycle. We report the structure of two sequences, designated G4 and G5, which are highly enriched in the matrix DNA. Sequence G4, of 152 bp, contains three 31-bp direct head-to-tail repeats. An 11-bp sequence at the end of each repeat is homologous to the first large tumor antigen recognition site of human papova virus. Sequence G5, of 135 bp, consists of two well-defined domains, in which the first domain is a fragment of the B1 repetitive sequence. The results suggest the possibility that the loops of histone-depleted chromatin are connected to the scaffold of the nuclear matrix, with specific DNA sequences at the anchorage sites. Images PMID:6580619

  1. Identification of pathway-based toxicity in the BALB/c 3T3 cell model.

    PubMed

    Vaccari, Monica; Mascolo, Maria Grazia; Rotondo, Francesca; Morandi, Elena; Quercioli, Daniele; Perdichizzi, Stefania; Zanzi, Cristina; Serra, Stefania; Poluzzi, Vanes; Angelini, Paola; Grilli, Sandro; Colacci, Annamaria

    2015-09-01

    The particulate matter represents one of the most complex environmental mixtures, whose effects on human health and environment vary according to particles characteristics and source of emissions. The present study describes an integrated approach, including in vitro tests and toxicogenomics, to highlight the effects of air particulate matter on toxicological relevant endpoints. Air samples (PM2.5) were collected in summer and winter at different sites, representative of different levels of air pollution. Samples organic extracts were tested in the BALB/c 3T3 CTA at a dose range 1-12m(3). The effect of the exposure to the samples at a dose of 8m(3) on the whole-genome transcriptomic profile was also assessed. All the collected samples induced dose-related toxic effects in the exposed cells. The modulated gene pathways confirmed that toxicity was related to sampling season and sampling site. The analysis of the KEGG's pathways showed modulation of several gene networks related to oxidative stress and inflammation. Even if the samples did not induce cell transformation in the treated cells, gene pathways related to the onset of cancer were modulated as a consequence of the exposure. This integrated approach could provide valuable information for predicting toxic risks in humans exposed to air pollution. PMID:25450744

  2. Serum-induced G0/G1 transition in chemically transformed 3T3 cells

    SciTech Connect

    Gray, H.E.; Buchou, T.; Mester, J.

    1987-03-01

    Quiescent, chemically transformed (benzo-a-pyrene) BALB/c 3T3 cells (BP A31) enter the cell division cycle when exposed to complete medium containing 10% fetal calf serum (FCS); the number of cells recruited is a function of the duration of serum exposure. The recruitment of cells by short (<4 h) serum pulses is not inhibited by simultaneous exposure to cycloheximide (CH), and therefore the initial commitment does not require protein synthesis. The cells enter S phase with a constant delay following the removal of CH, even if CH exposure has been continued for as long as 20 h after the end of the serum pulse. The cell recruitment by serum pulses was inhibited by 5,6-dichloro-1-..beta..-D-ribofuranosyl-benzimidazole (DRB), an inhibitor of cytoplasmic mRNA accumulation. These data suggest that serum exposure produces a stable memory that is necessary and sufficient for the eventual progression through G1 to S phase that occurs when protein synthesis is resumed after the removal of CH; this memory probably consists of mRNA species that are induced by serum and that are stable in the absence of protein synthesis. Unexpectedly, pretreatment of quiescent BP A31 cells with CH (8-24 h) dramatically increased the fraction of the total cell population that is recruited by a serum pulse of fixed duration.

  3. Osteogenic role of endosomal chloride channels in MC3T3-E1 cells.

    PubMed

    Wang, Huan; Huo, Na; Li, Feifei; Fu, Shanmin; Xue, Yang; Yang, Ting; Wen, Xuan; Ding, Yin; Duan, Xiaohong

    2010-09-01

    ClC-3, ClC-4, and ClC-5 belong to the voltage gated chloride channels (ClCs) and facilitate the endosomal acidification. The mutations of these endosomal chloride channel genes cause different genetic diseases with various bone disorders. We hypothesized that these endosomal ClCs might be involved in the bone development or osteoblast differentiation. Here we used MC3T3-E1 osteoprogenitor cell line and primarily cultured mouse osteoblasts and detected the expression of Clcn3, Clcn4, and Clcn5 in these cells. We analyzed the relationships between three endosomal ClCs and the osteogenic phenotype using osteoinductive treatment, overexpressing of ClCs and RNAi of ClCs. We found the increased mRNA levels of osteogenic markers [alkaline phosphatase (Alp), osteocalcin (Oc), bone sialoprotein (Bsp), and runt-related transcription factor 2 (Runx2)] were in parallel to that of Clcn3, Clcn4 and Clcn5 with osteoinductive treatment and overexpressed ClCs. Overexpressed ClCs were localized in intracellular periphery and also promoted the mineralization of cells in vitro. While RNAi mediated gene silencing of ClC-3, ClC-4, and ClC-5 down regulated the expression of the four osteogenic markers. The positive relationship between endosomal ClCs and the osteogenic markers suggested a new function of endosomal ClCs in osteogenic differentiation. PMID:20473779

  4. MC3T3-E1 Cells on Titanium Surfaces with Nanometer Smoothness and Fibronectin Immobilization

    PubMed Central

    Hayakawa, Tohru; Yoshida, Eiji; Yoshimura, Yoshitaka; Uo, Motohiro; Yoshinari, Masao

    2012-01-01

    The present study was aimed to evaluate the viability and total protein contents of osteoblast-like cells on the titanium surface with different surface mechanical treatment, namely, nanometer smoothing (Ra: approximately 2.0?nm) and sandblasting (Ra: approximately 1.0??m), and biochemical treatment, namely, with or without fibronectin immobilization. Fibronectin could be easily immobilized by tresyl chloride-activation technique. MC3T3-E1 cells were seeded on the different titanium surfaces. Cell viability was determined by MTT assay. At 1 day of cell culture, there were no significant differences in cell viability among four different titanium surfaces. At 11 days, sandblasted titanium surface with fibronectin immobilization showed the significantly highest cell viability than other titanium surface. No significant differences existed for total protein contents among four different titanium surfaces at 11 days of cell culture. Scanning electron microscopy observation revealed that smoothness of titanium surface produced more spread cell morphologies, but that fibronectin immobilization did not cause any changes of the morphologies of attached cells. Fibronectin immobilization provided greater amount of the number of attached cells and better arrangement of attached cells. In conclusion, the combination of sandblasting and fibronectin immobilization enhanced the cell viability and fibronectin immobilization providing better arrangements of attached cells. PMID:22675359

  5. Proliferative and morphological changes induced by vanadium compounds on Swiss 3T3 fibroblasts.

    PubMed

    Cortizo, A M; Sálice, V C; Vescina, C M; Etcheverry, S B

    1997-04-01

    Vanadium compounds are shown to have a mitogenic effect on fibroblast cells. The effects of vanadate, vanadyl and pervanadate on the proliferation and morphological changes of Swiss 3T3 cells in culture are compared. Vanadium derivatives induced cell proliferation in a biphasic manner, with a toxic-like effect at doses over 50 microM, after 24 h of incubation. Vanadyl and vanadate were equally potent at 2.5-10 microM. At 50 microM vanadate inhibited cell proliferation, whereas slight inhibition was observed at 100 microM of vanadyl. At 10 microM pervanadate was as potent as vanadate and vanadyl in stimulating fibroblast proliferation, but no effect was observed at lower concentrations. A pronounced cytotoxic-like effect was induced by pervanadate at 50 microM. All of these effects were accompanied by morphological changes: transformation of fibroblast shape from polygonal to fusiform; retraction with cytoplasm condensation; and loss of lamellar processes. The magnitude of these transformations correlates with the potency of vanadium derivatives to induce a cytotoxic-like effect: pervanadate > vanadate > vanadyl. These data suggest that the oxidation state and coordination geometry of vanadium determine the degree of the cytotoxicity. PMID:9210295

  6. Growth, cell cycle progression, and morphology of 3T3 cells following fibroin microsphere ingestion.

    PubMed

    Go, Nam Kyung; Lee, Jin Sil; Lee, Joon Ho; Hur, Won

    2015-04-01

    Cellular uptake of microspheres may cause physiological stress and toxicity. In this report, we investigated the effect of cellular uptake of fibroin microspheres on the growth, cell cycle progression, and morphology of 3T3 cells. The microspheres were prepared by physical cross-linking of fibroin molecules without any chemical modification. Fluorescent microspheres are comprised of fluorescein isothiocyanate-dextran core and fibroin shell. More than 90% of cells were determined to be fluorescence-positive following 24-h incubation with fluorescent microspheres (0.17 mg/mL). Microsphere localization in the cytoplasm was demonstrated using confocal and transmission electron microscopy. Cellular uptake of microspheres did not influence cellular viability, but microsphere concentrations above 0.1 mg/mL resulted in decreased cell proliferation. The proliferation inhibition was attributed to G2 /M phase delay in cell cycle progression and S-phase delay at higher microsphere concentrations (0.33 mg/mL). Although flow cytometry light-scattering data raised the possibility of morphological changes, Coulter counter analysis confirmed no significant size differences between cells incubated with and without microspheres. Accordingly, fibroin microspheres can be a potential vehicle for intracytoplasmic delivery of cargos, without affecting cell viability. PMID:25044553

  7. Attachment and spreadout study of 3T3 cells onto PP track etched films

    NASA Astrophysics Data System (ADS)

    Smolko, Eduardo; Mazzei, Ruben; Tadey, Daniel; Lombardo, Daniel

    2001-12-01

    Polymer surface modifications are obtained by the application of radiation treatments and other physico-chemical methods: fission fragment (ff) irradiation and etching. The biocompatibility of the surface is then observed by cell seeding and cell adhesion experiments. Approaches to improvement of the cell adhesion are obtained by different methods: for example, in PS, cell adhesion is improved after ion implantation; in PMMA, after bombarding the polymer, the surface is reconditioned with surfactants and proteins and in PVDF, cell adhesion is assayed on nuclear tracks membranes. In this work, we obtained important cell adhesion improvements in PP films by irradiation with swift heavy ions and subsequent etching of the nuclear tracks. We use BOPP (isotactic -25 ?m thickness). Irrradiations were performed with a Cf-252 californium ff source. The source has a heavy ff and a light one, with 160-200 MeV energy divided among them corresponding to ff energies between 1 and 2 MeV/amu. A chemical etching procedure consisting of a solution of sulphuric acid and chromium three oxide at 85 °C was used. The 3T3 NIH fibroblast cell line was used for the cell adhesion experiment. Here we report for the first time, the results of a series of experiments by varying the ff fluence and the etching time showing that attachment and spreadout of cells are very much improved in this cell line according to the number of pores and the pore size.

  8. Effect of Black Soybean Koji Extract on Glucose Utilization and Adipocyte Differentiation in 3T3-L1 Cells

    PubMed Central

    Huang, Chi-Chang; Huang, Wen-Ching; Hou, Chien-Wen; Chi, Yu-Wei; Huang, Hui-Yu

    2014-01-01

    Adipocyte differentiation and the extent of subsequent fat accumulation are closely related to the occurrence and progression of diseases such as insulin resistance and obesity. Black soybean koji (BSK) is produced by the fermentation of black soybean with Aspergilllus awamori. Previous study indicated that BSK extract has antioxidative and multifunctional bioactivities, however, the role of BSK in the regulation of energy metabolism is still unclear. We aimed to investigate the effect of glucose utilization on insulin-resistant 3T3-L1 preadipocytes and adipogenesis-related protein expression in differentiated adipocytes with BSK treatment. Cytoxicity assay revealed that BSK did not adversely affect cell viability at levels up to 200 ?g/mL. The potential for glucose utilization was increased by increased glucose transporter 1 (GLUT1), GLUT4 and protein kinase B (AKT) protein expression in insulin-resistant 3T3-L1 cells in response to BSK treatment. Simultaneously, BSK inhibited lipid droplet accumulation in differentiated 3T3-L1 cells. The inhibitory effect of adipogenesis was associated with downregulated peroxisome proliferator-activated receptor ? (PPAR?) level and upregulated Acrp30 protein expression. Our results suggest that BSK extract could improve glucose uptake by modulating GLUT1 and GLUT4 expression in a 3T3-L1 insulin-resistance cell model. In addition, BSK suppressed differentiation and lipid accumulation in mature 3T3-L1 adipocytes, which may suggest its potential for food supplementation to prevent obesity and related metabolic abnormalities. PMID:24821545

  9. Purple Sweet Potato Leaf Extract Induces Apoptosis and Reduces Inflammatory Adipokine Expression in 3T3-L1 Differentiated Adipocytes

    PubMed Central

    Lee, Shou-Lun; Chin, Ting-Yu; Tu, Ssu-Chieh; Wang, Yu-Jie; Hsu, Ya-Ting; Kao, Ming-Ching; Wu, Yang-Chang

    2015-01-01

    Background. Purple sweet potato leaves (PSPL) are widely grown and are considered a healthy vegetable in Taiwan. PSPL contain a high content of flavonoids, and the boiling water-extracted PSPL (PSPLE) is believed to prevent metabolic syndrome. However, its efficacy has not yet been verified. Therefore, we investigated the effect of PSPLE on adipocytes. Methods. The differentiated 3T3-L1 cells used in this study were derived from preadipocytes that were differentiated into adipocytes using an adipogenic agent (insulin, dexamethasone, and 3-isobutyl-1-methylxanthine); approximately 90% of the cells were differentiated using this method. Results. Treating the differentiated 3T3-L1 cells with PSPLE caused a dose-dependent decrease in the number of adipocytes rather than preadipocytes. In addition, treatment with PSPLE resulted in apoptosis of the differentiated 3T3-L1 cells as determined by DAPI analysis and flow cytometry. PSPLE also increased the expression of cleaved caspase-3 and poly ADP-ribose polymerase (PARP). Furthermore, PSPLE induced downregulation of interleukin-6 (IL-6) and tumor necrosis factor-? (TNF-?) gene expression in the differentiated 3T3-L1 cells. Conclusions. These results suggest that PSPLE not only induced apoptosis but also downregulated inflammation-associated genes in the differentiated 3T3-L1 cells. PMID:26170870

  10. Mouse osteoblastic cell line (MC3T3-E1) expresses extracellular calcium (Ca2+o)-sensing receptor and its agonists stimulate chemotaxis and proliferation of MC3T3-E1 cells

    NASA Technical Reports Server (NTRS)

    Yamaguchi, T.; Chattopadhyay, N.; Kifor, O.; Butters, R. R. Jr; Sugimoto, T.; Brown, E. M.; O'Malley, B. W. (Principal Investigator)

    1998-01-01

    The calcium-sensing receptor (CaR) is a G protein-coupled receptor that plays key roles in extracellular calcium ion (Ca2+o) homeostasis in parathyroid gland and kidney. Osteoblasts appear at sites of osteoclastic bone resorption during bone remodeling in the "reversal" phase following osteoclastic resorption and preceding bone formation. Bone resorption produces substantial local increases in Ca2+o that could provide a signal for osteoblasts in the vicinity, leading us to determine whether such osteoblasts express the CaR. In this study, we used the mouse osteoblastic, clonal cell line MC3T3-E1. Both immunocytochemistry and Western blot analysis, using an antiserum specific for the CaR, detected CaR protein in MC3T3-E1 cells. We also identified CaR transcripts in MC3T3-E1 cells by Northern analysis using a CaR-specific riboprobe and by reverse transcription-polymerase chain reaction with CaR-specific primers, followed by nucleotide sequencing of the amplified products. Exposure of MC3T3-E1 cells to high Ca2+o (up to 4.8 mM) or the polycationic CaR agonists, neomycin and gadolinium (Gd3+), stimulated both chemotaxis and DNA synthesis in MC3T3-E1 cells. Therefore, taken together, our data strongly suggest that the osteoblastic cell line MC3T3-E1 possesses both CaR protein and mRNA very similar, if not identical, to those in parathyroid and kidney. Furthermore, the CaR in these osteoblasts could play a key role in regulating bone turnover by stimulating the proliferation and migration of such cells to sites of bone resorption as a result of local release of Ca2+o.

  11. TR4 promotes fatty acid synthesis in 3T3-L1 adipocytes by activation of pyruvate carboxylase expression.

    PubMed

    Park, Sung-Soo; Kim, Seung-Jin; Choi, Hojung; Chang, Chawnshang; Kim, Eungseok

    2014-11-01

    We show that testicular orphan nuclear receptor 4 (TR4) increases the expression of pyruvate carboxylase (PC) gene in 3T3-L1 adipocytes by direct binding to a TR4 responsive element in the murine PC promoter. While TR4 overexpression increased PC activity, oxaloacetate (OAA) and glycerol levels with enhanced incorporation of (14)C from (14)C-pyruvate into fatty acids in 3T3-L1 adipocytes, PC knockdown by short interfering RNA (siRNA) or inhibition of PC activity by phenylacetic acid (PAA) abolished TR4-enhanced fatty acid synthesis. Moreover, TR4 microRNA reduced PC expression with decreased fatty acid synthesis in 3T3-L1 adipocytes, suggesting that TR4-mediated enhancement of fatty acid synthesis in adipocytes requires increased expression of PC gene. PMID:25240193

  12. Effects of continuous passaging on mineralization of MC3T3-E1 cells with improved osteogenic culture protocol.

    PubMed

    Yan, Xiang-Zhen; Yang, Wanxun; Yang, Fang; Kersten-Niessen, Monique; Jansen, John A; Both, Sanne K

    2014-03-01

    The murine-derived MC3T3-E1 cell line provided by the American Type Culture Collection (ATCC) is a well-known osteogenic cell culture model system to test materials in vitro. However, the effect of passaging on its mineralization capacity has never been described and their culture supplements can be further optimized. Therefore, we evaluated the influence of the passage number and different osteogenic culture supplements, including ascorbic acid (AsAP) and dexamethasone (Dex) on the osteogenic capacity of MC3T3-E1 cells. This capacity was measured by the deposited calcium, the alkaline phosphatase activity, and the expression of osteogenic-related genes, including bone sialoprotein (BSP), osteocalcin (OC), and osteopontin (OPN). The results indicated that the mineralization capacity of MC3T3-E1 cells significantly decreased during passaging and got exhausted at passage 34, as assessed by measuring calcium deposition after 28 days of osteogenic induction. Moreover, the combination of AsAP and Dex triggered significantly more mineralization in MC3T3-E1 cells than the ATCC recommended addition of AsAP alone, as indicated by increased calcium deposition and higher expression of BSP and OPN. However, Dex alone could not trigger this effect, but only in combination with the AsAP, which indicates that Dex has no direct effect on mineralization. In conclusion, the passage number of MC3T3-E1 cells is of great importance and the use of cells above 30 passages should be avoided. In addition, the favored osteogenic supplements providing an improved osteogenic differentiation of MC3T3-E1 cells are the combination of AsAP and Dex. PMID:23898861

  13. Inhibitory effects of garcinol and pterostilbene on cell proliferation and adipogenesis in 3T3-L1 cells.

    PubMed

    Hsu, Chin-Lin; Lin, Yu-Jyun; Ho, Chi-Tang; Yen, Gow-Chin

    2012-01-01

    The aim of this work was to study the effects of garcinol and pterostilbene on cell proliferation and adipogenesis in 3T3-L1 cells. The results showed that garcinol and pterostilbene decreased the cell population growth and caused cell cycle arrest at the G2/M phase in 3T3-L1 preadipocytes. During adipocyte differentiation, both garcinol and pterostilbene had inhibitory effects on fat droplet formation and triacylglycerol accumulation. The data indicated that garcinol and pterostilbene could inhibit the glycerol-3-phosphate dehydrogenase (GPDH) activity by 97.8 and 61.5%, respectively, as compared to the control. Both garcinol and pterostilbene significantly attenuated the protein expressions of PPAR? and C/EBP? during 3T3-L1 adipocyte differentiation. Moreover, garcinol and pterostilbene caused an inhibition of lipid accumulation in the 3T3-L1 adipocyte differentiation phase. Garcinol and pterostilbene also significantly up-regulated the gene expression of adiponectin as well as down-regulated the gene expressions of leptin, resistin, and fatty acid synthase (FAS) in 3T3-L1 adipocyte differentiation. In 3T3-L1 adipocytes, garcinol significantly down-regulated the protein expressions of PPAR? and FAS as well as up-regulated the protein expressions of adipose triglyceride lipase (ATGL) and adiponectin. Garcinol also significantly up-regulated the gene expression of adiponectin as well as down-regulated the gene expressions of leptin and FAS. These results suggest that garcinol and pterostilbene have anti-adipogenic effects on preadipocytes and adipocytes. PMID:22094440

  14. ATF3 inhibits adipocyte differentiation of 3T3-L1 cells.

    PubMed

    Jang, Min Kyung; Kim, Cho Hee; Seong, Je Kyung; Jung, Myeong Ho

    2012-04-27

    ATF3 is a stress-adaptive gene that regulates proliferation or apoptosis under stress conditions. However, the role of ATF3 is unknown in adipocyte cells. Therefore, in this study, we investigated the functional role of ATF3 in adipocytes. Both lentivirus-mediated overexpression of ATF3 and stably-overexpressed ATF3 inhibited adipocyte differentiation in 3T3-L1 cells, as revealed by decreased lipid staining with oil red staining and reduction in adipogenic genes. Thapsigargin treatment and overexpression of ATF3 decreased C/EBP? transcript and repressed the activity of the 3.6-kb mouse C/EBP? promoter, demonstrating that ATF3 downregulates C/EBP? expression. Transfection studies using mutant constructs containing 5'-deletions in the C/EBP? promoter revealed that a putative ATF/CRE element, GGATGTCA, is located between -1921 and -1914. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay demonstrated that ATF3 directly binds to mouse C/EBP? promoter spanning from -1928 to -1907. Both chemical hypoxia-mimetics or physical hypoxia led to reduce the C/EBP? mRNA and repress the promoter activity of the C/EBP? gene, whereas increase ATF3 mRNA, suggesting that ATF3 may contribute to the inhibition of adipocyte differentiation in hypoxia through downregulation of C/EBP? expression. Collectively, these results demonstrate that ATF3 represses the C/EBP? gene, resulting in inhibition of adipocyte differentiation, and thus plays a role in hypoxia-mediated inhibition of adipocyte differentiation. PMID:22475484

  15. A proteomic approach to investigate AuNPs effects in Balb/3T3 cells.

    PubMed

    Gioria, Sabrina; Chassaigne, Hubert; Carpi, Donatella; Parracino, Antonietta; Meschini, Stefania; Barboro, Paola; Rossi, François

    2014-07-15

    Although gold nanoparticles (AuNPs) are currently used in several industrial products and biomedical applications, information about their biological effects is very limited. Thus, it is becoming crucial to assess their safety and adequately investigate the complexity of cell-nanoparticles interactions. In this work, the Balb/3T3 mouse fibroblast cell line was selected as an in vitro model to study the effects of AuNPs. Alteration of cellular processes and biochemical pathways caused by AuNPs exposure was investigated by analysing the differentially expressed proteome. Of interest was the difference observed in the protein pattern expression of cells exposed to AuNPs. It was found that 88 and 83 proteins were de-regulated after exposure to 5 and 15nm AuNPs, respectively. Analysis of the proteome revealed that AuNPs triggers several pathways related to cellular growth and proliferation, cell morphology, cell cycle regulation, cellular function and maintenance, oxidative stress, and inflammatory response. Moreover, SPR analysis showed an increase of ECM proteins biosynthesis in cells exposed to AuNPs. We observed by TEM analysis that NPs are internalized and confined mainly in autophagosomes. Endoplasmic reticulum stressed and modification at mitochondrial level occurred. This study aims to improve existing knowledge necessary for a correct assessment of the balance between AuNPs potential adverse and beneficial effects and might have important implications for biomedical applications (e.g. nanomedicine). To conclude proteomics link to system biology analysis is a valuable tool to understand and predict nanoparticles' toxicity, furthermore it has the potential to reveal pathways that may not be immediately evident with classical toxicological assays. PMID:24780912

  16. Hydroxytyrosol promotes mitochondrial biogenesis and mitochondrial function in 3T3-L1 adipocytes.

    PubMed

    Hao, Jiejie; Shen, Weili; Yu, Guangli; Jia, Haiqun; Li, Xuesen; Feng, Zhihui; Wang, Ying; Weber, Peter; Wertz, Karin; Sharman, Edward; Liu, Jiankang

    2010-07-01

    Hydroxytyrosol (HT) in extra-virgin olive oil is considered one of the most important polyphenolic compounds responsible for the health benefits of the Mediterranean diet for lowering incidence of cardiovascular disease, the most common and most serious complication of diabetes. We propose that HT may prevent these diseases by a stimulation of mitochondrial biogenesis that leads to enhancement of mitochondrial function and cellular defense systems. In the present study, we investigated effects of HT that stimulate mitochondrial biogenesis and promote mitochondrial function in 3T3-L1 adipocytes. HT over the concentration range of 0.1-10 micromol/L stimulated the promoter transcriptional activation and protein expression of peroxisome proliferator-activated receptor (PPAR) coactivator 1 alpha (PPARGC1 alpha, the central factor for mitochondrial biogenesis) and its downstream targets; these included nuclear respiration factors 1 and 2 and mitochondrial transcription factor A, which leads to an increase in mitochondrial DNA (mtDNA) and in the number of mitochondria. Knockdown of Ppargc1 alpha by siRNA blocked HT's stimulating effect on Complex I expression and mtDNA copy number. The HT treatment resulted in an enhancement of mitochondrial function, including an increase in activity and protein expression of Mitochondrial Complexes I, II, III and V; increased oxygen consumption; and a decrease in free fatty acid contents in the adipocytes. The mechanistic study of the PPARGC1 alpha activation signaling pathway demonstrated that HT is an activator of 5'AMP-activated protein kinase and also up-regulates gene expression of PPAR alpha, CPT-1 and PPAR gamma. These data suggest that HT is able to promote mitochondrial function by stimulating mitochondrial biogenesis. PMID:19576748

  17. Mitogenic stimuli and phosphatidylinositol (PI) turnover in cultured 3T3 fibroblasts

    SciTech Connect

    Kohler, C.; Petersen, R.

    1986-03-01

    The hydrolysis of PI and polyphosphoinositides by phopholipase C is an early and rapid response to cell activation by a variety of neurotransmitters, hormones, growth factors and pharmacological agonists. The authors have examined the role of PI turnover and the generation of second messengers (diacylglycerol and inositol trisphosphate) in the mitogenic response of cultured Balb/c and Swiss 3T3 cells to polypeptide growth factors. Cells were prelabelled with /sup 3/H inositol for 18-20 hours, washed and suspended in Herpes + Li/sup +/ buffer, and stimulated with platelet-derived growth factor (PDGF), vasopressin, insulin, and other growth factors. PI turnover was measured as the increase in total inositol phosphate (IP) production. IP1, IP2, and IP3 were characterized by sequential elution from a Dowex column. Partially purified PDGF produced a 2-4 fold stimulation of total IP production. This was seen as early as 30 seconds after stimulation and increased for up to 1-2 hours. Balb/c cells were more sensitive than Swiss cells to the mitogenic and PI effects of PDGF. Other mitogenic stimuli had differential effects on PI turnover. Vasopressin (4-400 ng/ml) markedly stimulated PI turnover (3-6 fold) in Swiss, but not Balb/c cells. Insulin (100 ng/ml - 10 ..mu..g/ml) increased total IP to a greater degree in Balb/c cells. Epidermal growth factor (10 ng/ml - 10 ..mu..g/ml) had no effect on PI turnover and fibroblast growth factor (10 ng/ml - 10 ..mu..g/ml) only stimulated at the higher concentrations in Swiss cells. Thrombin (1U/ml - 10 U/ml) produced a 1.5 - 2 fold stimulation in Balb/c cells. Thus, various polypeptide growth factors have differential effects on PI turnover depending on their mitogenic potential and the effector cell type.

  18. Analysis of transcription factor network underlying 3T3-L1 adipocyte differentiation.

    PubMed

    Choi, KyungOh; Ghaddar, Bassel; Moya, Colby; Shi, Hai; Sridharan, Gautham V; Lee, Kyongbum; Jayaraman, Arul

    2014-01-01

    Lipid accumulation in adipocytes reflects a balance between enzymatic pathways leading to the formation and breakdown of esterified lipids, primarily triglycerides. This balance is extremely important, as both high and low lipid levels in adipocytes can have deleterious consequences. The enzymes responsible for lipid synthesis and breakdown (lipogenesis and lipolysis, respectively) are regulated through the coordinated actions of several transcription factors (TFs). In this study, we examined the dynamics of several key transcription factors (TFs) - PPAR?, C/EBP?, CREB, NFAT, FoxO1, and SREBP-1c - during adipogenic differentiation (week 1) and ensuing lipid accumulation. The activation profiles of these TFs at different times following induction of adipogenic differentiation were quantified using 3T3-L1 reporter cell lines constructed to secrete the Gaussia luciferase enzyme upon binding of a TF to its DNA binding element. The dynamics of the TFs was also modeled using a combination of logical gates and ordinary differential equations, where the logical gates were used to explore different combinations of activating inputs for PPAR?, C/EBP?, and SREBP-1c. Comparisons of the experimental profiles and model simulations suggest that SREBP-1c could be independently activated by either insulin or PPAR?, whereas PPAR? activation required both C/EBP? as well as a putative ligand. Parameter estimation and sensitivity analysis indicate that feedback activation of SREBP-1c by PPAR? is negligible in comparison to activation of SREBP-1c by insulin. On the other hand, the production of an activating ligand could quantitatively contribute to a sustained elevation in PPAR? activity. PMID:25075860

  19. Gene expression of MC3T3-E1 osteoblastic cells on titanium and zirconia surface

    PubMed Central

    Gong, Soon-Hyun; Lee, Heesu; Pae, Ahran; Noh, Kwantae; Shin, Yong-Moon; Lee, Jung-Haeng

    2013-01-01

    PURPOSE This study was performed to define attachment and growth behavior of osteoblast-like cells and evaluate the gene expression on zirconia compared to titanium. MATERIALS AND METHODS MC3T3-E1 cells were cultured on (1) titanium and (2) zirconia discs. The tetrazolium-based colorimetric assay (MTT test) was used for examining the attachment of cells. Cellular morphology was examined by scanning electron microscopy (SEM) and alkaline phosphatase (ALP) activity was measured to evaluate the cell differentiation rate. Mann-Whitney test was used to assess the significance level of the differences between the experimental groups. cDNA microarray was used for comparing the 20215 gene expressions on titanium and zirconia. RESULTS From the MTT assay, there was no significant difference between titanium and zirconia (P>.05). From the SEM image, after 4 hours of culture, cells on both discs were triangular or elongated in shape with formation of filopodia. After 24 hours of culture, cells on both discs were more flattened and well spread compared to 4 hours of culture. From the ALP activity assay, the optical density of E1 cells on titanium was slightly higher than that of E1 cells on zirconia but there was no significant difference (P>.05). Most of the genes related to cell adhesion showed similar expression level between titanium and zirconia. CONCLUSION Zirconia showed comparable biological responses of osteoblast-like cells to titanium for a short time during cell culture period. Most of the genes related to cell adhesion and signal showed similar expression level between titanium and zirconia. PMID:24353879

  20. The water-soluble matrix fraction from the nacre of Pinctada maxima produces earlier mineralization of MC3T3-E1 mouse pre-osteoblasts.

    PubMed

    Rousseau, Marthe; Pereira-Mouričs, Lucilia; Almeida, Maria José; Milet, Christian; Lopez, Evelyne

    2003-05-01

    Nacre or mother of pearl is a calcified structure that forms the lustrous inner layer of some shells. We studied the biological activity of the water-soluble matrix (WSM) extracted from powdered nacre from the shell of the pearl oyster, Pinctada maxima, on the MC3T3-E1 pre-osteoblast cell line from mouse calvaria. This cell line has the ability to differentiate into osteoblasts and to mineralize in the presence of beta-glycerophosphate and ascorbic acid. Cell proliferation and alkaline phosphatase activity were measured as markers of osteoblast differentiation, and mineralization was analyzed. These studies revealed that WSM stimulates osteoblast differentiation and mineralization by day 6 instead of the 21-day period required for cells grown in normal mineralizing media. We compared the activity of WSM with that of dexamethasone on this cell line. WSM can inhibit alkaline phosphatase (ALP) activity and the activity of dexamethasone on MC3T3-E1 cells. This study shows that nacre WSM could speed up the differentiation and mineralization of this cell line more effectively than dexamethasone. PMID:12781967

  1. Detecting Low Levels of Cytochalasin B in 3T3 Fibroblast Cells by Analysis of Electrical Noise Obtained from Cellular Micromotion

    NASA Astrophysics Data System (ADS)

    Lovelady, Douglas; Rabson, David; Lo, Chun-Min

    2008-03-01

    We performed several micromotion experiments using the electric cell-substrate impedance sensing (ECIS) apparatus on a confluent layer of 3T3 fibroblast cells exposed to differing, low-level amounts of the toxin cytochalasin B. We previously developed a technique to distinguish cancerous from non-cancerous cultures.ootnotetextD.C. Lovelady et alia, Phys. Rev. E 76, 041908 (2007) Our goal here is to see if the same technique can be used to distinguish toxin levels in a single cell type. The noise of the time series extracted from these experiments is characterized by the power spectrum, Hurst exponent, DFA (detrended fluctuation analysis) exponent, first zero and first 1/e crossing of the autocorrelation function. These measures describe the long- and short-term correlations in the signal, which tell us something about the average behavior of these cells in culture. A change in the behavior of these cells is clearly revealed by an examination of these measures. A principal-component analysis shows a separation of the different toxin levels in the multidimensional space. To our knowledge, this is the most sensitive technique for detecting such a low level of cytochalasin B in 3T3 fibroblast cells.

  2. Polyphosphates inhibit extracellular matrix mineralization in MC3T3-E1 osteoblast cultures

    PubMed Central

    Hoac, Betty; Kiffer-Moreira, Tina; Millán, José Luis; McKee, Marc D.

    2013-01-01

    Studies on various compounds of inorganic phosphate, as well as on organic phosphate added by post-translational phosphorylation of proteins, all demonstrate a central role for phosphate in biomineralization processes. Inorganic polyphosphates are chains of orthophosphates linked by phosphoanhydride bonds that can be up to hundreds of orthophosphates in length. The role of polyphosphates in mammalian systems, where they are ubiquitous in cells, tissues and bodily fluids, and are at particularly high levels in osteoblasts, is not well understood. In cell-free systems, polyphosphates inhibit hydroxyapatite nucleation, crystal formation and growth, and solubility. In animal studies, polyphosphate injections inhibit induced ectopic calcification. While recent work has proposed an integrated view of polyphosphate function in bone, little experimental data for bone are available. Here we show in osteoblast cultures producing an abundant collagenous matrix that normally shows robust mineralization, that two polyphosphates (PolyP5 and PolyP65, polyphosphates of 5 and 65 phosphate residues in length) are potent mineralization inhibitors. Twelve-day MC3T3-E1 osteoblast cultures with added ascorbic acid (for collagen matrix assembly) and ?-glycerophosphate (a source of phosphate for mineralization) were treated with either PolyP5 or PolyP65. Von Kossa staining and calcium quantification revealed that mineralization was inhibited in a dose-dependent manner by both polyphosphates, with complete mineralization inhibition at 10 ?M PolyP. Cell proliferation and collagen assembly were unaffected by polyphosphate treatment, indicating that polyphosphate inhibition of mineralization results not from cell and matrix effects but from direct inhibition of mineralization. This was confirmed by showing that PolyP5 and PolyP65 bound to synthetic hydroxyapatite in a concentration-dependent manner. Tissue-nonspecific alkaline phosphatase (TNAP, ALPL) efficiently hydrolyzed the two PolyPs as measured by Pi release. Importantly, at the concentrations of polyphosphates used in this study which inhibited bone cell culture mineralization, the polyphosphates competitively saturated TNAP, thus potentially interfering with its ability to hydrolyze mineralization-inhibiting pyrophosphate (PPi) and mineralizing-promoting ?-glycerophosphate (in cell culture). In the biological setting, TNAP may regulate mineralization by shielding the essential inhibitory substrate pyrophosphate from TNAP degradation, and in the same process, delay the release of phosphate from this source. In conclusion, the inhibition of mineralization by polyphosphates is shown to occur via direct binding to apatitic mineral and by mixed inhibition of TNAP. PMID:23337041

  3. Lipid droplets fusion in adipocyte differentiated 3T3-L1 cells: A Monte Carlo simulation

    SciTech Connect

    Boschi, Federico, E-mail: federico.boschi@univr.it [Department of Neurological and Movement Sciences, University of Verona, Strada Le Grazie 8, 37134 Verona (Italy); Department of Computer Science, University of Verona, Strada Le Grazie 15, 37134 Verona (Italy); Rizzatti, Vanni; Zamboni, Mauro [Department of Medicine, Geriatric Section, University of Verona, Piazzale Stefani 1, 37126 Verona (Italy); Sbarbati, Andrea [Department of Neurological and Movement Sciences, University of Verona, Strada Le Grazie 8, 37134 Verona (Italy)

    2014-02-15

    Several human worldwide diseases like obesity, type 2 diabetes, hepatic steatosis, atherosclerosis and other metabolic pathologies are related to the excessive accumulation of lipids in cells. Lipids accumulate in spherical cellular inclusions called lipid droplets (LDs) whose sizes range from fraction to one hundred of micrometers in adipocytes. It has been suggested that LDs can grow in size due to a fusion process by which a larger LD is obtained with spherical shape and volume equal to the sum of the progenitors’ ones. In this study, the size distribution of two populations of LDs was analyzed in immature and mature (5-days differentiated) 3T3-L1 adipocytes (first and second populations, respectively) after Oil Red O staining. A Monte Carlo simulation of interaction between LDs has been developed in order to quantify the size distribution and the number of fusion events needed to obtain the distribution of the second population size starting from the first one. Four models are presented here based on different kinds of interaction: a surface weighted interaction (R2 Model), a volume weighted interaction (R3 Model), a random interaction (Random model) and an interaction related to the place where the LDs are born (Nearest Model). The last two models mimic quite well the behavior found in the experimental data. This work represents a first step in developing numerical simulations of the LDs growth process. Due to the complex phenomena involving LDs (absorption, growth through additional neutral lipid deposition in existing droplets, de novo formation and catabolism) the study focuses on the fusion process. The results suggest that, to obtain the observed size distribution, a number of fusion events comparable with the number of LDs themselves is needed. Moreover the MC approach results a powerful tool for investigating the LDs growth process. Highlights: • We evaluated the role of the fusion process in the synthesis of the lipid droplets. • We compared the size distribution of the lipid droplets in immature and mature cells. • We used the Monte Carlo simulation approach, simulating 10 thousand of fusion events. • Four different interaction models between the lipid droplets were tested. • The best model which mimics the experimental measures was selected.

  4. Molecular mechanism of 9-cis-retinoic acid inhibition of adipogenesis in 3T3-L1 cells

    SciTech Connect

    Sagara, Chiaki; Takahashi, Katsuhiko [Laboratory of Physiological Chemistry, Institute of Medicinal Chemistry, Hoshi University, Shinagawa, Tokyo 142-8501 (Japan)] [Laboratory of Physiological Chemistry, Institute of Medicinal Chemistry, Hoshi University, Shinagawa, Tokyo 142-8501 (Japan); Kagechika, Hiroyuki [School of Biomedical Science, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Chiyoda, Tokyo 101-0062 (Japan)] [School of Biomedical Science, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Chiyoda, Tokyo 101-0062 (Japan); Takahashi, Noriko, E-mail: t-noriko@hoshi.ac.jp [Laboratory of Physiological Chemistry, Institute of Medicinal Chemistry, Hoshi University, Shinagawa, Tokyo 142-8501 (Japan)] [Laboratory of Physiological Chemistry, Institute of Medicinal Chemistry, Hoshi University, Shinagawa, Tokyo 142-8501 (Japan)

    2013-03-29

    Highlights: ? We examined the effects of 9-cis-RA on adipogenesis in mouse preadipocyte 3T3-L1. ? 9-cis-RA inhibited lipid accumulation in adipogenetically-induced 3T3-L1 cells. ? A RXR pan-antagonist suppressed the inhibitory effects of 9-cis-RA on adipogenesis. ? This antagonist had no effects on RXR? and PPAR? levels in 9-cis-RA-treated cells. ? 9-cis-RA-induced decrease in both RXR? and PPAR? was independent of RXR activation. -- Abstract: Retinoic acid (RA) signaling is mediated by specific nuclear hormone receptors. Here we examined the effects of 9-cis-RA on adipogenesis in mouse preadipocyte 3T3-L1 cells. 9-cis-RA inhibits the lipid accumulation of adipogenetically induced 3T3-L1 cells. The complex of retinoid X receptor ? (RXR?) with peroxisome proliferator-activated receptor ? (PPAR?) is a major transcription factor in the process of adipogenesis, and the levels of these molecules were decreased by 9-cis-RA treatment. A RXR pan-antagonist suppressed 9-cis-RA’s inhibitory effects on adipogenesis, but not on the intracellular levels of both RXR? and PPAR?. These results suggest that 9-cis-RA could inhibit adipogenesis by activating RXR, and decrease both RXR and PPAR?s levels in a RXR activation-independent manner.

  5. A SCANNING ELECTRON MICROSCOPE STUDY OF SURFACE FEATURES OF VIRAL AND SPONTANEOUS TRANSFORMANTS OF MOUSE BALB\\/3T3 CELLS

    Microsoft Academic Search

    KEITH R. PORTER; GEORGE J. TODARO; VIRGINIA FONTE

    1973-01-01

    Cells of the mouse line Balb\\/3T3 as well as three virus-induced transformants and two spontaneous transformants grown in vitro have been studied for their topography by scanning electron microscopy . The parent cell in confluent culture closely resembles an endothelial cell in its form and in the structure of its association with adjacent cells . The tumorigenic transformants produced by

  6. STAT 5 activators can replace the requirement of FBS in the adipogenesis of 3T3-L1 cells

    Microsoft Academic Search

    William C. Stewart; James E. Baugh Jr.; Z. Elizabeth Floyd; Jacqueline M. Stephens

    2004-01-01

    The 3T3-L1 cells differentiate into fat cells that have many properties of native adipocytes including: substantial lipid accumulation, insulin sensitivity, and the ability to secrete endocrine hormones. A substantial expense in using these cells is fetal bovine serum (FBS), a critical component of efficient adipogenesis. Our recent studies on STAT 5 proteins have revealed that these transcription factors are phosphorylated

  7. Antisense RNA--Induced Reduction in Murine TIMP Levels Confers Oncogenicity on Swiss 3T3 Cells

    Microsoft Academic Search

    Rama Khokha; Paul Waterhouse; Simcha Yagel; Peeyush K. Lala; Christopher M. Overall; Gill Norton; David T. Denhardt

    1989-01-01

    Mouse 3T3 cell lines capable of constitutively synthesizing an RNA complementary to the messenger RNA encoding TIMP, tissue inhibitor of metalloproteinases, were constructed by transfection with appropriate plasmid constructs. Many of the lines were down-modulated for TIMP messenger RNA levels and secreted less TIMP into the culture medium. In comparison to noninvasive, nontumorigenic controls, these cells not only were invasive

  8. Ghrelin inhibits the apoptosis of MC3T3-E1 cells through ERK and AKT signaling pathway

    SciTech Connect

    Liang, Qiu-Hua; Liu, Yuan; Wu, Shan-Shan; Cui, Rong-Rong; Yuan, Ling-Qing, E-mail: allenylq@hotmail.com; Liao, Er-Yuan, E-mail: eyliao@21cn.com

    2013-11-01

    Ghrelin is a 28-amino-acid peptide that acts as a natural endogenous ligand of the growth hormone secretagogue receptor (GHSR) and strongly stimulates the release of growth hormone from the hypothalamus–pituitary axis. Previous studies have identified the important physiological effects of ghrelin on bone metabolism, such as regulating proliferation and differentiation of osteoblasts, independent of GH/IGF-1 axis. However, research on effects and mechanisms of ghrelin on osteoblast apoptosis is still rare. In this study, we identified expression of GHSR in MC3T3-E1 cells and determined the effects of ghrelin on the apoptosis of osteoblastic MC3T3-E1 cells and the mechanism involved. Our data demonstrated that ghrelin inhibited the apoptosis of osteoblastic MC3T3-E1 cells induced by serum deprivation, as determined by terminal deoxynucleotidyl transferase-mediated deoxyribonucleotide triphosphate nick end-labeling (TUNEL) and ELISA assays. Moreover, ghrelin upregulated Bcl-2 expression and downregulated Bax expression in a dose-dependent manner. Our study also showed decreased activated caspase-3 activity under the treatment of ghrelin. Further study suggested that ghrelin stimulated the phosphorylation of ERK and AKT. Pretreatment of cells with the ERK inhibitor PD98059, PI3K inhibitor LY294002, and GHSR-siRNA blocked the ghrelin-induced activation of ERK and AKT, respectively; however, ghrelin did not stimulate the phosphorylation of p38 or JNK. PD90859, LY294002 and GHSR-siRNA attenuated the anti-apoptosis effect of ghrelin in MC3T3-E1 cells. In conclusion, ghrelin inhibits the apoptosis of osteoblastic MC3T3-E1 cells induced by serum deprivation, which may be mediated by activating the GHSR/ERK and GHSR/PI3K/AKT signaling pathways. - Highlights: • We explored the effects of ghrelin on serum deprivation-induced MC3T3-E1 cells apoptosis. • Both ELISA and TUNEL were used to detect the apoptosis. • The receptor of ghrelin, GHSR, was expressed in MC3T3-E1 cells. • Both Akt and ERK are critical adaptor molecules to mediate the effects of ghrelin.

  9. Two new approaches to improve the analysis of BALB/c 3T3 cell transformation assay data.

    PubMed

    Hoffmann, Sebastian; Hothorn, Ludwig A; Edler, Lutz; Kleensang, André; Suzuki, Masaya; Phrakonkham, Pascal; Gerhard, Daniel

    2012-04-11

    Validation activities of the BALB/c 3T3 cell transformation assay (CTA) - a test method used for the assessment of the carcinogenic potential of compounds - have revealed the need for statistical analysis tailored to specific features of BALB/c 3T3 CTA data. Whereas a standard statistical approach for the Syrian hamster embryo (SHE) CTA was considered sufficient, an international expert group was gathered by the European Centre for the Validation of Alternative Methods (ECVAM) to review commonly applied statistical approaches for BALB/c 3T3 CTA. As it was concluded that none of the commonly applied approaches is entirely appropriate, two novel statistical approaches were found to be recommended for the evaluation of BALB/c 3T3 CTA data accounting for possible non-monotone concentration-response relationship and variance heterogeneity: a negative binomial generalised linear model with William's-type downturn-protected trend tests and a normalisation of the data by a specific transformation allowing for application of a general linear model that estimates effects assuming a normal distribution with William's-type protected tests. Both approaches are described in this article and their performance and the quality of the results they generate is demonstrated using exemplary data. Our work confirmed that both approaches are suitable for the statistical analysis of BALB/c 3T3 CTA data and that each of them is superior to commonly used methods. Furthermore, a procedure dichotomising data into negatives and positives is proposed which allows re-testing in cases where inconclusive data are encountered. The scripts of the statistical evaluation programs written in R - a freely available statistical software - are appended including exemplary outputs (Appendix A). PMID:22178130

  10. Bovine Collagen Peptides Compounds Promote the Proliferation and Differentiation of MC3T3-E1 Pre-Osteoblasts

    PubMed Central

    Liu, JunLi; Zhang, Bing; Song, ShuJun; Ma, Ming; Si, ShaoYan; Wang, YiHu; Xu, BingXin; Feng, Kai; Wu, JiGong; Guo, YanChuan

    2014-01-01

    Objective Collagen peptides (CP) compounds, as bone health supplements, are known to play a role in the treatment of osteoporosis. However, the molecular mechanisms of this process remain unclear. This study aimed to investigate the effects of bovine CP compounds on the proliferation and differentiation of MC3T3-E1 cells. Methods Mouse pre-osteoblast cell line MC3T3-E1 subclone 4 cells were treated with bovine CP compounds. Cell proliferation was analyzed by MTT assays and the cell cycle was evaluated by flow cytometry scanning. Furthermore, MC3T3-E1 cell differentiation was analyzed at the RNA level by real-time PCR and at the protein level by western blot analysis for runt-related transcription factor 2 (Runx2), a colorimetric p-nitrophenyl phosphate assay for alkaline phosphatase (ALP), and ELISA for osteocalcin (OC). Finally, alizarin red staining for mineralization was measured using Image Software Pro Plus 6.0. Results Cell proliferation was very efficient after treatment with different concentrations of bovine CP compounds, and the best concentration was 3 mg/mL. Bovine CP compounds significantly increased the percentage of MC3T3-E1 cells in G2/S phase. Runx2 expression, ALP activity, and OC production were significantly increased after treatment with bovine CP compounds for 7 or 14 days. Quantitative analyses with alizarin red staining showed significantly increased mineralization of MC3T3-E1 cells after treatment with bovine CP compounds for 14 or 21 days. Conclusions Bovine CP compounds increased osteoblast proliferation, and played positive roles in osteoblast differentiation and mineralized bone matrix formation. Taking all the experiments together, our study indicates a molecular mechanism for the potential treatment of osteoarthritis and osteoporosis. PMID:24926875

  11. Distribution feeder reconfiguration for loss reduction

    Microsoft Academic Search

    S. Civanlar; J. J. Grainger; H. Yin; S. S. H. Lee

    1988-01-01

    Feeder reconfiguration is defined as altering the topological structures of distribution feeders by changing the open\\/closed states of the sectionalizing and tie switches. In this paper, a scheme is presented which utilizes feeder reconfiguration as a planning and\\/or real-time control tool in order to restructure the primary feeders for loss reduction. The mathematical foundation of the scheme is given; the

  12. Response of osteoblast-like MC3T3-E1 cells on bioactive titanium fabricated by a chemical treatment process using a calcium-phosphate slurry.

    PubMed

    Ohtsu, Naofumi; Hirano, Mitsuhiro; Arai, Hirofumi

    2014-11-01

    We recently developed a chemical treatment process using a calcium-phosphate slurry for fabricating new layers consisting of hydroxyapatite and titanium dioxide (TiO2) on titanium (Ti) substrate. In this study, the response of osteoblast-like MC3T3-E1 cells on Ti substrate treated with a calcium-phosphate slurry was investigated to elucidate its behavior in a biological environment. The cellular adhesiveness and proliferation capacity did not differ significantly between the treated and untreated Ti substrates, suggesting that the slurry treatment did not cause cytotoxicity. The slurry treatment did not affect the increase in alkaline phosphatase activity after the induction of cell differentiation, whereas it was found to be significantly advantageous for the calcification behavior on the slurry-treated Ti substrate. In consequence, the hard-tissue compatibility of Ti is expected to be improved by the chemical treatment process using a calcium-phosphate slurry. PMID:24307316

  13. Protein Kinase B/AKT 1 Plays a Pivotal Role in Insulin-like Growth Factor-1 Receptor Signaling Induced 3T3-L1

    E-print Network

    Tian, Weidong

    kinase system in IGF-1 receptor signal-induced 3T3-L1 cell adipogenesis is not clear. It is further complicated by the different effects exhibited by p38 MAP kinase and ERK1/2 on 3T3-L1 cell adipogenesis (5 and 3T3-L1 cell adipogenesis. PI 3-kinase-PKB/Akt as an important intracellular signal cascade has been

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

    PubMed

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

    1996-07-01

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

  15. Chemerin enhances insulin signaling and potentiates insulin-stimulated glucose uptake in 3T3-L1 adipocytes.

    PubMed

    Takahashi, Michiko; Takahashi, Yutaka; Takahashi, Kenichi; Zolotaryov, Fyodor N; Hong, Kyoung Su; Kitazawa, Riko; Iida, Keiji; Okimura, Yasuhiko; Kaji, Hidesuke; Kitazawa, Sohei; Kasuga, Masato; Chihara, Kazuo

    2008-03-01

    To explore a novel adipokine, we screened adipocyte differentiation-related gene and found that TIG2/chemerin was strongly induced during the adipocyte differentiation. Chemerin was secreted by the mature 3T3-L1 adipocytes and expressed abundantly in adipose tissue in vivo as recently described. Intriguingly, the expression of chemerin was differently regulated in the liver and adipose tissue in db/db mice. In addition, serum chemerin concentration was decreased in db/db mice. Chemerin and its receptor/ChemR23 were expressed in mature adipocytes, suggesting its function in autocrine/paracrine fashion. Finally, chemerin potentiated insulin-stimulated glucose uptake concomitant with enhanced insulin signaling in the 3T3-L1 adipocytes. These data establish that chemerin is a novel adipokine that regulates adipocyte function. PMID:18242188

  16. Arachidonic acid inhibits lipogenic gene expression in 3T3-L1 adipocytes through a prostanoid pathway

    Microsoft Academic Search

    Michelle K. Mater; David Pan; W. G. Bergen; Donald B. Jump

    This report examines the effect of polyunsatu- rated fatty acids (PUFA) on lipogenic gene expression in cultured 3T3-L1 adipocytes. Arachidonic acid (20:4, n-6) and eicosapentaenoic acid (20:5, n-3) suppressed mRNAs encoding fatty acid synthase (FAS) and S14, but had no ef- fect on b -actin. Using a clonal adipocyte cell line containing a stably integrated S14CAT fusion gene, oleic acid

  17. Bisphenol A Accelerates Terminal Differentiation of 3T3-L1 Cells into Adipocytes through the Phosphatidylinositol 3Kinase Pathway

    Microsoft Academic Search

    Hiroshi Masuno; Jun Iwanami; Teruki Kidani; Kenshi Sakayama; Katsuhisa Honda

    2005-01-01

    In order to identify whether bisphenol A (BPA) acts as an adipogenic agent, following the hormonal induction of differen- tiation into adipocytes, 3T3-L1 cells were treated for six days with BPA alone. Treatment with BPA increased the triacylglycerol (TG) content of the cultures, increased the percentage of Oil Red O-staining cells in the cultures, and increased the levels of lipoprotein

  18. In vitro BALB\\/3T3 cell transformation assay of nonoxynol-9 and 1,4-dioxane

    Microsoft Academic Search

    Chingju W. Sheu; Frances M. Moreland; Jung Keun Lee; Virginia C. Dunkel

    1988-01-01

    The spermicidal surfactant nonoxynol-9 (Igepal CO-630, GAF Corp.) and a potential impurity, 1,4-dioxane, were tested in the in vitro cell transformation assay using BALB\\/3T3 cells. Two treatment periods, 48 hr and 13 days, were used. Nonoxynol-9, tested at levels up to 10 \\/sup +\\/g\\/ml, did not induce transformation, whereas dioxane was very active in the induction type II foci in

  19. Quercetin, a bioflavonoid, accelerates TNF-?-induced growth inhibition and apoptosis in MC3T3-E1 osteoblastic cells

    Microsoft Academic Search

    Young-Ok Son; Sung-Ho Kook; Ki-Choon Choi; Yong-Suk Jang; Young-Mi Jeon; Jong-Ghee Kim; Kyung-Yeol Lee; Ju Kim; Mi-Sun Chung; Gook-Hyun Chung; Jeong-Chae Lee

    2006-01-01

    The bioflavonoid quercetin is believed to play an important role in preventing bone loss by affecting osteoclastogenesis and regulating many systemic and local factors including hormones and cytokines. This study examined how quercetin acts on tumor necrosis factor-alpha (TNF-?)-mediated growth inhibition and apoptosis in MC3T3-E1 osteoblastic cells. Tritium uptake assay showed that a quercetin treatment accelerated TNF-?-induced inhibition of DNA

  20. Effects of DLX2 overexpression on the osteogenic differentiation of MC3T3-E1 cells

    PubMed Central

    SUN, HAO; LIU, ZHIXU; LI, BIAO; DAI, JIEWEN; WANG, XUDONG

    2015-01-01

    Distal-less genes (DLX) play important roles in regulating organism development. DLX2 is crucial for the differentiation and development of the primordium, which determines the subsequent development and phenotype of the maxillofacial skeletal patterns, and is the primary candidate gene that regulates the development of the first branchial arch. The aim of the present study was to investigate the effects of DLX2 overexpression on the osteogenic differentiation of MC3T3-E1 cells in vitro. A DLX2-expression retrovirus vector was constructed by subcloning with a murine stem cell virus (MSCV) and verified by sequencing. MC3T3-E1 cells were transfected with pMSCV-DLX2 and stable clones were selected with puromycin. The mRNA and protein expression levels of DLX2 were determined using quantitative polymerase chain reaction (PCR) and western blot analysis, respectively. In addition, the expression levels of the osteogenic biomarkers, alkaline phosphatase (ALP), osteocalcin (OCN), runt-related transcription factor (RUNX)2 and Msh homeobox (MSX)2, were assessed by quantitative PCR. ALP detection and Alizarin red staining were conducted to evaluate the effect of DLX2 overexpression on osteogenic differentiation. The data were analyzed by analysis of variance using the Student-Newman-Keuls method. Successful pMSCV-DLX2 construction, as verified by direct sequencing, enabled DLX2 overexpression in vitro. Enhanced ALP activity and Alizarin red staining were observed in the MC3T3-E1-DLX2 cells when compared with the control group. During osteogenic induction, DLX2 overexpression was demonstrated to upregulate ALP and MSX2 expression at the early stage and OCN expression at the late stage, while no statistically significant difference was observed in RUNX2 expression when compared with the control group. Therefore, DLX2 overexpression in vitro induced the osteogenic differentiation of MC3T3-E1 cells via upregulating bone formation-associated genes, such as ALP and MSX2.

  1. Tyrosine phosphorylation and morphological transfordmation induced by four vanadium compounds on MC3T3E1 cells

    Microsoft Academic Search

    V. C. Sálice; A. M. Cortizo; C. L. Gómez Dumm; S. B. Etcheverry

    1999-01-01

    The present study was performed to determine the phosphotyrosine-protein levels induced by insulin and by four vanadium derivatives in MC3T3E1 osteoblast-like cells. We have also attempted to associate these patterns vath the vanadium-induced growth and morphological changes of such cells. Vanadate (Vi), vanadyl (VO), bis(maltolato)oxovanadium (IV) (BMOV) and bis(maltolato)dioxovanadium (V) (BMV) stimulate cell growth in a narrow range of concentration,

  2. Expression of Gene rrg is Associated with Reversion of NIH 3T3 Transformed by LTR-c-H-ras

    Microsoft Academic Search

    Sara Contente; Kaylene Kenyon; Donata Rimoldi; Robert M. Friedman

    1990-01-01

    A partial complementary DNA was isolated for a gene (rrg) that is normally expressed in mouse NIH 3T3 cells, but is down-regulated after cellular transformation by long terminal repeat (LTR)-activated c-H-ras (LTR-c-H-ras). This gene was reexpressed in a nontumorigenic persistent revertant cell line created by prolonged treatment of the transformed cells with mouse interferon alpha\\/beta. Persistent revertants stably transfected with

  3. Type 3 protein kinase C localization to the nuclear envelope of phorbol ester-treated NIH 3T3 cells

    Microsoft Academic Search

    Karen L. Leach; Elaine A. Powers; Valerie A. Ruff; Susan Jaken; Scott Kaufmann

    1989-01-01

    We have examined the immunocytochemical localization of protein kinase C (PKC) in NIH 3T3 cells using mAbs that recognize Type 3 PKC. In con- trol cells, the immunofluorescent staining was similar with mAbs directed to either the catalytic or the regulatory domain of PKC. Type 3 PKC localized in a diffuse cytoplasmic pattern, while the nuclei were ap- parently unstained.

  4. Induction of Cell Proliferation in Quiescent NIH 3T3 Cells by Oncogenic cRaf1

    Microsoft Academic Search

    EUGEN KERKHOFF; ANDULF R. RAPP

    1997-01-01

    The c-Raf-1 kinase is activated by different mitogenic stimuli and has been shown to be an important mediator of growth factor responses. Fusion of the catalytic domain of the c-Raf-1 kinase with the hormone binding domain of the estrogen receptor (DRaf-ER) provides a hormone-regulated form of oncogenic activated c-Raf-1. We have established NIH 3T3 cells stably expressing a c-Raf-1 deletion

  5. Expression of Human Epidermal Growth Factor Precursor cDNA in Transfected Mouse NIH 3T3 Cells

    Microsoft Academic Search

    Barbara Mroczkowski; Martha Reich; Jonathan Whittaker; Graeme I. Bell; Stanley Cohen

    1988-01-01

    Stable cell lines expressing the human epidermal growth factor (EGF) precursor have been prepared by transfection of mouse NIH 3T3 cells with a bovine papillomavirus-based vector in which the human kidney EGF precursor cDNA has been placed under the control of the inducible mouse metallothionein I promoter. Synthesis of the EGF precursor can be induced by culturing the cells in

  6. Effects of C-reactive protein on adipokines genes expression in 3T3-L1 adipocytes

    SciTech Connect

    Yuan, Guoyue, E-mail: yuanguoyue@hotmail.com [Department of Endocrinology, The Affiliated Hospital of Jiangsu University, Zhenjiang, Jiangsu 212001 (China)] [Department of Endocrinology, The Affiliated Hospital of Jiangsu University, Zhenjiang, Jiangsu 212001 (China); Jia, Jue; Di, Liangliang [Department of Endocrinology, The Affiliated Hospital of Jiangsu University, Zhenjiang, Jiangsu 212001 (China)] [Department of Endocrinology, The Affiliated Hospital of Jiangsu University, Zhenjiang, Jiangsu 212001 (China); Zhou, Libin [Ruijin Hospital, Center of Molecular Medicine, Shanghai Institute of Endocrine and Metabolic Diseases, State Key Laboratory of Medical Genomics, Shanghai Jiaotong University Medical School, 197, Ruijin Road II, Shanghai 200025 (China)] [Ruijin Hospital, Center of Molecular Medicine, Shanghai Institute of Endocrine and Metabolic Diseases, State Key Laboratory of Medical Genomics, Shanghai Jiaotong University Medical School, 197, Ruijin Road II, Shanghai 200025 (China); Dong, Sijing; Ye, Jingjing; Wang, Dong; Yang, Ling; Wang, Jifang [Department of Endocrinology, The Affiliated Hospital of Jiangsu University, Zhenjiang, Jiangsu 212001 (China)] [Department of Endocrinology, The Affiliated Hospital of Jiangsu University, Zhenjiang, Jiangsu 212001 (China); Li, Lianxi [Department of Endocrinology and Metabolism, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, 600, Yishan Road, Shanghai 200233 (China)] [Department of Endocrinology and Metabolism, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, 600, Yishan Road, Shanghai 200233 (China); Yang, Ying [Ruijin Hospital, Center of Molecular Medicine, Shanghai Institute of Endocrine and Metabolic Diseases, State Key Laboratory of Medical Genomics, Shanghai Jiaotong University Medical School, 197, Ruijin Road II, Shanghai 200025 (China)] [Ruijin Hospital, Center of Molecular Medicine, Shanghai Institute of Endocrine and Metabolic Diseases, State Key Laboratory of Medical Genomics, Shanghai Jiaotong University Medical School, 197, Ruijin Road II, Shanghai 200025 (China); Mao, Chaoming [Department of Endocrinology, The Affiliated Hospital of Jiangsu University, Zhenjiang, Jiangsu 212001 (China)] [Department of Endocrinology, The Affiliated Hospital of Jiangsu University, Zhenjiang, Jiangsu 212001 (China); Chen, Mingdao, E-mail: mingdaochensh@yahoo.com [Ruijin Hospital, Center of Molecular Medicine, Shanghai Institute of Endocrine and Metabolic Diseases, State Key Laboratory of Medical Genomics, Shanghai Jiaotong University Medical School, 197, Ruijin Road II, Shanghai 200025 (China)] [Ruijin Hospital, Center of Molecular Medicine, Shanghai Institute of Endocrine and Metabolic Diseases, State Key Laboratory of Medical Genomics, Shanghai Jiaotong University Medical School, 197, Ruijin Road II, Shanghai 200025 (China)

    2012-08-03

    Highlights: Black-Right-Pointing-Pointer CRP increases TNF-{alpha} and IL-6 genes expression in matured 3T3-L1 adipocytes. Black-Right-Pointing-Pointer CRP suppresses adiponectin, leptin and PPAR-{gamma} mRNA levels in matured 3T3-L1 cells. Black-Right-Pointing-Pointer Wortmannin reverses effects of CRP on adiponectin, TNF-{alpha} and leptin mRNA levels. Black-Right-Pointing-Pointer CRP may regulate IR, obesity and metabolic syndrome by this mechanism. -- Abstract: Adipose tissue is now recognized to be an important endocrine organ, secreting a variety of adipokines that are involved in the regulation of energy metabolism, insulin resistance and metabolic syndrome. C-reactive protein (CRP) is considered as one of the most sensitive markers of inflammation. A number of studies have shown that elevation of CRP concentrations is an independent predictive parameter of type 2 diabetes mellitus, which is also strongly associated with various components of the metabolic syndrome. The aim of the present study is to investigate the effects of CRP on adipokines genes expression in 3T3-L1 adipocytes. Quantitative real-time PCR analysis revealed that CRP inhibited adiponectin, leptin and peroxisome proliferator-activated receptor-gamma (PPAR-{gamma}) genes expression and raised tumor necrosis factor-{alpha} (TNF-{alpha}) and interleukin-6 (IL-6) mRNA levels in matured 3T3-L1 adipocytes in a dose and time-dependent manner. Pharmacological inhibition of phosphatidylinositol (PI)-3 kinase by wortmannin partially reversed the effects of CRP on adiponectin, TNF-{alpha} and leptin genes expression. These results collectively suggest that CRP regulates adiponectin, TNF-{alpha}, leptin, IL-6 and PPAR-{gamma} genes expression, and that might represent a mechanism by which CRP regulates insulin resistance, obesity and metabolic syndrome.

  7. Control of DNA synthesis in growing BALB/c 3T3 mouse cells by a fibroblast growth regulatory factor

    PubMed Central

    Natraj, C. V.; Datta, Prasanta

    1978-01-01

    A cell surface component from quiescent BALB/c 3T3 mouse cells that inhibits DNA synthesis and cell division when added to a culture of growing 3T3 cells has been detected. The inhibition of DNA synthesis by this factor was dependent on concentration and time of incubation; a transient exposure of cells to the factor followed by incubation in its absence for 20 hr was sufficient to elicit its inhibitory effect. The active component appears to be protein in nature, as judged by heat inactivation and trypsin sensitivity. Extracts obtained in an identical manner from quiescent 3T3 cells that had been preincubated in situ with uridine diphosphate N-acetyl-D-glucosamine (UDP-GlcNAc) did not inhibit DNA synthesis. The effect was specific for UDP-GlcNAc: incubation with three other nucleotide sugars yielded active component. Incubation of the inactive component from UDP-GlcNAc-treated cells with purified N-acetyl-?-D-glucosaminidase in vitro restored its inhibitory property. Extracts from growing cells failed to inhibit DNA synthesis. These results suggest that reversible glycosylation with N-acetyl-D-glucosamine residues may serve as a regulatory signal for the conversion of the active factor to its inactive form. We propose that the onset of quiescence of 3T3 cells is due to a casual relationship between depletion of growth factors in the culture medium and the presence of the active regulatory factor on the cell surface that inhibits DNA synthesis; conversion of the regulatory factor to its inactive form under favorable nutritional status may be viewed as a switch that allows DNA synthesis to resume. PMID:282629

  8. Carnosic Acid Inhibits Lipid Accumulation in 3T3-L1 Adipocytes Through Attenuation of Fatty Acid Desaturation

    PubMed Central

    Park, Mi-Young; Sung, Mi-Kyung

    2015-01-01

    Background: Excess body fat accumulation contributes to the development of metabolic disorders that can cause adverse health effects. Carnosic acid (CA), a major bioactive component of rosemary (Rosemarinus officinalis), has been suggested to possess anti-adipogenic properties. The present study was conducted to elucidate the mechanism underlying the anti-adipogenic effects of CA. Methods: 3T3-L1 pre-adipocytes were treated with CA (0.1, 1, and 10 ?M) from day 0 to day 8 of differentiation. On day 8, biochemical markers of lipid accumulation and the degree of fatty acid desaturation were measured. Results: Oil Red O staining results, triglyceride (TG) accumulation, and glycerol 3-phosphate dehydrogenase activity suggested that CA significantly inhibited lipid accumulation in 3T3-L1 adipocytes. CA significantly decreased mRNA expression of peroxisome proliferator-activated receptor-?, sterol regulatory element-binding protein 1, and CCAAT/enhancer binding protein-? in a dose-dependent manner. Moreover, it decreased the ratio of both C16:1/C16:0 and C18:1/C18:0, with reduced expression of stearoyl CoA desaturase 1 mRNA and protein. Conclusions: These results suggest that CA efficiently suppressed adipogenesis in 3T3-L1 adipocytes and its action, at least in part, is associated with the downregulation of adipogenesis-related genes and the fatty acid composition of TG accumulated in adipocytes. PMID:25853102

  9. Magnetic resonance detects changes in phosphocholine associated with Ras activation and inhibition in NIH 3T3 cells

    PubMed Central

    Ronen, S M; Jackson, L E; Beloueche, M; Leach, M O

    2001-01-01

    Ras is frequently mutated in cancer, and novel therapies are being developed to target Ras signalling. To identify non-invasive surrogate markers of Ras activation and inhibition, we used31P magnetic resonance spectroscopy (MRS) and investigated NIH 3T3 cells compared to a mutant ras transfected counterpart. The MR spectra indicated that phosphocholine (PC) levels increased significantly from 3 ± 2 fmol cell?1in NIH 3T3 cells to 13?±?4 ?fmol cell?1in the transfected cells. The PC/NTP ratio increased significantly from 0.3?±?0.1 to 0.7 ± 0.3. This could not be explained by either a faster proliferation rate or by alterations in cell cycle distribution. Both cell lines were treated with simvastatin, 17-AAG and R115777, agents which inhibit Ras signalling. Cell proliferation was inhibited in both cell lines. The spectrum of NIH 3T3 cells was not affected by treatment. In contrast, in the ras transfected cells growth inhibition was associated with an average 35?±?5% drop in PC levels and a comparable drop in PC/NTP. Thus the MRS visible increase in phosphocholine is associated with Ras activation, and response to treatment is associated with partial reversal of phosphocholine increase in ras transfected cells. MRS might therefore be a useful tool in detecting Ras activation and its inhibition following targeted therapies. © 2001 Cancer Research Campaign http://www.bjcancer.com PMID:11237392

  10. K+ Efflux in NIH Mouse 3T3 Cells and Transformed Derivatives: Dependence on Extracellular Ca2+ and Phorbol Esters

    NASA Astrophysics Data System (ADS)

    Lubin, Martin

    1988-07-01

    In culture medium deficient in Ca2+, NIH mouse 3T3 cells lose K+, gain Na+, and stop growing. A marked increase in the rate of K+ efflux accounts for this loss; Na+,K+-ATPase pump activity increases but does not fully compensate for enhanced K+ efflux. Phorbol esters and cycloheximide inhibit K+ loss in Ca2+-deficient medium. Phorbol esters inhibit K+ efflux from human fibroblasts as well, even at physiological levels of Ca2+. Two cell lines derived from NIH-3T3, one transformed by a simian virus 40 deletion mutant, the other by the polyoma virus oncogene encoding the middle-sized tumor antigen, retain K+ and can multiply in medium with low Ca2+. Efflux of K+ from these cells is relatively insensitive to reduced Ca2+ concentration, phorbol esters, and cycloheximide. The results suggest the following hypothesis: a channel, nonselective for K+ and Na+, opens when NIH-3T3 cells are in Ca2+-deficient medium; the channel is controlled by the receptor for phorbol ester (protein kinase C) and may also be regulated by a short-lived protein.

  11. Temperature distribution during ICG-dye-enhanced laser photocoagulation of feeder vessels in treatment of AMD-related choroidal neovascularization.

    PubMed

    Zhu, Liang; Banerjee, Rupak K; Salloum, Maher; Bachmann, Albert; Flower, Robert W

    2008-06-01

    Laser photocoagulation of the feeder vessels of age-related macula degeneration-related choroidal neovascularization (CNV) membranes is a compelling treatment modality, one important reason being that the treatment site is removed from the fovea in cases of sub- or juxtafoveal CNV. To enhance the energy absorption in a target feeder vessel, an indocyanine green dye bolus is injected intravenously, and the 805 nm wavelength diode laser beam is applied when the dye bolus transits the feeder vessel; this tends to reduce concomitant damage to adjacent tissue. A 3D theoretical simulation, using the Pennes bioheat equation, was performed to study the temperature distribution in the choroidal feeder vessel and its vicinity during laser photocoagulation. The results indicate that temperature elevation in the target feeder vessel increases by 20% in dye-enhanced photocoagulation, compared to just photocoagulation alone. The dye bolus not only increases the laser energy absorption in the feeder vessel but also shifts the epicenter of maximum temperature away from the sensitive sensory retina and retinal pigment epithelial layers and toward the feeder vessel. Two dominant factors in temperature elevation of the feeder vessel are location of the feeder vessel and blood flow velocity through it. Feeder vessel temperature elevation becomes smaller as distance between it and the choriocapillaris layer increases. The cooling effect of blood flow through the feeder vessel can reduce the temperature elevation by up to 21% of the maximum that could be produced. Calculations were also performed to examine the effect of the size of the laser spot. To achieve the same temperature elevation in the feeder vessel when the laser spot diameter is doubled, the laser power level has to be increased by only 60%. In addition, our results have suggested that more studies are needed to measure the constants in the Arrhenius integral for assessing thermal damage in various tissues. PMID:18532859

  12. Radicicol, a heat shock protein 90 inhibitor, inhibits differentiation and adipogenesis in 3T3-L1 preadipocytes

    SciTech Connect

    He, Yonghan [Department of Nutrition and Food Hygiene, Public Health College, Harbin Medical University, 157 Baojian Road, Harbin 150081 (China) [Department of Nutrition and Food Hygiene, Public Health College, Harbin Medical University, 157 Baojian Road, Harbin 150081 (China); Aquatic and Crop Resource Development, Life Sciences Branch, National Research Council Canada, Charlottetown, PE, Canada C1A 4P3 (Canada); State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223 (China); Li, Ying [Department of Nutrition and Food Hygiene, Public Health College, Harbin Medical University, 157 Baojian Road, Harbin 150081 (China)] [Department of Nutrition and Food Hygiene, Public Health College, Harbin Medical University, 157 Baojian Road, Harbin 150081 (China); Zhang, Shuocheng [Aquatic and Crop Resource Development, Life Sciences Branch, National Research Council Canada, Charlottetown, PE, Canada C1A 4P3 (Canada)] [Aquatic and Crop Resource Development, Life Sciences Branch, National Research Council Canada, Charlottetown, PE, Canada C1A 4P3 (Canada); Perry, Ben [Aquatic and Crop Resource Development, Life Sciences Branch, National Research Council Canada, Charlottetown, PE, Canada C1A 4P3 (Canada) [Aquatic and Crop Resource Development, Life Sciences Branch, National Research Council Canada, Charlottetown, PE, Canada C1A 4P3 (Canada); Department of Biomedical Sciences, University of Prince Edward Island, 550 University Avenue, Charlottetown, PE, Canada C1A 4P3 (Canada); Zhao, Tiantian [Aquatic and Crop Resource Development, Life Sciences Branch, National Research Council Canada, Charlottetown, PE, Canada C1A 4P3 (Canada) [Aquatic and Crop Resource Development, Life Sciences Branch, National Research Council Canada, Charlottetown, PE, Canada C1A 4P3 (Canada); Department of Psychology, University of Toronto, 1265 Military Trail, Toronto, ON, Canada M1C 1A4 (Canada); Wang, Yanwen, E-mail: yanwen.wang@nrc.ca [Aquatic and Crop Resource Development, Life Sciences Branch, National Research Council Canada, Charlottetown, PE, Canada C1A 4P3 (Canada) [Aquatic and Crop Resource Development, Life Sciences Branch, National Research Council Canada, Charlottetown, PE, Canada C1A 4P3 (Canada); Department of Biomedical Sciences, University of Prince Edward Island, 550 University Avenue, Charlottetown, PE, Canada C1A 4P3 (Canada); Sun, Changhao, E-mail: sun2002changhao@yahoo.com [Department of Nutrition and Food Hygiene, Public Health College, Harbin Medical University, 157 Baojian Road, Harbin 150081 (China)] [Department of Nutrition and Food Hygiene, Public Health College, Harbin Medical University, 157 Baojian Road, Harbin 150081 (China)

    2013-06-28

    Highlights: •Radicicol suppressed intracellular fat accumulation in 3T3-L1 adipocytes. •Radicicol inhibited the expression of FAS and FABP4. •Radicicol blocked cell cycle at the G1-S phase during cell differentiation. •Radicicol inhibited the PDK1/Akt pathway in adipocyte differentiation. -- Abstract: Heat shock protein 90 (Hsp90) is involved in various cellular processes, such as cell proliferation, differentiation and apoptosis. As adipocyte differentiation plays a critical role in obesity development, the present study investigated the effect of an Hsp90 inhibitor radicicol on the differentiation of 3T3-L1 preadipocytes and potential mechanisms. The cells were treated with different concentrations of radicicol during the first 8 days of cell differentiation. Adipogenesis, the expression of adipogenic transcriptional factors, differentiation makers and cell cycle were determined. It was found that radicicol dose-dependently decreased intracellular fat accumulation through down-regulating the expression of peroxisome proliferator-activated receptor ? (PPAR{sub ?}) and CCAAT element binding protein ? (C/EBP{sub ?}), fatty acid synthase (FAS) and fatty acid-binding protein 4 (FABP4). Flow cytometry analysis revealed that radicicol blocked cell cycle at G1-S phase. Radicicol redcued the phosphorylation of Akt while showing no effect on ?-catenin expression. Radicicol decreased the phosphorylation of phosphoinositide-dependent kinase 1 (PDK1). The results suggest that radicicol inhibited 3T3-L1 preadipocyte differentiation through affecting the PDK1/Akt pathway and subsequent inhibition of mitotic clonal expansion and the expression/activity of adipogenic transcriptional factors and their downstream adipogenic proteins.

  13. Growth hormone promoted tyrosyl phosphorylation of growth hormone receptors in murine 3T3-F442A fibroblasts and adipocytes

    SciTech Connect

    Foster, C.M.; Shafer, J.A.; Rozsa, F.W.; Wang, X.; Lewis, S.D.; Renken, D.A.; Natale, J.E.; Schwartz, J.; Carter-Su, C.

    1988-01-12

    Because many growth factor receptors are ligand-activated tyrosine protein kinases, the possibility that growth hormone (GH), a hormone implicated in human growth, promotes tyrosyl phosphorylation of its receptor was investigated. /sup 125/I-Labeled human GH was covalently cross-linked to receptors in intact 3T3-F442A fibroblasts, a cell line which differentiates into adipocytes in response to GH. The cross-linked cells were solubilized and passed over a column of phosphotyrosyl binding antibody immobilized on protein A-Sepharose. Immunoadsorbed proteins were eluted with a hapten (p-nitrophenyl phosphate) and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The eluate from the antibody column contained in M/sub r/ 134,000 /sup 125/I-GH-receptor complex. A similar result was obtained when the adipocyte form of 3T3-F442A cells was used in place of fibroblast form. O-Phosphotyrosine prevented /sup 125/I-GH-receptor complexes from binding to the antibody column, whereas O-phosphoserine and O-phosphothreonine did not. In studies of GH-promoted phosphorylation in 3T3-F442A fibroblasts labeled metabolically with (/sup 32/P)P/sub i/, GH was shown to stimulate formation of a /sup 32/P-labeled protein which bound to immobilized phosphotyrosyl binding antibodies. The molecular weight of 114,000 obtained for this protein is similar to that expected for non-cross-linked GH receptor. These observations provide strong evidence that binding of GH to its receptor stimulates phosphorylation of tyrosyl residues in the GH receptor.

  14. The role of the coating and aggregation state in the interactions between iron oxide nanoparticles and 3T3 fibroblasts

    NASA Astrophysics Data System (ADS)

    Safi, Malak; Berret, Jean-François

    Recent nanotoxicity studies revealed that the physico-chemical characteristics of engineered nanomaterials play an important role in the interactions with living cells. Here, we report on the toxicity and uptake of the iron oxide sub-10 nm nanoparticles by NIH/3T3 mouse fibroblasts. Coating strategies include low-molecular weight ligands (citric acid) and polymers (poly(acrylic acid), MW = 2000 gmol-1). We find that most particles were biocompatible, as exposed cells remained 100% viable relative to controls. The strong uptake shown by the citrate-coated particles is related to the destabilization of the dispersions in the cell culture medium and their sedimentation down to the cell membranes.

  15. Stimulative effect of ginsenosides Rg5:Rk1 on murine osteoblastic MC3T3-E1 cells.

    PubMed

    Siddiqi, Muhammad Hanif; Siddiqi, Muhammad Zubair; Ahn, Sungeun; Kang, Sera; Kim, Yeon-Ju; Veerappan, Karpagam; Yang, Dong-Uk; Yang, Deok-Chun

    2014-10-01

    Panax ginseng C.A. Meyer (P.?ginseng), hereafter referred to as P. ginseng, is known to exert a wide range of pharmacological effects both in vitro and in vivo; however, few studies have investigated the effects of ginseng on bone metabolism. We therefore investigated the potential antiosteoporotic properties of ginseng on the growth and differentiation of murine MC3T3-E1 cells. Rg5:Rk1 is a mixture of protopanaxadiol-type ginsenosides, isolated from fresh P.?ginseng root, via a repetitive steaming and drying process. In this study, we examined the stimulatory effects of Rg5:Rk1 on the differentiation and mineralization of MC3T3-E1 cells. Undifferentiated cells were treated with a range of concentrations of Rg5:Rk1 (1-50?µg/mL), and cell viability was measured with the 3-(4,5-dimethyl-thiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Treatment with Rg5:Rk1 significantly increased cell viability in a dose-dependent manner. To investigate the possible mechanisms by which Rg5:Rk1 affects the early differentiation phase of MC3T3-E1 cells, the cells were treated with Rg5:Rk1 for 14-24?days before assessing the levels of multiple osteoblastic markers. The markers examined included alkaline phosphatase (ALP) activity type I collagen content (Coll-I), calcium deposition (by Alizarin Red S staining), extracellular mRNA expression of bone morphogenetic protein-2 (BMP-2), and the level of Runt-related transcription factor 2 (Runx2). Rg5:Rk1 treatment also increased the activities of proteins associated with osteoblast growth and differentiation in a dose-dependent manner. Overall, we found that the Rg5:Rk1 mixture of ginsenosides improved the osteoblastic function of MC3T3-E1 cells by increasing their proliferative capacity. This improvement is due to the action of Rg5:Rk1 on BMP-2, which is mediated by Runx2-dependent pathways. PMID:24643957

  16. Water extracts from Momordica charantia increase glucose uptake and adiponectin secretion in 3T3-L1 adipose cells

    Microsoft Academic Search

    Ben W. C. Roffey; Avtar S. Atwal; Timothy Johns; Stan Kubow

    2007-01-01

    To examine the effects of Momordica charantia on glucose uptake and adiponectin secretion in adipose cells, 3T3-L1 adipocytes were treated with three concentrations (0.2, 0.3 and 0.4mg\\/ml) of water and ethanol extracts of Momordica charantia fruit and seeds alone and in combination with either 0.5nM or 50nM insulin. The treatment combination of 0.2mg\\/ml water extract and 0.5nM insulin was associated

  17. Macrophage-conditioned medium inhibits differentiation-induced Rb phosphorylation in 3T3-L1 preadipocytes

    SciTech Connect

    Yarmo, Michelle N.; Landry, Anne; Molgat, Andre S.D.; Gagnon, AnneMarie [Department of Medicine, University of Ottawa, Chronic Disease Program, Ottawa Health Research Institute, Ottawa, Ontario (Canada); Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Chronic Disease Program, Ottawa Health Research Institute, Ottawa, Ontario (Canada); Sorisky, Alexander [Department of Medicine, University of Ottawa, Chronic Disease Program, Ottawa Health Research Institute, Ottawa, Ontario (Canada); Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Chronic Disease Program, Ottawa Health Research Institute, Ottawa, Ontario (Canada)], E-mail: asorisky@ohri.ca

    2009-02-01

    This study examines the mechanisms underlying the anti-adipogenic effect of macrophage-secreted products. 3T3-L1 preadipocytes were induced to differentiate over 8 days in medium conditioned by murine J774 macrophages (MacCM). The inhibitory effect on lipid accumulation and expression of adipogenic markers was diminished when addition of MacCM was delayed to day 2 of differentiation. Clonal expansion, an early event required for 3T3-L1 adipogenesis, was reduced in the presence of MacCM (89%; n = 3; p < 0.001), and BrdU incorporation was impaired by 55% (n = 3; p < 0.01). Activation of ERK1/2 was not affected by MacCM, and neither was the expression of p27{sup kip1}, a cyclin-dependent kinase inhibitor. However, phosphorylation of the retinoblastoma protein (Rb), required for cell cycle progression, was impaired by MacCM (94% inhibition; n = 3; p < 0.01). Differentiation-dependent expression, nuclear localization, and DNA binding ability of C/EBP{beta} were not inhibited by MacCM. Alterations in cell cycle-associated proteins may be important with respect to the anti-adipogenic action of MacCM.

  18. Suppression of lipin-1 expression increases monocyte chemoattractant protein-1 expression in 3T3-L1 adipocytes

    SciTech Connect

    Takahashi, Nobuhiko, E-mail: ntkhs@hoku-iryo-u.ac.jp [Department of Internal Medicine, School of Dentistry, Health Sciences University of Hokkaido, 1757 Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan) [Department of Internal Medicine, School of Dentistry, Health Sciences University of Hokkaido, 1757 Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan); Division of Gastroenterology and Hematology/Oncology, Department of Medicine, Asahikawa Medical University, 2-1-1-1 Midorigaoka-Higashi, Asahikawa, Hokkaido 078-8510 (Japan); Yoshizaki, Takayuki [Innovation Center, Kagoshima University, 1-21-40 Korimoto, Kagoshima 890-0065 (Japan)] [Innovation Center, Kagoshima University, 1-21-40 Korimoto, Kagoshima 890-0065 (Japan); Hiranaka, Natsumi; Suzuki, Takeshi [Department of Internal Medicine, School of Dentistry, Health Sciences University of Hokkaido, 1757 Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan)] [Department of Internal Medicine, School of Dentistry, Health Sciences University of Hokkaido, 1757 Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan); Yui, Tomoo; Akanuma, Masayasu; Oka, Kazuya [Department of Fixed Prosthodontics and Oral Implantology, School of Dentistry, Health Sciences University of Hokkaido, 1757 Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan)] [Department of Fixed Prosthodontics and Oral Implantology, School of Dentistry, Health Sciences University of Hokkaido, 1757 Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan); Kanazawa, Kaoru [Department of Dental Anesthesiology, School of Dentistry, Health Sciences University of Hokkaido, 1757 Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan)] [Department of Dental Anesthesiology, School of Dentistry, Health Sciences University of Hokkaido, 1757 Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan); Yoshida, Mika; Naito, Sumiyoshi [Department of Clinical Laboratory, Health Sciences University of Hokkaido, 1757 Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan)] [Department of Clinical Laboratory, Health Sciences University of Hokkaido, 1757 Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan); Fujiya, Mikihiro; Kohgo, Yutaka [Division of Gastroenterology and Hematology/Oncology, Department of Medicine, Asahikawa Medical University, 2-1-1-1 Midorigaoka-Higashi, Asahikawa, Hokkaido 078-8510 (Japan)] [Division of Gastroenterology and Hematology/Oncology, Department of Medicine, Asahikawa Medical University, 2-1-1-1 Midorigaoka-Higashi, Asahikawa, Hokkaido 078-8510 (Japan); Ieko, Masahiro [Department of Internal Medicine, School of Dentistry, Health Sciences University of Hokkaido, 1757 Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan)] [Department of Internal Medicine, School of Dentistry, Health Sciences University of Hokkaido, 1757 Kanazawa, Ishikari-Toubetsu, Hokkaido 061-0023 (Japan)

    2011-11-11

    Highlights: Black-Right-Pointing-Pointer Lipin-1 affects lipid metabolism, adipocyte differentiation, and transcription. Black-Right-Pointing-Pointer Adipose lipin-1 expression is reduced in obesity. Black-Right-Pointing-Pointer Lipin-1 depletion using siRNA in 3T3-L1 adipocytes increased MCP-1 expression. Black-Right-Pointing-Pointer Lipin-1 is involved in adipose inflammation. -- Abstract: Lipin-1 plays a crucial role in the regulation of lipid metabolism and cell differentiation in adipocytes. Expression of adipose lipin-1 is reduced in obesity, and metabolic syndrome. However, the significance of this reduction remains unclear. This study investigated if and how reduced lipin-1 expression affected metabolism. We assessed mRNA expression levels of various genes related to adipocyte metabolism in lipin-1-depleted 3T3-L1 adipocytes by introducing its specific small interfering RNA. In lipin-1-depleted adipocytes, mRNA and protein expression levels of monocyte chemoattractant protein-1 (MCP-1) were significantly increased, although the other genes tested were not altered. The conditioned media from the cells promoted monocyte chemotaxis. The increase in MCP-1 expression was prevented by treatment with quinazoline or salicylate, inhibitors of nuclear factor-{kappa}B activation. Because MCP-1 is related to adipose inflammation and systemic insulin resistance, these results suggest that a reduction in adipose lipin-1 in obesity may exacerbate adipose inflammation and metabolism.

  19. Negative regulation of adipogenesis by kaempferol, a component of Rhizoma Polygonati falcatum in 3T3-L1 cells.

    PubMed

    Park, Ui-Hyun; Jeong, Ji-Cheon; Jang, Jae-Sik; Sung, Mi-Ran; Youn, HyeSook; Lee, Sook-Jeong; Kim, Eun-Joo; Um, Soo-Jong

    2012-01-01

    Rhizoma Polygonati falcatum (RPF) has been used as a traditional herbal medicine in Asia, because of its anti-hyperglycemic, anti-triglycemic, and anti-tumor activity. In this study, we determined the anti-adipogenic potential of RPF extract and its component kaempferol in 3T3-L1 adipocytes, and the underlying molecular mechanism(s) using microarray analysis. Adipocyte differentiation of 3T3-L1 cells was significantly impaired by RPF extract and kaempferol as monitored by Oil Red O staining and quantitative measurement of lipid accumulation. Additionally, the mRNA expression of adipogenesis genes decreased on treatment with kaempferol. The role of kaempferol at the genome-wide level was further assessed by a microarray approach. Our analysis indicated that kaempferol decreased the expression of adipogenic transcription factors (Ppar?, Cebp?, Srebp1, Rxr?, Lxr?, Ror?) and genes involved in triglyceride biosynthesis (Gpd1, Agpat2, Dgat2), while increasing lipolysis-related genes, such as Tnf?, Lsr, and Cel. Finally, co-transfection assays using luciferase reporter gene and reverse transcription-polymerase chain reaction (RT-PCR) analysis using peroxisome proliferator-activated receptor-? (PPAR?) target genes indicated that kaempferol significantly repressed rosiglitazone-induced PPAR? transcriptional activity. Overall, our data suggests that kaempferol, a major component of RPF, may be beneficial in obesity, by reducing adipogenesis and balancing lipid homeostasis partly through the down-regulation of PPAR?. PMID:22975504

  20. Ajoene exerts potent effects in 3T3-L1 adipocytes by inhibiting adipogenesis and inducing apoptosis.

    PubMed

    Ambati, Suresh; Yang, Jeong-Yeh; Rayalam, Srujana; Park, Hea Jin; Della-Fera, Mary Anne; Baile, Clifton A

    2009-04-01

    This paper describes effects of several sulfur-containing compounds from garlic on the cell viability, apoptosis and adipogenesis in 3T3-L1 adipocytes. In both preadipocytes and mature adipocytes, 100 and 200 microM ajoene significantly decreased cell viability and increased apoptosis. The effect on apoptosis was further confirmed with Hoechst staining. In contrast, diallyl sulfide, diallyl disulfide, diallyl trisulfide, deoxyalliin, and allyl methyl sulfide had no significant effect on cell viability or apoptosis in either preadipocytes or mature adipocytes. In maturing preadipocytes ajoene significantly decreased lipid accumulation in a dose-dependent manner and these results were further confirmed by a decrease in lipid droplet number and lipid content through Oil Red O staining. There was no significant change in lipid accumulation in maturing preadipocytes treated with other garlic derivatives. Thus, despite the same source of origin, garlic, ajoene was the only one with potent effects on cell viability, apoptosis and adipogenesis in 3T3-L1 adipocytes. PMID:19051208

  1. Tyrosine phosphorylation and morphological transformation induced by four vanadium compounds on MC3T3E1 cells.

    PubMed

    Sálice, V C; Cortizo, A M; Gómez Dumm, C L; Etcheverry, S B

    1999-08-01

    The present study was performed to determine the phosphotyrosine-protein levels induced by insulin and by four vanadium derivatives in MC3T3E1 osteoblast-like cells. We have also attempted to associate these patterns with the vanadium-induced growth and morphological changes of such cells. Vanadate (Vi), vanadyl (VO), bis(maltolato)oxovanadium (IV) (BMOV) and bis(maltolato)dioxovanadium (V) (BMV) stimulate cell growth in a narrow range of concentration, but are also inhibitors for the cells at high concentrations. Vanadium-treated cells displayed clear changes in their morphology after overnight incubation. However, BMV was the least cytotoxic and the weakest inducer of morphological changes. All the compounds promote the phosphorylation of tyrosine residues in several proteins. This effect was more pronounced at low than at high doses. At low doses (10 microM), BMV showed a phosphorylation pattern similar to that of insulin, while Vi, VO and BMOV induced strong phosphorylation of cell proteins. The present findings suggest that the vanadium-induced growth regulation and morphological changes in MC3T3E1 osteoblast-like cells are associated with the ability of these agents to increase the phosphotyrosine protein levels and to inhibit phosphotyrosine phosphatases. These properties are dependent on the oxidation state as well as on the organic ligand which coordinates the vanadium atom. PMID:10497886

  2. Hirsutenone Directly Targets PI3K and ERK to Inhibit Adipogenesis in 3T3-L1 Preadipocytes.

    PubMed

    Cheong, Lai Yee; Suk, Sujin; Thimmegowda, N R; Chung, Min-Yu; Yang, Hee; Seo, Sang Gwon; Shwetha, B; Kim, Jong-Eun; Kwon, Jung Yeon; Kim, Bo Yeon; Lee, Ki Won

    2015-07-01

    Adipogenesis is a key driver of the expansion of adipose tissue mass that causes obesity. Hirsutenone (HST) is an active botanical diarylheptanoid present in Alnus species. In this study, we evaluated the effects of HST on adipogenesis, its mechanisms of action and the molecular targets involved. Using Oil Red O staining, we observed that HST dose-dependently suppresses lipid accumulation during adipogenesis in 3T3-L1 preadipocytes, concomitant with a decrease in peroxisome proliferator-activated receptor-? (PPAR?), CCAAT/enhancer-binding protein ? (C/EBP?) and fatty acid synthase (FAS) protein expression. This inhibitory effect was largely limited to the early stage of adipogenesis, which includes mitotic clonal expansion (MCE), as evidenced by delayed cell cycle entry of preadipocytes from G1 to S phase. Furthermore, the regulation of MCE was accompanied by suppression of phosphatidylinositol 3-kinase (PI3K) and extracellular-regulated kinase (ERK) activity. HST was also shown to bind directly to PI3K and ERK1 in a non-ATP competitive manner. Our results suggest that HST attenuates adipogenesis by directly targeting PI3K and ERK during MCE in 3T3-L1 preadipocytes, underscoring the potential therapeutic application of HST in preventing obesity. J. Cell. Biochem. 116: 1361-1370, 2015. © 2015 Wiley Periodicals, Inc. PMID:25756947

  3. Anti-obesity effects of hispidin and Alpinia zerumbet bioactives in 3T3-L1 adipocytes.

    PubMed

    Tu, Pham Thi Be; Tawata, Shinkichi

    2014-01-01

    Obesity and its related disorders have become leading metabolic diseases. In the present study, we used 3T3-L1 adipocytes to investigate the anti-obesity activity of hispidin and two related compounds that were isolated from Alpinia zerumbet (alpinia) rhizomes. The results showed that hispidin, dihydro-5,6-dehydrokawain (DDK), and 5,6-dehydrokawain (DK) have promising anti-obesity properties. In particular, all three compounds significantly increased intracellular cyclic adenosine monophosphate (cAMP) concentrations by 81.2% ± 0.06%, 67.0% ± 1.62%, and 56.9% ± 0.19%, respectively. Hispidin also stimulated glycerol release by 276.4% ± 0.8% and inhibited lipid accumulation by 47.8% ± 0.16%. Hispidin and DDK decreased intracellular triglyceride content by 79.5% ± 1.37% and 70.2% ± 1.4%, respectively, and all three compounds inhibited glycerol-3-phosphate dehydrogenase (GPDH) and pancreatic lipase, with hispidin and DDK being the most potent inhibitors. Finally, none of the three compounds reduced 3T3-L1 adipocyte viability. These results highlight the potential for developing hispidin and its derivatives as anti-obesity compounds. PMID:25322285

  4. Characterization of RNA from Noninfectious Virions Produced by Sarcoma Positive-Leukemia Negative Transformed 3T3 Cells

    PubMed Central

    Phillips, Leo A.; Hollis, Vincent W.; Bassin, Robert H.; Fischinger, Peter J.

    1973-01-01

    RNA from noninfectious virions produced by two established clonal lines of sarcoma positive-leukemia negative (S+L-)-transformed 3T3 cells has been characterized. RNA from virions or nucleoids of S+L--(C243) cells consisted of three to four sizes: ±44 S (6%), 28 S (17%), 18 S (38%), and <18 S (39%). 28S virion RNA contained some virus-specific information demonstrable by RNA·DNA hybridization with a DNA probe derived from the RNA-directed DNA polymerase product of murine sarcoma-leukemia virus, while parallel studies indicated that 28S ribosomal RNA from ribosomal subunits of transformed and nontransformed 3T3 cells did not contain virus-specific information. In contrast to the S+L-(C243) virions, RNA from virions or nucleoids of S+L-(D56) cells consisted of five sizes: 80 S (6%), 68 S (8%), 56 S (5%), 28 S (28%), and <28 S (53%). Thermal dissociation studies suggested that this S+L- genome is comprised of 28S RNA subunits. From these studies we postulate that the 28S viral RNA is most probably the monomeric genome of S+L- virions. PMID:4355380

  5. In combination, nucleoside reverse transcriptase inhibitors have significant effects on 3T3-L1 adipocyte lipid accumulation and survival.

    PubMed

    Kosmiski, Lisa A; Miller, Heidi L; Klemm, Dwight J

    2006-01-01

    The use of nucleoside reverse transcriptase inhibitors (NRTIs) for the treatment of HIV infection is clearly linked to the development of subcutaneous fat atrophy. Until recently, however, in vitro studies of adipocytes have shown no or only modest and inconsistent effects of these agents on adipocyte biology. This is in contrast to the protease inhibitors (PIs), which are also linked to the development of HIV lipodystrophy. These agents have relatively consistent inhibitory effects on the differentiation of cultured adipocytes, and have occasionally been found to have other effects on adipocyte biology as well. We aimed to explore more thoroughly the effects of NRTIs and combinations of antiretroviral agents commonly used in clinical practice on multiple aspects of adipocyte biology using the 3T3-L1 adipocyte cell line. We found that when used individually, NRTIs decrease cell survival but only lamivudine significantly alters lipid accumulation. However, NRTI and dual NRTI-PI combinations do significantly decrease lipid accumulation in 3T3-L1 adipocytes, have a much greater detrimental impact on cell survival and decrease adipocyte differentiation. PMID:16640100

  6. 46 CFR 58.25-65 - Feeder circuits.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ...HOMELAND SECURITY (CONTINUED) MARINE ENGINEERING MAIN AND AUXILIARY MACHINERY AND RELATED SYSTEMS Steering Gear § 58.25-65...feeder. (c) Each feeder circuit must have a disconnect switch in the steering-gear compartment. (d) Each feeder...

  7. 46 CFR 58.25-65 - Feeder circuits.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ...HOMELAND SECURITY (CONTINUED) MARINE ENGINEERING MAIN AND AUXILIARY MACHINERY AND RELATED SYSTEMS Steering Gear § 58.25-65...feeder. (c) Each feeder circuit must have a disconnect switch in the steering-gear compartment. (d) Each feeder...

  8. 46 CFR 58.25-65 - Feeder circuits.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ...HOMELAND SECURITY (CONTINUED) MARINE ENGINEERING MAIN AND AUXILIARY MACHINERY AND RELATED SYSTEMS Steering Gear § 58.25-65...feeder. (c) Each feeder circuit must have a disconnect switch in the steering-gear compartment. (d) Each feeder...

  9. 46 CFR 58.25-65 - Feeder circuits.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ...HOMELAND SECURITY (CONTINUED) MARINE ENGINEERING MAIN AND AUXILIARY MACHINERY AND RELATED SYSTEMS Steering Gear § 58.25-65...feeder. (c) Each feeder circuit must have a disconnect switch in the steering-gear compartment. (d) Each feeder...

  10. 46 CFR 58.25-65 - Feeder circuits.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ...HOMELAND SECURITY (CONTINUED) MARINE ENGINEERING MAIN AND AUXILIARY MACHINERY AND RELATED SYSTEMS Steering Gear § 58.25-65...feeder. (c) Each feeder circuit must have a disconnect switch in the steering-gear compartment. (d) Each feeder...

  11. Decellularized Feeders: An Optimized Method for Culturing Pluripotent Cells

    PubMed Central

    Lim, Mei Ling; Jungebluth, Philipp; Sjöqvist, Sebastian; Nikdin, Hero; Kjartansdóttir, Kristín Rós; Unger, Christian; Vassliev, Ivan

    2013-01-01

    Pluripotent cells such as human embryonic stem cells and human induced pluripotent stem cells are useful in the field of regenerative medicine because they can proliferate indefinitely and differentiate into all cell types. However, a limiting factor for maintaining and propagating stem cells is the need for inactivated fibroblasts as a growth matrix, since these may potentially cause cross-contamination. In this study, we aimed to maintain stem cells on the extracellular matrix (ECM) of either nonirradiated or ?-irradiated fibroblasts. It has been demonstrated that the ECM contains factors and proteins vital for the adhesion, proliferation, and differentiation of pluripotent cells. In order to preserve the ECM, the cell layers of the fibroblasts were decellularized by treatment with 0.05% sodium dodecyl sulfate (SDS), which resulted in an absence of DNA as compared with conventional feeder culture. However, SDS treatment did not cause a detectable change in the ECM architecture and integrity. Furthermore, immunohistochemistry demonstrated that expressions of major ECM proteins, such as fibronectin, collagen, and laminin, remained unaltered. The human pluripotent cells cultured on this decellularized matrix maintained gene expression of the pluripotency markers NANOG and OCT4 and had the potency to differentiate to three germ layers. The in vitro culture system shown here has an excellent potential since the main allogeneic components (i.e., DNA of the feeder cells) are removed. It is also a technically easy, fast, safe, and cheap method for maintaining a refined feeder-free stem cell culture for further cell differentiation studies. PMID:24167316

  12. Structural Assessment of Small Bore Feeder Piping

    E-print Network

    PIPES AND VALIDATE COMPUTATIONAL METHODS COMPARED TO FULL SCALE TESTS TO FAILURE. THE CLIENT CANDUBACKGROUND Structural Assessment of Small Bore Feeder Piping Kathryn Tang, Janos Mann, Skerdi. Supervisor: A. N. Sinclair CASE ONE CANDU REACTORS HAVE 380+ SMALL BORE FEEDER PIPES. THE PIPES

  13. A Prunus mume extract stimulated the proliferation and differentiation of osteoblastic MC3T3-E1 cells.

    PubMed

    Kono, Ryohei; Okuno, Yoshiharu; Inada, Ken-ichi; Tokuda, Akihiko; Hashizume, Hiroshi; Yoshida, Munehito; Nakamura, Misa; Utsunomiya, Hirotoshi

    2011-01-01

    Osteoporosis is a serious disease caused by decreased bone mass. There is constant matrix remodeling in bones, by which bone formation is performed by osteoblastic cells, whereas bone resorption is accomplished by osteoclast cells. We investigated the effect of a Japanese apricot (Prunus mume SIBE. et ZUCC.) extract on the proliferation and osteoblastic differentiation in pre-osteoblastic MC3T3-E1 cells. An alkaline phosphatase (ALP) activity assay, cell proliferation assay, alizarin red staining and expression analysis of osteoblastic genes were carried out to assess the proliferation and osteoblastic differentiation. The water-soluble fraction of Prunus mume (PWF) increased the ALP activity, cell proliferation and mineralization. The gene expression of osteopontin and bone morphogenetic protein-2, which are markers in the early period of osteoblastic differentiation, were significantly enhanced by the PWF treatment. PWF therefore stimulated the proliferation and osteoblastic differentiation of cells and may have potential to prevent osteoporosis. PMID:21979066

  14. Lactobacillus plantarum LG42 Isolated from Gajami Sik-Hae Inhibits Adipogenesis in 3T3-L1 Adipocyte

    PubMed Central

    Park, Jeong-Eun; Oh, Suk-Heung; Cha, Youn-Soo

    2013-01-01

    We investigated whether lactic acid bacteria isolated from gajami sik-hae (GLAB) are capable of reducing the intracellular lipid accumulation by downregulating the expression of adipogenesis-related genes in differentiated 3T3-L1 cells. The GLAB, Lactobacillus plantarum LG42, significantly decreased the intracellular triglyceride storage and the glycerol-3-phosphate dehydrogenase (GPDH) activity in a dose-dependent manner. mRNA expression of transcription factors like peroxisome proliferator-activated receptor (PPAR) ? and CCAAT/enhancer-binding protein (C/EBP) ? involved in adipogenesis was markedly decreased by the GLAB treatment. Moreover, the GLAB also decreased the expression level of adipogenic markers like adipocyte fatty acid binding protein (aP2), leptin, GPDH, and fatty acid translocase (CD36) significantly. These results suggest that the GLAB inhibits lipid accumulation in the differentiated adipocyte through downregulating the expression of adipogenic transcription factors and other specific genes involved in lipid metabolism. PMID:23555088

  15. Cultured 3T3L1 adipocytes dispose of excess medium glucose as lactate under abundant oxygen availability

    NASA Astrophysics Data System (ADS)

    Sabater, David; Arriarán, Sofía; Romero, María Del Mar; Agnelli, Silvia; Remesar, Xavier; Fernández-López, José Antonio; Alemany, Mariŕ

    2014-01-01

    White adipose tissue (WAT) produces lactate in significant amount from circulating glucose, especially in obesity;Under normoxia, 3T3L1 cells secrete large quantities of lactate to the medium, again at the expense of glucose and proportionally to its levels. Most of the glucose was converted to lactate with only part of it being used to synthesize fat. Cultured adipocytes were largely anaerobic, but this was not a Warburg-like process. It is speculated that the massive production of lactate, is a process of defense of the adipocyte, used to dispose of excess glucose. This way, the adipocyte exports glucose carbon (and reduces the problem of excess substrate availability) to the liver, but the process may be also a mechanism of short-term control of hyperglycemia. The in vivo data obtained from adipose tissue of male rats agree with this interpretation.

  16. Antidiabetic screening of commercial botanical products in 3T3-L1 adipocytes and db/db mice.

    PubMed

    Babish, John G; Pacioretty, Linda M; Bland, Jeffrey S; Minich, Deanna M; Hu, Jeffrey; Tripp, Matthew L

    2010-06-01

    Numerous botanicals are purported to improve glucose metabolism and diabetic risk factors with varying degrees of supportive evidence. We investigated 203 commercially available botanical products representing 90 unique botanical species for effects on lipogenic activity in differentiating 3T3-L1 adipocytes. Anti-inflammatory activity of 21 of these products was further assessed in tumor necrosis factor alpha (TNFalpha)-stimulated, mature 3T3-L1 adipocytes. From these results, rho-isoalpha acids, Acacia nilotica bark, fennel, and wasabi were tested in the db/db mouse model. Fifty-nine percent of the 90 unique botanicals increased adipogenesis as did the standard troglitazone relative to the solvent controls. Botanical species with the greatest percentage of positive products were Centella asiatica, Panax quinquefolius, and Phyllanthus amarus at 100%, Vitis vinifera at 80%, Humulus lupulus at 71%, Aloe barbadensis at 66%, and Momordica charantia, Phaseolus vulgaris, and Punica granatum at 60%. All 21 subset samples inhibited TNFalpha-stimulated free fatty acid release and attenuated TNFalpha inhibition of adiponectin secretion. Both rho-isoalpha acids and A. nilotica reduced nonfasting glucose in the db/db mouse model, whereas A. nilotica also decreased nonfasting insulin levels. A post hoc analysis of the screening results indicated that the positive predictive value of the lipogenesis assay alone was 72%, while adding the criterion of a positive response in the anti-inflammatory assays increased this figure to 82%. Moreover, this large-scale evaluation demonstrates that antidiabetic, in vitro efficacy of botanicals is more a function of manufacturing or quality control differences than the presence of marker compounds and further underscores the need to develop functional as well as analytical bases for standardization of dietary supplements. PMID:20521979

  17. Ginsenoside Rb1 stimulates glucose uptake through insulin-like signaling pathway in 3T3-L1 adipocytes.

    PubMed

    Shang, Wenbin; Yang, Ying; Zhou, Libin; Jiang, Boren; Jin, Hua; Chen, Mingdao

    2008-09-01

    A series of clinical trials and animal experiments have demonstrated that ginseng and its major active constituent, ginsenosides, possess glucose-lowering action. In our previous study, ginsenoside Rb(1) has been shown to regulate peroxisome proliferator-activated receptor gamma activity to facilitate adipogenesis of 3T3-L1 cells. However, the effect of Rb(1) on glucose transport in insulin-sensitive cells and its molecular mechanism need further elucidation. In this study, Rb(1) significantly stimulated basal and insulin-mediated glucose uptake in a time- and dose-dependent manner in 3T3-L1 adipocytes and C2C12 myotubes; the maximal effect was achieved at a concentration of 1 microM and a time of 3 h. In adipocytes, Rb(1) promoted GLUT1 and GLUT4 translocations to the cell surface, which was examined by analyzing their distribution in subcellular membrane fractions, and enhanced translocation of GLUT4 was confirmed using the transfection of GLUT4-green fluorescence protein in Chinese Hamster Ovary cells. Meanwhile, Rb(1) increased the phosphorylation of insulin receptor substrate-1 and protein kinase B (PKB), and stimulated phosphatidylinositol 3-kinase (PI3K) activity in the absence of the activation of the insulin receptor. Rb(1)-induced glucose uptake as well as GLUT1 and GLUT4 translocations was inhibited by the PI3K inhibitor. These results suggest that ginsenoside Rb(1) stimulates glucose transport in insulin-sensitive cells by promoting translocations of GLUT1 and GLUT4 by partially activating the insulin signaling pathway. These findings are useful in understanding the hypoglycemic and anti-diabetic properties of ginseng and ginsenosides. PMID:18550785

  18. Protein phosphatase 2A inactivates the mitogen-stimulated S6 kinase from Swiss mouse 3T3 cells.

    PubMed

    Ballou, L M; Jenö, P; Thomas, G

    1988-01-25

    Treatment of quiescent 3T3 cells with sodium orthovanadate induces a 10-fold stimulation of a kinase that phosphorylates ribosomal protein S6. The kinase in crude extracts is extremely labile and rapidly loses activity when incubated at 37 degrees C. This reaction is blocked by phosphatase inhibitors such as p-nitrophenyl phosphate and beta-glycerophosphate, suggesting that dephosphorylation of the kinase leads to its inactivation (Novak-Hofer, I., and Thomas, G. (1985) J. Biol. Chem. 260, 10314-10319). After three steps of purification the kinase can be separated from greater than 99% of the cellular phosphorylase a phosphatases. At this stage the kinase preparation is almost completely stable but can be inactivated by readdition of specific column fractions that contain both phosphorylase phosphatase and protease activity. However, employing a number of specific inhibitors it is shown that the inactivating agent in these fractions is a protein phosphatase. Furthermore, the physical and enzymatic properties of the kinase inactivator argue that it can be classified as a type 2A phosphatase. These results are consistent with the finding that the purified catalytic subunits of phosphatase type 1 and type 2A also inactivate the kinase. At equivalent phosphorylase a phosphatase activities, the type 2A catalytic subunit is 3 times more potent than the type 1 enzyme in carrying out this reaction. These data indicate that the major S6 kinase inactivator in 3T3 cell extracts is a type 2A phosphatase, supporting the hypothesis that the orthovanadate-stimulated S6 kinase is regulated in vivo by a phosphorylation-dephosphorylation mechanism. PMID:2826472

  19. Helenalin-mediated Post-transcriptional Regulation of p21(Cip1) Inhibits 3T3-L1 Preadipocyte Proliferation

    PubMed Central

    Fernandes, Karishma M.; Auld, Corinth A.; Hopkins, Robin G.; Morrison, Ron F.

    2008-01-01

    We have previously shown that post-transcriptional mechanisms involving the 26S proteasome regulate the cyclin-dependent kinase inhibitors (CKIs), p21(Cip1) and p27(Kip1) during preadipocyte proliferation. Earlier studies further demonstrated that the anti-inflammatory, anti-carcinogenic phytochemical, helenalin is a potent inhibitor of periodic Skp2 accumulation, an F-box protein mediating SCF E3 ligase ubiquitylation and degradation of both CKIs during S phase progression. Data presented here demonstrate that helenalin dose-dependently induced G1 arrest of synchronously replicating 3T3-L1 preadipocytes. This effect occurred in the absence of discernable indices of cell toxicity or apoptosis under the conditions used in this study. Our results demonstrate that helenalin markedly increased p21 protein accumulation in both density-arrested and proliferating preadipocytes in a dose-dependent manner. This increase in p21 protein abundance occurred without change in mRNA transcript demonstrating that post-transcriptional mechanisms were involved. This notion was further supported by the modest accumulation of polyubiquitylated p21 following treatment with helenalin suggesting that suppression of targeted p21 proteolysis by the 26S proteasome contributed to helenalin-mediated p21 accumulation. The increase in p21 protein was compartmentalized to the nucleus where p21 is known to inhibit cell cycle progression. Finally, helenalin increased protein-protein interactions between p21 and cyclin-dependent kinase 2 (Cdk2) which may account in part for the anti-proliferative effect in 3T3-L1 preadipocytes. PMID:18729080

  20. 1-Deoxynojirimycin isolated from a Bacillus subtilis stimulates adiponectin and GLUT4 expressions in 3T3-L1 adipocytes.

    PubMed

    Lee, Seung-Min; Do, Hyun Ju; Shin, Min-Jeong; Seong, Su-Il; Hwang, Kyo Yeol; Lee, Jae Yeon; Kwon, Ohsuk; Jin, Taewon; Chung, Ji Hyung

    2013-05-01

    We have demonstrated that 1-deoxynojirimycin (DNJ) isolated from Bacillus subtilis MORI could enhance the levels of adiponectin and its receptors in differentiated 3T3-L1 adipocytes, which has been shown to be effective in lowering blood glucose levels and enhancing insulin sensitivity. DNJ was not toxic to differentiated 3T3-L1 adipocytes for up to a concentration of 5 microM. In terms of expression levels of adiponectin and its receptors (AdipoR1 and AdipoR2), DNJ in concentrations as low as 0.5 microM elevated both mRNA and protein levels of adiponectin and transcript levels of AdipoR1 and AdipoR2. In addition, DNJ increased phosphorylation of 5' adenosine monophosphateactivated protein kinase (AMPK) in a statistically significant manner. Finally, treatment with DNJ resulted in increased mRNA expression of glucose transporter 4 (GLUT4), which encodes for a glucose transporter, along with a significant increase in glucose uptake into the adipocytes based on results of a 2-deoxy-D-[3H] glucose uptake assay. Our findings indicate that DNJ may greatly facilitate glucose uptake into adipose tissues by increasing the action of adiponectin via its up-regulated expression as well as its receptor genes. In addition, the glucose-lowering effects of DNJ may be achieved by an increased abundance of GLUT4 protein in the plasma membrane, as a consequence of the increased transcript levels of the GLUT4 gene and the activation of AMPK. PMID:23648852

  1. Identification of suitable reference genes for quantitative RT-PCR during 3T3-L1 adipocyte differentiation.

    PubMed

    Zhang, Juan; Tang, Hongju; Zhang, Yuqing; Deng, Ruyuan; Shao, Li; Liu, Yun; Li, Fengying; Wang, Xiao; Zhou, Libin

    2014-05-01

    Quantitative reverse transcription PCR (qRT-PCR) is becoming increasingly important in the effort to gain insight into the molecular mechanisms underlying adipogenesis. However, the expression profile of a target gene may be misinterpreted due to the unstable expression of the reference genes under different experimental conditions. Therefore, in this study, we investigated the expression stability of 10 commonly used reference genes during 3T3-L1 adipocyte differentiation. The mRNA expression levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and transferrin receptor (TFRC) significantly increased during the course of 3T3-L1 adipocyte differentiation, which was decreased by berberine, an inhibitor of adipogenesis. Three popular algorithms, GeNorm, NormFinder and BestKeeper, identified 18 ribosomal RNA and hydroxymethylbilane synthase (HMBS) as the most stable reference genes, while GAPDH and TFRC were the least stable ones. Peptidylprolyl isomerase A [PIPA (cyclophilin A)], ribosomal protein, large, P0 (36-B4), beta-2-microglobulin (B2M), ?1-tubulin, hypoxanthine-guanine phosphoribosyltransferase (HPRT) and ?-actin showed relatively stable expression levels. The choice of reference genes with various expression stabilities exerted a profound influence on the expression profiles of 2 target genes, peroxisome proliferator-activated receptor (PPAR)?2 and C/EBP?. In addition, western blot analysis revealed that the increased protein expression of GAPDH was markedly inhibited by berberine during adipocyte differentiation. This study highlights the importance of selecting suitable reference genes for qRT-PCR studies of gene expression during the process of adipogenesis. PMID:24626784

  2. Genomic instability in silica- and cadmium chloride-transformed BALB\\/c-3T3 and tumor cell lines by random amplified polymorphic DNA analysis

    Microsoft Academic Search

    Channa Keshava; Nagalakshmi Keshava; Gu Zhou; Wen-Zong Whong; Tong-man Ong

    1999-01-01

    Our earlier studies using random amplified polymorphic DNA (RAPD) analysis have shown genetic instability in human lung cancer tissues. Here we have investigated the potential for genetic instability in silica- and cadmium chloride (CdCl2)-transformed BALB\\/c-3T3 cell lines. Non-transformed, transformed BALB\\/c-3T3 cells, and tumor cell lines (obtained by injecting nude mice with transformed cell lines) were analyzed for genomic changes. DNAs

  3. Endrin Inhibits Adipocyte Differentiation by Selectively Altering Expression Pattern of CCAAT\\/Enhancer Binding Protein-a in 3T3-L1 Cells

    Microsoft Academic Search

    MARIA J. MORENO-ALIAGA; FUMIO MATSUMURA

    The effects of selected chlorinated cyclodiene pesticides on the adipocyte differentiation process were examined using the 3T3-L1 adipocyte model in vitro. Endrin was found to cause a dose-dependent inhibition of adipocyte differentiation in 3T3-L1 cells. Aldrin and dieldrin were less potent than endrin in interfering with the adipogenic process. Endrin's inhibitory ef- fect was effective only when the pesticide was

  4. Improved medium and culture conditions for clonal growth with minimal serum protein and for enhanced serum-free survival of Swiss 3T3 cells

    Microsoft Academic Search

    Gary D. Shipley; Richard G. Ham

    1981-01-01

    Summary  Improved culture conditions have been developed that will support clonal growth of Swiss mouse embryo 3T3 cells at concentrations\\u000a of serum protein as low as 125?g\\/ml. Survival of the cells under completely protein-free conditions also is enhanced greatly.\\u000a The improvements that made these results possible include: (a) use of medium MCDB 402, which was developed specifically for\\u000a Swiss 3T3 cells

  5. Calcium-sensing receptor-mediated activation of phospholipase C-?1 is downstream of phospholipase C-? and protein kinase C in MC3T3-E1 osteoblasts

    Microsoft Academic Search

    S. L Godwin; S. P Soltoff

    2002-01-01

    Elevated extracellular calcium (Cae) stimulates both chemotaxis and mitogenesis of MC3T3-E1 osteoblasts via a calcium-sensing receptor (CasR). Cae-mediated chemotaxis of these bone-forming cells is dependent on phospholipase C (PLC) and blocked by the Gi-protein inhibitor pertussis toxin. In this study, we examine the signaling mechanisms by which the CasR stimulates PLC activity in MC3T3-E1 osteoblasts. We found that elevated Cae

  6. A possible role of oxidative stress in the vanadium-induced cytotoxicity in the MC3T3E1 osteoblast and UMR106 osteosarcoma cell lines

    Microsoft Academic Search

    Ana Mar??a Cortizo; Liliana Bruzzone; Silvina Molinuevo; Susana Beatriz Etcheverry

    2000-01-01

    The cytotoxicity and free radical production induced by vanadium compounds were investigated in an osteoblast (MC3T3E1) and an osteosarcoma (UMR106) cell lines in culture. Vanadate induced cell toxicity, reactive oxygen species (ROS) formation and thiobarbituric acid reactive substances (TBARS) increased in a concentration-dependent manner (0.1–10 mM) after 4 h. The concentration–response curve of vanadate-induced cytotoxicity and oxidative stress in MC3T3E1

  7. Mouse Balb/c3T3 cell mutant with low epidermal growth factor receptor activity: induction of stable anchorage-independent growth by transforming growth factor. beta

    SciTech Connect

    Kuratomi, Y.; Ono, M.; Yasutake, C.; Mawatari, M.; Kuwano, M.

    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 in 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.

  8. Anthocyanidins-enriched bilberry extracts inhibit 3T3-L1 adipocyte differentiation via the insulin pathway

    PubMed Central

    2011-01-01

    Background Obesity and metabolic syndrome are important public concerns, and there is increasing demand for effective therapeutic strategies. Flavonoids are expected to improve the risk factors associated with metabolic syndrome. Anthocyanidins are a kind of flavonoids; well known for their anti-oxidative, anti-inflammatory and anti-tumor properties. However, their effects on adipocytes and molecular systems are not well defined. In this study, we examined the effects of anthocyanidins-enriched bilberry extracts on adipocyte differentiation. Methods Utilizing 3T3-L1 cell line, we investigated that bilberry extracts and anthocyanidins induced inhibition of lipid accumulation during adipogenesis. To identify what is the most important bilberry mediated-effect, we analyzed the expressions of key transcriptional factors associated with adipocyte differentiation by Real Time (RT)-PCR. From the results of RT-PCR, we hypothesized that bilberry extracts and anthocyanidins blocks insulin signal, we determined the phosphorylation of tyrosine residues of insulin receptor substrate 1 (IRS1) protein by western blotting analysis. In addition, we compared the whole-genome expression profiles of early stage of adipocyte differentiation under four different growth conditions (DMSO, bilberry, two anthocyanidins) by microarray analyses and Gene Set Enrichment Analysis (GSEA). Results Exposure to bilberry extracts and anthocyanidins during adipocyte differentiation inhibited 3T3-L1 differentiation. During this period, bilberry extracts and anthocyanidin significantly decreased a key adipocyte differentiation-associated marker, peroxisome proliferator-activated receptor- ? (Ppar ? ) and sterol regulatory element-binding protein 1c (Srebp1c). Western blotting analysis showed that bilberry extracts and anthocyanidin decreased the phosphorylation of tyrosine residues of IRS1. In addition, microarray experiments and GSEA data revealed significantly altered expression of the known genes of the insulin pathway in cells treated with bilberry extracts or anthocyanidins in the early differentiation stages. Conclusions Our data demonstrate that anthocyanidin enriched bilberry extracts strongly inhibit the adipocyte differentiation via the insulin pathway. Furthermore, bilberry extracts might be used as a potential complementary treatment for the obese patients with metabolic syndrome. PMID:21385419

  9. FEEDER PIPING NDE - CURRENT CAPABILITIES AND FUTURE DIRECTION

    Microsoft Academic Search

    G. Rousseau; K. Chaplin; T. Hazelton; P. Martin; E. Choi

    The primary heat transport system of a typical CANDU®-6 nuclear power reactor contains 760 feeder pipes. These feeders carry the coolant between the inlet or outlet headers and the individual fuel channels. Inspection requirements that developed in feeders in the 1990's led to rapid development of NDE technology to evaluate feeder integrity. The NDE technology had to deal with the

  10. Water Usage in Finishing Facilities: Wet\\/Dry Feeders Versus Dry Feeders with Nipple Waterers

    Microsoft Academic Search

    Jay D. Harmon

    1998-01-01

    Water usage was measured in swine finishing facilities containing wet\\/dry tube feeders and more traditional dry feeders with nipple waterers. Comparisons of groups finished with the two systems show a trend toward a reduction in water wastage of 17.2%. A water efficiency is introduced that shows a reduction of 0.27 gal\\/lb gain. Pigs finished using wet\\/dry feeders also had a

  11. Manually Operated Welding Wire Feeder

    NASA Technical Reports Server (NTRS)

    Rybicki, Daniel J. (Inventor)

    2001-01-01

    A manual welding wire feeder apparatus comprising a bendable elongate metal frame with a feed roller mounted at the center thereof for rotation about an axis transverse to the longitudinal axis of the frame. The frame ends are turned up as tabs and each provided with openings in alignment with each other and the mid-width center of the roller surface. The tab openings are sized to accommodate welding wire and each extends to a side edge of the tab, both opening on the same side of the frame, whereby welding wire can be side-loaded onto the frame. On the side of the frame, opposite the roller a lock ring handle is attached tangentially and is rotatable about the attachment point and an axis perpendicular to the frame. The device is grasped in the hand normally used to hold the wire. A finger is placed through the loop ring and the frame positioned across the palm and lower fingers. The thumb is positioned atop the wire so it can be moved from the back of the frame across the roller, and towards the front. In doing so, the wire is advanced at a steady rate in axial alignment with the tab openings and roller. To accommodate different wire diameters the frame is bendable about its center in the plane of the frame axis and wire so as to keep the wire in sufficient tension against the roller and to keep the wire fixed when the frame is tilted and thumb pressure released.

  12. Dual role for myosin II in GLUT4-mediated glucose uptake in 3T3-L1 adipocytes

    SciTech Connect

    Fulcher, F. Kent; Smith, Bethany T.; Russ, Misty [Department of Biology, University of North Carolina at Greensboro, Greensboro, North Carolina 27402 (United States); Patel, Yashomati M. [Department of Biology, University of North Carolina at Greensboro, Greensboro, North Carolina 27402 (United States)], E-mail: ympatel@uncg.edu

    2008-10-15

    Insulin-stimulated glucose uptake requires the activation of several signaling pathways to mediate the translocation and fusion of GLUT4 vesicles to the plasma membrane. Our previous studies demonstrated that GLUT4-mediated glucose uptake is a myosin II-dependent process in adipocytes. The experiments described in this report are the first to show a dual role for the myosin IIA isoform specifically in regulating insulin-stimulated glucose uptake in adipocytes. We demonstrate that inhibition of MLCK but not RhoK results in impaired insulin-stimulated glucose uptake. Furthermore, our studies show that insulin specifically stimulates the phosphorylation of the RLC associated with the myosin IIA isoform via MLCK. In time course experiments, we determined that GLUT4 translocates to the plasma membrane prior to myosin IIA recruitment. We further show that recruitment of myosin IIA to the plasma membrane requires that myosin IIA be activated via phosphorylation of the RLC by MLCK. Our findings also reveal that myosin II is required for proper GLUT4-vesicle fusion at the plasma membrane. We show that once at the plasma membrane, myosin II is involved in regulating the intrinsic activity of GLUT4 after insulin stimulation. Collectively, our results are the first to reveal that myosin IIA plays a critical role in mediating insulin-stimulated glucose uptake in 3T3-LI adipocytes, via both GLUT4 vesicle fusion at the plasma membrane and GLUT4 activity.

  13. The Effect of OSM on MC3T3-E1 Osteoblastic Cells in Simulated Microgravity with Radiation

    PubMed Central

    Goyden, Jake; Tawara, Ken; Hedeen, Danielle; Willey, Jeffrey S.; Thom Oxford, Julia; Jorcyk, Cheryl L.

    2015-01-01

    Bone deterioration is a challenge in long-term spaceflight with significant connections to patients experiencing disuse bone loss. Prolonged unloading and radiation exposure, defining characteristics of space travel, have both been associated with changes in inflammatory signaling via IL-6 class cytokines in bone. While there is also evidence for perturbed IL-6 class signaling in spaceflight, there has been scant examination of the connections between microgravity, radiation, and inflammatory stimuli in bone. Our lab and others have shown that the IL-6 class cytokine oncostatin M (OSM) is an important regulator of bone remodeling. We hypothesize that simulated microgravity alters osteoblast OSM signaling, contributing to the decoupling of osteolysis and osteogenesis in bone homeostasis. To test this hypothesis, we induced OSM signaling in murine MC3T3-E1 pre-osteoblast cells cultured in modeled microgravity using a rotating wall vessel bioreactor with and without exposure to radiation typical of a solar particle event. We measured effects on inflammatory signaling, osteoblast activity, and mineralization. Results indicated time dependent interactions among all conditions in the regulation of IL-6 production. Furthermore, OSM induced the transcription of OSM receptor ß, IL 6 receptor ? subunits, collagen ?1(I), osteocalcin, sclerostin, RANKL, and osteoprotegerin. Measurements of osteoid mineralization suggest that the spatial organization of the osteoblast environment is an important consideration in understanding bone formation. Taken together, these results support a role for altered OSM signaling in the mechanism of microgravity-induced bone loss. PMID:26030441

  14. Effects of yerba maté, a plant extract formulation ("YGD") and resveratrol in 3T3-L1 adipogenesis.

    PubMed

    Santos, Juliana C; Gotardo, Erica M F; Brianti, Mitsue T; Piraee, Mahmood; Gambero, Alessandra; Ribeiro, Marcelo L

    2014-01-01

    We aimed to evaluate the in vitro effects of yerba maté, YGD (a herbal preparation containing yerba maté, guarana and damiana), and resveratrol on adipogenesis. The anti-adipogenic effects of yerba mate, YGD, resveratrol and YGD + resveratrol and yerba mate + resveratrol combinations were evaluated in 3T3-L1 cells by Oil Red staining, cellular triglyceride content, and PCR quantitative array. The results demonstrated that all of the tested compounds inhibited adipogenesis. Yerba maté extract significantly down-regulated the expression of genes that play an important role in regulating adipogenesis, such as Adig, Axin, Cebpa, Fgf10, Lep, Lpl, and Ppar?2. In addition, these genes, YGD also repressed Bmp2, Ccnd1, Fasn, and Srebf1. Resveratrol also modulated the expression of Adig, Bmp2, Ccnd1, C/EBP?, Fasn, Fgf10, Lep, Lpl, and Ppar?2. Moreover, resveratrol repressed Cebpb, Cdk4, Fgf2, and Klf15. The yerba maté extract and YGD up-regulated the expression of genes involved in inhibiting adipogenesis, such as Dlk-1, Klf2, and Ucp1. Resveratrol also induced the expression of Klf2 and Ucp1. In addition resveratrol modulated the Ddit3, Foxo1, Sirt1, and Sirt2. The combined effects of these compounds on gene expression showed similar results observed from individual treatments. Our data indicates that the synergy between the compounds favors the inhibition of adipogenesis. PMID:25338179

  15. Effect of ginsenosides Rg3 and Re on glucose transport in mature 3T3-L1 adipocytes.

    PubMed

    Lee, Ok-Hwan; Lee, Hee-Hyun; Kim, Ji-Hyup; Lee, Boo-Yong

    2011-05-01

    Ginsenosides, the active component of Panax ginseng, have been shown to evidence a variety of biological activities associated with hyperglycemia, obesity and type 2 diabetes mellitus. This study evaluated the effects of the ginsenosides, Rg3 and Re, on glucose uptake and the glucose transport system in mature 3T3-L1 cells. The results demonstrated that the glucose uptake of ginsenosides Rg3 and Re at concentrations of 1-10?µM significantly increased by approximately ?10% and ?12%, respectively. Furthermore, the glucose transporter 4 (GLUT4) mRNA expression of ginsenosides Rg3 and Re at 10?µM was increased by approximately ?1.73 and 1.43 fold, respectively. It was further confirmed in a series of experiments that ginsenosides Rg3 and Re stimulated the mRNA expression of insulin receptor substrate (IRS-1) and the expression of phosphatidylinositol 3-kinase (PI3K)-110? protein, which is involved in downstream events in the insulin signaling pathway. These findings demonstrate that ginsenosides Rg3 and Re may stimulate glucose uptake via the PI3K pathways involving IRS-1. Further, our results suggest that both of these ginsenosides might prove useful as effective antidiabetic and antihyperglycemic agents. PMID:21520470

  16. Caveolar structure and protein sorting are maintained in NIH 3T3 cells independent of glycosphingolipid depletion.

    PubMed

    Shu, L; Lee, L; Chang, Y; Holzman, L B; Edwards, C A; Shelden, E; Shayman, J A

    2000-01-01

    Glycosphingolipids have been proposed to be critical components of clustered lipids within cell membranes that serve as rafts for the attachment and sorting of proteins to the cell membrane. Density gradient centrifugation was used to isolate and to ascertain the lipid composition of caveolin-enriched membranes. These membranes demonstrated a significant enrichment of sphingolipids and cholesterol containing up to 20 and 30%, respectively, of the cellular glucosylceramide and lactosylceramide. A specific inhibitor of glucosylceramide synthase, d-threo-1-phenyl-2-palmitoyl-3-pyrrolidino-propanol, was used to test the hypothesis that glycosphingolipids are required for the sorting of proteins to caveolae. When NIH 3T3 cells were depleted of their glucosylceramide based glycosphingolipid mass, the caveolar structure remained intact as determined by electron microscopy and confocal microscopy. The caveolar proteins caveolin and annexin II sorted normally to caveolae, as determined by immunoblotting and confocal microscopy. When the GPI-linked protein B61 was inducibly expressed in these cells, sorting to caveolar membranes occurred normally, even in the presence of glucosylceramide depletion. These observations suggest that protein sorting to caveolae in fibroblasts occurs independently of glycosphingolipid synthesis. PMID:10620326

  17. Chromosomal rearrangements involving telomeric DNA sequences in Balb/3T3 cells transfected with the Ha-ras oncogene.

    PubMed

    Peitl, Paulo; Mello, Stephano S; Camparoto, Marjori L; Passos, Geraldo A S; Hande, Manoor P; Cardoso, Renato S; Sakamoto-Hojo, Elza T

    2002-01-01

    Chromosomal instability involving telomeric DNA sequences was studied in mouse Balb/3T3 fibroblasts transfected with a mutated human c-Ha-ras-1 gene (B61 cells) and spontaneously immortalized normal parental cells (A31 cells), using fluorescence in situ hybridization (FISH). FISH analysis with a telomeric probe revealed high frequencies of chromosome alterations involving telomeric regions, mainly stable and unstable Robertsonian fusion-like configurations (RLC) (0.25 and 1.95/cell in A31 and B61 cells, respectively) and chromosome ends lacking telomeric signals in one (LTS') or both chromatids (LTS") (5.9 and 17.5/cell for A31 and B61 cells, respectively). Interstitial telomeric sequences (ITS) were also detected at both non-telomeric sites and in the centromeres of RLC. The frequencies of RLCs with ITS located in the centromeres were 3-fold higher in B61 compared with A31 cells. We demonstrated a high level of chromosome instability involving telomeric DNA sequences in ras-transfected cells overexpressing ras mRNA, which could be a consequence of rapid cell cycle progression associated with a deficient telomere capping mechanism. PMID:11752236

  18. Retinol encapsulated into amorphous Ca(2+) polyphosphate nanospheres acts synergistically in MC3T3-E1 cells.

    PubMed

    Müller, Werner E G; Tolba, Emad; Schröder, Heinz C; Diehl-Seifert, Bärbel; Wang, Xiaohong

    2015-06-01

    Both the quality and quantity of collagen, the major structural component of the skin, decrease in aging skin. We succeeded to encapsulate retinol into amorphous inorganic polyphosphate (polyP) nanoparticles together with calcium ions ("aCa-polyP-NP"), under formation of amorphous Ca-polyP/retinol nanospheres ("retinol/aCa-polyP-NS"). The globular nanospheres are not cytotoxic, show an almost uniform size of ?45nm and have a retinol content of around 25%. Both components of those nanospheres, retinol and the aCa-polyP-NP, if administered together, caused a strong increase in proliferation of mouse calvaria MC3T3 cells. The expressions of collagen types I, II and III genes, but not the expression of collagen type V gene, were significantly enhanced if retinol is added together with aCa-polyP-NP. This synergistic effect was especially pronounced for the expression of the collagen type III gene. We propose that the synergistic effect of the retinol/aCa-polyP-NS on cell growth and collagen type III expression is induced via two routes, first through cellular uptake of the 45nm nanospheres by clathrin-mediated endocytosis and second through extracellular disintegration of the nanospheres resulting in the release of retinol which is then taken up into the cells after binding to the retinal binding protein receptor. PMID:25900862

  19. IGF 2 expression in 3T3 adipocytes in response to serum from hypophysectomized or diabetic swine

    SciTech Connect

    Srinivas, V.; White, M.E.; Ramsay, T.G. (Ohio State Univ., Columbus (United States))

    1990-02-26

    Expression of IGF-2 and changes in its expression in response to systemic endocrine alterations have not been demonstrated for adipocytes. Adipocytes were induced to develop within cultures of 3T3-L1 cells using a medium containing 0.5mM isobutylmethylxanthine, 1uM insulin and 100ng hydrocortisone/ml for 48 hours of exposure. Cultures containing developing adipocytes were incubated with 10% pig serum and 1 uM insulin for several days. The resultant adipocyte cultures were then treated with either 10% pig serum, diabetic pig serum or hypophysectomized pig serum in DMEM for 48 hours. Adipocytes within the cultures were separated from undifferentiated cells using percoll density gradient centrifugation. Total RNA was isolated from adipocytes and dot blotted. Blots were probed with a {sup 32}P-cDNA probe for rat IGF-2. IGF-2 was expressed by the adipocytes and the pattern of expression showed specific differences between serum treatments; IGF-2 expression was highest in cells exposed to normal pig serum, less expressed in cells exposed to diabetic serum and with minimal expression in adipocytes incubated with hypophysectomized pig serum. These data suggest that adipocyte expression of IGF-2 is influenced by endocrine factors present in pig serum.

  20. The protective effects of guaraná extract (Paullinia cupana) on fibroblast NIH-3T3 cells exposed to sodium nitroprusside.

    PubMed

    Bittencourt, L S; Machado, D C; Machado, M M; Dos Santos, G F F; Algarve, T D; Marinowic, D R; Ribeiro, E E; Soares, F A A; Barbisan, F; Athayde, M L; Cruz, I B M

    2013-03-01

    The antioxidant effects of the hydro-alcoholic guaraná extract (Paullinia cupana var. sorbilis Mart.) on nitric oxide (NO) and other compounds generated from the degradation of sodium nitroprusside (SNP) in an embryonic fibroblast culture (NIH-3T3 cells) were evaluated. The guaraná bioactive compounds were initially determined by high-performance liquid chromatography: caffeine=12.240 mg/g, theobromine=6.733 mg/g and total catechins=4.336 mg/g. Cells were exposed to 10 ?M SNP during a 6 h period because the cells exhibited >90% mortality at this concentration. Guaraná was added to the cultures in five concentrations (0.5, 1, 5, 10 and 20 mg/mL). The guaraná antioxidant effect was evaluated by viability assays, biochemical oxidation [lipid peroxidation, catalase and superoxide dismutase (SOD) activity] and genotoxicity (DNA Comet assay) analysis. Additionally, oxidative stress was evaluated by a 2,7-dihydrodichlorofluorescein diacetate fluorescence assay. Guaraná reverted the SNP toxicity mainly at lower concentrations (<5 mg), which decreased cell mortality, lipid peroxidation, DNA damage and cell oxidative stress as well as increased the SOD levels. These results demonstrate that guaraná has an antioxidant effect on NO metabolism in situations with higher cellular NO levels. PMID:23220610

  1. Fluid shear-induced mechanical signaling in MC3T3-E1 osteoblasts requires cytoskeleton-integrin interactions

    NASA Technical Reports Server (NTRS)

    Pavalko, F. M.; Chen, N. X.; Turner, C. H.; Burr, D. B.; Atkinson, S.; Hsieh, Y. F.; Qiu, J.; Duncan, R. L.

    1998-01-01

    Mechanical stimulation of bone induces new bone formation in vivo and increases the metabolic activity and gene expression of osteoblasts in culture. We investigated the role of the actin cytoskeleton and actin-membrane interactions in the transmission of mechanical signals leading to altered gene expression in cultured MC3T3-E1 osteoblasts. Application of fluid shear to osteoblasts caused reorganization of actin filaments into contractile stress fibers and involved recruitment of beta1-integrins and alpha-actinin to focal adhesions. Fluid shear also increased expression of two proteins linked to mechanotransduction in vivo, cyclooxygenase-2 (COX-2) and the early response gene product c-fos. Inhibition of actin stress fiber development by treatment of cells with cytochalasin D, by expression of a dominant negative form of the small GTPase Rho, or by microinjection into cells of a proteolytic fragment of alpha-actinin that inhibits alpha-actinin-mediated anchoring of actin filaments to integrins at the plasma membrane each blocked fluid-shear-induced gene expression in osteoblasts. We conclude that fluid shear-induced mechanical signaling in osteoblasts leads to increased expression of COX-2 and c-Fos through a mechanism that involves reorganization of the actin cytoskeleton. Thus Rho-mediated stress fiber formation and the alpha-actinin-dependent anchorage of stress fibers to integrins in focal adhesions may promote fluid shear-induced metabolic changes in bone cells.

  2. Mechanically induced c-fos expression is mediated by cAMP in MC3T3-E1 osteoblasts

    NASA Technical Reports Server (NTRS)

    Fitzgerald, J.; Hughes-Fulford, M.

    1999-01-01

    In serum-deprived MC3T3-E1 osteoblasts, mechanical stimulation caused by mild (287 x g) centrifugation induced a 10-fold increase in mRNA levels of the proto-oncogene, c-fos. Induction of c-fos was abolished by the cAMP-dependent protein kinase inhibitor H-89, suggesting that the transient c-fos mRNA increase is mediated by cAMP. Down-regulation of protein kinase C (PKC) activity by chronic TPA treatment failed to significantly reduce c-fos induction, suggesting that TPA-sensitive isoforms of PKC are not responsible for c-fos up-regulation. In addition, 287 x g centrifugation increased intracellular prostaglandin E2 (PGE2) levels 2.8-fold (P<0. 005). Since we have previously shown that prostaglandin E2 (PGE2) can induce c-fos expression via a cAMP-mediated mechanism, we asked whether the increase in c-fos mRNA was due to centrifugation-induced PGE2 release. Pretreatment with the cyclooxygenase inhibitors indomethacin and flurbiprofen did not hinder the early induction of c-fos by mechanical stimulation. We conclude that c-fos expression induced by mild mechanical loading is dependent primarily on cAMP, not PKC, and initial induction of c-fos is not necessarily dependent on the action of newly synthesized PGE2.

  3. The Effect of OSM on MC3T3-E1 Osteoblastic Cells in Simulated Microgravity with Radiation.

    PubMed

    Goyden, Jake; Tawara, Ken; Hedeen, Danielle; Willey, Jeffrey S; Thom Oxford, Julia; Jorcyk, Cheryl L

    2015-01-01

    Bone deterioration is a challenge in long-term spaceflight with significant connections to patients experiencing disuse bone loss. Prolonged unloading and radiation exposure, defining characteristics of space travel, have both been associated with changes in inflammatory signaling via IL-6 class cytokines in bone. While there is also evidence for perturbed IL-6 class signaling in spaceflight, there has been scant examination of the connections between microgravity, radiation, and inflammatory stimuli in bone. Our lab and others have shown that the IL-6 class cytokine oncostatin M (OSM) is an important regulator of bone remodeling. We hypothesize that simulated microgravity alters osteoblast OSM signaling, contributing to the decoupling of osteolysis and osteogenesis in bone homeostasis. To test this hypothesis, we induced OSM signaling in murine MC3T3-E1 pre-osteoblast cells cultured in modeled microgravity using a rotating wall vessel bioreactor with and without exposure to radiation typical of a solar particle event. We measured effects on inflammatory signaling, osteoblast activity, and mineralization. Results indicated time dependent interactions among all conditions in the regulation of IL-6 production. Furthermore, OSM induced the transcription of OSM receptor ß, IL 6 receptor ? subunits, collagen ?1(I), osteocalcin, sclerostin, RANKL, and osteoprotegerin. Measurements of osteoid mineralization suggest that the spatial organization of the osteoblast environment is an important consideration in understanding bone formation. Taken together, these results support a role for altered OSM signaling in the mechanism of microgravity-induced bone loss. PMID:26030441

  4. Expression of human epidermal growth factor pressures cDNA in transfected mouse NIH 3T3 cells

    SciTech Connect

    Mroczkowski, B.; Reich, M.; Whittaker, J.; Bell, G.I.; Cohen, S.

    1988-01-01

    Stable cell lines expressing the human epidermal growth factor (EGF) precursor have been prepared by transfection of mouse NIH 3T3 cells with a bovine papillomavirus-based vector in which the human kidney EGF precursor cDNA has been placed under the control of the inducible mouse metallothionein I promoter. Synthesis of the EGF precursor can be induced by culturing the cells in 5 mM butyric acid or 100 ..mu..M ZnCl/sub 2/. The EGF precursor synthesized by these cells appears to be membrane associated; none is detectable in the cytoplasm. The size of the EGF precursor expressed by these cells is approx. = 150-180 kDa, which is larger than expected from its amino acid sequence, suggesting that it is posttranslationally modified, presumably by glycosylation. The EGF precursor was also detected in the conditioned medium from these cells, indicating that some fraction of the EGF precursor synthesized by these transfected cells may be secreted. Preliminary data suggest that this soluble form of the EGF precursor may compete with /sup 125/I-labeled EGF for binding to the EGF receptor. These cell lines should be useful for studying the processing of the EGF precursor to EGF as well as determining the properties and possible functions of the EGF precursor itself.

  5. Stimulation of osteoblastic differentiation and mineralization in MC3T3-E1 cells by yeast hydrolysate.

    PubMed

    Lee, Hyun-Sun; Jung, Eun-Young; Bae, Song Hwan; Kwon, Ki Han; Kim, Jin-Man; Suh, Hyung Joo

    2011-05-01

    In a previous study, it was reported that yeast hydrolysate (YH) was effective in promoting bone growth in Sprague-Dawley (SD) rats. To further clarify the mechanism of YH, the effects of YH on proliferation, differentiation and gene expression in vitro were investigated using osteoblastic cell lines (MC3T3-E1). Cell proliferation increased significantly as much as 110% of the basal value when cells were treated with 100?µg/mL of YH. Alkaline phosphatase (ALP) activity increased significantly with a YH concentration of 25-100?µg/mL, and the activity increased 152% that of the control at 100?µg/mL. The calcium content increased as much as 129% at 100?µg/mL YH. The gene expression levels of ALP and collagen type II (COL II) significantly increased approximately 1.3-fold and 1.7-fold of control, respectively, at 100?µg/mL. YH increased significantly the mRNA level of bone sialoprotein (BSP) but not in a dose-dependent manner. The mRNA levels of bone morphogenetic proteins (BMP)-2, BMP-4, collagen type I (COL I) and osteonectin (ON) did not increase. In summary, YH increased the proliferation of osteoblasts and directly stimulated ALP and bone matrix proteins (e.g. BSP, COL II), and these increases trigger osteoblastic differentiation (e.g. mineralized nodule formation). PMID:21077261

  6. Fas activates lipolysis in a Ca2+-CaMKII-dependent manner in 3T3-L1 adipocytes.

    PubMed

    Rapold, Reto A; Wueest, Stephan; Knoepfel, Adrian; Schoenle, Eugen J; Konrad, Daniel

    2013-01-01

    Fas (CD95) is a member of the tumor necrosis factor (TNF) receptor superfamily and plays a crucial role in the induction of apoptosis. However, like TNF, Fas can induce nonapoptotic signaling pathways. We previously demonstrated that mice lacking Fas specifically in adipocytes are partly protected from diet-induced insulin resistance, potentially via decreased delivery of FAs to the liver, as manifested by lower total liver ceramide content. In the present study, we aimed to delineate the signaling pathway involved in Fas-mediated adipocyte lipid mobilization. Treatment of differentiated 3T3-L1 adipocytes with membrane-bound Fas ligand (FasL) significantly increased lipolysis after 12 h without inducing apoptosis. In parallel, Fas activation increased phosphorylation of ERK1/2, and FasL-induced lipolysis was blunted in the presence of the ERK-inhibitor U0126 or in ERK1/2-depleted adipocytes. Furthermore, Fas activation increased phosphorylation of the Ca(2+)/calmodulin-dependent protein kinases II (CaMKII), and blocking of the CaMKII-pathway (either by the Ca(2+) chelator BAPTA or by the CaMKII inhibitor KN62) blunted FasL-induced ERK1/2 phosphorylation and glycerol release. In conclusion, we propose a novel role for CaMKII in promoting lipolysis in adipocytes. PMID:23089915

  7. Activation of inositol phospholipid breakdown by prostaglandin F2 alpha without any stimulation of proliferation in quiescent NIH-3T3 fibroblasts.

    PubMed Central

    Black, F M; Wakelam, M J

    1990-01-01

    Stimulation of NIH-3T3 cells with prostaglandin F2 alpha (PGF2 alpha) caused a dose- and time-dependent generation of inositol phosphates. The first detectable changes were in the levels of Ins(1,4,5)P3 and Ins(1,3,4,5)P4. Increases in Ins(1,3,4)P3, InsP2 and InsP were detected later, and only minor changes were observed in putative InsP5 or InsP6. The accumulation of inositol phosphates was synergistically increased by the addition of calf serum, whereas PGF2 alpha had no effects on cell proliferation in either the presence or the absence of calf serum. Stimulation of a different clone of NIH-3T3 cells (AmNIH-3T3) or Swiss 3T3 cells with PGF2 alpha resulted in both inositol phospholipid breakdown and cell proliferation. No differences were found in the characteristics of PGF2 alpha-stimulated inositol phosphate generation between the two clones of NIH-3T3 cells, nor was there any difference in receptor number of Kd. These results question the role of inositol phospholipid breakdown in mitogenesis and demonstrate significant differences in the biochemical properties of apparently the 'same' cells. PMID:2327955

  8. Visfatin is involved in TNF?-mediated insulin resistance via an NAD+/Sirt1/PTP1B pathway in 3T3-L1 adipocytes

    PubMed Central

    Gouranton, Erwan; Romier, Béatrice; Marcotorchino, Julie; Tourniaire, Franck; Astier, Julien; Peiretti, Franck; Landrier, Jean-François

    2014-01-01

    Tumor necrosis factor ? (TNF?) is a well-known mediator of inflammation in the context of obesity in adipose tissue. Its action appears to be directly linked to perturbations of the insulin pathway, leading to the development of insulin resistance. Visfatin has been suspected to be linked to insulin sensitivity, but the mechanism involved is still partly unknown. The aim of this study was to evaluate the role of visfatin in the impairment of the insulin pathway by TNF? activity in 3T3-L1 adipocytes and to unveil the mechanisms involved in such impairment. We demonstrated in 3T3-L1 adipocytes that visfatin was involved in TNF?-mediated insulin resistance in adipocytes. Indeed, after TNF? treatment in 3T3-L1 cells, visfatin was downregulated, leading to decreased nicotinamide adenine dinucleotide (NAD+) concentrations in cells. This decrease was followed by a decrease in Sirt1 activity, which was linked to an increase in PTP1B expression. The modulation of PTP1B by visfatin was likely responsible for the observed decreases in glucose uptake and Akt phosphorylation in 3T3-L1 adipocytes. Here, we demonstrated a complete pathway involving visfatin, NAD+, Sirt1, and PTP1B that led to the perturbation of insulin signaling by TNF? in 3T3-L1 adipocytes. PMID:25068084

  9. Heterologous expression of C. elegans fat-1 decreases the n-6/n-3 fatty acid ratio and inhibits adipogenesis in 3T3-L1 cells

    SciTech Connect

    An, Lei, E-mail: anleim@yahoo.com.cn [Ministry of Agriculture Key Laboratory of Animal Genetics, Breeding and Reproduction, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing 100193 (China)] [Ministry of Agriculture Key Laboratory of Animal Genetics, Breeding and Reproduction, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing 100193 (China); Pang, Yun-Wei, E-mail: yunweipang@126.com [Ministry of Agriculture Key Laboratory of Animal Genetics, Breeding and Reproduction, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing 100193 (China)] [Ministry of Agriculture Key Laboratory of Animal Genetics, Breeding and Reproduction, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing 100193 (China); Gao, Hong-Mei, E-mail: Gaohongmei_123@yahoo.cn [Ministry of Agriculture Key Laboratory of Animal Genetics, Breeding and Reproduction, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing 100193 (China) [Ministry of Agriculture Key Laboratory of Animal Genetics, Breeding and Reproduction, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing 100193 (China); Research Unit for Animal Life Sciences, Animal Resource Science Center, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Ibaraki-Iwama 319-0206 (Japan); Tao, Li, E-mail: Eunice8023@yahoo.cn [Ministry of Agriculture Key Laboratory of Animal Genetics, Breeding and Reproduction, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing 100193 (China) [Ministry of Agriculture Key Laboratory of Animal Genetics, Breeding and Reproduction, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing 100193 (China); College of Animal Science and Technology, Jilin Agricultural University, Changchun, Jilin 130118 (China); Miao, Kai, E-mail: miaokai7@163.com [Ministry of Agriculture Key Laboratory of Animal Genetics, Breeding and Reproduction, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing 100193 (China)] [Ministry of Agriculture Key Laboratory of Animal Genetics, Breeding and Reproduction, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing 100193 (China); Wu, Zhong-Hong, E-mail: wuzhh@cau.edu.cn [Ministry of Agriculture Key Laboratory of Animal Genetics, Breeding and Reproduction, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing 100193 (China)] [Ministry of Agriculture Key Laboratory of Animal Genetics, Breeding and Reproduction, National Engineering Laboratory for Animal Breeding, College of Animal Science and Technology, China Agricultural University, Beijing 100193 (China); and others

    2012-11-23

    Highlights: Black-Right-Pointing-Pointer Expression of C. elegans fat-1 reduces the n-6/n-3 PUFA ratio in 3T3-L1 cells. Black-Right-Pointing-Pointer fat-1 inhibits the proliferation and differentiation of 3T3-L1 preadipocytes. Black-Right-Pointing-Pointer fat-1 reduces lipid deposition in 3T3-L1 adipocytes. Black-Right-Pointing-Pointer The lower n-6/n-3 ratio induces apoptosis in 3T3-L1 adipocytes. -- Abstract: In general, a diet enriched in polyunsaturated fatty acids (PUFAs) inhibits the development of obesity and decreases adipose tissue. The specific impacts of n-3 and n-6 PUFAs on adipogenesis, however, have not been definitively determined. Traditional in vivo and in vitro supplementation studies have yielded inconsistent or even contradictory results, which likely reflect insufficiently controlled experimental systems. Caenorhabditiselegans fat-1 gene encodes an n-3 fatty acid desaturase, and its heterologous expression represents an effective method both for altering the n-6/n-3 PUFA ratio and for evaluating the biological effects of n-3 and n-6 PUFAs. We sought to determine whether a reduced n-6/n-3 ratio could influence adipogenesis in 3T3-L1 cells. Lentivirus-mediated introduction of the fat-1 gene into 3T3-L1 preadipocytes significantly reduced the n-6/n-3 ratio and inhibited preadipocyte proliferation and differentiation. In mature adipocytes, fat-1 expression reduced lipid deposition, as measured by Oil Red O staining, and induced apoptosis. Our results indicate that a reduced n-6/n-3 ratio inhibits adipogenesis through several mechanisms and that n-3 PUFAs more effectively inhibit adipogenesis (but not lipogenesis) than do n-6 PUFAs.

  10. Differentially Expressed Proteins in Nitric Oxide-Stimulated NIH/3T3 Fibroblasts: Implications for Inhibiting Cancer Development

    PubMed Central

    Shim, Dong Hwi

    2015-01-01

    Purpose 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. Materials and Methods 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. Results Seventeen differentially expressed proteins were identified in NOC-18-treated cells. Nine proteins [vinculin protein, keratin 19, ubiquitous tropomodulin, F-actin capping protein (?1 subunit), tropomyosin 3, 26S proteasome-associated pad1 homolog, T-complex protein 1 (? subunit) NG-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 NG-dimethylarginine dimethylaminohydrolase) are related to carcinogenesis. The functions of 150 kD oxygen-regulated protein precursor and T-complex protein 1 (? subunit) are unknown in relation to carcinogenesis. Conclusion Most proteins differentially expressed by NOC-18 are involved in inhibiting cancer development. PMID:25684010

  11. Regulation of Adipogenesis and Key Adipogenic Gene Expression by 1, 25-Dihydroxyvitamin D in 3T3-L1 Cells

    PubMed Central

    Ji, Shuhan; Doumit, Matthew E.; Hill, Rodney A.

    2015-01-01

    The functions of 1, 25-dihydroxyvitamin D (1, 25-(OH)2D3) in regulating adipogenesis, adipocyte differentiation and key adipogenic gene expression were studied in 3T3-L1 preadipocytes. Five concentrations (0.01, 0.1, 1, 10, 100nM) of 1, 25-(OH)2D3 were studied and lipid accumulation measured by Oil Red O staining and expression of adipogenic genes quantified using quantitative real-time PCR. Adipogenic responses to 1, 25-(OH)2D3 were determined on 6, and 12 h, and days 1-10 after induction of adipogenesis by a hormonal cocktail with or without 1, 25-(OH)2D3. In response to 1, 25-(OH)2D3 (1, 10, and 100 nM), lipid accumulation and the expression of PPAR?, C/EBP?, FABP4 and SCD-1 were inhibited through day 10, and vitamin D receptor expression was inhibited in the early time points. The greatest inhibitory effect was upon expression of FABP4. Expression of SREBP-1c was only affected on day 2. The lowest concentrations of 1, 25-(OH)2D3 tested did not affect adipocyte differentiation or adipogenic gene expression. The C/EBP? promoter activity response to 1, 25-(OH)2D3 was also tested, with no effect detected. These results indicate that 1, 25-(OH)2D3 inhibited adipogenesis via suppressing adipogenic-specific genes, and is invoked either during PPAR? activation or immediately up-stream thereof. Gene expression down-stream of PPAR? especially FABP4 was strongly inhibited, and we suggest that the role of 1, 25-(OH)2D3 in regulating adipogenesis will be informed by further studies of adipogenic-specific gene promoter activity. PMID:26030589

  12. Biological effects of THC and a lipophilic cannabis extract on normal and insulin resistant 3T3-L1 adipocytes.

    PubMed

    Gallant, M; Odei-Addo, F; Frost, C L; Levendal, R-A

    2009-10-01

    Type 2 diabetes, a chronic disease, affects about 150 million people world wide. It is characterized by insulin resistance of peripheral tissues such as liver, skeletal muscle, and fat. Insulin resistance is associated with elevated levels of tumor necrosis factor alpha (TNF-alpha), which in turn inhibits insulin receptor tyrosine kinase autophosphorylation. It has been reported that cannabis is used in the treatment of diabetes. A few reports indicate that smoking cannabis can lower blood glucose in diabetics. Delta(9)-tetrahydrocannabinol (THC) is the primary psychoactive component of cannabis. This study aimed to determine the effect of a lipophilic cannabis extract on adipogenesis, using 3T3-L1 cells, and to measure its effect on insulin sensitivity in insulin resistant adipocytes. Cells were cultured in Dulbecco's modified eagle medium (DMEM) with 10% fetal bovine serum (FBS) and differentiated over a 3 day period for all studies. In the adipogenesis studies, differentiated cells were exposed to the extract in the presence and absence of insulin. Lipid content and glucose uptake was subsequently measured. Insulin-induced glucose uptake increased, while the rate of adipogenesis decreased with increasing THC concentration. Insulin-resistance was induced using TNF-alpha, exposed to the extract and insulin-induced glucose uptake measured. Insulin-induced glucose was increased in these cells after exposure to the extract. Semiquantitative real time polymerase chain reaction (RT-PCR) was performed after ribonucleic acid (RNA) extraction to evaluate the effects of the extract on glucose transporter isotype 4 (GLUT-4), insulin receptor substrate-1 (IRS-1) and IRS-2 gene expression. PMID:19345076

  13. Flavonoids from Triticum aestivum inhibit adipogenesis in 3T3-L1 cells by upregulating the insig pathway.

    PubMed

    Poudel, Barun; Nepali, Sarmila; Xin, Mingjie; Ki, Hyeon-Hui; Kim, Young-Ho; Kim, Dae-Ki; Lee, Young-Mi

    2015-08-01

    The present study aimed to compare the potential anti-adipogenic effects and underlying mechanisms of the luteolin, isoscoparin and isoorientin flavonoids, purified from Triticum aestivum sprout (TA) in 3T3?L1 cells. The cells were treated with different concentrations of flavonoids for 8 days and the lipid accumulation was assessed using Oil?Red?O staining. The expression levels of the transcription factors and the genes involved in adipogenesis in the cells were assessed by reverse transcription?quantitative polymerase chain reaction and western blotting. The results demonstrated that 10 µM luteolin, isoscoparin or isoorientin inhibited lipid deposition in the cells by 74, 63 and 65%, respectively. The flavonoids also significantly inhibited the transcriptional regulators of adipogenesis, including peroxisome proliferator?activated receptor??, CAAT/enhancer binding protein?? and sterol regulatory element binding protein (SREBP)?1c, compared with the control cells. Similarly, there was a significant downregulation of the adipocyte specific markers associated with lipid metabolism, including activating protein?2, fatty acid synthase, hormone?sensitive lipase and lipoprotein lipase, in the flavonoid treated cells. Notably, the cells treated with the flavonoids demonstrated increased expression levels of the insulin?induced genes, insig?1 and insig?2, which may have inhibited the activation of the adipogenic transcription factor, SREBP, eventually leading to the inhibition of adipogenesis. Taken together, these results revealed that the flavonoids from TA possessed an inhibitory effect on adipogenesis through downregulation of adipogenic transcription factors and genes associated with lipid metabolism, and the upregulation of insig 1 and 2, suggesting that the flavonoids from TA may be potential therapeutic agents for the prevention and treatment of obesity. PMID:25936595

  14. Type 3 protein kinase C localization to the nuclear envelope of phorbol ester-treated NIH 3T3 cells

    PubMed Central

    1989-01-01

    We have examined the immunocytochemical localization of protein kinase C (PKC) in NIH 3T3 cells using mAbs that recognize Type 3 PKC. In control cells, the immunofluorescent staining was similar with mAbs directed to either the catalytic or the regulatory domain of PKC. Type 3 PKC localized in a diffuse cytoplasmic pattern, while the nuclei were apparently unstained. Cytoskeletal components also were Treatment of the cells with phorbol 12-myristate 13-acetate (PMA) resulted in a redistribution of PKC with a specific increase in nuclear PKC. Compared to control cells, the staining with the anticatalytic domain mAbs changed markedly, covering the entire cell surface. In contrast, the staining by the antiregulatory domain mAb did not cover the cell surface and the nuclei remained unstained; these results suggest that PKC activation leads to a conformational change of the regulatory domain such that the epitope recognized by the antiregulatory domain mAb is not readily accessible. We have demonstrated by three criteria that PMA treatment specifically increased PKC in the nucleus: (a) immunofluorescent staining in isolated nuclei increased; (b) Western blots showed that our mAbs detected only one protein, the 82-kD PKC, whose level increased in nuclear lysates from PMA-treated cells; and (c) PKC activity increased in nuclear lysates. In fractionation studies we demonstrated that PKC specifically localized to the nuclear envelope fraction. These results demonstrate that PMA activation leads to a rapid redistribution of Type 3 PKC to the nuclear envelope, and suggests that this isozyme may play a role in mediating PKC-induced changes in gene expression. PMID:2668302

  15. Engagement of the insulin-sensitive pathway in the stimulation of glucose transport by ? -lipoic acid in 3T3-L1 adipocytes

    Microsoft Academic Search

    K. Yaworsky; R. Somwar; T. Ramlal; H. J. Tritschler; A. Klip

    2000-01-01

    Aims\\/hypothesis. A natural cofactor of mitochondrial dehydrogenase complexes and a potent antioxidant, ?-lipoic acid improves glucose metabolism\\u000a in people with Type II (non-insulin-dependent) diabetes mellitus and in animal models of diabetes. In this study we investigated\\u000a the cellular mechanism of action of ?-lipoic acid in 3T3-L1 adipocytes.¶Methods. We treated 3T3-L1 adipocytes with 2.5 mmol\\/l R (+) ?-lipoic acid for 2

  16. Prototypes for Application of Choice Feeding in Caged Laying Hens Using Flat Chain Feeders

    Microsoft Academic Search

    Ragnar Tauson; Klas Elwinger

    1986-01-01

    In two experiments with caged layers lasting from 22 to 82 weeks of age, three designs of prototypes for choice feeding (CF) and semi-choice feeding (SCF) using flat chain feeders in battery rows of 15 m length, were compared with conventional all mash (CAM) flat chain ad lib. feeding. Expt. I totally involved 3 888 Shaver Starcross 288 birds distributed

  17. Power budget analysis of dual/single feeder fiber WDMPON

    NASA Astrophysics Data System (ADS)

    Imtiaz, Waqas A.; Khan, Yousaf; Qamar, Affaq; Khan, Jehanzeb; Khan, Noaman Ahmed

    2014-03-01

    This paper investigates how to reduce the cost of wavelength division multiplexing passive optical network (WDMPON) by comparing the transmission performance of bidirectional single feeder fiber and dual feeder fiber. Comparison is performed on the basis of power budgeting and cost of both arrangements. Simulation results using Optisystem show that the performance of a single feeder fiber is almost equivalent to that of a dual feeder fiber. Therefore, the single feeder fiber WDM-PON can efficiently replace the dual feeder fiber WDM-PON with the minimum deterioration in system performance and reduction in cost.

  18. Carnosic acid inhibits TLR4-MyD88 signaling pathway in LPS-stimulated 3T3-L1 adipocytes

    PubMed Central

    Park, Mi-Young

    2014-01-01

    BACKGROUND/OBJECTIVES Carnosic acid (CA), found in rosemary (Rosemarinus officinalis) leaves, is known to exhibit anti-obesity and anti-inflammatory activities. However, whether its anti-inflammatory potency can contribute to the amelioration of obesity has not been elucidated. The aim of the current study was to investigate the effect of CA on Toll-like receptor 4 (TLR4) pathways in the presence of lipopolysaccharide (LPS) in 3T3-L1 adipocytes. MATERIALS/METHODS 3T3-L1 adipocytes were treated with CA (0-20 µM) for 1 h, followed by treatment with LPS for 30 min; mRNA expression of adipokines and protein expression of TLR4-related molecules were then measured. RESULTS LPS-stimulated 3T3-L1 adipocytes showed elevated mRNA expression of tumor necrosis factor (TNF)-?, interleukin-6, and monocyte chemoattractant protein-1, and CA significantly inhibited the expression of these adipokine genes. LPS-induced up regulation of TLR4, myeloid differentiation factor 88, TNF receptor-associated factor 6, and nuclear factor-?B, as well as phosphorylated extracellular receptor-activated kinase were also suppressed by pre-treatment of 3T3-L1 adipocytes with CA. CONCLUSIONS Results of this study suggest that CA directly inhibits TLR4-MyD88-dependent signaling pathways and decreases the inflammatory response in adipocytes. PMID:25324930

  19. Inhibition of MMP-13 prevents diet-induced obesity in mice and suppresses adipogenesis in 3T3-L1 preadipocytes.

    PubMed

    Shih, Chia-Li M; Ajuwon, Kolapo M

    2015-07-01

    Adipose tissue remodeling by the matrix metalloproteases (MMPs) is critical for tissue hypertrophy and obesity. MMP-13 is an important protein that is highly expressed in adipose tissue but whose potential role in adipose tissue expansion is poorly characterized. We investigated the effect of pharmacological inhibition of MMP-13 with a selective inhibitor, CP-544439, on adipose tissue mass in mice on a high fat diet, and determined the effect of the inhibitor during in vitro adipocyte differentiation of 3T3-L1 cells. CP-544439 was administered for 6 weeks to mice on a high fat diet. Body adiposity and glucose tolerance was determined. Differentiating 3T3-L1 adipocytes were also treated with the inhibitor for a maximum of 8 days and adipogenesis assessed. Treatment of mice with the inhibitor resulted in reduction in body adiposity and improvement in glucose clearance. Histological examination of epididymal adipose showed reduced adipocyte hypertrophy accompanied by increased staining for collagen in the inhibitor treated mice. Treatment of differentiating 3T3-L1 cells with the inhibitor resulted in reduced adipocyte differentiation. Knockdown of MMP-13 using small interfering RNA in differentiating 3T3-L1 cells reduced adipocyte differentiation indicated by reduced expression of PPAR?. These results suggest that MMP-13 may play a major role in adipose development and its inhibition could be a potential strategy to prevent obesity. PMID:25682268

  20. Correlation between the rates of aerobic glycolysis and glucose transport, unrelated to neoplastic transformation, in a series of balb 3t3-derived cell lines

    Microsoft Academic Search

    Beverly Peterkofsky; Willie Prather

    1982-01-01

    The relationship between transformation, lactate production, and glucose transport was examined in a series of ten cell lines consisting of subclones of BALB 3T3 A31 cells and viral and chemical transformants of either the subclones or the original A31 line. Comparisons were made over a relatively narrow range of cell densities to minimize changes in the biochemical parameters during growth.

  1. [Construction of adeno-associated virus vector carried mutated dihydrofolate reductase and green fluorescent protein and its expression in NIH3T3 cells].

    PubMed

    Li, Li-Bo; Feng, Ru; Zhou, Shu-Yun

    2002-06-01

    The aim of this study was to construct recombinant mDHFR-GFP/AAV vector containing mutated dihydrofolate reductase (mDHFR) and green fluorescent protein (GFP) fusion genes and its expression in NIH3T3 cells, to investigate the resistance of the cells to methotrexate. Amplified cDNA of mDHFR and GFP segmented from their plasmid separately were linked by PCR with the aminoacetic acid linker. The fusion gene was inserted into T vector, and after enzyme cutting the fusion gene fragment was inserted into AAV vector, then packaging the vector into recombined AAV and infected NIH3T3 cells. Expression of gene fusion was observed by PCR, fluorescent microscopy and flow cytometry. mDHFR and GFP cDNA were found in NIH3T3 genomic DNA, the GFP expression rate was about 25%, and resistance of the transferred cells to MTX was increased markedly. The results showed that AAV vector can transfer mDHFR and GFP fusion gene into NIH3T3 cells and increase resistance to MTX in gene modified cells. This data provided a basis for application of mDHFR and AAV vector in gene therapy. PMID:12513788

  2. Estrogen stimuli promote osteoblastic differentiation via the subtilisin-like proprotein convertase PACE4 in MC3T3-E1 cells.

    PubMed

    Kim, Hyejin; Tabata, Atsushi; Tomoyasu, Toshifumi; Ueno, Tomomi; Uchiyama, Shigeto; Yuasa, Keizo; Tsuji, Akihiko; Nagamune, Hideaki

    2015-01-01

    Estrogenic compounds include endogenous estrogens such as estradiol as well as soybean isoflavones, such as daidzein and its metabolite equol, which are known phytoestrogens that prevent osteoporosis in postmenopausal women. Indeed, mineralization of MC3T3-E1 cells, a murine osteoblastic cell line, was significantly decreased in medium containing fetal bovine serum treated with charcoal-dextran to deplete endogenous estrogens, but estradiol and these soybean isoflavones dose-dependently restored the differentiation of MC3T3-E1 cells; equol was tenfold more effective than daidzein. These differentiation-promoting effects were inhibited by the addition of fulvestrant, which is a selective downregulator of estrogen receptors. Analysis of the expression pattern of bone-related genes by reverse transcription PCR (RT-PCR)/quantitative real-time PCR (qRT-PCR), which focused on responsiveness to the estrogen stimuli, revealed that the transcription of PACE4, a subtilisin-like proprotein convertase, was tightly linked with the differentiation of MC3T3-E1 cells induced by estrogen stimuli. Moreover, treatment with RNAi of PACE4 in MC3T3-E1 cells resulted in a drastic decrease of mineralization in the presence of estrogen stimuli. These results strongly suggest that PACE4 participates in bone formation at least in osteoblast differentiation, and estrogen receptor-mediated stimuli induce osteoblast differentiation through the upregulation of PACE4 expression. PMID:24557631

  3. Epidermal growth factor-nonresponsive 3T3 variants do not contain epidermal growth factor receptor-related antigens or mRNA

    SciTech Connect

    Schneider, C.A.; Lim, R.W.; Terwilliger, E.; Herschman, H.R.

    1986-01-01

    The authors have previously isolated three independent variants of Swiss 3T3 cells that are unable to generate a mitogenic response to epidermal growth factor (EGF). Each of the variants is unable to bind /sup 125/I-labeled EGF; each lacks a functional EGF receptor. They used an antiserum to murine EGF receptor to look for an EGF-receptor gene product in wild-type 3T3 cells and in the three EGF-nonresponsive variants. No cross-reactive material could be detected in any of the three variants, either in /sup 125/I-labeled cell extracts or in (/sup 35/S)methionine metabolically labeled cells. 3T3 cells contained mRNA molecules homologous to a cDNA probe for the human EGF-receptor coding region. In contrast, no homologous RNA could be detected in any of the three variants. Analysis of genomic Southern blots of the DNA from 3T3 cells and the three EGF-nonresponsive variants indicated sequences from the EGF-receptor gene are present in the DNA of all four cell lines. These EGF-nonresponsive lines, which demonstrate proliferative responses to a variety of mitogens, will be ideal recipients for structure-function studies of the EGF receptor by transfection of the cloned gene.

  4. Effects of Lipophilic Extract of Viscum album L. and Oleanolic Acid on Migratory Activity of NIH/3T3 Fibroblasts and on HaCat Keratinocytes

    PubMed Central

    Kuonen, R.; Weissenstein, U.; Urech, K.; Kunz, M.; Hostanska, K.; Estko, M.; Heusser, P.; Baumgartner, S.

    2013-01-01

    Viscum album L. lipophilic extract (VALE) contains pharmacologically active pentacyclic triterpenes that are known to exhibit immunomodulatory, antitumor, and wound healing activity. Preliminary clinical observations indicate that VALE was able to influence cutaneous wound healing in vivo. The objective of this study was to investigate wound closure related properties of VALE in vitro. As measured in a wound healing assay, VALE and its predominant triterpene oleanolic acid (OA) significantly and dose dependently promoted the migration of NIH/3T3 fibroblasts in vitro, thereby leading to an enhanced wound closure. Compared to the negative control, maximal stimulation by 26.1% and 26.2%, respectively, was attained with 10??g/mL VALE and 1??g/mL OA. Stimulation of proliferation in NIH/3T3 fibroblasts by VALE and OA could be excluded. At higher concentrations both substances affected proliferation and viability of NIH/3T3 fibroblasts and HaCat keratinocytes. In the toxic range of concentrations of VALE and OA, migration of NIH/3T3 fibroblasts was suppressed. The extent of the stimulatory effect on cell migration of VALE quite closely corresponded to the effect expected by the concentrations of OA contained in the crude extract VALE. These data support the casual observation that Viscum album L. lipophilic extract might modulate wound healing related processes in vivo. PMID:24379890

  5. Injectable calcium phosphate-alginate-chitosan microencapsulated MC3T3-E1 cell paste for bone tissue engineering in vivo.

    PubMed

    Qiao, Pengyan; Wang, Juan; Xie, Qiufei; Li, Fangfang; Dong, Limin; Xu, Tao

    2013-12-01

    Osteoblasts or stem cells have been delivered into injectable calcium phosphate cement (CPC) to improve its effectiveness and biological function. However, the osteogenic potential of the new construct in vivo has been rarely reported, and there are no reports on alginate-chitosan microencapsulated osteoblasts mixed with CPC. This study aimed to develop alginate-chitosan microencapsulated mouse osteoblast MC3T3-E1 cells (AC-cells), evaluate the osteogenic potential of a calcium phosphate cement complex with these AC-cells (CPC-AC-cell), and trace the implanted MC3T3-E1 cells in vivo. MC3T3-E1 cells were embedded in alginate microcapsules, cultured in osteogenic medium for 7 days, and then covered with chitosan before mixing with a paste of ?-tricalcium phosphate/calcium phosphate cement (?-TCP/CPC). The construct was injected into the dorsal subcutaneous area of nude mice. Lamellar-bone-like mineralization, newly formed collagen and angiogenesis were observed at 4 weeks. At 8 weeks, areas of newly formed collagen expanded; further absorption of ?-TCP/CPC and osteoid-like structures could be seen. Cell tracing in vivo showed that implanted MC3T3-E1 cells were clearly visible at 2 weeks. These in vivo results indicate that the novel injectable CPC-AC-cell construct is promising for bone tissue engineering applications. PMID:24094170

  6. Effects of Lipophilic Extract of Viscum album L. and Oleanolic Acid on Migratory Activity of NIH/3T3 Fibroblasts and on HaCat Keratinocytes.

    PubMed

    Kuonen, R; Weissenstein, U; Urech, K; Kunz, M; Hostanska, K; Estko, M; Heusser, P; Baumgartner, S

    2013-01-01

    Viscum album L. lipophilic extract (VALE) contains pharmacologically active pentacyclic triterpenes that are known to exhibit immunomodulatory, antitumor, and wound healing activity. Preliminary clinical observations indicate that VALE was able to influence cutaneous wound healing in vivo. The objective of this study was to investigate wound closure related properties of VALE in vitro. As measured in a wound healing assay, VALE and its predominant triterpene oleanolic acid (OA) significantly and dose dependently promoted the migration of NIH/3T3 fibroblasts in vitro, thereby leading to an enhanced wound closure. Compared to the negative control, maximal stimulation by 26.1% and 26.2%, respectively, was attained with 10??g/mL VALE and 1??g/mL OA. Stimulation of proliferation in NIH/3T3 fibroblasts by VALE and OA could be excluded. At higher concentrations both substances affected proliferation and viability of NIH/3T3 fibroblasts and HaCat keratinocytes. In the toxic range of concentrations of VALE and OA, migration of NIH/3T3 fibroblasts was suppressed. The extent of the stimulatory effect on cell migration of VALE quite closely corresponded to the effect expected by the concentrations of OA contained in the crude extract VALE. These data support the casual observation that Viscum album L. lipophilic extract might modulate wound healing related processes in vivo. PMID:24379890

  7. Coal gasification: New challenge for the Beaumont rotary feeder

    NASA Technical Reports Server (NTRS)

    Stelian, J.

    1977-01-01

    The use of rotary feeders in the coal gasification process is described with emphasis on the efficient conversion of coal to clean gaseous fuels. Commercial applications of the rotary feeder system are summarized.

  8. Feeder and Stocker Health and Management Practices

    E-print Network

    Liskiewicz, Maciej

    Feeder and Stocker Health and Management Practices W. Dee Whittier, Extension Specialist of disease loss. Goals for herd health management in a stocker setting should be centered around in its cause. The first is stress which decreases the animal's ability to fight disease invasion

  9. ELECTRICAL ENERGY LOSS IN RURAL DISTRIBUTION FEEDERS- A CASE STUDY

    Microsoft Academic Search

    K. V. S. Ramachandra; M. Ramalinga Raju

    This paper presents the analysis of electrical energy loss in rural distribution feeders. Eastern Power Distribution Corporation Limited, APEPDCL of Andhra Pradesh State in India has been implementing some methods to reduce technical losses on rural distribution feeders. Statistical Data of two years on 80 rural distribution feeders of Visakhapatnam district has been analyzed and the results were presented. Field

  10. Cytotoxic effects in 3T3-L1 mouse and WI-38 human fibroblasts following 72 hour and 7 day exposures to commercial silica nanoparticles

    SciTech Connect

    St?pnik, Maciej, E-mail: mstep@imp.lodz.pl [Nofer Institute of Occupational Medicine, ?ód? (Poland)] [Nofer Institute of Occupational Medicine, ?ód? (Poland); Arkusz, Joanna; Smok-Pieni??ek, Anna [Nofer Institute of Occupational Medicine, ?ód? (Poland)] [Nofer Institute of Occupational Medicine, ?ód? (Poland); Bratek-Skicki, Anna; Salvati, Anna; Lynch, Iseult; Dawson, Kenneth A. [Centre for BioNano Interactions, School of Chemistry and Chemical Biology, University College Dublin, Belfield, Dublin 4 (Ireland)] [Centre for BioNano Interactions, School of Chemistry and Chemical Biology, University College Dublin, Belfield, Dublin 4 (Ireland); Gromadzi?ska, Jolanta [Nofer Institute of Occupational Medicine, ?ód? (Poland)] [Nofer Institute of Occupational Medicine, ?ód? (Poland); De Jong, Wim H. [National Institute for Public Health and the Environment, Antonie van Leeuwenhoeklaan 9 NL?3720, Bilthoven (Netherlands)] [National Institute for Public Health and the Environment, Antonie van Leeuwenhoeklaan 9 NL?3720, Bilthoven (Netherlands); Rydzy?ski, Konrad [Nofer Institute of Occupational Medicine, ?ód? (Poland)] [Nofer Institute of Occupational Medicine, ?ód? (Poland)

    2012-08-15

    The potential toxic effects in murine (3T3-L1) and human (WI-38) fibroblast cell lines of commercially available silica nanoparticles (NPs), Ludox CL (nominal size 21 nm) and CL-X (nominal size of 30 nm) were investigated with particular attention to the effect over long exposure times (the tests were run after 72 h exposure up to 7 days). These two formulations differed in physico-chemical properties and showed different stabilities in the cell culture medium used for the experiments. Ludox CL silica NPs were found to be cytotoxic only at the higher concentrations to the WI-38 cells (WST-1 and LDH assays) but not to the 3T3-L1 cells, whereas the Ludox CL-X silica NPs, which were less stable over the 72 h exposure, were cytotoxic to both cell lines in both assays. In the clonogenic assay both silica NPs induced a concentration dependent decrease in the surviving fraction of 3T3-L1 cells, with the Ludox CL-X silica NPs being more cytotoxic. Cell cycle analysis showed a trend indicating alterations in both cell lines at different phases with both silica NPs tested. Buthionine sulfoximine (?-glutamylcysteine synthetase inhibitor) combined with Ludox CL-X was found to induce a strong decrease in 3T3-L1 cell viability which was not observed for the WI-38 cell line. This study clearly indicates that longer exposure studies may give important insights on the impact of nanomaterials on cells. However, and especially when investigating nanoparticle effects after such long exposure, it is fundamental to include a detailed physico-chemical characterization of the nanoparticles and their dispersions over the time scale of the experiment, in order to be able to interpret eventual impacts on cells. -- Highlights: ? Ludox CL silica NPs are cytotoxic to WI-38 fibroblasts but not to 3T3-L1 fibroblasts. ? Ludox CL-X silica NPs are cytotoxic to both cell lines. ? In clonogenic assay both silica NPs induce cytotoxicity, higher for CL-X silica. ? Cell cycle analysis shows alterations in both cell lines with both silica NP tested. ? Buthionine sulfoximine enhances cytotoxicity of Ludox CL-X in 3T3-L1 cells.

  11. Prostaglandin F2 alpha decreases the affinity of epidermal growth factor receptors in Swiss mouse 3T3 cells via protein kinase C activation.

    PubMed

    Jimenez de Asua, L; Goin, M

    1992-03-16

    Prostaglandin F2 alpha (PGF2 alpha) selectively decreases the binding of 125I-labelled epidermal growth factor ([125I]EGF) to intact Swiss 3T3 cells. Scatchard analysis reveals that PGF2 alpha decreases the number of high-affinity EGF binding sites without changing the total number of receptors. Prostaglandins E1 (PGE1), E2 (PGE2) or F2 beta (PGF2 beta) do not alter the EGF binding to these cells and do not enhance the PGF2 alpha effect. R-59022 and R-59949, two diacylglycerol kinase inhibitors, enhance the inhibitory effect of PGF2 alpha, whereas down-modulation of protein kinase C (PKC) abolishes the effect. These results indicate that PGF2 alpha decreases EGF binding in Swiss 3T3 cells via PKC activation. PMID:1312042

  12. Pentacyclic triterpenoids from spikes of Prunella vulgaris L. inhibit glycogen phosphorylase and improve insulin sensitivity in 3T3-L1 adipocytes.

    PubMed

    Yu, Qian; Qi, Jin; Wang, Lu; Liu, Shou-Jin; Yu, Bo-Yang

    2015-01-01

    Phytochemical investigation of methanol extract from the spikes of Prunella vulgaris L. led to the isolation of two new pentacyclic triterpenoid glycosides Vulgasides I (1) and II (2) along with 13 known compounds (3-15). Their structures were established on the basis of nuclear magnetic resonance (1D and 2D) and mass spectroscopic data analysis. All the isolated compounds were screened for glycogen phosphorylase inhibitory activity and also evaluated for their effect on insulin sensitivity in 3T3-L1 adipocytes. Two new compounds (1, 2) did not demonstrate the glycogen phosphorylase inhibitory activity, but other compounds (3-11) exhibited varying degrees of glycogen phosphorylase inhibitory activity with IC50 values in the range from 30.69 to 68.85??M. Compounds 3, 6, 7, 11, and 13 demonstrated markedly increased insulin-mediated glucose consumption in 3T3-L1 adipocytes. PMID:25278372

  13. Characterization of Extracellular Matrix (ECM) Produced by MC3T3 Cells Using Thickness Shear Mode (TSM) Resonators

    Microsoft Academic Search

    Fang Li; Qing-Ming Wang; James H.-C. Wang

    2006-01-01

    Quartz thickness shear mode (TSM) resonators for monitoring the attachment and spreading of mammalian cells have been investigated in the past years. Recent studies have shown that the TSM resonator signal is not only contributed by cellular body closed to the resonator substrate, but also contributed by the extracellular matrix (ECM), which is a protein layer between the cellular body

  14. CFD Application to Flow-Accelerated Corrosion in Feeder Bends

    SciTech Connect

    Pietralik, John M.; Smith, Bruce A.W. [Atomic Energy of Canada, Ltd. (Canada)

    2006-07-01

    Feeder piping in CANDU{sup R} plants experiences a thinning degradation mechanism called Flow-Accelerated Corrosion (FAC). The piping is made of carbon steel and has high water flow speeds. Although the water chemistry is highly alkaline with room-temperature pH in a range of 10.0-10.5, the piping has FAC rates exceeding 0.1 mm/year in some locations, e.g., in bends. One of the most important parameters affecting the FAC rate is the mass transfer coefficient for convective mass transport of ferrous ions. The ions are created at the pipe wall as a result of corrosion, diffuse through the oxide layer, and are transported from the oxide-layer/water interface to the bulk water by mass transport. Consequently, the local flow characteristics contribute to the highly turbulent convective mass transfer. Plant data and laboratory experiments indicate that the mass transfer step dominates FAC under feeder conditions. In this study, the flow and mass transfer in a feeder bend under operating conditions were simulated using the Fluent{sup TM} computer code. Because the flow speed is very high, with the Reynolds numbers in a range of several millions, and because the geometry is complex, experiments in a 1:1 scale were conducted with the main objective to validate flow simulations. The experiments measured pressure at several key locations and visualized the flow. The flow and mass transfer models were validated using available friction-factor and mass transfer correlations and literature experiments on mass transfer in a bend. The validation showed that the turbulence model that best predicts the experiments is the realizable k-{epsilon} model. Other two-equation turbulence models, as well as one-equation models and Reynolds stress models were tried. The near-wall treatment used the non-equilibrium wall functions. The wall functions were modified for surface roughness when necessary. A comparison of the local mass transfer coefficient with measured FAC rate in plant specimens shows very good agreement. Visualization experiments indicate secondary flows in the bends. No boundary layer separation was observed in experiments or in simulations. (authors)

  15. Osteogenic differentiation of MC3T3-E1 cells on poly(l-lactide)/Fe3O4 nanofibers with static magnetic field exposure.

    PubMed

    Cai, Qing; Shi, Yuzhou; Shan, Dingying; Jia, Wenkai; Duan, Shun; Deng, Xuliang; Yang, Xiaoping

    2015-10-01

    Proliferation and differentiation of bone-related cells are modulated by many factors such as scaffold design, growth factor, dynamic culture system, and physical simulation. Nanofibrous structure and moderate-intensity (1mT-1T) static magnetic field (SMF) have been identified as capable of stimulating proliferation and differentiation of osteoblasts. Herein, magnetic nanofibers were prepared by electrospinning mixture solutions of poly(l-lactide) (PLLA) and ferromagnetic Fe3O4 nanoparticles (NPs). The PLLA/Fe3O4 composite nanofibers demonstrated homogeneous dispersion of Fe3O4 NPs, and their magnetism depended on the contents of Fe3O4 NPs. SMF of 100mT was applied in the culture of MC3T3-E1 osteoblasts on pure PLLA and PLLA/Fe3O4 composite nanofibers for the purpose of studying the effect of SMF on osteogenic differentiation of osteoblastic cells on magnetic nanofibrous scaffolds. On non-magnetic PLLA nanofibers, the application of external SMF could enhance the proliferation and osteogenic differentiation of MC3T3-E1 cells. In comparison with pure PLLA nanofibers, the incorporation of Fe3O4 NPs could also promote the proliferation and osteogenic differentiation of MC3T3-E1 cells in the absence or presence of external SMF. The marriage of magnetic nanofibers and external SMF was found most effective in accelerating every aspect of biological behaviors of MC3T3-E1 osteoblasts. The findings demonstrated that the magnetic feature of substrate and microenvironment were applicable ways in regulating osteogenesis in bone tissue engineering. PMID:26117751

  16. Enhanced Nuclear Diacylglycerol Kinase Activity in Response to a Mitogenic Stimulation of Quiescent Swiss 3T3 Cells with Insulin-like Growth Factor I1

    Microsoft Academic Search

    Alberto M. Martelli; Giovanna Tabellini; Roberta Bortul; Lucia Manzoli; Renato Bareggi; Giovanna Baldini; Vittorio Grill; Marina Zweyer; Paola Narducci; Lucio Cocco

    2000-01-01

    Results from several laboratories have established the existence in the nucleus of an autonomous polyphosphoinositide cycle, which is involved in both cell proliferation and differentiation. A key step of intranuclear polyphosphoinositide metabolism is the phospholipase C-mediated gener- ation of diacylglycerol (DAG). In insulin-like growth factor (IGF)-I-stim- ulated Swiss 3T3 cells, a transient elevation of intranuclear DAG levels is essential for

  17. ARF1 Mediates Paxillin Recruitment to Focal Adhesions and Potentiates Rho-stimulated Stress Fiber Formation in Intact and Permeabilized Swiss 3T3 Fibroblasts

    Microsoft Academic Search

    J. C. Norman; D. Jones; S. T. Barry; M. R. Holt; S. Cockcroft; D. R. Critchley

    1998-01-01

    Focal adhesion assembly and actin stress fi- ber formation were studied in serum-starved Swiss 3T3 fibroblasts permeabilized with streptolysin-O. Perme- abilization in the presence of GTP g S stimulated rho- dependent formation of stress fibers, and the redis- tribution of vinculin and paxillin from a perinuclear location to focal adhesions. Addition of GTP g S at 8 min after permeabilization

  18. Red yeast rice extracts suppress adipogenesis by down-regulating adipogenic transcription factors and gene expression in 3T3-L1 cells

    Microsoft Academic Search

    Taeil Jeon; Seong Gu Hwang; Shizuka Hirai; Tohru Matsui; Hideo Yano; Teruo Kawada; Beoung Ou Lim; Dong Ki Park

    2004-01-01

    The effects of red yeast rice extracts (RE) on adipocyte differentiation of 3T3-L1 cells were studied. RE were extracted from embryonic rice fermented with red yeast (Monascus ruber). These extracts significantly decreased glycerol-3-phosphate dehydrogenase (GPDH) activity and lipid accumulation, a marker of adipogenesis, in a dose-dependent manner. Moreover, mRNA expression levels of both CCAAT\\/enhancer-binding protein (C\\/EBP) ? and peroxisome proliferator-activated

  19. Stimulatory effect of ?-alanyl-L-histidinato zinc on cell proliferation is dependent on protein synthesis in osteoblastic MC3T3-E1 cells

    Microsoft Academic Search

    Masayuki Hashizume; Masayoshi Yamaguchi

    1993-01-01

    The effect of ß-alanyl-L-histidinato zinc (AHZ) on bone metabolism was investigated in osteoblastic MC3T3-El cells. Cells were cultured for 3 days at 37°C in a CO2 incubator in plastic dishes containing a-modified minimum essential medium supplemented with 10% fetal bovine serum. After the cultures, the medium was exchanged for that containing 0.1% bovine serum albumin plus various concentrations of AHZ

  20. G Alpha-q\\/11 Protein Plays a Key Role in Insulin-Induced Glucose Transport in 3T3-L1 Adipocytes

    Microsoft Academic Search

    TAKESHI IMAMURA; PETER VOLLENWEIDER; KATSUYA EGAWA; MARTIN CLODI; KENICHI ISHIBASHI; NAOKI NAKASHIMA; SATOSHI UGI; JOHN W. ADAMS; JOAN HELLER BROWN; JERROLD M. OLEFSKY

    1999-01-01

    We evaluated the role of the G alpha-q (Gaq) subunit of heterotrimeric G proteins in the insulin signaling pathway leading to GLUT4 translocation. We inhibited endogenous Gaq function by single cell microinjection of anti-Gaq\\/11 antibody or RGS2 protein (a GAP protein for Gaq), followed by immunostaining to assess GLUT4 translocation in 3T3-L1 adipocytes. Gaq\\/11 antibody and RGS2 inhibited insulin-induced GLUT4

  1. Effects of aqueous extracts of raw pu-erh tea and ripened pu-erh tea on proliferation and differentiation of 3T3-L1 preadipocytes.

    PubMed

    Cao, Zhen-Hui; Yang, Hui; He, Zhan-Long; Luo, Cheng; Xu, Zhi-Qiang; Gu, Da-Hai; Jia, Jun-Jing; Ge, Chang-Rong; Lin, Qiu-Ye

    2013-08-01

    Pu-erh tea has shown anti-obesity effects but little is known about its effect on proliferation and differentiation of preadipocytes. This study investigated the effects of the aqueous extracts of raw pu-erh tea and ripened pu-erh tea on proliferation and differentiation of murine 3T3-L1 preadiopocytes. We examined dose and time effects of both aqueous extracts on proliferation of 3T3-L1 preadipocytes. The contents of triglycerides in cytoplasm and the mRNA expression of critical transcriptional factors involved in differentiation were determined. Cytotoxicity and apoptosis rate of preadipocytes by pu-erh tea extracts treatment were test for toxic and pro-apoptotic effects. Both aqueous extracts of pu-erh tea inhibited the proliferation of 3T3-L1 preadipocytes at the selected time points. At lower concentration of raw pu-erh tea extracts (less than 300 µg/ml) and ripened pu-erh tea extracts (less than 350 µg/ml), no significant cytotoxic and pro-apoptotic were observed. Ripened pu-erh tea was more effective with lower IC50 than raw pu-erh tea. Both extracts suppressed the differentiation and down-regulated the gene expression of peroxisome proliferator-activated receptor-? and CCAAT/enhancer binding proteins-?. Therefore, these results indicate that both aqueous extracts of pu-erh tea can inhibit proliferation and differentiation with ripened pu-erh tea more potent. Polyphenol rich in both extracts may play a role in the inhibition of proliferation and differentiation of 3T3-L1 preadipocytes. PMID:23027678

  2. Differentially expressed genes and signalling pathways are involved in mouse osteoblast-like MC3T3-E1 cells exposed to 17-? estradiol

    PubMed Central

    Shang, Zhen-Zhen; Li, Xin; Sun, Hui-Qiang; Xiao, Guo-Ning; Wang, Cun-Wei; Gong, Qi

    2014-01-01

    Oestrogen is essential for maintaining bone mass, and it has been demonstrated to induce osteoblast proliferation and bone formation. In this study, complementary DNA (cDNA) microarrays were used to identify and study the expression of novel genes that may be involved in MC3T3-E1 cells' response to 17-? estradiol. MC3T3-E1 cells were inoculated in minimum essential media alpha (?-MEM) cell culture supplemented with 17-? estradiol at different concentrations and for different time periods. MC3T3-E1 cells treated with 10?8 mol?L?1 17-? estradiol for 5 days exhibited the highest proliferation and alkaline phosphatase (ALP) activity; thus, this group was chosen for microarray analysis. The harvested RNA was used for microarray hybridisation and subsequent real-time reverse transcription polymerase chain reaction (RT-PCR) to validate the expression levels for selected genes. The microarray results were analysed using both functional and pathway analysis. In this study, microarray analysis detected 5?403 differentially expressed genes, of which 1?996 genes were upregulated and 3?407 genes were downregulated, 1?553 different functional classifications were identified by gene ontology (GO) analysis and 53 different pathways were involved based on pathway analysis. Among the differentially expressed genes, a portion not previously reported to be associated with the osteoblast response to oestrogen was identified. These findings clearly demonstrate that the expression of genes related to osteoblast proliferation, cell differentiation, collagens and transforming growth factor beta (TGF-?)-related cytokines increases, while the expression of genes related to apoptosis and osteoclast differentiation decreases, following the exposure of MC3T3-E1 cells to ?-MEM supplemented with 17-? estradiol. Microarray analysis with functional gene classification is critical for a complete understanding of complementary intracellular processes. This microarray analysis provides large-scale gene expression data that require further confirmatory studies. PMID:24556956

  3. Requirement of Phosphatidylinositol 3-Kinase-Dependent Pathway and Src for Gas6-Axl Mitogenic and Survival Activities in NIH 3T3 Fibroblasts

    Microsoft Academic Search

    SANDRO GORUPPI; ELISABETTA RUARO; BRIAN VARNUM; CLAUDIO SCHNEIDER

    1997-01-01

    Gas6 is a secreted protein previously identified as the ligand of the Axl receptor tyrosine kinase. We have shown that Gas6 is able to induce cell cycle reentry of serum-starved NIH 3T3 cells and to efficiently prevent apoptosis after complete growth factor removal, a survival effect uncoupled from Gas6-induced mitogenesis. Here we report that the mitogenic effect of Gas6 requires

  4. Ras Transformation Results in an Elevated Level of Cyclin D1 and Acceleration of G1Progression in NIH 3T3 cells

    Microsoft Academic Search

    JAN-JAN LIU; JYH-RONG CHAO; MING-CHUNG JIANG; SUN-YU NG; JEFFREY JONG-YOUNG YEN; ANDHSIN-FANG YANG-YEN

    1995-01-01

    Ectopic overexpression of v-H-Ras protein in NIH 3T3 cells resulted in cellular transformation and an acceleration of G1 progression of these cells. A shortened G1 phase was found to be associated with an increased level of cyclin D1 but not cyclin E protein. Using an antisense blocking method, reduced synthesis of cyclin D1 in v-H-Ras transformants resulted in a slower

  5. Invasiveness and Metastasis of NIH 3T3 Cells Induced by Met-Hepatocyte Growth Factor\\/Scatter Factor Autocrine Stimulation

    Microsoft Academic Search

    Sing Rong; Shraga Segal; Miriam Anver; James H. Resau; George F. Vande Woude

    1994-01-01

    The met protooncogene product, Met, is the tyrosine kinase growth factor receptor for hepatocyte growth factor\\/scatter factor (HGF\\/SF). NIH 3T3 cells express HGF\\/SF endogenously and become tumorigenic in nude mice via an autocrine mechanism when murine Met is expressed ectopically (Metmu cells) or when human Met and human HGF\\/SF are coexpressed (HMH cells). Here, we show that Metmu and HMH

  6. Reversion of v-H- ras-Trasformed NIH 3T3 Cells by Apigenin Through Inhibiting Mitogen-Activated Protein Kinase and Its Downstream Oncogenes

    Microsoft Academic Search

    M. L. Kuo; N. C. Yang

    1995-01-01

    Apigenin, a plant flavonoid, induced the reversion of transformed phenotypes of v-H-ras-transformed NIH 3T3 cells at a quite low concentration of 12.5 ?M. In the present study, we have examined the components of this Ras-mediated signaling transduction to determine whether they were involved in the apigenin-induced reversion process. Interestingly, the consitutively activated mitogen activated protein kinase (MAPK) in the ras

  7. Cytoprotective role of the fatty acid binding protein 4 against oxidative and endoplasmic reticulum stress in 3T3-L1 adipocytes

    PubMed Central

    Kajimoto, Kazuaki; Minami, Yoshitaka; Harashima, Hideyoshi

    2014-01-01

    The fatty acid binding protein 4 (FABP4), one of the most abundant proteins in adipocytes, has been reported to have a proinflammatory function in macrophages. However, the physiological role of FABP4, which is constitutively expressed in adipocytes, has not been fully elucidated. Previously, we demonstrated that FABP4 was involved in the regulation of interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) production in 3T3-L1 adipocytes. In this study, we examined the effects of FABP4 silencing on the oxidative and endoplasmic reticulum (ER) stress in 3T3-L1 adipocytes. We found that the cellular reactive oxygen species (ROS) and 8-nitro-cyclic GMP levels were significantly elevated in the differentiated 3T3-L1 adipocytes transfected with a small interfering RNA (siRNA) against Fabp4, although the intracellular levels or enzyme activities of antioxidants including reduced glutathione (GSH), superoxide dismutase (SOD) and glutathione S-transferase A4 (GSTA4) were not altered. An in vitro evaluation using the recombinant protein revealed that FABP4 itself functions as a scavenger protein against hydrogen peroxide (H2O2). FABP4-knockdown resulted in a significant lowering of cell viability of 3T3-L1 adipocytes against H2O2 treatment. Moreover, four kinds of markers related to the ER stress response including the endoplasmic reticulum to nucleus signaling 1 (Ern1), the signal sequence receptor ? (Ssr1), the ORM1-like 3 (Ormdl3), and the spliced X-box binding protein 1 (Xbp1s), were all elevated as the result of the knockdown of FABP4. Consequently, FABP4 might have a new role as an antioxidant protein against H2O2 and contribute to cytoprotection against oxidative and ER stress in adipocytes. PMID:25161868

  8. Epidermal growth factor receptor is activated by hyposmolarity and is an early signal modulating osmolyte efflux pathways in Swiss 3T3 fibroblasts

    Microsoft Academic Search

    Rodrigo Franco; Ruth Lezama; Benito Ordaz; Herminia Pasantes-Morales

    2004-01-01

    Exposure of cultured Swiss 3T3 fibroblasts to 35% hyposmotic solution activated epidermal growth factor receptor (EGFR) phosphorylation to a greater extent than the ligand, EGF. Concanavalin A (Con A) and wheat-germ agglutinin (WGA) had the same effect. EGFR phosphorylation seems to be involved in the transduction signalling for hyposmotically induced taurine release, as suggested by the latter’s reduction when EGFR

  9. Interactions between sub-10-nm iron and cerium oxide nanoparticles and 3T3 fibroblasts: the role of the coating and aggregation state

    Microsoft Academic Search

    M. Safi; H. Sarrouj; O. Sandre; N. Mignet; J.-F. Berret

    2010-01-01

    Recent nanotoxicity studies revealed that the physico-chemical characteristics of engineered nanomaterials play an important role in the interactions with living cells. Here, we report on the toxicity and uptake of cerium and iron oxide sub-10-nm nanoparticles by NIH\\/3T3 mouse fibroblasts. Coating strategies include low-molecular weight ligands (citric acid) and polymers (poly(acrylic acid), MW = 2000 g mol - 1). Electrostatically

  10. Regulated expression of an insulin-responsive glucose transporter (GLUT4) minigene in 3T3-L1 adipocytes and transgenic mice.

    PubMed Central

    Ezaki, O; Flores-Riveros, J R; Kaestner, K H; Gearhart, J; Lane, M D

    1993-01-01

    Preliminary studies showed that up to 7 kb of 5' flanking sequence of the insulin-responsive glucose transporter (GLUT4) gene are insufficient to mediate differentiation-induced reporter gene expression in mouse 3T3-L1 preadipocytes. To locate the regulatory element(s) responsible for this function, a minigene containing the entire GLUT4 gene with substantial 5' and 3' flanking sequence and a short segment of foreign DNA (for transcript identification) was constructed and transfected into mice and 3T3-L1 preadipocytes at relatively low copy number. In transgenic mice the GLUT4 minigene exhibited a pattern of tissue-specific expression similar, but not identical, to that of the endogenous gene. In 3T3-L1 cells expression of minigene mRNA occurred upon differentiation into adipocytes, with kinetics virtually identical to that of endogenous GLUT4 mRNA. In both cultured adipocytes and transgenic mice, the level of expression of the minigene was low relative to that of the endogenous gene. Treatment of minigene-transfected 3T3-L1 adipocytes with 8-bromo-cAMP, which represses transcription of the endogenous GLUT4 gene, also repressed expression of the GLUT4 minigene. However, insulin, which down-regulates transcription of the endogenous GLUT4 gene, failed to normally down-regulate expression of the GLUT4 minigene. These findings indicate that the cis-acting elements required for directing tissue-specific expression (in heart, skeletal muscle, and brown adipose tissue), differentiation-induced activation of transcription, and cAMP-induced repression of transcription are located within the 14-kb GLUT4 minigene. However, the cis elements necessary for maximal tissue-specific expression and for insulin-induced down-regulation of expression are not located in the minigene. Images Fig. 2 Fig. 3 Fig. 5 PMID:8475079

  11. Comparison of sensitivity to arsenic compounds between a Bhas 42 cell transformation assay and a BALB\\/c 3T3 cell transformation assay

    Microsoft Academic Search

    Dai Muramatsu; Kiyoshi Sasaki; Sachiko Kuroda; Kumiko Hayashi; Noriho Tanaka; Ayako Sakai

    2009-01-01

    A short-term cell transformation assay has recently been developed, using Bhas 42 cells which were established from BALB\\/c 3T3 cells transfected by v-Ha-ras gene and postulated to be initiated in the two-stage carcinogenesis theory. The Bhas 42 cell transformation assay has been reported to be capable of detecting initiating and promoting activities of chemical carcinogens, according to the different protocols,

  12. A novel role for fatty acid transport protein 1 in the regulation of tricarboxylic acid cycle and mitochondrial function in 3T3-L1 adipocytes

    PubMed Central

    Wiczer, Brian M.; Bernlohr, David A.

    2009-01-01

    Fatty acid transport proteins (FATPs) are integral membrane acyl-CoA synthetases implicated in adipocyte fatty acid influx and esterification. Whereas some FATP1 translocates to the plasma membrane in response to insulin, the majority of FATP1 remains within intracellular structures and bioinformatic and immunofluorescence analysis of FATP1 suggests the protein primarily resides in the mitochondrion. To evaluate potential roles for FATP1 in mitochondrial metabolism, we used a proteomic approach following immunoprecipitation of endogenous FATP1 from 3T3-L1 adipocytes and identified mitochondrial 2-oxoglutarate dehydrogenase. To assess the functional consequence of the interaction, purified FATP1 was reconstituted into phospholipid-containing vesicles and its effect on 2-oxoglutarate dehydrogenase activity evaluated. FATP1 enhanced the activity of 2-oxoglutarate dehydrogenase independently of its acyl-CoA synthetase activity whereas silencing of FATP1 in 3T3-L1 adipocytes resulted in decreased activity of 2-oxoglutarate dehydrogenase. FATP1 silenced 3T3-L1 adipocytes exhibited decreased tricarboxylic acid cycle activity, increased cellular NAD+/NADH, increased fatty acid oxidation, and increased lactate production indicative of altered mitochondrial energy metabolism. These results reveal a novel role for FATP1 as a regulator of tricarboxylic acid cycle activity and mitochondrial function. PMID:19535819

  13. 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. PMID:23066647

  14. Euphorbiasteroid, a component of Euphorbia lathyris L., inhibits adipogenesis of 3T3-L1 cells via activation of AMP-activated protein kinase.

    PubMed

    Park, Su-Jin; Park, Jae Ho; Han, Anna; Davaatseren, Munkhtugs; Kim, Hyun Jin; Kim, Myung-Sunny; Hur, Haeng Jeon; Sung, Mi-Jeong; Hwang, Jin-Taek; Yang, Hye Jeong; Kwon, Dae Young

    2015-06-01

    The purpose of this study is to investigate the effects of euphorbiasteroid, a component of Euphorbia lathyris L., on adipogenesis of 3T3-L1 pre-adipocytes and its underlying mechanisms. Euphorbiasteroid decreased differentiation of 3T3-L1 cells via reduction of intracellular triglyceride (TG) accumulation at concentrations of 25 and 50??M. In addition, euphorbiasteroid altered the key regulator proteins of adipogenesis in the early stage of adipocyte differentiation by increasing the phosphorylation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase. Subsequently, levels of adipogenic proteins, including fatty acid synthase, peroxisome proliferator-activated receptor-? and CCAAT/enhancer-binding protein ?, were decreased by euphorbiasteroid treatment at the late stage of adipocyte differentiation. The anti-adipogenic effect of euphorbiasteroid may be derived from inhibition of early stage of adipocyte differentiation. Taken together, euphorbiasteroid inhibits adipogenesis of 3T3-L1 cells through activation of the AMPK pathway. Therefore, euphorbiasteroid and its source plant, E.?lathyris L., could possibly be one of the fascinating anti-obesity agent. Copyright © 2015 John Wiley & Sons, Ltd. PMID:25914364

  15. Regulation of the levels of Smad1 and Smad5 in MC3T3-E1 cells by Icariine in vitro.

    PubMed

    Zhou, Huifang; Wang, Shuo; Xue, Yuan; Shi, Nianke

    2014-02-01

    The purpose of this study was to investigate the role of Icariine on the expression of Smadl and Smad5 mRNA and protein levels in MC3T3-E1 cells in vitro. MC3T3-E1 cells were cultured in the presence of different concentrations of Icariine (0, 10, 40 and 80 ng/ml). Smad1 and Smad5 mRNA levels were detected by reverse transcription-polymerase chain reaction (RT-PCR) and the expression of proteins was determined by western blotting, immunohistochemistry staining and immunofluorescence. Smad1 and Smad5 mRNA levels continuously increased in 10, 40 and 80 ng/ml of Icariine with time and the differences indicated statistical significance. Western blot analysis demonstrated that the Smad1 and Smad5 protein levels in the 10, 40 and 80 ng/ml groups were higher compared with the 0 ng/ml group at 24, 48 and 72 h, and the difference was statistically significant. Immunohistochemistry staining and immunofluorescence showed that the expression of the Smad1 and Smad5 proteins was higher in the cytoplasm and nuclei in the 10, 40 and 80 ng/ml groups compared with the 0 ng/ml group. Icariine has a direct stimulatory function on the differentiation of MC3T3-E1 osteoblastic cells in vitro, which may be mediated by increasing the production of Smad1 and Smad5 in osteoblasts. PMID:24297369

  16. Persicaria hydropiper (L.) spach and its flavonoid components, isoquercitrin and isorhamnetin, activate the Wnt/?-catenin pathway and inhibit adipocyte differentiation of 3T3-L1 cells.

    PubMed

    Lee, Soung-Hoon; Kim, Bora; Oh, Myoung Jin; Yoon, Juyong; Kim, Hyun Yi; Lee, Kye Jong; Lee, Joo Dong; Choi, Kang-Yell

    2011-11-01

    Obesity, which is related to metabolic syndrome and is associated with liver disease, represents an epidemic problem demanding effective therapeutic strategies. Evidence shows that the Wnt/?-catenin pathway is closely associated with obesity and that small molecules regulating the Wnt/?-catenin pathway can potentially control adipogenesis related to obesity. Eleven plant extracts activating the Wnt/?-catenin pathway were screened by using HEK 293-TOP cells retaining the Wnt/?-catenin signaling reporter gene. An extract of Persicaria hydropiper (L.) Spach was found to activate Wnt/?-catenin signaling. P. hydropiper is grown worldwide in temperate climates and is found widely in Southeast Asia. The P. hydropiper extract inhibited the differentiation of adipocyte 3T3-L1 cells. Isoquercitrin and isorhamnetin, constituents of P. hydropiper, also activated Wnt/?-catenin signaling and suppressed the differentiation of 3T3-L1 cells. These results indicate that isoquercitrin in P. hydropiper suppresses the adipogenesis of 3T3-L1 cells via the inhibition of Wnt/?-catenin signaling. P. hydropiper and isoquercitrin may therefore be potential therapeutic agents for obesity and its associated disorders. PMID:21413092

  17. Effect and mechanism of ginsenosides CK and Rg1 on stimulation of glucose uptake in 3T3-L1 adipocytes.

    PubMed

    Huang, Yu-Chuan; Lin, Cheng-Yu; Huang, Su-Fen; Lin, Han-Ching; Chang, Wen-Liang; Chang, Tsu-Chung

    2010-05-26

    The glucoregulatory activities of ginsenosides compound K (CK) and Rg1 were investigated in 3T3-L1 adipocytes. Both compounds significantly enhanced glucose uptake in 3T3-L1 adipocytes in a dose-response manner, which is correlated with increased GLUT4 translocation from intracellular vesicles to the plasma membrane in adipocytes. The stimulating effects of CK and Rg1 on glucose uptake and GLUT4 translocation are associated with activation of AMP-activated protein kinase (AMPK) and phosphatidylinositol 3-kinase (PI3K) signaling pathways; both are key pathways in mediating glucose uptake in animal cells. In addition to the acute stimulus effect of glucose uptake, prolonged incubation of CK and Rg1 significantly induced GLUT4, but not GLUT1, expression at both the mRNA and protein levels in 3T3-L1 adipocytes. Further studies showed that CK inhibited and Rg1 enhanced triglyceride accumulation in adipocytes, suggesting that the mechanism of action of these ginsenosides may not be completely identical. In summary, this study demonstrated insulin-like activities of CK and Rg1 in adipocytes. These findings are important in understanding the hypoglycemic properties and potential applications of ginseng and ginsenosides. PMID:20441170

  18. Effect of Metformin on Viability, Morphology, and Ultrastructure of Mouse Bone Marrow-Derived Multipotent Mesenchymal Stromal Cells and Balb/3T3 Embryonic Fibroblast Cell Line

    PubMed Central

    Czyrek, Aleksandra; Basinska, Katarzyna; Trynda, Justyna; Skaradzi?ska, Aneta; Siudzi?ska, Anna; Marycz, Krzysztof

    2015-01-01

    Metformin, a popular drug used to treat diabetes, has recently gained attention as a potentially useful therapeutic agent for treating cancer. In our research metformin was added to in vitro cultures of bone marrow-derived multipotent mesenchymal stromal cells (BMSCs) and Balb/3T3 fibroblast at concentration of 1?mM, 5?mM, and 10?mM. Obtained results indicated that metformin negatively affected proliferation activity of investigated cells. The drug triggered the formation of autophagosomes and apoptotic bodies in all tested cultures. Additionally, we focused on determination of expression of genes involved in insulin-like growth factor 2 (IGF2) signaling pathway. The most striking finding was that the mRNA level of IGF2 was constant in both BMSCs and Balb/3T3. Further, the analysis of IGF2 concentration in cell supernatants showed that it decreased in BMSC cultures after 5 and 10?mM metformin treatments. In case of Balb/3T3 the concentration of IGF2 in culture supernatants decreased after 1 and 5?mM and increased after 10?mM of metformin. Our results suggest that metformin influences the cytophysiology of somatic cells in a dose- and time-dependent manner causing inhibition of proliferation and abnormalities of their morphology and ultrastructure. PMID:26064951

  19. Bixin regulates mRNA expression involved in adipogenesis and enhances insulin sensitivity in 3T3-L1 adipocytes through PPAR{gamma} activation

    SciTech Connect

    Takahashi, Nobuyuki; Goto, Tsuyoshi; Taimatsu, Aki; Egawa, Kahori; Katoh, Sota; Kusudo, Tatsuya; Sakamoto, Tomoya; Ohyane, Chie; Lee, Joo-Young; Kim, Young-il; Uemura, Taku; Hirai, Shizuka [Laboratory of Molecular Function of Food, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji 611-0011 (Japan)] [Laboratory of Molecular Function of Food, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji 611-0011 (Japan); Kawada, Teruo, E-mail: fat@kais.kyoto-u.ac.jp [Laboratory of Molecular Function of Food, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji 611-0011 (Japan)] [Laboratory of Molecular Function of Food, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji 611-0011 (Japan)

    2009-12-25

    Insulin resistance is partly due to suppression of insulin-induced glucose uptake into adipocytes. The uptake is dependent on adipocyte differentiation, which is controlled at mRNA transcription level. The peroxisome proliferator-activated receptor (PPAR), a ligand-regulated nuclear receptor, is involved in the differentiation. Many food-derived compounds serve as ligands to activate or inactivate PPAR. In this study, we demonstrated that bixin and norbixin (annatto extracts) activate PPAR{gamma} by luciferase reporter assay using GAL4-PPAR chimera proteins. To examine the effects of bixin on adipocytes, 3T3-L1 adipocytes were treated with bixin or norbixin. The treatment induced mRNA expression of PPAR{gamma} target genes such as adipocyte-specific fatty acid-binding protein (aP2), lipoprotein lipase (LPL), and adiponectin in differentiated 3T3-L1 adipocytes and enhanced insulin-dependent glucose uptake. The observations indicate that bixin acts as an agonist of PPAR{gamma} and enhances insulin sensitivity in 3T3-L1 adipocytes, suggesting that bixin is a valuable food-derived compound as a PPAR ligand to regulate lipid metabolism and to ameliorate metabolic syndrome.

  20. Theobromine inhibits differentiation of 3T3-L1 cells during the early stage of adipogenesis via AMPK and MAPK signaling pathways.

    PubMed

    Jang, Yeon Jeong; Koo, Hyun Jung; Sohn, Eun-Hwa; Kang, Se Chan; Rhee, Dong-Kwon; Pyo, Suhkneung

    2015-07-01

    Obesity is characterized by hypertrophy and/or by the differentiation or adipogenesis of pre-existing adipocytes. In this study, we investigated the inhibitory effects of theobromine, a type of alkaloid in cocoa, on adipocyte differentiation of 3T3-L1 preadipocytes and its mechanisms of action. Theobromine inhibited the accumulation of lipid droplets, the expression of PPAR? and C/EBP?, and the mRNA expression of aP2 and leptin. The inhibition of adipogenic differentiation by theobromine occurred primarily in the early stages of differentiation. In addition, theobromine arrested the cell cycle at the G0/G1 phase and regulated the expressions of CDK2, p27, and p21. Theobromine treatment increased AMPK phosphorylation and knockdown of AMPK?1/?2 prevented the ability of theobromine to inhibit PPAR? expression in the differentiating 3T3-L1 cells. Theobromine reduced the phosphorylation of ERK and JNK. Moreover, the secretion and the mRNA level of TNF-? and IL-6 were inhibited by theobromine treatment. These data suggest that theobromine inhibits adipocyte differentiation during the early stages of adipogenesis by regulating the expression of PPAR? and C/EBP? through the AMPK and ERK/JNK signaling pathways in 3T3-L1 preadipocytes. PMID:26085110

  1. Differentiation to adipocytes in accompanied by an increase in the amounts of Gi- and Go-proteins in 3T3-L1 cells

    SciTech Connect

    Watkins, D.C.; Northup, J.K.; Malbon, C.C.

    1986-05-01

    Treatment of cultures of 3T3-L1 cells with methylisobutyl-xanthine and dexamethasone has been shown to result in accumulation of lipid and conversion to the morphology of adipocytes in more than 90% of the cells. The status of the stimulatory (Gs), inhibitory (Gi) and Go-proteins during the course of 3T3-L1 differentiation was examined. The amount of alpha subunit of Gs (..cap alpha..Gs), assayed by radiolabeling in the presence of cholera toxin and (/sup 32/P)NAD/sup +/, increased upon differentiation as previously described by others. The amounts of ..cap alpha..Gi and ..cap alpha..Go assayed by radiolabeling in the presence of pertussis toxin and (/sup 32/P)NAD/sup +/ increased 3-fold upon differentiation. Immunoblots of cell membranes subjected to gel electrophoresis in sodium dodecyl sulfate were probed with two rabbit antisera raised against bovine brain ..cap alpha..Go and with one raised against the..beta..-subunit of the bovine rod-outer-segment G-protein, referred to as transducin. The immunoblotting data confirm the increase upon differentiation of ..cap alpha..Go and also demonstrate an increase in the amount of the ..beta..-subunit. Thus differentiation of 3T3-L1 cells is accompanied by dramatic changes in the complexion of G-proteins in the membranes.

  2. ?? adrenergic receptor activation suppresses bone morphogenetic protein (BMP)-induced alkaline phosphatase expression in osteoblast-like MC3T3E1 cells.

    PubMed

    Yamada, Takayuki; Ezura, Yoichi; Hayata, Tadayoshi; Moriya, Shuichi; Shirakawa, Jumpei; Notomi, Takuya; Arayal, Smriti; Kawasaki, Makiri; Izu, Yayoi; Harada, Kiyoshi; Noda, Masaki

    2015-06-01

    ? adrenergic stimulation suppresses bone formation in vivo while its actions in osteoblastic differentiation are still incompletely understood. We therefore examined the effects of ?2 adrenergic stimulation on osteoblast-like MC3T3-E1 cells focusing on BMP-induced alkaline phosphatase expression. Morphologically, isoproterenol treatment suppresses BMP-induced increase in the numbers of alkaline phosphatase-positive small foci in the cultures of MC3T3-E1 cells. Biochemically, isoproterenol treatment suppresses BMP-induced enzymatic activity of alkaline phosphatase in a dose-dependent manner. Isoproterenol suppression of alkaline phosphatase activity is observed even when the cells are treated with high concentrations of BMP. With respect to cell density, isoproterenol treatment tends to suppress BMP-induced increase in alkaline phosphatase expression more in osteoblasts cultured at higher cell density. In terms of treatment protocol, continuous isoproterenol treatment is compared to cyclic treatment. Continuous isoproterenol treatment is more suppressive against BMP-induced increase in alkaline phosphatase expression than cyclic regimen. At molecular level, isoproterenol treatment suppresses BMP-induced enhancement of alkaline phosphatase mRNA expression. Regarding the mode of isoproterenol action, isoproterenol suppresses BMP-induced BRE-luciferase activity. These data indicate that isoproterenol regulates BMP-induced alkaline phosphatase expression in osteoblast-like MC3T3E1 cells. PMID:25536656

  3. Bis(alpha-furancarboxylato)oxovanadium(IV) prevents and improves dexamethasone-induced insulin resistance in 3T3-L1 adipocytes.

    PubMed

    Zuo, Yi-Qing; Liu, Wei-Ping; Niu, Yan-Fen; Tian, Chang-Fu; Xie, Ming-Jin; Chen, Xi-Zhu; Li, Ling

    2008-10-01

    Previous studies showed that bis(alpha-furancarboxylato)oxovanadium(IV) (BFOV), an orally active anti-diabetic organic vanadium complex, could improve insulin resistance in animals with type 2 diabetes. The present study has been carried out to evaluate the effects of BFOV on insulin-resistant glucose metabolism using dexamethasone-treated 3T3-L1 adipocytes as an in-vitro model of insulin resistance. The results showed that BFOV, similar to vanadyl sulfate and rosiglitazone, caused a concentration-dependent increase in glucose consumption by insulin-resistant adipocytes. Moreover, BFOV enhanced the action of insulin and completely prevented the development of insulin resistance induced by dexamethasone, leading to glucose consumption equal to that by normal cells. In addition, dexamethasone reduced the mRNA expression of insulin receptor substrate 1 (IRS-1) and glucose transporter 4 (GLUT4) in 3T3-L1 adipocytes, while BFOV normalized the expression of IRS-1 and GLUT4. These findings suggest that BFOV prevents and improves dexamethasone-induced insulin resistance in 3T3-L1 adipocytes by enhancing expression of IRS-1 and GLUT4 mRNA. PMID:18812026

  4. The ?-SiC Nanowires (~100 nm) Induce Apoptosis via Oxidative Stress in Mouse Osteoblastic Cell Line MC3T3-E1

    PubMed Central

    Xie, Weili; Xie, Qi; Jin, Meishan; Huang, Xiaoxiao; Zhang, Xiaodong; Shao, Zhengkai; Wen, Guangwu

    2014-01-01

    Silicon carbide (SiC), a compound of silicon and carbon, with chemical formula SiC, the beta modification (?-SiC), with a zinc blende crystal structure (similar to diamond), is formed at temperature below 1700°C. ?-SiC will be the most suitable ceramic material for the future hard tissue replacement, such as bone and tooth. The in vitro cytotoxicity of ?-SiC nanowires was investigated for the first time. Our results indicated that 100?nm long SiC nanowires could significantly induce the apoptosis in MC3T3-E1 cells, compared with 100??m long SiC nanowires. And 100?nm long SiC nanowires increased oxidative stress in MC3T3-E1 cells, as determined by the concentrations of MDA (as a marker of lipid peroxidation) and 8-OHdG (indicator of oxidative DNA damage). Moreover, transmission electron microscopy (TEM) was performed to evaluate the morphological changes of MC3T3-E1 cells. After treatment with 100?nm long SiC nanowires, the mitochondria were swelled and disintegrated, and the production of ATP and the total oxygen uptake were also decreased significantly. Therefore, ?-SiC nanowires may have limitations as medical material. PMID:24967352

  5. Ginsenoside Rh2(S) induces differentiation and mineralization of MC3T3-E1 cells through activation of the PKD/AMPK signaling pathways.

    PubMed

    Kim, Do Yeon; Park, Ki Ho; Jung, Mi Song; Huang, Bo; Yuan, Hai-Dan; Quan, Hai-Yan; Chung, Sung Hyun

    2011-11-01

    As part of our search for biologically active anti-osteoporotic agents that enhance differentiation and mineralization of osteoblastic MC3T3-E1 cells, we identified the ginsenoside Rh2(S). Mostly known to exhibit beneficial effects in cancer prevention and metabolic diseases, Rh2(S) is one of the most active ginsenosides. Here, we show that Rh2(S) stimulates osteoblastic differentiation and mineralization, manifested by the up-regulation of differentiation markers (alkaline phosphatase and osteogenic genes) and von Kossa/Alizarin Red staining, respectively. Rh2(S) also activated protein kinase D (PKD) and AMP-activated protein kinase (AMPK) in a time- and concentration-dependent manner, and Rh2(S)-induced differentiation and mineralization of osteoblastic cells were significantly abolished in the presence of specific inhibitors; Go6976 for PKD and Ara-A for AMPK. Furthermore, Go6976 suppressed Rh2(S)-mediated activation of AMPK, indicating that PKD may be an upstream signal for AMPK in Rh2(S)-induced differentiation and mineralization of MC3T3-E1 cells. Taken together, these results indicate that Rh2(S) induces the differentiation and mineralization of MC3T3-E1 cells through activation of PKD/AMPK signaling pathways. These findings provide a molecular basis for the osteogenic effect of Rh2(S). PMID:21769419

  6. Ginsenoside Rh2(S) induces the differentiation and mineralization of osteoblastic MC3T3-E1 cells through activation of PKD and p38 MAPK pathways.

    PubMed

    Kim, Do Yeon; Jung, Mi Song; Park, Young Guk; Yuan, Hai Dan; Quan, Hai Yan; Chung, Sung Hyun

    2011-10-01

    As part of the search for biologically active anti-osteoporotic agents that enhance differentiation and mineralization of osteoblastic MC3T3-E1 cells, we identified the ginsenoside Rh2(S), which is an active component in ginseng. Rh2(S) stimulates osteoblastic differentiation and mineralization, as manifested by the up-regulation of differentiation markers (alkaline phosphatase and osteogenic genes) and Alizarin Red staining, respectively. Rh2(S) activates p38 mitogen-activated protein kinase (MAPK) in time- and concentration-dependent manners, and Rh2(S)-induced differentiation and mineralization of osteoblastic cells were totally inhibited in the presence of the p38 MAPK inhibitor, SB203580. In addition, pretreatment with Go6976, a protein kinase D (PKD) inhibitor, significantly reversed the Rh2(S)-induced p38 MAPK activation, indicating that PKD might be an upstream kinase for p38 MAPK in MC3T3-E1 cells. Taken together, these results suggest that Rh2(S) induces the differentiation and mineralization of MC3T3-E1 cells through activation of PKD/p38 MAPK signaling pathways, and these findings provide a molecular basis for the osteogenic effect of Rh2(S). PMID:22026999

  7. Maximum Photovoltaic Penetration Levels on Typical Distribution Feeders: Preprint

    SciTech Connect

    Hoke, A.; Butler, R.; Hambrick, J.; Kroposki, B.

    2012-07-01

    This paper presents simulation results for a taxonomy of typical distribution feeders with various levels of photovoltaic (PV) penetration. For each of the 16 feeders simulated, the maximum PV penetration that did not result in steady-state voltage or current violation is presented for several PV location scenarios: clustered near the feeder source, clustered near the midpoint of the feeder, clustered near the end of the feeder, randomly located, and evenly distributed. In addition, the maximum level of PV is presented for single, large PV systems at each location. Maximum PV penetration was determined by requiring that feeder voltages stay within ANSI Range A and that feeder currents stay within the ranges determined by overcurrent protection devices. Simulations were run in GridLAB-D using hourly time steps over a year with randomized load profiles based on utility data and typical meteorological year weather data. For 86% of the cases simulated, maximum PV penetration was at least 30% of peak load.

  8. Blueberry Peel Extracts Inhibit Adipogenesis in 3T3-L1 Cells and Reduce High-Fat Diet-Induced Obesity

    PubMed Central

    Jang, Sun-Hee; Lee, Soo-Jung; Ko, Yeoung-Gyu; Kim, Gon-Sup; Cho, Jae-Hyeon

    2013-01-01

    This study examined the anti-obesity effect and mechanism of action of blueberry peel extracts (BPE) in 3T3-L1 cells and high-fat diet (HFD)-induced obese rats. The levels of lipid accumulation were measured, along with the changes in the expression of genes and proteins associated with adipocyte differentiation in 3T3-L1 cells. Evidenced by Oil-red O staining and triglyceride assay, BPE dose-dependently inhibited lipid accumulation at concentrations of 0, 50, and 200 µg/ml. BPE decreased the expression of the key adipocyte differentiation regulator C/EBP?, as well as the C/EBP? and PPAR? genes, during the differentiation of preadipocytes into adipocytes. Moreover, BPE down-regulated adipocyte-specific genes such as aP2 and FAS compared with control adipocytes. The specific mechanism mediating the effects of BP revealed that insulin-stimulated phosphorylation of Akt was strongly decreased, and its downstream substrate, phospho-GSK3?, was downregulated by BPE treatment in 3T3-L1 cells. Together, these data indicated that BP exerted anti-adipogenic activity by inhibiting the expression of PPAR? and C/EBP? and the Akt signaling pathway in 3T3-L1 adipocytes. Next, we investigated whether BP extracts attenuated HFD-induced obesity in rats. Oral administration of BPE reduced HFD-induced body weight gain significantly without affecting food intake. The epididymal or perirenal adipose tissue weights were lower in rats on an HFD plus BPE compared with the tissue weights of HFD-induced obese rats. Total cholesterol and triglyceride levels in the rats fed BPE were modestly reduced, and the HDL-cholesterol level was significantly increased in HFD plus BP-fed rats compared with those of HFD-fed rats. Taken together, these results demonstrated an inhibitory effect of BP on adipogenesis through the down-regulation of C/EBP?, C/EBP?, and PPAR? and the reduction of the phospho-Akt adipogenic factor in 3T3-L1 cells. Moreover, BPE reduced body weight gain and inhibited fat accumulation in an HFD-induced animal model of obesity. PMID:23936120

  9. Effects of DNA methylation and histone modification on differentiation-associated gene expression in ES, NIH3T3, and NIT-1.

    PubMed

    Fang, Aiping; Zhang, Yue; Li, Mingyue; Guo, Hui; Yu, Xiaofang; Li, Furong; Hu, Hong

    2011-02-01

    The effects of epigenetic modification on the differentiation of islet cells and the expression of associated genes (Pdx-1, Pax4, MafA, and Nkx6.1, etc) were investigated. The promoter methylation status of islet differentiation-associated genes (Pdx-1, Pax4, MafA and Nkx6.1), Oct4 and MLH1 genes of mouse embryonic stem cells, NIH3T3 cells and NIT-1 cells were profiled by methylated DNA immunoprecipitation, real-time quantitative PCR (MeDIP-qPCR) techniques. The histone modification status of these genes promoter region in different cell types was also measured by using chromatin immunoprecipitation real-time quantitative PCR methods. The expression of these genes in these cells was detected by using real-time quantitative PCR. The relationship between the epigenetic modification (DNA methylation, H3 acetylation, H3K4m3 and H3K9m3) of these genes and their expression was analyzed. The results showed that: (1) the transcription-initiation-sites of Pdx-1, MafA and Nkx6.1 were highly methylated in NIH3T3 cells; (2) NIH3T3 cells showed a significantly higher level of DNA methylation modification in the transcription-initiation-site of Pdx-1, Pax4, MafA and Nkx6.1 genes than that in mES cells and NIT-1 cells (P<0.05); (3) NIT-1 cells had a significantly higher level of H3K4m3 modification in the transcription-initiation-site of Pdx-1, Pax4, MafA and Nkx6.1 genes than that in mES cells and NIH3T3 cells (P<0.05), with significantly increased level of gene expression; (4) NIH3T3 cell had a significantly higher level of H3K9m3 modification in the transcription-initiation-site of Pdx-1, Pax4, MafA and Nkx6.1 genes than that in mES cells and with NIT-1 cell (P<0.05), with no detectable mRNA expression of these genes. It was concluded that histone modification (H3K4m3 and H3K9m3) and DNA methylation might have an intimate communication between each other in the differentiation process from embryonic stem cells into islet cells. PMID:21336716

  10. OSM is overexpressed in knee osteoarthritis and Notch signaling is involved in the effects of OSM on MC3T3-E1 cell proliferation and differentiation.

    PubMed

    Ni, J; Yuan, X M; Yao, Q; Peng, L B

    2015-06-01

    Knee osteoarthritis (OA) is the most prevalent type of OA and the cytokine, oncostatin M (OSM), may contribute to the pathogenesis of OA. However, the exact role of OSM in the development of knee OA and the underlying mechanisms are not yet fully understood. This study was designed to detect the expression of OSM in the synovial tissue of patients with knee OA. Furthermore, we investigated whether Notch signaling is involved in the effects of OSM on MC3T3?E1 cell proliferation and differentiation. The synovial tissue of the knee joint was collected from 32 patients with knee OA. We detected OSM mRNA and protein expression (by RT-qPCR and western blot analysis, respectively) in the synovial tissue of the knee joint, and the expression level of OSM was higher in the patients with knee OA compared with the controls. MTT assay was used in the in vitro experiments to determine MC3T3?E1 cell proliferation, and cell differentiation was determined by measuring alkaline phosphatase (ALP) activity and osteocalcin (OCN) expression. The results from our in vitro experiments revealed that OSM induced bone formation by increasing osteoblast cell proliferation and differentiation. In addition, the expression levels of Notch ligand, receptor and target gene, including Delta-like 1 (Dll1), Notch homolog 1 (Notch1) and Hes family bHLH transcription factor 1 (Hes1) were decreased following treatment with OSM in a time-dependent manner in the MC3T3?E1 cells. A Dll1 overexpression vector was transfected into the cells to activate Notch signaling, and the results revealed that the activation of Notch signaling attenuated the effects of OSM on MC3T3?E1 cell proliferation and differentiation. In conclusion, our data demonstrate that elevated levels of OSM in synovial tissue induce bone formation by increasing osteoblast cell proliferation and differentiation. The Notch signaling pathway was found to be one of the signaling pathways that inhibit OSM-induced MC3T3?E1 cell proliferation and differentiation. The findings of this study may broaden our understanding of the mechanisms behing the role of OSM in the development of knee OA. PMID:25845347

  11. Detection of arcing faults on distribution feeders

    NASA Astrophysics Data System (ADS)

    Russell, B. D.

    1982-12-01

    The problem of detecting high impedance faults is examined from the perspective of current utility protection practices and it is shown why conventional overcurrent protection systems may not detect such faults. A microcomputer based prototype of an arcing, high impedance fault detector was tested. The fault detection technique is based on an increase in the high frequency component of distribution feeder current caused by the arcing associated with many high impedance faults. This theory is supported by field data measurements and analysis of a large number of staged distribution primary faults and normal system conditions. The design and demonstration of the prototype is explained. The device successfully detected many faults of greater than 5 to 10 A on a typical distribution feeder without false trips. General application of this fault detection techniques is considered.

  12. Omega-3 polyunsaturated fatty acid has an anti-oxidant effect via the Nrf-2/HO-1 pathway in 3T3-L1 adipocytes

    SciTech Connect

    Kusunoki, Chisato, E-mail: yosizaki@belle.shiga-med.ac.jp [Department of Medicine, Shiga University of Medical Science, Seta Tsukinowa-Cho, Otsu, Shiga 520-2192 (Japan)] [Department of Medicine, Shiga University of Medical Science, Seta Tsukinowa-Cho, Otsu, Shiga 520-2192 (Japan); Yang, Liu; Yoshizaki, Takeshi; Nakagawa, Fumiyuki; Ishikado, Atsushi; Kondo, Motoyuki; Morino, Katsutaro; Sekine, Osamu; Ugi, Satoshi [Department of Medicine, Shiga University of Medical Science, Seta Tsukinowa-Cho, Otsu, Shiga 520-2192 (Japan)] [Department of Medicine, Shiga University of Medical Science, Seta Tsukinowa-Cho, Otsu, Shiga 520-2192 (Japan); Nishio, Yoshihiko [Division of Diabetes, Metabolism and Endocrinology, Department of Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8544 (Japan)] [Division of Diabetes, Metabolism and Endocrinology, Department of Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8544 (Japan); Kashiwagi, Atsunori; Maegawa, Hiroshi [Department of Medicine, Shiga University of Medical Science, Seta Tsukinowa-Cho, Otsu, Shiga 520-2192 (Japan)] [Department of Medicine, Shiga University of Medical Science, Seta Tsukinowa-Cho, Otsu, Shiga 520-2192 (Japan)

    2013-01-04

    Highlights: Black-Right-Pointing-Pointer Omega-3 PUFA has a direct anti-oxidant effect in adipocytes. Black-Right-Pointing-Pointer EPA and DHA induce HO-1 expression in 3T3-L1 adipocytes. Black-Right-Pointing-Pointer Omega-3 PUFA and its end-product, 4-HHE, activates the Nrf-2/HO-1 pathway. Black-Right-Pointing-Pointer Omega-3 PUFA protects against oxidative stress-induced cytotoxicity. -- Abstract: Oxidative stress is produced in adipose tissue of obese subjects and has been associated with obesity-related disorders. Recent studies have shown that omega-3 polyunsaturated fatty acid ({omega}3-PUFA) has beneficial effects in preventing atherosclerotic diseases and insulin resistance in adipose tissue. However, the role of {omega}3-PUFA on adipocytes has not been elucidated. In this study, 3T3-L1 adipocytes were treated with {omega}3-PUFA and its metabolites, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), or 4-hydroxy hexenal (4-HHE). {omega}3-PUFA and its metabolites dose-dependently increased mRNA and protein levels of the anti-oxidative enzyme, heme oxygenase-1 (HO-1); whereas no changes in the well-known anti-oxidant molecules, superoxide dismutase, catalase, and glutathione peroxidase, were observed. Knockdown of nuclear factor erythroid 2-related factor 2 (Nrf-2) significantly reduced EPA, DHA or 4-HHE-induced HO-1 mRNA and protein expression. Also, pretreatment with {omega}3-PUFA prevented H{sub 2}O{sub 2}-induced cytotoxicity in a HO-1 dependent manner. In conclusion, treatment with EPA and DHA induced HO-1 through the activation of Nrf-2 and prevented oxidative stress in 3T3-L1 adipocytes. This anti-oxidant defense may be of high therapeutic value for clinical conditions associated with systemic oxidative stress.

  13. Enhanced nuclear diacylglycerol kinase activity in response to a mitogenic stimulation of quiescent Swiss 3T3 cells with insulin-like growth factor I.

    PubMed

    Martelli, A M; Tabellini, G; Bortul, R; Manzoli, L; Bareggi, R; Baldini, G; Grill, V; Zweyer, M; Narducci, P; Cocco, L

    2000-02-15

    Results from several laboratories have established the existence in the nucleus of an autonomous polyphosphoinositide cycle, which is involved in both cell proliferation and differentiation. A key step of intranuclear polyphosphoinositide metabolism is the phospholipase C-mediated generation of diacylglycerol (DAG). In insulin-like growth factor (IGF)-I-stimulated Swiss 3T3 cells, a transient elevation of intranuclear DAG levels is essential for attracting the alpha isoform of protein kinase C (PKC) to the nucleus. Previous evidence has shown that the nucleus also contains DAG kinase, i.e., the enzyme that yields phosphatidic acid from DAG, thus terminating PKC-mediated signaling events. Here we show that IGF-I treatment of quiescent Swiss 3T3 cells results in the stimulation of nuclear DAG kinase activity. Time course analysis showed an inverse relationship between nuclear DAG mass and DAG kinase activity levels. After IGF-I treatment, maximal enhancement of DAG kinase activity was measured in the internal matrix domain of the nucleus. PKC-alpha remained within the nuclear compartment, even when nuclear DAG mass returned to basal levels. This was conceivably due to interactions with specific nuclear PKC-binding proteins, some of which were identified as lamins A, B, and C and protein C23/nucleolin. Treatment of cells with two DAG kinase inhibitors, R59022 and R59949, blocked the IGF-I-dependent rise in nuclear DAG kinase activity and maintained elevated intranuclear levels of DAG. The two inhibitors also markedly potentiated the mitogenic effect of IGF-I. These results suggest that nuclear DAG kinase plays a key role in regulating the levels of DAG present in the nucleus and that DAG is a key molecule for the mitogenic effect that IGF-I exerts on Swiss 3T3 cells. PMID:10706086

  14. Anti-adipogenic effects of extracts of Ficus deltoidea var. deltoidea and var. angustifolia on 3T3-L1 adipocytes*

    PubMed Central

    Woon, Shiau Mei; Seng, Yew Wei; Ling, Anna Pick Kiong; Chye, Soi Moi; Koh, Rhun Yian

    2014-01-01

    Objective: This study examined the anti-adipogenic effects of extracts of Ficus deltoidea var. deltoidia and var. angustifolia, a natural slimming aid, on 3T3-L1 adipocytes. Methods: Methanol and water extracts of leaves of the F. deltoidea varieties were analyzed to determine their total flavonoid content (TFC) and total phenolic content (TPC), respectively. The study was initiated by determining the maximum non-toxic dose (MNTD) of the methanol and water extracts for 3T3-L1 preadipocytes. Possible anti-adipogenic effects were then examined by treating 2-d post confluent 3T3-L1 preadipocytes with either methanol extract or water extract at MNTD and half MNTD (˝MNTD), after which the preadipocytces were induced to form mature adipocytes. Visualisation and quantification of lipid content in mature adipocytes were carried out through oil red O staining and measurement of optical density (OD) at 520 nm, respectively. Results: The TFCs of the methanol extracts were 1.36 and 1.97 g quercetin equivalents (QE)/100 g dry weight (DW), while the TPCs of the water extracts were 5.61 and 2.73 g gallic acid equivalents (GAE)/100 g DW for var. deltoidea and var. angustilofia, respectively. The MNTDs determined for methanol and water extracts were (300.0±28.3) and (225.0±21.2) ?g/ml, respectively, for var. deltoidea, while much lower MNTDs [(60.0±2.0) ?g/ml for methanol extracts and (8.0±1.0) ?g/ml for water extracts] were recorded for var. angustifolia. Studies revealed that the methanol extracts of both varieties and the water extracts of var. angustifolia at either MNTD or ˝MNTD significantly inhibited the maturation of preadipocytes. Conclusions: The inhibition of the formation of mature adipocytes indicated that leaf extracts of F. deltoidea could have potential anti-obesity effects. PMID:24599694

  15. Sustained release of Semaphorin 3A from ?-tricalcium phosphate based cement composite contributes to osteoblastic differentiation of MC3T3-E1 cells

    NASA Astrophysics Data System (ADS)

    Wang, Jin-Ning; Pi, Bin; Wang, Peng; Li, Xue-Feng; Yang, Hui-Lin; Zhu, Xue-Song

    2015-06-01

    The reinforcement of calcium phosphate materials with silk fibroin (SF) has been one of the strategies to overcome the brittleness. However, the lack of osteoinductivity may still restrict their further use. This study aimed to investigate the biocompatibility and osteogenesis capacity of a novel Semaphorin 3A-loaded chitosan microspheres/SF/?-tricalcium phosphate composite (Sema3A CMs/SF/?-TCP) in vitro. Sema3A was first incorporated into CMs, and the Sema3A CMs/SF/?-TCP composite was then prepared. The morphology of the CMs was observed using SEM. The in vitro release kinetics, cytotoxicity, and cell compatibility were evaluated, and the real-time quantitative polymerase chain reaction (RT-qPCR) and activity of alkaline phosphatase (ALP) were used to evaluate the osteogenesis capacity of the composite. The in vitro release of Sema3A from the Sema3A CMs/SF/?-TCP composite showed a temporally controlled manner. The extract of the Sema3A CMs/SF/?-TCP composite presented no obvious side effect on the MC3T3-E1 cell proliferation, nor promote cell proliferation. The MC3T3-E1 cells were well-spread and presented an elongated shape on the Sema3A CMs/SF/?-TCP composite surface; the ALP activity and the osteogenic-related gene expression were higher than those seeded on the surface of the CMs/SF/?-TCP and SF/?-TCP composites. In conclusion, Sema3A CMs/SF/?-TCP has excellent biocompatibility and contributes to the osteoblastic differentiation of MC3T3-E1 cells.

  16. Novel ATP-binding heat-inducible protein of Mr = 37,000 that is sensitive to transformation in BALB/3T3 cells

    SciTech Connect

    Nakai, A.; Hirayama, C.; Ohtsuka, K.; Hirayoshi, K.; Nagata, K. (Kyoto Univ. (Japan))

    1990-06-01

    Using affinity chromatography on ATP-agarose, we have identified a major ATP-binding protein in Nonidet P-40 extracts of avian and mammalian cells labeled with (35S)methionine. After washing ATP-agarose beads with high-ionic-strength buffer (0.4 M NaCl), the 37-kD protein was shown to be one of the major ATP-binding proteins while p72 and grp78, which are members of the hsp70 family, also bound to ATP-agarose. This protein consisted of several spots on two-dimensional gel electrophoresis. The isoelectric point of the most basic spot was approximately 9.2 in chick embryo fibroblasts, whereas it was about 8.8 in mouse 3T3 cells. The identities of these proteins in mouse and chick cells were confirmed by peptide mapping. After heat-shock treatment of BALB/3T3 cells, the major heat-shock protein, hsp70, was shown to be induced very rapidly after heat shock and was recovered in the ATP-binding fraction. Besides hsp70, a 37-kD protein was also found to be induced by heat shock. This protein was drastically induced by treating the cells with alpha,alpha'-dipyridyl, an iron chelating reagent, but not with sodium arsenite, calcium ionophore, or tunicamycin. The synthesis and the total amount of this ATP-binding protein increased in mouse 3T3 cells transformed by simian virus 40, methylcholanthrene, or activated c-Ha-ras oncogene compared to their normal counterparts. The incorporation of (32P)orthophosphate was not detected in either normal or transformed cells. These studies established that a major ATP-binding protein of Mr = 37,000 is a heat-inducible protein and that the synthesis of this protein is regulated by malignant transformation.

  17. Regulation of the beta-adrenergic receptor-adenylate cyclase complex of 3T3-L1 fibroblasts by sodium butyrate

    SciTech Connect

    Stadel, J.M.; Poksay, K.S.; Nakada, M.T.; Crooke, S.T.

    1986-05-01

    Mouse 3T3-L1 fibroblasts contain beta-adrenergic receptors (BAR), predominantly of the B/sub 1/ subtype. Incubation of these cells with 2-10 mM sodium butyrate (SB) for 24-48 hr results in a switch in the BAR subtype from B/sub 1/ to B/sub 2/ and promotes a 1.5 to 2.5 fold increase in total BAR number. Other short chain acids were not as effective as SB in promoting changes in BAR. BAR were assayed in membranes prepared from the 3T3-L1 cells using the radiolabeled antagonist (/sup 125/I)-cyanopindolol and the B/sub 2/ selective antagonist ICI 118.551. BAR subtype switch was confirmed functionally by measuring cellular cAMP accumulation in response to agonists. The structure and amount of the alpha subunits of the guanine nucleotide regulatory proteins N/sub s/ and N/sub i/ were determined by ADP-ribosylation using /sup 32/P-NAD and either cholera toxin or pertussis toxin for labeling of the respective subunits. Preincubation of cells with 5 mM SB for 48 hr resulted in a 2-3 fold increase in the labeling of the alpha subunits of both N/sub s/ and N/sub i/. A protein of M/sub r/ = 44,000 showed enhanced labeling by cholera toxin following SB treatment of the cells. These data indicate SB concomitantly regulates expression of BAR subtype and components of the adenylate cyclase in 3T3-L1 cells.

  18. The effects of bone morphogenetic protein-2 and enamel matrix derivative on the bioactivity of mineral trioxide aggregate in MC3T3-E1cells

    PubMed Central

    Jeong, Youngdan; Yang, Wonkyung; Ko, Hyunjung

    2014-01-01

    Objectives The effects of bone morphogenetic protein-2 (BMP-2) and enamel matrix derivative (EMD) respectively with mineral trioxide aggregate (MTA) on hard tissue regeneration have been investigated in previous studies. This study aimed to compare the osteogenic effects of MTA/BMP-2 and MTA/EMD treatment in MC3T3-E1 cells. Materials and Methods MC3T3-E1 cells were treated with MTA (ProRoot, Dentsply), BMP-2 (R&D Systems), EMD (Emdogain, Straumann) separately and MTA/BMP-2 or MTA/EMD combination. Mineralization was evaluated by staining the calcium deposits with alkaline phosphatase (ALP, Sigma-Aldrich) and Alizarin red (Sigma-Aldrich). The effects on the osteoblast differentiation were evaluated by the expressions of osteogenic markers, including ALP, bone sialoprotein (BSP), osteocalcin (OCN), osteopontin (OPN) and osteonectin (OSN), as determined by reverse-transcription polymerase chain reaction analysis (RT-PCR, AccuPower PCR, Bioneer). Results Mineralization increased in the BMP-2 and MTA/BMP-2 groups and increased to a lesser extent in the MTA/EMD group but appeared to decrease in the MTA-only group based on Alizarin red staining. ALP expression largely decreased in the EMD and MTA/EMD groups based on ALP staining. In the MTA/BMP-2 group, mRNA expression of OPN on day 3 and BSP and OCN on day 7 significantly increased. In the MTA/EMD group, OSN and OCN gene expression significantly increased on day 7, whereas ALP expression decreased on days 3 and 7 (p < 0.05). Conclusions These results suggest the MTA/BMP-2 combination promoted more rapid differentiation in MC3T3-E1 cells than did MTA/EMD during the early mineralization period. PMID:25110642

  19. Insulin stimulation of K+ uptake in 3T3-L1 fibroblasts involves phosphatidylinositol 3-kinase and protein kinase C-zeta

    Microsoft Academic Search

    G. Sweeney; R. Somwar; T. Ramlal; P. Martin-Vasallo; A. Klip

    1998-01-01

    Summary   Despite the important physiological role of insulin in the regulation of ionic homeostasis, primarily mediated by the Na+\\/K+-ATPase and Na+\\/K+\\/2Cl– cotransporter, the intracellular signalling molecules mediating this effect of insulin have not been elucidated. Treatment\\u000a of 3T3-L1 fibroblasts with insulin increased total 86Rb+ (K+) uptake from 0.8 ± 0.04 to 1.02 ± 0.05 nmol · mg–1· protein–1· min–1 (p

  20. Effects of DNA methylation and histone modification on differentiation-associated gene expression in ES, NIH3T3, and NIT1

    Microsoft Academic Search

    Aiping Fang; Yue Zhang; Mingyue Li; Hui Guo; Xiaofang Yu; Furong Li; Hong Hu

    2011-01-01

    Summary  The effects of epigenetic modification on the differentiation of islet cells and the expression of associated genes (Pdx-1,\\u000a Pax4, MafA, and Nkx6.1, etc) were investigated. The promoter methylation status of islet differentiation-associated genes\\u000a (Pdx-1, Pax4, MafA and Nkx6.1), Oct4 and MLH1 genes of mouse embryonic stem cells, NIH3T3 cells and NIT-1 cells were profiled\\u000a by methylated DNA immunoprecipitation, real-time quantitative

  1. Arctiin inhibits adipogenesis in 3T3-L1 cells and decreases adiposity and body weight in mice fed a high-fat diet

    PubMed Central

    Min, Byulchorong; Lee, Heejin; Song, Ji Hye; Han, Myung Joo

    2014-01-01

    BACKGROUND/OBJECTIVES The purpose of this study was to examine the effects and associated mechanisms of arctiin, a lignan compound found in burdock, on adipogenesis in 3T3-L1 cells. Also, the effects of arctiin supplementation in obese mice fed a high-fat diet on adiposity were examined. MATERIALS/METHODS 3T3-L1 cells were treated with arctiin (12.5 to 100 µM) during differentiation for 8 days. The accumulation of lipid droplets was determined by Oil Red O staining and intracellular triglyceride contents. The expressions of genes related to adipogenesis were measured by real-time RT-PCR and Western blot analyses. For in vivo study, C57BL/6J mice were first fed either a control diet (CON) or high-fat diet (HF) to induce obesity, and then fed CON, HF, or HF with 500 mg/kg BW arctiin (HF + AC) for four weeks. RESULTS Arctiin treatment to 3T3-L1 pre-adipocytes markedly decreased adipogenesis in a dose-dependent manner. The arctiin treatment significantly decreased the protein levels of the key adipogenic regulators PPAR? and C/EBP?, and also significantly inhibited the expression of SREBP-1c, fatty acid synthase, fatty acid-binding protein and lipoprotein lipase. Also, arctiin greatly increased the phosphorylation of AMP-activated protein kinase (AMPK) and its downstream target phosphorylated-acetyl CoA carboxylase. Furthermore, administration of arctiin significantly decreased the body weight in obese mice fed with the high-fat diet. The epididymal, perirenal or total visceral adipose tissue weights of mice were all significantly lower in the HF + AC than in the HF. Arctiin administration also decreased the sizes of lipid droplets in the epididymal adipose tissue. CONCLUSIONS Arctiin inhibited adipogenesis in 3T3-L1 adipocytes through the inhibition of PPAR? and C/EBP? and the activation of AMPK signaling pathways. These findings suggest that arctiin has a potential benefit in preventing obesity. PMID:25489405

  2. Conditionally Oncogenic Forms of the A-Raf and B-Raf Protein Kinases Display Different Biological and Biochemical Properties in NIH 3T3 Cells

    Microsoft Academic Search

    CATRIN A. PRITCHARD; MICHAEL L. SAMUELS; ELIZABETH BOSCH; ANDMARTIN MCMAHON

    1995-01-01

    The protein kinase domains of mouse A-Raf and B-Raf were expressed as fusion proteins with the hormone bindingdomainofthehumanestrogenreceptorinmammaliancells.Intheabsenceofestradiol,3T3andrat1a cells expressing DA-Raf:ER and DB-Raf:ER were nontransformed, but upon the addition of estradiol the cells becameoncogenicallytransformed.Morphologicaloncogenictransformationwasmorerapidanddistinctivein cells expressing DB-Raf:ER compared with cells expressing DA-Raf:ER. Biochemical analysis of cells trans- formed by DA-Raf:ER and DB-Raf:ER revealed several interesting differences. The activation of DB-Raf:ER

  3. Thrombin increases hyposmotic taurine efflux and accelerates and RVD in 3T3 fibroblasts by a src-dependent EGFR transactivation

    Microsoft Academic Search

    E. Vázquez-Juárez; G. Ramos-Mandujano; R. A. Lezama; S. Cruz-Rangel; L. D. Islas; H. Pasantes-Morales

    2008-01-01

    The present study in Swiss3T3 fibroblasts examines the effect of thrombin on hyposmolarity-induced osmolyte fluxes and RVD,\\u000a and the contribution of the src\\/EGFR pathway. Thrombin (5 U\\/ml) added to a 30% hyposmotic medium markedly increased hyposmotic\\u000a 3H-taurine efflux (285%), accelerated the volume-sensitive Cl? current ($$ {\\\\text{ICI}}^{ - }_{{{\\\\text{swell}}}} $$) and increased RVD rate. These effects were reduced (50–65%) by preventing the

  4. Effect of methylated tea catechins from Chinese oolong tea on the proliferation and differentiation of 3T3-L1 preadipocyte.

    PubMed

    Yang, Yang; Qiao, Longliang; Zhang, Xin; Wu, Zufang; Weng, Peifang

    2015-07-01

    As the important component of tea catechins in oolong tea, (-)-epigallocatechin 3-O-(3-O-methyl) gallate (EGCG3?Me) has exhibited various beneficial effects, however, little attention about its obesity prevention effect is available. In this study, the inhibitory effects of tea catechin monomers, including their methylated forms on the proliferation and differentiation of 3T3-L1 preadipocyte were studied. The major methylated tea catechins in oolong tea were identified as EGCG3?Me and ECG3?Me. The accumulation of triglyceride was significantly reduced in a concentration-dependent manner in groups treated with EGCG3?Me at concentrations of 20, 40 and 80?g/mL, and the accumulation of lipid was decreased to 89.42±2.66%, 64.36±3.13% and 39.37±2.79%, respectively. Both EGCG3?Me and EGCG treatments showed a significant inhibitory effect on adipogenesis, while EGCG3?Me showed a relatively higher effect than EGCG, which was contrary to the results of cytotoxic activity. For ECG and ECG3?Me, ECG3?Me also showed a relatively higher antiobesity effect and lower cytotoxic activity. The results of activity screening showed that methylated tea catechins, including EGCG3?Me and ECG3?Me inhibited the proliferation and differentiation of 3T3-L1 preadipocyte. The difference of inhibitory effects for tested compounds may be due to their structural difference (the hydroxyl group at C-3 in D ring substituted by methoxy group). PMID:26002426

  5. Piperine, a component of black pepper, decreases eugenol-induced cAMP and calcium levels in non-chemosensory 3T3-L1 cells.

    PubMed

    Yoon, Yeo Cho; Kim, Sung-Hee; Kim, Min Jung; Yang, Hye Jeong; Rhyu, Mee-Ra; Park, Jae-Ho

    2015-01-01

    This study investigated the effects of an ethanol extract of black pepper and its constituent, piperine, on odorant-induced signal transduction in non-chemosensory cells. An ethanol extract of black pepper decreased eugenol-induced cAMP and calcium levels in preadipocyte 3T3-L1 cells with no toxicity. Phosphorylation of CREB (cAMP response element-binding protein) was down-regulated by the black pepper extract. The concentration (133.8 mg/g) and retention time (5.5 min) of piperine in the ethanol extract were quantified using UPLC-MS/MS. Pretreatment with piperine decreased eugenol-induced cAMP and calcium levels in 3T3-L1 cells. Piperine also decreased the phosphorylation of CREB, which is up-regulated by eugenol. These results suggest that piperine inhibits the eugenol-induced signal transduction pathway through modulation of cAMP and calcium levels and phosphorylation of CREB in non-chemosensory cells. PMID:25685661

  6. Effects of Panicum miliaceum L. extract on adipogenic transcription factors and fatty acid accumulation in 3T3-L1 adipocytes.

    PubMed

    Park, Mi-Young; Seo, Dong-Won; Lee, Jin-Young; Sung, Mi-Kyung; Lee, Young-Min; Jang, Hwan-Hee; Choi, Hae-Yeon; Kim, Jae-Hyn; Park, Dong-Sik

    2011-06-01

    The dietary intake of whole grains is known to reduce the incidence of chronic diseases such as obesity, diabetes, cardiovascular disease, and cancer. To investigate whether there are anti-adipogenic activities in various Korean cereals, we assessed water extracts of nine cereals. The results showed that treatment of 3T3-L1 adipocytes with Sorghum bicolor L. Moench, Setaria italica Beauvois, or Panicum miliaceum L. extract significantly inhibited adipocyte differentiation, as determined by measuring oil red-O staining, triglyceride accumulation, and glycerol 3-phosphate dehydrogenase activity. Among the nine cereals, P. miliaceum L. showed the highest anti-adipogenic activity. The effects of P. miliaceum L. on mRNA expression of peroxisome proliferator-activated receptor-?, sterol regulatory element-binding protein 1, and the CCAAT/enhancer binding protein-? were evaluated, revealing that the extract significantly decreased the expression of these genes in a dose-dependent manner. Moreover, P. miliaceum L. extract changed the ratio of monounsaturated fatty acids to saturated fatty acids in adipocytes, which is related to biological activity and cell characteristics. These results suggest that some cereals efficiently suppress adipogenesis in 3T3-L1 adipocytes. In particular, the effect of P. miliaceum L. on adipocyte differentiation is associated with the downregulation of adipogenic genes and fatty acid accumulation in adipocytes. PMID:21779521

  7. Invasiveness and metastasis of NIH 3T3 cells induced by Met-hepatocyte growth factor/scatter factor autocrine stimulation.

    PubMed Central

    Rong, S; Segal, S; Anver, M; Resau, J H; Vande Woude, G F

    1994-01-01

    The met protooncogene product, Met, is the tyrosine kinase growth factor receptor for hepatocyte growth factor/scatter factor (HGF/SF). NIH 3T3 cells express HGF/SF endogenously and become tumorigenic in nude mice via an autocrine mechanism when murine Met is expressed ectopically (Metmu cells) or when human Met and human HGF/SF are coexpressed (HMH cells). Here, we show that Metmu and HMH cells are invasive in vitro and display enhanced protease activity necessary for the invasive phenotype. In experimental and spontaneous metastasis assays, Metmu or HMH cells metastasize to the lung, but lower numbers of subcutaneously injected Metmu and HMH cells produced invasive tumors in the heart, diaphragm, salivary gland, and retroperitoneum. It has been reported elsewhere that Met expression increased with tumor passage in athymic nude mice, and these tumor explants show enhanced activity in the metastasis assays. Autocrine-mediated Met-HGF/SF signal transduction in NIH 3T3 mesenchymal cells may provide an important system for understanding the biological process of metastasis. Images PMID:8197126

  8. Regulation of collagenase-3 and osteocalcin gene expression by collagen and osteopontin in differentiating MC3T3-E1 cells

    NASA Technical Reports Server (NTRS)

    D'Alonzo, Richard C.; Kowalski, Aaron J.; Denhardt, David T.; Nickols, G. Allen; Partridge, Nicola C.

    2002-01-01

    Both collagenase-3 and osteocalcin mRNAs are expressed maximally during the later stages of osteoblast differentiation. Here, we demonstrate that collagenase-3 mRNA expression in differentiating MC3T3-E1 cells is dependent upon the presence of ascorbic acid, is inhibited in the presence of the collagen synthesis inhibitor, 3,4-dehydroproline, and is stimulated by growth on collagen in the absence of ascorbic acid. Transient transfection studies show that collagenase-3 promoter activity increases during cell differentiation and requires the presence of ascorbic acid. Additionally, we show that, in differentiating MC3T3-E1 cells, collagenase-3 gene expression increases in the presence of an anti-osteopontin monoclonal antibody that binds near the RGD motif of this protein, whereas osteocalcin expression is inhibited. Furthermore, an RGD peptidomimetic compound, designed to block interaction of ligands to the alpha(v) integrin subunit, increases osteocalcin expression and inhibits collagenase-3 expression, suggesting that the RGD peptidomimetic initiates certain alpha(v) integrin signaling in osteoblastic cells. Overall, these studies demonstrate that stimulation of collagenase-3 expression during osteoblast differentiation requires synthesis of a collagenous matrix and that osteopontin and alpha(v) integrins exert divergent regulation of collagenase-3 and osteocalcin expression during osteoblast differentiation.

  9. Variation in capacity for anchorage-independent growth among agar-derived clones of spontaneously transformed BALB/3T3 cells

    SciTech Connect

    Romerdahl, C.A.; Rubin, H.

    1984-12-01

    A subline of cloned spontaneously transformed BALB/3T3 cells had a colony-forming efficiency (CFE) in agar of 5 to 20%. Individual agar colonies isolated and reseeded into agar were not significantly more efficient at initiating colonies than the original unselected subline. Four successive cycles of agar growth and selection also failed to increase the mean CFE in agar. Randomly selected clones isolated on a plastic surface all had the capacity to grow in agar. These results suggest that the failure of the majority of the cells to grow in agar is not the result of an intrinsic or heritable inability to do so. The ability to initiate a colony in agar seems to vary phenotypically from cell to cell. In contrast, agar colonies isolated from some tumor cell lines (originating from related spontaneously transformed 3T3 cells) and reseeded in agar had a higher CFE than the unselected tumor cell lines. In one case, this increased CFE in agar was lost when the cells were passaged on plastic without further selection for agar growth. Thus, expression of the anchorage-independent phenotype may vary, even among related cloned populations of transformed cells. 39 references, 3 tables.

  10. Antiobesity effects of quercetin-rich onion peel extract on the differentiation of 3T3-L1 preadipocytes and the adipogenesis in high fat-fed rats.

    PubMed

    Moon, Jiyoung; Do, Hyun-Ju; Kim, Oh Yoen; Shin, Min-Jeong

    2013-08-01

    The aim of the present study was to examine the effect of quercetin-rich onion peel extract (OPE) on anti-differentiation in 3T3-L1 preadipocytes and the antiobesity in high-fat fed rats. We found that lipid accumulations and TG contents in 3T3-L1 cells were markedly suppressed by OPE. The mRNA levels of activating protein (AP2) were down-regulated and those of carnitine palmitoyl transferase-1 ? (CPT-1?) and fatty acid binding protein 4 (FABP4) were up-regulated by 75 and 100 ?g/ml OPE. Body weight, retroperitoneal and mesenteric fat weights of SD rats were significantly lower in the 8 week high fat (HF) diet+0.72% OPE group than in the HF group. Peroxisome proliferator-activated receptor (PPAR)? mRNA levels were down-regulated in the epididymal fat of OPE than those of control and HF, and significant down-regulation of CCAAT/enhancer binding protein (C/EBP)? mRNA levels in OPE was also observed than the control. The mRNA levels of CPT-1? and uncoupling protein-1 (UCP-1) were up-regulated by the OPE, while those of fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC) were down-regulated in HF and OPE groups compared to control group. These results suggest that quercentin-enriched OPE may have antiobesity effects by suppressing preadipocyte differentiation and inhibiting adipogenesis. PMID:23684756

  11. Targeting the Autophagy Pathway Using Ectopic Expression of Beclin 1 in Combination with Rapamycin in Drug-Resistant v-Ha-ras-Transformed NIH 3T3 Cells

    PubMed Central

    Eum, Ki-Hwan; Lee, Michael

    2011-01-01

    The effectiveness of an apoptosis-targeting therapy may be limited in tumor cells with defects in apoptosis. Recently, considerable attention in the field of cancer therapy has been focused on the mammalian rapamycin target (mTOR), inhibition of which results in autophagic cell death. In our study using multidrug-resistant v-Ha-ras-transformed NIH3T3 (Ras-NIH 3T3/Mdr) cells, we demonstrated that rapamycin-induced cell death may result from 2 different mechanisms. At high rapamycin concentrations (? 100 nM), cell death may occur via an autophagy-dependent pathway, whereas at lower concentrations (?10 nM), cell death may occur after G1-phase cell cycle arrest. This effect was accompanied by upregulation of p21Cip1 and p27Kip1 expression via an autophagy-independent pathway. We also tested whether inhibition of mTOR with low concentrations of rapamycin and ectopic Beclin-1 expression would further sensitize multidrug resistance (MDR)-positive cancer cells by upregulating autophagy. Rapamycin at low concentrations might be insufficient to initiate autophagosome formation in autophagy but Beclin-1 overexpression triggered additional processes downstream of mTOR during G1 cell cycle arrest by rapamycin. Our findings suggest that these combination strategies targeting autophagic cell death may yield significant benefits for cancer patients, because lowering rapamycin concentration for cancer treatment minimizes its side effects in patients undergoing chemotherapy. PMID:21350938

  12. Distinct Roles of the Phosphatidate Phosphatases Lipin 1 and 2 during Adipogenesis and Lipid Droplet Biogenesis in 3T3-L1 Cells*

    PubMed Central

    Sembongi, Hiroshi; Miranda, Merce; Han, Gil-Soo; Fakas, Stylianos; Grimsey, Neil; Vendrell, Joan; Carman, George M.; Siniossoglou, Symeon

    2013-01-01

    Lipins are evolutionarily conserved Mg2+-dependent phosphatidate phosphatase (PAP) enzymes with essential roles in lipid biosynthesis. Mammals express three paralogues: lipins 1, 2, and 3. Loss of lipin 1 in mice inhibits adipogenesis at an early stage of differentiation and results in a lipodystrophic phenotype. The role of lipins at later stages of adipogenesis, when cells initiate the formation of lipid droplets, is less well characterized. We found that depletion of lipin 1, after the initiation of differentiation in 3T3-L1 cells but before the loading of lipid droplets with triacylglycerol, results in a reciprocal increase of lipin 2, but not lipin 3. We generated 3T3-L1 cells where total lipin protein and PAP activity levels are down-regulated by the combined depletion of lipins 1 and 2 at day 4 of differentiation. These cells still accumulated triacylglycerol but displayed a striking fragmentation of lipid droplets without significantly affecting their total volume per cell. This was due to the lack of the PAP activity of lipin 1 in adipocytes after day 4 of differentiation, whereas depletion of lipin 2 led to an increase of lipid droplet volume per cell. We propose that in addition to their roles during early adipogenesis, lipins also have a role in lipid droplet biogenesis. PMID:24133206

  13. Extracellular matrix mineralization in murine MC3T3-E1 osteoblast cultures: an ultrastructural, compositional and comparative analysis with mouse bone.

    PubMed

    Addison, W N; Nelea, V; Chicatun, F; Chien, Y-C; Tran-Khanh, N; Buschmann, M D; Nazhat, S N; Kaartinen, M T; Vali, H; Tecklenburg, M M; Franceschi, R T; McKee, M D

    2015-02-01

    Bone cell culture systems are essential tools for the study of the molecular mechanisms regulating extracellular matrix mineralization. MC3T3-E1 osteoblast cell cultures are the most commonly used in vitro model of bone matrix mineralization. Despite the widespread use of this cell line to study biomineralization, there is as yet no systematic characterization of the mineral phase produced in these cultures. Here we provide a comprehensive, multi-technique biophysical characterization of this cell culture mineral and extracellular matrix, and compare it to mouse bone and synthetic apatite mineral standards, to determine the suitability of MC3T3-E1 cultures for biomineralization studies. Elemental compositional analysis by energy-dispersive X-ray spectroscopy (EDS) showed calcium and phosphorus, and trace amounts of sodium and magnesium, in both biological samples. X-ray diffraction (XRD) on resin-embedded intact cultures demonstrated that similar to 1-month-old mouse bone, apatite crystals grew with preferential orientations along the (100), (101) and (111) mineral planes indicative of guided biogenic growth as opposed to dystrophic calcification. XRD of crystals isolated from the cultures revealed that the mineral phase was poorly crystalline hydroxyapatite with 10 to 20nm-sized nanocrystallites. Consistent with the XRD observations, electron diffraction patterns indicated that culture mineral had low crystallinity typical of biological apatites. Fourier-transform infrared spectroscopy (FTIR) confirmed apatitic carbonate and phosphate within the biological samples. With all techniques utilized, cell culture mineral and mouse bone mineral were remarkably similar. Scanning (SEM) and transmission (TEM) electron microscopy showed that the cultures had a dense fibrillar collagen matrix with small, 100nm-sized, collagen fibril-associated mineralization foci which coalesced to form larger mineral aggregates, and where mineralized sites showed the accumulation of the mineral-binding protein osteopontin. Light microscopy, confocal microscopy and three-dimensional reconstructions showed that some cells had dendritic processes and became embedded within the mineral in an osteocyte-like manner. In conclusion, we have documented characteristics of the mineral and matrix phases of MC3T3-E1 osteoblast cultures, and have determined that the structural and compositional properties of the mineral are highly similar to that of mouse bone. PMID:25460184

  14. Chondroitin sulphate proteoglycan in the substratum adhesion sites of Balb/c 3T3 cells. Fractionation on various ion-exchange and affinity columns.

    PubMed Central

    Wightman, B C; Weltman, E A; Culp, L A

    1986-01-01

    Proteoglycans on the cell surface play critical roles in the adhesion of fibroblasts to a fibronectin-containing extracellular matrix, including the model mouse cell line Balb/c 3T3. In order to evaluate the biochemistry of these processes, long-term [35S]sulphate-labelled proteoglycans were extracted quantitatively from the adhesion sites of 3T3 cells, after their EGTA-mediated detachment from the substratum, by using an extractant containing 1% octyl glucoside, 1 M-NaCl and 0.5 M-guanidinium chloride (GdnHCl) in buffer with many proteinase inhibitors. Greater than 90% of the material was identified as a large chondroitin sulphate proteoglycan (Kav. = 0.4 on a Sepharose CL2B column), and the remainder was identified as a smaller heparan sulphate proteoglycan; only small amounts of free chains of glycosaminoglycan were observed in these sites. These extracts were fractionated on DEAE-Sepharose columns under two different sets of elution conditions: with acetate buffer (termed DEAE-I) or with acetate buffer supplemented with 8 M-urea (termed DEAE-II). Under DEAE-I conditions about one-half of the material was eluted as a single peak and the remainder required 4 M-GdnHCl in order to recover it from the column; in contrast, greater than 90% of the material was eluted as a single peak from DEAE-II columns. Comparison of the elution of [35S]sulphate-labelled proteoglycan with that of 3H-labelled proteins from these two columns, as well as mixing experiments, indicated that the GdnHCl-sensitive proteoglycans were trapped at the top of columns, partially as a consequence of their association with proteins in these adhesion-site extracts. Affinity chromatography of these proteoglycans on columns of either immobilized platelet factor 4 or immobilized plasma fibronectin revealed that most of the chondroitin sulphate proteoglycan and the heparan sulphate proteoglycan bound to platelet factor 4 but that only the heparan sulphate proteoglycan bound to fibronectin, providing a ready means of separating the two proteoglycan classes. Affinity chromatography on octyl-Sepharose columns to test for hydrophobic domains in their core proteins demonstrated that a high proportion of the heparan sulphate proteoglycan but none of the chondroitin sulphate proteoglycan bound to the hydrophobic matrix. These results are discussed in light of the possible functional importance of the chondroitin sulphate proteoglycan in the detachment of cells from extracellular matrix and in light of previous affinity fractionations of proteoglycans from the substratum-adhesion sites of simian-virus-40-transformed 3T3 cells. PMID:3741402

  15. Prolonged inorganic arsenite exposure suppresses insulin-stimulated AKT S473 phosphorylation and glucose uptake in 3T3-L1 adipocytes: Involvement of the adaptive antioxidant response

    SciTech Connect

    Xue, Peng [The Hamner Institutes for Health Sciences, Research Triangle Park, NC 27709 (United States) [The Hamner Institutes for Health Sciences, Research Triangle Park, NC 27709 (United States); School of Public Health, China Medical University, Shenyang 110001 (China); Hou, Yongyong; Zhang, Qiang; Woods, Courtney G.; Yarborough, Kathy; Liu, Huiyu [The Hamner Institutes for Health Sciences, Research Triangle Park, NC 27709 (United States)] [The Hamner Institutes for Health Sciences, Research Triangle Park, NC 27709 (United States); Sun, Guifan [School of Public Health, China Medical University, Shenyang 110001 (China)] [School of Public Health, China Medical University, Shenyang 110001 (China); Andersen, Melvin E. [The Hamner Institutes for Health Sciences, Research Triangle Park, NC 27709 (United States)] [The Hamner Institutes for Health Sciences, Research Triangle Park, NC 27709 (United States); Pi, Jingbo, E-mail: jpi@thehamner.org [The Hamner Institutes for Health Sciences, Research Triangle Park, NC 27709 (United States)] [The Hamner Institutes for Health Sciences, Research Triangle Park, NC 27709 (United States)

    2011-04-08

    Highlights: {yields} In 3T3-L1 adipocytes iAs{sup 3+} decreases insulin-stimulated glucose uptake. {yields} iAs{sup 3+} attenuates insulin-induced phosphorylation of AKT S473. {yields} iAs{sup 3+} activates the cellular adaptive oxidative stress response. {yields} iAs{sup 3+} impairs insulin-stimulated ROS signaling. {yields} iAs{sup 3+} decreases expression of adipogenic genes and GLUT4. -- Abstract: There is growing evidence that chronic exposure of humans to inorganic arsenic, a potent environmental oxidative stressor, is associated with the incidence of type 2 diabetes (T2D). One critical feature of T2D is insulin resistance in peripheral tissues, especially in mature adipocytes, the hallmark of which is decreased insulin-stimulated glucose uptake (ISGU). Despite the deleterious effects of reactive oxygen species (ROS), they have been recognized as a second messenger serving an intracellular signaling role for insulin action. Nuclear factor erythroid 2-related factor 2 (NRF2) is a central transcription factor regulating cellular adaptive response to oxidative stress. This study proposes that in response to arsenic exposure, the NRF2-mediated adaptive induction of endogenous antioxidant enzymes blunts insulin-stimulated ROS signaling and thus impairs ISGU. Exposure of differentiated 3T3-L1 cells to low-level (up to 2 {mu}M) inorganic arsenite (iAs{sup 3+}) led to decreased ISGU in a dose- and time-dependent manner. Concomitant to the impairment of ISGU, iAs{sup 3+} exposure significantly attenuated insulin-stimulated intracellular ROS accumulation and AKT S473 phosphorylation, which could be attributed to the activation of NRF2 and induction of a battery of endogenous antioxidant enzymes. In addition, prolonged iAs{sup 3+} exposure of 3T3-L1 adipocytes resulted in significant induction of inflammatory response genes and decreased expression of adipogenic genes and glucose transporter type 4 (GLUT4), suggesting chronic inflammation and reduction in GLUT4 expression may also be involved in arsenic-induced insulin resistance in adipocytes. Taken together our studies suggest that prolonged low-level iAs{sup 3+} exposure activates the cellular adaptive oxidative stress response, which impairs insulin-stimulated ROS signaling that is involved in ISGU, and thus causes insulin resistance in adipocytes.

  16. Effects of uremic toxin p-cresol on proliferation, apoptosis, differentiation, and glucose uptake in 3T3-L1 cells.

    PubMed

    Tanaka, Sayuri; Yano, Shozo; Sheikh, Abdullah M; Nagai, Atsushi; Sugimoto, Toshitsugu

    2014-07-01

    Malnutrition is a common feature seen in chronic dialysis patients, and the survival rate of obese patients receiving such treatment is higher than that of lean patients. Irrespective of obesity or diabetes, dialysis patients commonly have insulin resistance, and the leading cause of death is cardiovascular (CV) disease. It has been reported that the concentration of p-cresol, a uremic toxin, is highly associated with CV events. As uremic toxin levels are high in dialysis patients, they may be involved in the pathogenesis of insulin resistance and CV disease in this population. However, little is known so far. Thus, we focused on this uremic toxin to examine its effects on adipocytes and their precursors. 3T3-L1 cells, a mouse preadipocyte cell line, were cultured until 90% confluency. The cells were then differentiated with 500??M 3-isobutyl-methylxanthine, 250?nM dexamethasone, and 10??g/mL insulin. Cell proliferation was evaluated by cell counting and bromodeoxyuridine (Brd-U) incorporation assay. Glucose uptake was estimated using radiolabeled 2-deoxyglucose. The range of concentrations of p-cresol used in the experiments was from 2 to 200??M. The investigation of cell proliferation by cell counting revealed that, compared with control, 3T3-L1 cells treated with 100 and 200??M p-cresol were significantly decreased in number at day 3 and day 7 of culture. The Brd-U incorporation assay also demonstrated similar inhibitory effects on cell proliferation, suggesting that p-cresol affected the normal cell cycle. Oil Red O staining at day 7 showed that the number of mature adipocytes was decreased by treatment with 200??M p-cresol. Consistent with that finding, the number of apoptotic cells at day 7 was increased by treatment with 100 and 200??M p-cresol. Peroxisome proliferator-activated receptor ? (PPAR?) mRNA expression increased time-dependently during the differentiation process of 3T3-L1 cells. p-Cresol dose-dependently decreased differentiation-induced mRNA expression of PPAR?. Uptake of 3H-labeled 2-deoxyglucose was markedly decreased by 200??M p-cresol in the presence or in the absence of insulin, mainly because of the decreased number of mature adipocytes. High concentrations of p-cresol disturbed the cell cycle, induced apoptosis, inhibited the differentiation of preadipocytes into mature adipocytes, and decreased glucose uptake at baseline and after insulin stimulation. These findings indicate that accumulated p-cresol may induce reduction in adipose tissue, insulin resistance, and malnutrition, eventually leading to poor outcomes in chronic dialysis patients. PMID:24417700

  17. Predicting Electricity Distribution Feeder Failures Using Machine Learning Susceptibility Analysis

    E-print Network

    Tomkins, Andrew

    }@coned.com Abstract A Machine Learning (ML) System known as ROAMS (Ranker for Open-Auto Maintenance Scheduling system makes seasonal predictions of failure susceptibility. These feeder failures, known as "Open Autos the monitoring and maintenance of primary feeders, as well as their speedy repair on failure. In the specific

  18. 103. CANAL AT THE ENTRANCE TO THE POMPTON FEEDER NEAR ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    103. CANAL AT THE ENTRANCE TO THE POMPTON FEEDER NEAR MOUNTAIN VIEW, NEW JERSEY. THE POMPTON FEEDER BOATS TO TRAVEL NORTH 4.26 MILES TO TO POMPTON AS WELL AS PROVIDING THE MAIN CANAL WITH WATER FROM GREENWOOD LAKE. A MODIFIED QUEEN POST TRUSS BRIDGE SPANS THE CANAL IN THE FOREGROUND. - Morris Canal, Phillipsburg, Warren County, NJ

  19. MC3T3-E1 cell response of amorphous phase/TiO2 nanocrystal composite coating prepared by microarc oxidation on titanium.

    PubMed

    Zhou, Rui; Wei, Daqing; Yang, Haoyue; Feng, Wei; Cheng, Su; Li, Baoqiang; Wang, Yaming; Jia, Dechang; Zhou, Yu

    2014-06-01

    Bioactive amorphous phase/TiO2 nanocrystal (APTN) composite coatings were fabricated by microarc oxidation (MAO) on Ti. The APTN coatings are composed of much amorphous phase with Si, Na, Ca, Ti and O elements and a few TiO2 nanocrystals. With increasing applied voltage, the micropore density of the APTN coating decreases and the micropore size of the APTN coating increases. The results indicate that less MC3T3-E1 cells attach on the APTN coatings as compared to Ti. However, the APTN coatings greatly enhance the cell proliferation ability and the activity of alkaline phosphatase. The amorphous phase and the concentrations of the released Ca and Si from the APTN coatings during cell culture have significant effects on the cell response. PMID:24863215

  20. Adipogenesis stimulates the nuclear localization of EWS with an increase in its O-GlcNAc glycosylation in 3T3-L1 cells.

    PubMed

    Li, Qiang; Kamemura, Kazuo

    2014-07-18

    Although the Ewing sarcoma (EWS) proto-oncoprotein is found in the nucleus and cytosol and is associated with the cell membrane, the regulatory mechanisms of its subcellular localization are still unclear. Here we found that adipogenic stimuli induce the nuclear localization of EWS in 3T3-L1 cells. Tyrosine phosphorylation in the C-terminal PY-nuclear localization signal of EWS was negative throughout adipogenesis. Instead, an adipogenesis-dependent increase in O-linked ?-N-acetylglucosamine (O-GlcNAc) glycosylation of EWS was observed. Pharmacological inactivation of O-GlcNAcase in preadipocytes promoted perinuclear localization of EWS. Our findings suggest that the nuclear localization of EWS is partly regulated by the glycosylation. PMID:24928395

  1. Adipogenic effects of piperlonguminine in 3T3-L1 cells and plasma concentrations of several amide constituents from Piper chaba extracts after treatment of mice.

    PubMed

    Yamaguchi, Itadaki; Matsuda, Hisashi; Zhang, Hailong; Hamao, Makoto; Yamashita, Chihiro; Kogami, Yuichiro; Kon'I, Haruka; Murata, Megumi; Nakamura, Seikou; Yoshikawa, Masayuki

    2014-01-01

    In our previous study, piperlonguminine from the fruit of Piper chaba was reported to promote adipogenesis in 3T3-L1 cells like the peroxisome proliferator-activated receptor-? (PPAR?) agonist, troglitazone. In the present study, the mode of action of piperlonguminine in cells was examined. Piperlonguminine increased mRNA levels of adiponectin, glucose transporter 4, and fatty acid-binding protein (aP2). It also increased mRNA levels of PPAR?2 but, unlike troglitazone, piperlonguminine did not activate PPAR? directly in a nuclear receptor cofactor assay. Analyses of plasma from mice treated with piperlonguminine, piperine, and retrofractamide A, and an extract of the fruit, showed that concentrations of piperlonguminine were higher than those of piperine and retrofractamide A, and that the "area-under-the-curve" of piperine increased following in vivo administration of the extract. PMID:23584920

  2. Covalent Immobilization of Collagen on Titanium through Polydopamine Coating to Improve Cellular Performances of MC3T3-E1 Cells

    PubMed Central

    Yu, Xiaohua; Walsh, John; Wei, Mei

    2014-01-01

    Surface modification of orthopedic implants is critical for improving the clinical performance of these medical devices. Herein, collagen was covalently immobilized onto a titanium implant surface via a novel adherent polydopamine coating inspired by mussel adhesive proteins. The formation and composition of the collagen coating was characterized using X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM). Fluorescent labeled collagen was also used to examine the formation and uniformity of the collagen coating. The resultant collagen coating with a polydopamine supporting substrate demonstrated better uniformity and distribution on the titanium surface compared to a physical adsorption of collagen. The covalent immobilized collagen coating is biologically active, as evidenced by its ability to enhance MC3T3-E1 cell adhesion, support cell proliferation and promote early stage osteogenic differentiation of pre-osteoblasts. Our study suggests covalent immobilization of collagen through the polydopamine coating might be an efficient way to improve the cellular performance of implant surfaces. PMID:24932406

  3. A quantitative study of MC3T3-E1 cell adhesion, morphology and biomechanics on chitosan-collagen blend films at single cell level.

    PubMed

    Wang, Chuang; Xie, Xu-Dong; Huang, Xun; Liang, Zhi-Hong; Zhou, Chang-Ren

    2015-08-01

    The interaction between cells and biomaterials plays a key role in cell proliferation and differentiation in tissue engineering. However, a quantitative analysis of those interactions has been less well studied. The objective of this study was to quantitative recapitulate the difference of MC3T3-E1 cell adhesion, morphological and biomechanical properties on chitosan-collagen films in terms of chemical composition. Here, the unbinding force between MC3T3-E1 cell and a series of chitosan-collagen films was probed by a real-time and in situ atomic force microscopy-single cell force spectroscopy (AFM-SCFS). Meanwhile, changes in cell morphology and Young's modulus on different chitosan-collagen films were detected by AFM. The cell area and CCK-8 results showed that cell spreading and proliferation increased with increasing collagen content. AFM observations clearly showed cell height decreased and pseudopod fusion with the collagen content increased. Cell adhesive force increased from 0.76±0.17nN to 1.70±0.19nN. On the contrary, cells Young's modulus, which reflected biophysical changes of cells decreased from 11.94±3.19kPa to 1.81±0.52kPa, respectively. It suggested that stronger cell-substrate interactions benefit cell adhesion, and better cell flexibility improve cell spreading. The findings indicate that cell morphology, adhesive force and Young's modulus are significant affected by various chitosan-collagen substrates. Those methods and quantitative results have guiding significance for investigating the mechanism of chitosan and/or collagen based cell-targeting drug carrier and the preparation of chitosan-collagen composite biomaterials. PMID:25996415

  4. Fatty Acid 2-Hydroxylase Mediates Diffusional Mobility of Raft-associated Lipids, GLUT4 Level, and Lipogenesis in 3T3-L1 Adipocytes*

    PubMed Central

    Guo, Lin; Zhou, Dequan; Pryse, Kenneth M.; Okunade, Adewole L.; Su, Xiong

    2010-01-01

    Straight chain fatty acid ?-oxidation increases during differentiation of 3T3-L1 adipocytes, leading to a marked accumulation of odd chain length fatty acyl moieties. Potential roles of this pathway in adipocyte differentiation and lipogenesis are unknown. Mammalian fatty acid 2-hydroxylase (FA2H) was recently identified and suggested to catalyze the initial step of straight chain fatty acid ?-oxidation. Accordingly, we examined whether FA2H modulates adipocyte differentiation and lipogenesis in mature adipocytes. FA2H level markedly increases during differentiation of 3T3-L1 adipocytes, and small interfering RNAs against FA2H inhibit the differentiation process. In mature adipocytes, depletion of FA2H inhibits basal and insulin-stimulated glucose uptake and lipogenesis, which are partially rescued by the enzymatic product of FA2H, 2-hydroxy palmitic acid. Expression of fatty-acid synthase and SCD1 was decreased in FA2H-depleted cells, and levels of GLUT4 and insulin receptor proteins were reduced. 2-Hydroxy fatty acids are enriched in cellular sphingolipids, which are components of membrane rafts. Accelerated diffusional mobility of raft-associated lipids was shown to enhance degradation of GLUT4 and insulin receptor in adipocytes. Consistent with this, depletion of FA2H appeared to increase raft lipid mobility as it significantly accelerated the rates of fluorescence recovery after photobleaching measurements of lipid rafts labeled with Alexa 488-conjugated cholera toxin subunit B. Moreover, the enhanced recovery rates were partially reversed by treatment with 2-hydroxy palmitic acid. In conclusion, our findings document the novel role of FA2H in adipocyte lipogenesis possibly by modulation of raft fluidity and level of GLUT4. PMID:20519515

  5. 31P NMR analysis of intracellular pH of Swiss Mouse 3T3 cells: effects of extracellular Na+ and K+ and mitogenic stimulation.

    PubMed

    Civan, M M; Williams, S R; Gadian, D G; Rozengurt, E

    1986-01-01

    Swiss mouse 3T3 cells grown on microcarrier beads were superfused with electrolyte solution during continuous NMR analysis. Conventional 31P and 19F probes of intracellular pH (pHc) were found to be impracticable. Cells were therefore superfused with 1 to 4 mM 2-deoxyglucose, producing a large intracellular, pH-sensitive signal of 2-deoxyglucose phosphate (2DGP). The intracellular incorporation of 2DGP inhibited the Embden-Meyerhof pathway. However, intracellular ATP was at least in part retained and the cellular responsivity to changes in extracellular ionic composition and to the application of growth factors proved intact. Transient replacement of external Na+ with choline or K+ reversibly acidified the intracellular fluids. Quiescent cells and mitogenically stimulated cells displayed the same dependence of shifts in pHc on external Na+ concentration (CoNa). PHc also depended on intracellular Na+ concentration (CcNa). Increasing ccNa by withdrawing external K+ (thereby inhibiting the Na,K-pump) caused reversible intracellular acidification; subsequently reducing CoNa produced a larger acid shift in pHc than with external K+ present. Comparison of separate preparations indicated that pHc was higher in stimulated than in quiescent cells. Transient administration of mitogens also reversibly alkalinized quiescent cells studied continuously. This study documents the feasibility of monitoring pHc of Swiss mouse 3T3 cells using 31P NMR analysis of 2DGP. The results support the concept of a Na/H antiport operative in these cells, both in quiescence and after mitogenic stimulation. The data document by an independent technique that cytoplasmic alkalinization is an early event in mitogenesis, and that full activity of the Embden-Meyerhof pathway is not required for the expression of this event. PMID:3543375

  6. ToF-SIMS Depth Profiling of Cells: Z-correction, 3D Imaging, and Sputter Rate of Individual NIH/3T3 Fibroblasts

    PubMed Central

    Robinson, Michael A.; Graham, Daniel J.; Castner, David G.

    2012-01-01

    Proper display of three-dimensional time-of-flight secondary ion mass spectrometry (ToF-SIMS) imaging data of complex, non-flat samples requires a correction of the data in the z-direction. Inaccuracies in displaying three dimensional ToF-SIMS data arise from projecting data from a non-flat surface onto a 2D image plane, as well as possible variations in the sputter rate of the sample being probed. The current study builds on previous studies by creating software written in Matlab to apply the z-correction to entire 3D data sets, the ZCorrectorGUI (available at http://mvsa.nb.uw.edu/). Three dimensional image data sets were acquired from NIH/3T3 fibroblasts by collecting ToF-SIMS images using a dual beam approach (25 keV Bi3+ for analysis cycles and 20 keV C60++ for sputter cycles). The entire data cube was then corrected using the new ZCorrectorGUI software, producing accurate chemical information from single cells in 3D. For the first time, a three dimensional corrected view of a lipid-rich subcellular region, possibly the nuclear membrane, is presented. Additionally, the key assumption of a constant sputter rate throughout the data acquisition was tested using ToF-SIMS and atomic force microscopy (AFM) analysis of the same cells. For the dried NIH/3T3 fibroblasts examined in this study, the sputter rate was found to not change appreciably in x, y or z, and the cellular material was sputtered at approximately 10 nm per 1.25×1013 ions C60++/cm2. PMID:22530745

  7. The cytotoxicity of the autonomous parvovirus minute virus of mice nonstructural proteins in FR3T3 rat cells depends on oncogene expression.

    PubMed Central

    Mousset, S; Ouadrhiri, Y; Caillet-Fauquet, P; Rommelaere, J

    1994-01-01

    The nonstructural (NS) proteins of the autonomous parvovirus minute virus of mice are involved in viral DNA replication and in the regulation of homologous and heterologous promoters. Moreover, NS products have proved to be cytotoxic, especially for transformed cells. We show here that intracellular accumulation of NS products is not sufficient to kill rat fibroblasts from the established cell line FR3T3, which is phenotypically normal in several respects. FRNS cell lines were obtained by stable transfection of FR3T3 cells by a vector carrying the NS genes under the control of the hormone-inducible long terminal repeat promoter of the mouse mammary tumor virus. In the presence of dexamethasone, the NS proteins were synthesized without associated cell death. Transformation of FRNS cells with the c-Ha-ras oncogene or polyomavirus oncogenes had little effect on their capacity for NS induction, as measured at both concentration and transactivating activity levels, yet the transformants were now dying within a few days in the presence of the inducer. The same results were obtained with cells stably transfected by a vector expressing the NS1 product alone, suggesting that in this system there is no cooperation between NS1 and NS2 for maximal cytopathic effect. Cell mortality after NS protein induction was quantitatively related to the yield of oncogene expression, while NS-1 was not limiting in this respect. Our results show that the NS1 protein is not lethal unless cellular factors that may depend on oncogene expression trigger its cytotoxicity. Images PMID:8083981

  8. PPAR? agonist fenofibrate attenuates TNF-?-induced CD40 expression in 3T3-L1 adipocytes via the SIRT1-dependent signaling pathway

    SciTech Connect

    Wang, Weirong [Department of Pharmacology, Cardiovascular Research Center, School of Medicine, Xi'an Jiaotong University, Xi'an, Shaanxi 710061 (China); Lin, Qinqin [Physical Education College, Yanshan University, Qinhuangdao, Hebei 066004 (China); Lin, Rong, E-mail: linrong63@yahoo.com.cn [Department of Pharmacology, Cardiovascular Research Center, School of Medicine, Xi'an Jiaotong University, Xi'an, Shaanxi 710061 (China); Zhang, Jiye [Faculty of Pharmacy, School of Medicine, Xi'an Jiaotong University, Xi'an, Shaanxi 710061 (China); Ren, Feng; Zhang, Jianfeng; Ji, Meixi; Li, Yanxiang [Department of Pharmacology, Cardiovascular Research Center, School of Medicine, Xi'an Jiaotong University, Xi'an, Shaanxi 710061 (China)

    2013-06-10

    The ligand-activated transcription factor peroxisome proliferator-activated receptor-? (PPAR?) participates in the regulation of cellular inflammation. More recent studies indicated that sirtuin1 (SIRT1), a NAD{sup +}-dependent deacetylase, regulates the inflammatory response in adipocytes. However, whether the role of PPAR? in inflammation is mediated by SIRT1 remains unclear. In this study, we aimed to determine the effect of PPAR? agonist fenofibrate on the expressions of SIRT1 and pro-inflammatory cytokine CD40 and underlying mechanisms in 3T3-L1 adipocytes. We found that fenofibrate inhibited CD40 expression and up-regulated SIRT1 expression in tumor necrosis factor-? (TNF-?)-stimulated adipocytes, and these effects of fenofibrate were reversed by PPAR? antagonist GW6471. Moreover, SIRT1 inhibitors sirtinol/nicotinamide (NAM) or knockdown of SIRT1 could attenuate the effect of fenofibrate on TNF-?-induced CD40 expression in adipocytes. Importantly, NF-?B inhibitor pyrrolidine dithiocarbamate (PDTC) augmented the effect of fenofibrate on CD40 expression in adipocytes. Further study found that fenofibrate decreased the expression of acetylated-NF-?B p65 (Ac-NF-?B p65) in TNF-?-stimulated adipocytes, and the effect of fenofibrate was abolished by SIRT1 inhibition. In addition, fenofibrate up-regulated SIRT1 expression through AMPK in TNF-?-stimulated adipocytes. Taken together, these findings indicate that PPAR? agonist fenofibrate inhibits TNF-?-induced CD40 expression in 3T3-L1 adipocytes via the SIRT1-dependent signaling pathway. -- Highlights: • Fenofibrate up-regulates SIRT1 expression in TNF-?-stimulated adipocytes. • Fenofibrate inhibits CD40 expression through SIRT1 in adipocytes. • The effects of fenofibrate on CD40 and SIRT1 expressions are dependent on PPAR?. • Fenofibrate inhibits CD40 expression via SIRT1-dependent deacetylation of NF-?B. • Fenofibrate increases SIRT1 expression through PPAR? and AMPK in adipocytes.

  9. Alpha-Lipoic Acid Promotes Osteoblastic Formation in H2 O2 -Treated MC3T3-E1 Cells and Prevents Bone Loss in Ovariectomized Rats.

    PubMed

    Fu, Chao; Xu, Dong; Wang, Chang-Yuan; Jin, Yue; Liu, Qi; Meng, Qiang; Liu, Ke-Xin; Sun, Hui-Jun; Liu, Mo-Zhen

    2015-09-01

    Alpha-lipoic acid (ALA), a naturally occurring compound and dietary supplement, has been established as a potent antioxidant that is a strong scavenger of free radicals. Recently, accumulating evidences has indicated the relationship between oxidative stress and osteoporosis (OP). Some studies have investigated the possible beneficial effects of ALA on OP both in vivo and in vitro; however, the precise mechanism(s) underlying the bone-protective action of ALA remains unclear. Considering this, we focused on the anti-oxidative capacity of ALA to exert bone-protective effects in vitro and in vivo. In the present study, the effects of ALA on osteoblastic formation in H2 O2 -treated MC3T3-E1 pre-osteoblasts and ovariectomy (OVX)-induced bone loss in rats were investigated. The results showed that ALA promoted osteoblast differentiation, mineralization and maturation and inhibited osteoblast apoptosis, thus increasing the OPG/receptor activator of nuclear factor-?B (NF-?B) ligand (RANKL) ratio and leading to enhanced bone formation in vitro and inhibited bone loss in vivo. Further study revealed that ALA exerted its bone-protective effects by inhibiting reactive oxygen species (ROS) generation by down-regulating Nox4 gene expression and protein synthesis and attenuating the transcriptional activation of NF-?B. In addition, ALA might exert its bone-protective effects by activating the Wnt/Lrp5/?-catenin signaling pathway. Taken together, the present study indicated that ALA promoted osteoblastic formation in H2 O2 -treated MC3T3-E1 cells and prevented OVX-induced bone loss in rats by regulating Nox4/ROS/NF-?B and Wnt/Lrp5/?-catenin signaling pathways, which provided possible mechanisms of bone-protective effects in regulating osteoblastic formation and preventing bone loss. Taken together, the results suggest that ALA may be a candidate for clinical OP treatment. J. Cell. Physiol. 230: 2184-2201, 2015. © 2015 Wiley Periodicals, Inc. PMID:25655087

  10. A water-soluble extract of Petalonia binghamiae inhibits the expression of adipogenic regulators in 3T3-L1 preadipocytes and reduces adiposity and weight gain in rats fed a high-fat diet

    Microsoft Academic Search

    Seong-Il Kang; Moo-Han Kim; Hye-Sun Shin; Hyo-Min Kim; Youn-Suk Hong; Ji-Gweon Park; Hee-Chul Ko; Nam-Ho Lee; Wan-Seok Chung; Se-Jae Kim

    2010-01-01

    We previously showed that an ethanolic extract of the edible brown algae Petalonia binghamiae promotes the differentiation of 3T3-L1 preadipocytes and decreases hyperglycemia in streptozotocin-induced diabetic mice. Here, we report that a water-soluble extract of P. binghamiae thalli, prepared by enzymatic digestion, inhibits preadipocyte differentiation and adipogenesis in a dose-dependent manner. In differentiating 3T3-L1 preadipocytes, the extract (designated PBEE) decreased

  11. Feeder-independent derivation of induced-pluripotent stem cells from peripheral blood endothelial progenitor cells.

    PubMed

    Chang, Wing Y; Lavoie, Jessie R; Kwon, Sarah Y; Chen, Zhaoyi; Manias, Janet L; Behbahani, John; Ling, Vicki; Kandel, Rita A; Stewart, Duncan J; Stanford, William L

    2013-03-01

    Induced-pluripotent stem cells (iPSCs) are a potential alternative cell source in regenerative medicine, which includes the use of differentiated iPSCs for cell therapies to treat coronary artery and/or peripheral arterial diseases. Late-outgrowth endothelial progenitor cells (late-EPCs) are a unique primary cell present in peripheral blood that exhibit high proliferative capacity, are being used in a wide variety of clinical trials, and have the ability to differentiate into mature endothelial cells. The objective of this study was to reprogram peripheral blood-derived late-EPCs to a pluripotent state under feeder-free and defined culture conditions. Late-EPCs that were retrovirally transduced with OCT4, SOX2, KLF4, c-MYC, and iPSC colonies were derived in feeder-free and defined media conditions. EPC-iPSCs expressed pluripotent markers, were capable of differentiating to cells from all three germ-layers, and retained a normal karyotype. Transcriptome analyses demonstrated that EPC-iPSCs exhibit a global gene expression profile similar to human embryonic stem cells (hESCs). We have generated iPSCs from late-EPCs under feeder-free conditions. Thus, peripheral blood-derived late-outgrowth EPCs represent an alternative cell source for generating iPSCs. PMID:23291290

  12. Real-time monitoring of inflammation status in 3T3-L1 adipocytes possessing a secretory Gaussia luciferase gene under the control of nuclear factor-kappa B response element

    SciTech Connect

    Nagasaki, Haruka; Yoshimura, Takeshi [Department of Life Sciences, Graduate School of Bioresources, Mie University, Tsu 514-8507 (Japan)] [Department of Life Sciences, Graduate School of Bioresources, Mie University, Tsu 514-8507 (Japan); Aoki, Naohito, E-mail: n-aoki@bio.mie-u.ac.jp [Department of Life Sciences, Graduate School of Bioresources, Mie University, Tsu 514-8507 (Japan)] [Department of Life Sciences, Graduate School of Bioresources, Mie University, Tsu 514-8507 (Japan)

    2012-04-13

    Highlights: Black-Right-Pointing-Pointer Inflammation status in adipocytes can be monitored by the new assay system. Black-Right-Pointing-Pointer Only an aliquot of conditioned medium is required without cell lysis. Black-Right-Pointing-Pointer Inflammation-attenuating compounds can be screened more conveniently. -- Abstract: We have established 3T3-L1 cells possessing a secretory Gaussia luciferase (GLuc) gene under the control of nuclear factor-kappa B (NF-{kappa}B) response element. The 3T3-L1 cells named 3T3-L1-NF-{kappa}B-RE-GLuc could differentiate into adipocyte as comparably as parental 3T3-L1 cells. Inflammatory cytokines such as tumor necrosis factor (TNF)-{alpha} and interleukin (IL)-1{beta} induced GLuc secretion of 3T3-L1-NF-{kappa}B-RE-GLuc adipocytes in a concentration- and time-dependent manner. GLuc secretion of 3T3-L1-NF-{kappa}B-RE-GLuc adipocytes was also induced when cultured with RAW264.7 macrophages and was dramatically enhanced by lipopolysaccharide (LPS)-activated macrophages. An NF-{kappa}B activation inhibitor BAY-11-7085 and an antioxidant N-acetyl cysteine significantly suppressed GLuc secretion induced by macrophages. Finally, we found that rosemary-derived carnosic acid strongly suppressed GLuc secretion induced by macrophages and on the contrary up-regulated adiponectin secretion. Collectively, by using 3T3-L1-NF-{kappa}B-RE-GLuc adipocytes, inflammation status can be monitored in real time and inflammation-attenuating compounds can be screened more conveniently.

  13. 11. MOVABLE BED SEDIMENTATION MODELS. AUTOMATIC SEDIMENT FEEDER DESIGNED AND ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    11. MOVABLE BED SEDIMENTATION MODELS. AUTOMATIC SEDIMENT FEEDER DESIGNED AND BUILT BY WES. - Waterways Experiment Station, Hydraulics Laboratory, Halls Ferry Road, 2 miles south of I-20, Vicksburg, Warren County, MS

  14. 46 CFR 111.75-1 - Lighting feeders.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ...OF HOMELAND SECURITY (CONTINUED) ELECTRICAL ENGINEERING ELECTRIC SYSTEMS-GENERAL REQUIREMENTS Lighting Circuits and Protection § 111.75-1 ...emergency lighting, feeders, and branch circuits are in subpart 112.43 of this...

  15. 46 CFR 111.75-1 - Lighting feeders.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ...OF HOMELAND SECURITY (CONTINUED) ELECTRICAL ENGINEERING ELECTRIC SYSTEMS-GENERAL REQUIREMENTS Lighting Circuits and Protection § 111.75-1 ...emergency lighting, feeders, and branch circuits are in subpart 112.43 of this...

  16. 46 CFR 111.75-1 - Lighting feeders.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ...OF HOMELAND SECURITY (CONTINUED) ELECTRICAL ENGINEERING ELECTRIC SYSTEMS-GENERAL REQUIREMENTS Lighting Circuits and Protection § 111.75-1 ...emergency lighting, feeders, and branch circuits are in subpart 112.43 of this...

  17. 46 CFR 111.75-1 - Lighting feeders.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ...OF HOMELAND SECURITY (CONTINUED) ELECTRICAL ENGINEERING ELECTRIC SYSTEMS-GENERAL REQUIREMENTS Lighting Circuits and Protection § 111.75-1 ...emergency lighting, feeders, and branch circuits are in subpart 112.43 of this...

  18. 46 CFR 111.75-1 - Lighting feeders.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ...OF HOMELAND SECURITY (CONTINUED) ELECTRICAL ENGINEERING ELECTRIC SYSTEMS-GENERAL REQUIREMENTS Lighting Circuits and Protection § 111.75-1 ...emergency lighting, feeders, and branch circuits are in subpart 112.43 of this...

  19. 2014 Cattle Feeder Days U of M Beef Team

    E-print Network

    Minnesota, University of

    2014 Cattle Feeder Days U of M Beef Team Value of oil-extracted distillers grains in feedlot cattle in the feedlot Dr. Alvaro Garcia, SDSU Mineral nutrition of feedlot cattle Nicole Kenney Rambo, U of M Beef Team

  20. 46 CFR 112.43-15 - Emergency lighting feeders.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 2010-10-01 2010-10-01 false Emergency lighting feeders. 112.43-15 Section...SECURITY (CONTINUED) ELECTRICAL ENGINEERING EMERGENCY LIGHTING AND POWER SYSTEMS Emergency Lighting Systems § 112.43-15...

  1. 46 CFR 112.43-15 - Emergency lighting feeders.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 2014-10-01 2014-10-01 false Emergency lighting feeders. 112.43-15 Section...SECURITY (CONTINUED) ELECTRICAL ENGINEERING EMERGENCY LIGHTING AND POWER SYSTEMS Emergency Lighting Systems § 112.43-15...

  2. 46 CFR 112.43-15 - Emergency lighting feeders.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 2013-10-01 2013-10-01 false Emergency lighting feeders. 112.43-15 Section...SECURITY (CONTINUED) ELECTRICAL ENGINEERING EMERGENCY LIGHTING AND POWER SYSTEMS Emergency Lighting Systems § 112.43-15...

  3. One of two rotodip feeders used to control addition of ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    One of two rotodip feeders used to control addition of alum solution into the water - Division Avenue Pumping Station & Filtration Plant, West 45th Street and Division Avenue, Cleveland, Cuyahoga County, OH

  4. VIEW OF FEEDER TABLE WITH THE BOOM OF THE UNLOADER ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    VIEW OF FEEDER TABLE WITH THE BOOM OF THE UNLOADER IN BACKGROUND. THE CASE HYDRAULIC BOOM HOIST IS TO THE RIGHT. VIEW FROM THE SOUTHWEST - Kekaha Sugar Company, Sugar Mill Building, 8315 Kekaha Road, Kekaha, Kauai County, HI

  5. 39. CLOSE UP DETAIL OF THE FEEDER AND STAMP CONNECTION. ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    39. CLOSE UP DETAIL OF THE FEEDER AND STAMP CONNECTION. THE STAMP AN MORTAR BOX ARE ON THE LEFT AND THE FEEDER WITH ITS FEEDER DISK IS ON THE RIGHT. NOTE THE COLLAR ON THE CENTER STAMP STEM (UPPER LEFT CORNER OF THE IMAGE) THAT ACTIVATES THE LEVER IN THE CENTER OF THE PHOTO. THE COLLAR IS POSITIONED SUCH THAT WHEN THE LEVEL OF THE MATERIAL REACHES A LOW POINT IN THE MORTAR BOX IT PUSHES DOWN ON THE LEVER WHICH IN TURN ACTIVATES THE AUTOMATIC FEEDER DRIVE MECHANISM WHICH THEM DELIVERS ORE INTO THE BACKSIDE OF THE MORTAR BOX. - Standard Gold Mill, East of Bodie Creek, Northeast of Bodie, Bodie, Mono County, CA

  6. 46 CFR 112.43-15 - Emergency lighting feeders.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ...Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) ELECTRICAL ENGINEERING EMERGENCY LIGHTING AND POWER SYSTEMS Emergency Lighting Systems § 112.43-15 Emergency lighting feeders. For a vessel with...

  7. 7 CFR 58.625 - Fruit or syrup feeders.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ...material into the semi-frozen product. Product contact surfaces shall be constructed of stainless steel or equally corrosion resistant metal and all pumps shall be in accordance to 3-A Sanitary Standards for dairy equipment. The feeder shall be...

  8. 7 CFR 58.625 - Fruit or syrup feeders.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ...material into the semi-frozen product. Product contact surfaces shall be constructed of stainless steel or equally corrosion resistant metal and all pumps shall be in accordance to 3-A Sanitary Standards for dairy equipment. The feeder shall be...

  9. 7 CFR 58.625 - Fruit or syrup feeders.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ...material into the semi-frozen product. Product contact surfaces shall be constructed of stainless steel or equally corrosion resistant metal and all pumps shall be in accordance to 3-A Sanitary Standards for dairy equipment. The feeder shall be...

  10. 7 CFR 58.625 - Fruit or syrup feeders.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ...material into the semi-frozen product. Product contact surfaces shall be constructed of stainless steel or equally corrosion resistant metal and all pumps shall be in accordance to 3-A Sanitary Standards for dairy equipment. The feeder shall be...

  11. 7 CFR 58.625 - Fruit or syrup feeders.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ...material into the semi-frozen product. Product contact surfaces shall be constructed of stainless steel or equally corrosion resistant metal and all pumps shall be in accordance to 3-A Sanitary Standards for dairy equipment. The feeder shall be...

  12. Peptide modulators of Src activity in G{sub 1} regulate entry into S phase and proliferation of NIH 3T3 cells

    SciTech Connect

    Mamidipudi, Vidya [Department of Medicine, Stanford University School of Medicine, Stanford, CA 94305-5187 (United States); Miller, Laura D. [Department of Medicine, Stanford University School of Medicine, Stanford, CA 94305-5187 (United States); Mochly-Rosen, Daria [Department of Molecular Pharmacology, Stanford University School of Medicine, Stanford, CA 94305-5174 (United States); Cartwright, Christine A. [Department of Medicine, Stanford University School of Medicine, Stanford, CA 94305-5187 (United States)]. E-mail: chris.cartwright@stanford.edu

    2007-01-12

    Cascades of kinases and phosphatases are regulated by selective protein-protein interactions that are essential for signal transduction. Peptide modulators of these interactions have been used to dissect the function of individual components of the signaling cascade, without relying on either the over- or underexpression of proteins. Previously, we identified RACK1 as an endogenous substrate, binding partner and inhibitor of Src tyrosine kinases. Here, we utilized cell-permeable peptides that selectively disrupt or enhance the interaction of RACK1 and Src to further examine the function of RACK1. Our results provide direct physiologic evidence that RACK1 regulates growth of NIH3T3 cells by suppressing the activity of Src and other cell cycle regulators in G{sub 1}, and delaying entry into S phase. They also demonstrate the potential for using peptide modulators of Src activity as a tool for regulating cell growth, and for designing new strategies for cancer therapy that target specific protein-protein interactions.

  13. Toddaculin, Isolated from of Toddalia asiatica (L.) Lam., Inhibited Osteoclastogenesis in RAW 264 Cells and Enhanced Osteoblastogenesis in MC3T3-E1 Cells

    PubMed Central

    Watanabe, Akio; Kumagai, Momochika; Mishima, Takashi; Ito, Junya; Otoki, Yurika; Harada, Teppei; Kato, Tsuyoshi; Yoshida, Mikihiko; Suzuki, Misora; Yoshida, Izumi; Fujita, Kazuhiro; Watai, Masatoshi; Nakagawa, Kiyotaka; Miyazawa, Teruo

    2015-01-01

    Osteoporosis with bone loss is widely recognized as a major health problem. Bone homeostasis is maintained by balancing bone formation and bone resorption. The imbalance caused by increased bone resorption over bone formation can lead to various bone-related diseases such as osteoporosis and rheumatoid arthritis. Osteoclasts are the principal cells responsible for bone resorption and the main targets of anti-resorptive therapies. However, excessive inhibition of osteoclast differentiation may lead to inhibition of osteoblast differentiation. Therefore, it is important to screen for new compounds capable of inhibiting bone resorption and enhancing bone formation. Toddalia asiatica (L.) Lam. has been utilized traditionally for medicinal purposes such as the treatment of rheumatism. Currently, the extract is considered to be a good source of pharmacological agents for the treatment of bone-related diseases, but the active compounds have yet to be identified. We investigated whether toddaculin, derived from Toddalia asiatica (L.) Lam., affects both processes by inhibiting bone resorption and enhancing bone formation. Towards this end, we used pre-osteoclastic RAW 264 cells and pre-osteoblastic MC3T3-E1 cells. We found that toddaculin not only inhibited the differentiation of osteoclasts via activation of the NF-?B, ERK 1/2, and p38 MAPK signaling pathways, but it also induced differentiation and mineralization of osteoblasts by regulating differentiation factors. Thus, toddaculin might be beneficial for the prevention and treatment of osteoporosis. PMID:25993011

  14. Cinnamon Extract Enhances Glucose Uptake in 3T3-L1 Adipocytes and C2C12 Myocytes by Inducing LKB1-AMP-Activated Protein Kinase Signaling

    PubMed Central

    Shen, Yan; Honma, Natsumi; Kobayashi, Katsuya; Jia, Liu Nan; Hosono, Takashi; Shindo, Kazutoshi; Ariga, Toyohiko; Seki, Taiichiro

    2014-01-01

    We previously demonstrated that cinnamon extract (CE) ameliorates type 1 diabetes induced by streptozotocin in rats through the up-regulation of glucose transporter 4 (GLUT4) translocation in both muscle and adipose tissues. This present study was aimed at clarifying the detailed mechanism(s) with which CE increases the glucose uptake in vivo and in cell culture systems using 3T3-L1 adipocytes and C2C12 myotubes in vitro. Specific inhibitors of key enzymes in insulin signaling and AMP-activated protein kinase (AMPK) signaling pathways, as well as small interference RNA, were used to examine the role of these kinases in the CE-induced glucose uptake. The results showed that CE stimulated the phosphorylation of AMPK and acetyl-CoA carboxylase. An AMPK inhibitor and LKB1 siRNA blocked the CE-induced glucose uptake. We also found for the first time that insulin suppressed AMPK activation in the adipocyte. To investigate the effect of CE on type 2 diabetes in vivo, we further performed oral glucose tolerance tests and insulin tolerance tests in type 2 diabetes model rats administered with CE. The CE improved glucose tolerance in oral glucose tolerance tests, but not insulin sensitivity in insulin tolerance test. In summary, these results indicate that CE ameliorates type 2 diabetes by inducing GLUT4 translocation via the AMPK signaling pathway. We also found insulin antagonistically regulates the activation of AMPK. PMID:24551069

  15. Screening of medicinal plants for PPAR? and PPAR? activation and evaluation of their effects on glucose uptake and 3T3-L1 adipogenesis.

    PubMed

    Yang, Min Hye; Avula, Bharathi; Smillie, Troy; Khan, Ikhlas A; Khan, Shabana I

    2013-08-01

    Medicinal plants are a rich source of ligands for nuclear receptors. The present study was aimed to screen a collection of plant extracts for PPAR?/?-activating properties and identify the active extract that can stimulate cellular glucose uptake without enhancing the adipogenesis. A reporter gene assay was performed to screen ethanolic extracts of 263 plant species, belonging to 94 families, for activation of PPAR? and PPAR?. Eight extracts showed activation of PPAR?, while 22 extracts showed activation of PPAR?. The extracts of five plants (Daphne gnidium, Illicium anisatum, Juniperus virginiana, Terminalia chebula, and Thymelaea hirsuta) showed activation of both PPAR? and PPAR? and out of them, D. gnidium and T. hirsuta markedly increased PPAR?/? protein expression. All five extracts showed an increase in cellular glucose uptake. Of the five dual agonists, T. chebula and T. hirsuta did not show any increase in differentiation of 3T3-L1 preadipocytes, but I. anisatum caused an increase in adipogenesis, while D. gnidium and J. virginiana were toxic to adipocytes. The adipogenic effect of rosiglitazone was antagonized by T. chebula and T. hirsuta. It was concluded that T. hirsuta and T. chebula retain the property of elevating glucose uptake as PPAR?/? dual agonists without the undesired side effect of adipogenesis. This is the first report to reveal the PPAR?/? dual agonistic action and glucose uptake enhancing property of T. hirsuta and T. chebula. PMID:23877921

  16. Ginsenoside 20S-protopanaxatriol (PPT) activates peroxisome proliferator-activated receptor gamma (PPARgamma) in 3T3-L1 adipocytes.

    PubMed

    Han, Kyu Lee; Jung, Myeong Ho; Sohn, Jong Hee; Hwang, Jae-Kwan

    2006-01-01

    Peroxisome proliferator-activated receptor gamma (PPARgamma), a member of the nuclear receptor of ligand-activated transcription factors, regulates the expression of key genes involved in lipid and glucose metabolism or adipocyte differentiation. Ligands for this receptor have emerged as potent insulin sensitizers used in the treatment of Type2 diabetes. Ginseng saponins or ginsenosides are reported to provide anti-diabetic activity as well as to modulate glucose metabolism, although the mechanism remains unclear. In this study, we examined the effect of ginsenosides on activation of PPARgamma and adipogenes in 3T3-L1. Using a GAL-4/PPARgamma transactivation assay, 20(S)-protopanaxatriol (PPT), one of the ginsenoside metabolites, was found to increase PPARgamma-transactivation activity dose-dependently with similar activity as troglitazone, a well-known PPARgamma agonist. PPT enhanced adipogenesis by increasing the expression of PPARgamma target genes such as aP2, LPL and PEPCK. Furthermore, PPT significantly increased expression of glucose transporter 4 (GLUT4). These results indicate that PPT can be developed as a PPARgamma agonist for the improvement of insulin resistance associated with diabetes. PMID:16394521

  17. (-)-Hydroxycitric acid attenuates endoplasmic reticulum stress-mediated alterations in 3T3-L1 adipocytes by protecting mitochondria and downregulating inflammatory markers.

    PubMed

    Nisha, V M; Priyanka, A; Anusree, S S; Raghu, K G

    2014-11-01

    Endoplasmic reticulum (ER) stress is an emerging potential therapeutic target for metabolic syndrome due to its role in synthesis, secretion, and folding of proteins. It leads to an increased production of reactive oxygen species (ROS) which, along with mitochondrial dysfunction and reduced antioxidant defense, causes chronic cell injury. The present investigation aims to observe the alterations in adipocytes due to ER stress and the protective effect of hydroxycitric acid (HCA), a bioactive from Garcinia species, to develop the same as a nutraceutical. ER stress was induced in mature 3T3-L1 adipocytes by treating them with tunicamycin (2?g/ml) for 18 h. Alterations in cell viability, innate antioxidant system (superoxide dismutase, glutathione peroxidase, and glutathione reductase), mitochondria (membrane potential, biogenesis, and transition pore opening), and inflammatory cytokines (tumor necrosis factor, monocyte chemoattractant protein, interferon-?, interleukin (IL)-10, IL-6, and IL-1?) during ER stress, and co-treatment with HCA were analyzed. Endocrine function of adipocytes was also assessed by measuring adiponectin and leptin secretion levels. HCA protected the cells from ER stress by improving the antioxidant status and mitochondrial functions. The results validate nutraceutical properties of the edible bioactive, commonly used for culinary purpose. A more detailed study on the mechanism of action of HCA is required for developing it as a therapeutic agent for metabolic syndrome. PMID:25175938

  18. Interactions between sub-10-nm iron and cerium oxide nanoparticles and 3T3 fibroblasts: the role of the coating and aggregation state

    NASA Astrophysics Data System (ADS)

    Safi, M.; Sarrouj, H.; Sandre, O.; Mignet, N.; Berret, J.-F.

    2010-04-01

    Recent nanotoxicity studies revealed that the physico-chemical characteristics of engineered nanomaterials play an important role in the interactions with living cells. Here, we report on the toxicity and uptake of cerium and iron oxide sub-10-nm nanoparticles by NIH/3T3 mouse fibroblasts. Coating strategies include low-molecular weight ligands (citric acid) and polymers (poly(acrylic acid), MW = 2000 g mol - 1). Electrostatically adsorbed on the surfaces, the organic moieties provide a negatively charged coating in physiological conditions. We find that most particles were biocompatible, as exposed cells remained 100% viable relative to controls. Only the bare and the citrate-coated nanoceria exhibit a slight decrease in mitochondrial activity at very high cerium concentrations (>1 g l - 1). We also observe that the citrate-coated particles are internalized/adsorbed by the cells in large amounts, typically 250 pg/cell after 24 h incubation for iron oxide. In contrast, the polymer-coated particles are taken up at much lower rates (<30 pg/cell). The strong uptake shown by the citrated particles is related to the destabilization of the dispersions in the cell culture medium and their sedimentation down to the cell membranes. In conclusion, we show that the uptake of nanomaterials by living cells depends on the coating of the particles and on its ability to preserve the colloidal nature of the dispersions.

  19. In Vitro and In Vivo Enhancement of Adipogenesis by Italian Ryegrass (Lolium multiflorum) in 3T3-L1 Cells and Mice

    PubMed Central

    Kim, Da Hye; Gun Roh, Sang; Lee, Jeong-Chae; Choi, Ki Choon

    2014-01-01

    Adipogenesis is very much important in improving the quality of meat in animals. The aim of the present study was to investigate the in vitro and in vivo adipogenesis regulation properties of Lolium multiflorum on 3T3-L1 pre-adipocytes and mice. Chemical composition of petroleum ether extract of L. multiflorum (PET-LM) confirmed the presence of fatty acids, such as ?-linolenic acid, docosahexaenoic acid, oleic acid, docosatetraenoic acid, and caprylic acid, as the major compounds. PET-LM treatment increased viability, lipid accumulation, lipolysis, cell cycle progression, and DNA synthesis in the cells. PET-LM treatment also augmented peroxysome proliferator activated receptor (PPAR)-?2, CCAAT/enhancer binding protein-?, adiponectin, adipocyte binding protein, glucose transporter-4, fatty acid synthase, and sterol regulatory element binding protein-1 expression at mRNA and protein levels in differentiated adipocytes. In addition, mice administered with 200 mg/kg body weight PET-LM for 8 weeks showed greater body weight than control mice. These findings suggest that PET-LM facilitates adipogenesis by stimulating PPAR?-mediated signaling cascades in adipocytes which could be useful for quality meat development in animals. PMID:24454838

  20. In vitro and in vivo enhancement of adipogenesis by Italian ryegrass (Lolium multiflorum) in 3T3-L1 cells and mice.

    PubMed

    Valan Arasu, Mariadhas; Ilavenil, Soundharrajan; Kim, Da Hye; Gun Roh, Sang; Lee, Jeong-Chae; Choi, Ki Choon

    2014-01-01

    Adipogenesis is very much important in improving the quality of meat in animals. The aim of the present study was to investigate the in vitro and in vivo adipogenesis regulation properties of Lolium multiflorum on 3T3-L1 pre-adipocytes and mice. Chemical composition of petroleum ether extract of L. multiflorum (PET-LM) confirmed the presence of fatty acids, such as ?-linolenic acid, docosahexaenoic acid, oleic acid, docosatetraenoic acid, and caprylic acid, as the major compounds. PET-LM treatment increased viability, lipid accumulation, lipolysis, cell cycle progression, and DNA synthesis in the cells. PET-LM treatment also augmented peroxysome proliferator activated receptor (PPAR)-?2, CCAAT/enhancer binding protein-?, adiponectin, adipocyte binding protein, glucose transporter-4, fatty acid synthase, and sterol regulatory element binding protein-1 expression at mRNA and protein levels in differentiated adipocytes. In addition, mice administered with 200 mg/kg body weight PET-LM for 8 weeks showed greater body weight than control mice. These findings suggest that PET-LM facilitates adipogenesis by stimulating PPAR?-mediated signaling cascades in adipocytes which could be useful for quality meat development in animals. PMID:24454838

  1. Bisphenol-A Impairs Insulin Action and Up-Regulates Inflammatory Pathways in Human Subcutaneous Adipocytes and 3T3-L1 Cells

    PubMed Central

    Valentino, Rossella; D’Esposito, Vittoria; Passaretti, Federica; Liotti, Antonietta; Cabaro, Serena; Longo, Michele; Perruolo, Giuseppe; Oriente, Francesco; Beguinot, Francesco; Formisano, Pietro

    2013-01-01

    Current evidence indicates that chemical pollutants may interfere with the homeostatic control of nutrient metabolism, thereby contributing to the increased prevalence of metabolic disorders. Bisphenol-A (BPA) is a lipophilic compound contained in plastic which is considered a candidate for impairing energy and glucose metabolism. We have investigated the impact of low doses of BPA on adipocyte metabolic functions. Human adipocytes derived from subcutaneous adipose tissue and differentiated 3T3-L1 cells were incubated with BPA, in order to evaluate the effect on glucose utilization, insulin sensitivity and cytokine secretion. Treatment with 1nM BPA significantly inhibited insulin-stimulated glucose utilization, without grossly interfering with adipocyte differentiation. Accordingly, mRNA levels of the adipogenic markers PPAR? and GLUT4 were unchanged upon BPA exposure. BPA treatment also impaired insulin-activated receptor phosphorylation and signaling. Moreover, adipocyte incubation with BPA was accompanied by increased release of IL-6 and IFN-?, as assessed by multiplex ELISA assays, and by activation of JNK, STAT3 and NFkB pathways. Treatment of the cells with the JNK inhibitor SP600125 almost fully reverted BPA effect on insulin signaling and glucose utilization. In conclusion, low doses of BPA interfere with inflammatory/insulin signaling pathways, leading to impairment of adipose cell function. PMID:24349194

  2. Isomeric C12-alkamides from the roots of Echinacea purpurea improve basal and insulin-dependent glucose uptake in 3T3-L1 adipocytes.

    PubMed

    Kotowska, Dorota; El-Houri, Rime B; Borkowski, Kamil; Petersen, Rasmus K; Fretté, Xavier C; Wolber, Gerhard; Grevsen, Kai; Christensen, Kathrine B; Christensen, Lars P; Kristiansen, Karsten

    2014-12-01

    Echinacea purpurea has been used in traditional medicine as a remedy for the treatment and prevention of upper respiratory tract infections and the common cold. Recent investigations have indicated that E. purpurea also has an effect on insulin resistance. A dichloromethane extract of E. purpurea roots was found to enhance glucose uptake in adipocytes and to activate peroxisome proliferator-activated receptor ?. The purpose of the present study was to identify the bioactive compounds responsible for the potential antidiabetic effect of the dichloromethane extract using a bioassay-guided fractionation approach. Basal and insulin-dependent glucose uptake in 3T3-L1 adipocytes were used to assess the bioactivity of extract, fractions and isolated metabolites. A peroxisome proliferator-activated receptor ? transactivation assay was used to determine the peroxisome proliferator-activated receptor ? activating properties of the extract, active fractions and isolated metabolites. Two novel isomeric dodeca-2E,4E,8Z,10E/Z-tetraenoic acid 2-methylbutylamides together with two known C12-alkamides and ?-linolenic acid were isolated from the active fractions. The isomeric C12-alkamides were found to activate peroxisome proliferator-activated receptor ?, to increase basal and insulin-dependent glucose uptake in adipocytes in a dose-dependent manner, and to exhibit characteristics of a peroxisome proliferator-activated receptor ? partial agonist. PMID:25371981

  3. Differentiation of MC3T3-E1 on poly(4-styrenesulfonic acid-co-maleic acid)sodium salt-coated films.

    PubMed

    Angwarawong, Thidarat; Dubas, Stephan T; Arksornnukit, Mansuang; Pavasant, Prasit

    2011-01-01

    Polyelectrolyte multilayer (PEM) film can modify the surface properties of materials to improve cellular responses. In this study poly(diallyldimethylammonium chloride) (PDADMAC), poly(sodium 4-styrene sulfonate) (PSS) and poly(4-styrenesulfonic acid-co-maleic acid) sodium salt (PSS-co-MA) were assembled into PEM {(PDADMAC/PSS)(4)/PDADMAC+PSS-co-MA} film on glass surfaces and its ability on affecting osteoblast functions were examined. PSS-co-MA film showed an increase roughness and more wettable surface as compared to glass. When the osteoblast cell line, MC3T3-E1, was seeded on the surfaces, no differences were observed in cell attachment or spreading on either PSS-co-MA film or glass at 4-16 hours. However, increases in alkaline phosphatase activity (day-5 and 7) and the expression of osteocalcin mRNA/protein at day-13 were observed. Cells cultured on PSS-co-MA film developed a faster rate of calcium deposition at day-15 compared to control. In conclusion, PSS-co-MA film enhanced osteoblast differentiation and could be used to promote mineralization and improve osseointegration for dental implants. PMID:21422666

  4. Assessment of cytocompatibility of surface-modified CdSe/ZnSe quantum dots for BALB/3T3 fibroblast cells.

    PubMed

    Mahto, Sanjeev Kumar; Park, Chansik; Yoon, Tae Hyun; Rhee, Seog Woo

    2010-06-01

    With the widespread use of quantum dots (QDs), the likelihood of exposure to QDs has been assumed to have increased substantially. Recently, QDs have been employed in numerous biological and medical applications. However, there is a lack of toxicological data pertaining to QDs. In this study, we aimed to investigate the cytocompatibility of surface-modified CdSe/ZnSe QDs for BALB/3T3 fibroblast cells. The ligands used for surface modification are mercaptopropionic acid (MPA) and Gum arabic (GA)/tri-n-octylphosphine oxide (TOPO). Cells were exposed to different concentrations of QDs followed by illustrative cytotoxicity analyses. Furthermore, we used a confocal microscope to assess intracellular uptake of QDs. Confocal images showed that MPA-coated QDs were distributed inside the cytoplasmic region of cells. In contrast, GA/TOPO-coated QDs were not found inside cells. MPA-coated QDs were highly cytocompatible, whereas GA/TOPO-coated QDs were toxic to the cells. Cells treated with GA/TOPO-coated QDs showed altered morphology, decreased viability, significant concentrations of intracellular free cadmium, detectable reactive oxygen species (ROS) formation, depolymerized cytoskeleton, and irregular-shaped nuclei. This study suggests that surface modification by ligands plays a significant role in the prevention of cytotoxicity of QDs. PMID:20362659

  5. Response of newly established mouse myeloid leukemic cell lines to MC3T3-G2/PA6 preadipocytes and hematopoietic factors

    SciTech Connect

    Kodama, H.; Iizuka, M.; Tomiyama, T.; Yoshida, K.; Seki, M.; Suda, T.; Nishikawa, S. (Ohu Univ. School of Dentistry, Koriyama (Japan))

    1991-01-01

    Some mouse myeloid leukemias induced by X-irradiation and serially transplanted into syngenic mice do not proliferate in vitro even in the presence of hematopoietic factors. To examine whether such leukemic cells can proliferate in response to stromal cells, we cocultured them with MC3T3-G2/PA6 (PA6) preadipocytes, cells that can support the growth of hematopoietic stem cells. All leukemias developed into in vitro cell lines, showing a dependence on contact with the PA6 cells. Two cell lines responded to none of the known hematopoietic factors including interleukin-3 (IL-3), IL-4, IL-5, IL-6, GM-CSF, G-CSF, M-CSF, and Epo. These results demonstrate that the mechanism of the action of PA6 cells is different from that of any of the known hematopoietic factors, and that, because these two leukemic cell lines retained the ability to grow in vivo, responsiveness to the known hematopoietic factors is not essential for the leukemic cell growth in vivo. Furthermore, all leukemic cell lines could respond also to the preadipocytes fixed with formalin, paraformaldehyde, or glutaraldehyde, suggesting that some molecule(s) associated with the surface of PA6 cells or with extracellular matrix secreted by the preadipocytes is responsible for the leukemic cell growth.

  6. 3T3 fibroblasts induce cloned interleukin 3-dependent mouse mast cells to resemble connective tissue mast cells in granular constituency

    SciTech Connect

    Dayton, E.T.; Pharr, P.; Ogawa, M.; Serafin, W.E.; Austen, K.F.; Levi-Schaffer, F.; Stevens, R.L.

    1988-01-01

    As assessed by ultrastructure, histochemical staining, and T-cell dependency, in vitro-differentiated interleukin 3-dependent mouse mast cells are comparable to the mast cells that reside in the gastrointestinal mucosa but not in the skin or the serosal cavity of the mouse. The authors now demonstrate that when cloned interleukin 3-dependent mast cells are cocultured with mouse skin-derived 3T3 fibroblasts in the presence of WEHI-3 conditioned medium for 28 days, the mast cells acquire the ability to stain with safranin, increase their histamine content approx. 50-fold and their carboxypeptidase. A content approx. 100-fold, and augment approx. their biosynthesis of proteoglycans bearing /sup 35/S-labeled haparin relative to /sup 35/S-labeled chondroitin sulfate glycosaminoglycans. Thus, fibroblasts induce interleukin 3-dependent mouse mast cells to change phenotype from mucosal-like to connective tissue-like, indicating that the biochemical and functional characteristics of this mast cell type are strongly influenced by the connective tissue microenvironment.

  7. Screening of dried plant seed extracts for adiponectin production activity and tumor necrosis factor-alpha inhibitory activity on 3T3-L1 adipocytes.

    PubMed

    Okada, Yoshinori; Okada, Mizue; Sagesaka, Yumi

    2010-09-01

    To search for dried plant seeds with potent anti-diabetes activity, we conducted a large scale screening for inhibitory activity on tumor necrosis factor-alpha and facilitating activity on adiponectin production in vitro. These activities in 3T3-L1 adipocytes were screened from ethanol extracts of 20 kinds of dried plant seed marketed in Japan. komatsuna (Brassica rapa var. perviridis), common bean (Phaseolus vulgaris L.), qing geng cai (Brassica rapa var. chinensis), green soybean (Glycine max), spinach (Spinacia oleracea L.) and sugar snap pea (Pisum sativum L.) markedly enhanced adiponectin production (11.3?~?12.7 ng/ml) but Japanese radish (Raphanus sativus), edible burdock (Arctium lappa L.), bitter melon (Momordica charantia) and broccoli (Brassica oleracea var. italica) did not (0.9?~?2.7 ng/ml). All adiponectin-production-enhancing seeds except spinach (2.7 pg/ml) and okra (Abelmoschus esculentus) (6.6 pg/ml) effectively decreased tumor necrosis factor-alpha levels (0.0 pg/ml). We further examined the effects on free radical scavenging activities in the dried seed extracts. Although scavenging activity correlated well with total phenolic content of samples, no correlation was observed with adiponectin production. These results point to the potential of dried seed extracts as a means to modify the activity of tumor necrosis factor-alpha for the adiponectin production. PMID:20717728

  8. Muscarinic receptors transform NIH 3T3 cells through a Ras-dependent signalling pathway inhibited by the Ras-GTPase-activating protein SH3 domain.

    PubMed Central

    Mattingly, R R; Sorisky, A; Brann, M R; Macara, I G

    1994-01-01

    Expression of certain subtypes of human muscarinic receptors in NIH 3T3 cells provides an agonist-dependent model of cellular transformation by formation of foci in response to carbachol. Although focus formation correlates with the ability of the muscarinic receptors to activate phospholipase C, the actual mitogenic signal transduction pathway is unknown. Through cotransfection experiments and measurement of the activation state of native and epitope-tagged Ras proteins, the contributions of Ras and Ras GTPase-activating protein (Ras-GAP) to muscarinic receptor-dependent transformation were defined. Transforming muscarinic receptors were able to activate Ras, and such activation was required for transformation because focus formation was inhibited by coexpression of either Ras with a dominant-negative mutation or constructs of Ras-GAP that include the catalytic domain. Coexpression of the N-terminal region of GAP or of its isolated SH3 (Src homology 3) domain, but not its SH2 domain, was also sufficient to suppress muscarinic receptor-dependent focus formation. Point mutations at conserved residues in the Ras-GAP SH3 domain reversed its action, leading to an increase in carbachol-dependent transformation. The inhibitory effect of expression of the Ras-GAP SH3 domain occurs proximal to Ras activation and is selective for the mitogenic pathway activated by carbachol, as cellular transformation by either v-Ras or trkA/nerve growth factor is unaffected. Images PMID:7969134

  9. Protection of fibroblasts (NIH-3T3) against oxidative damage by cyanidin-3-rhamnoglucoside isolated from fig fruits (Ficus carica L.).

    PubMed

    Solomon, Anat; Golubowicz, Sara; Yablowicz, Zeev; Bergman, Margalit; Grossman, Shlomo; Altman, Arie; Kerem, Zohar; Flaishman, Moshe A

    2010-06-01

    Anthocyanins, plant secondary metabolites, have been recognized for their health-promoting properties when consumed by humans. In this study, the antioxidant properties of a major anthocyanin in fresh fig fruits, cyanidin-3-rhamnoglucoside (C3R), were evaluated by various assays in vitro and correlated with the protection afforded by C3R to cultured NIH-3T3 fibroblast cells. C3R inhibited lipid peroxidation from producing peroxy radicals (ROO(*)) and MDA in a dose-dependent manner, and a high calculated stoichiometric coefficient [n] for peroxy radicals was demonstrated. In addition to its scavenging of reactive oxygen species (ROS), C3R showed a strong chelating activity toward the Fe(2+) ion. Finally, pretreatment with C3R inhibited proapoptotic processes that were initiated by the oxidation of lysosome membranes in fibroblast cells. The high antioxidant potential, with several modes of action of purified C3R, may contribute to health benefits gained by the consumption of fresh fig fruits. PMID:20443626

  10. Extract from Edible Red Seaweed (Gelidium amansii) Inhibits Lipid Accumulation and ROS Production during Differentiation in 3T3-L1 Cells

    PubMed Central

    Seo, Min-Jung; Lee, Ok-Hwan; Choi, Hyeon-Son; Lee, Boo-Yong

    2012-01-01

    Gelidium (G.) amansii is a red alga widely distributed in the shallow waters around East Asian countries. We investigated the effect of G. amansii on lipid accumulation and ROS (Reactive Oxygen Species) production in 3T3-L1 cells. G. amansii extracts dose-dependently inhibited lipid formation and ROS generation in cultured cells. Our results showed that anti-adipogenic effect of G. amansii was due to the reduction in mRNA expressions of PPAR? peroxisome proliferator-activated receptor-? and aP2 (adipocyte protein 2). G. amansii extracts significantly decreased mRNA levels of a ROS-generator, NOX4 (nicotinamide adenine dinucleotide phosphate hydrogen oxidase 4), and increased the protein levels of antioxidant enzymes including SOD1/2 (superoxide dis-mutases), Gpx (glutathione peroxidase), and GR (glutathione reductase), which can lead to the reduction of ROS in the cell. In addition, the G. amansii extract enhanced mRNA levels of adiponectin, one of the adipokines secreted from adipocytes, and GLUT4, glucose uptake protein. Taken together, our study shows that G. amansii extract inhibited lipid accumulation and ROS production by controlling adipogenic signals and ROS regulating genes. PMID:24471074

  11. Extract from Edible Red Seaweed (Gelidium amansii) Inhibits Lipid Accumulation and ROS Production during Differentiation in 3T3-L1 Cells.

    PubMed

    Seo, Min-Jung; Lee, Ok-Hwan; Choi, Hyeon-Son; Lee, Boo-Yong

    2012-06-01

    Gelidium (G.) amansii is a red alga widely distributed in the shallow waters around East Asian countries. We investigated the effect of G. amansii on lipid accumulation and ROS (Reactive Oxygen Species) production in 3T3-L1 cells. G. amansii extracts dose-dependently inhibited lipid formation and ROS generation in cultured cells. Our results showed that anti-adipogenic effect of G. amansii was due to the reduction in mRNA expressions of PPAR? peroxisome proliferator-activated receptor-? and aP2 (adipocyte protein 2). G. amansii extracts significantly decreased mRNA levels of a ROS-generator, NOX4 (nicotinamide adenine dinucleotide phosphate hydrogen oxidase 4), and increased the protein levels of antioxidant enzymes including SOD1/2 (superoxide dis-mutases), Gpx (glutathione peroxidase), and GR (glutathione reductase), which can lead to the reduction of ROS in the cell. In addition, the G. amansii extract enhanced mRNA levels of adiponectin, one of the adipokines secreted from adipocytes, and GLUT4, glucose uptake protein. Taken together, our study shows that G. amansii extract inhibited lipid accumulation and ROS production by controlling adipogenic signals and ROS regulating genes. PMID:24471074

  12. Neoplastic transformation and tumorigenesis associated with overexpression of imup-1 and imup-2 genes in cultured NIH/3T3 mouse fibroblasts

    SciTech Connect

    Ryoo, Zae Young [School of Life Science and Biotechnology, Kyungpook National University, Daegu 702-701 (Korea, Republic of)]. E-mail: jaewoong64@hanmail.net; Jung, Boo Kyoung [School of Life Science and Biotechnology, Kyungpook National University, Daegu 702-701 (Korea, Republic of); Department of Laboratory Animal Medicine, College of Veterinary Medicine, Seoul National University, Seoul 110-799 (Korea, Republic of); Lee, Sang Ryeul [School of Life Science and Biotechnology, Kyungpook National University, Daegu 702-701 (Korea, Republic of); Kim, Myoung Ok [School of Life Science and Biotechnology, Kyungpook National University, Daegu 702-701 (Korea, Republic of); Kim, Sung Hyun [School of Life Science and Biotechnology, Kyungpook National University, Daegu 702-701 (Korea, Republic of); Kim, Hyo Jin [CHA Research Institute, Pochon CHA University, Sungnam 463-836 (Korea, Republic of); Ahn, Jung Yong [CHA Research Institute, Pochon CHA University, Sungnam 463-836 (Korea, Republic of); Lee, Tae-Hoon [Department of Oral Biochemistry, College of Dentistry, Chonnam National University, Gwangju 500-757 (Korea, Republic of); Cho, Youl Hee [Department of Medical Genetics, College of Medicine, Hanyang University, Seoul 133-791 (Korea, Republic of); Park, Jae Hak [Department of Laboratory Animal Medicine, College of Veterinary Medicine, Seoul National University, Seoul 110-799 (Korea, Republic of); Kim, Jin Kyeoung [CHA Research Institute, Pochon CHA University, Sungnam 463-836 (Korea, Republic of); Graduate School of Life Science and Biotechnology, College of Medicine, Pochon CHA University, Sungnam 463-836 (Korea, Republic of)

    2006-10-27

    Immortalization-upregulated protein 1 (IMUP-1) and immortalization-upregulated protein 2 (IMUP-2) genes have been recently cloned and are known to be involved in SV40-mediated immortalization. IMUP-1 and IMUP-2 genes were strongly expressed in various cancer cell lines and tumors, suggesting the possibility that they might be involved in tumorigenicity. To directly elucidate the functional role of IMUP-1 and IMUP-2 on neoplastic transformation and tumorigenicity, we stably transfected IMUP-1 and IMUP-2 into NIH/3T3 mouse fibroblast cells. Cellular characteristics of the neoplastic transformation were assessed by transformation foci, growth in soft agar, and tumor development in nude mice. We found that IMUP-1 and IMUP-2 overexpressing cells showed altered growth properties, anchorage-independent growth in soft agar and inducing tumor in nude mice. Furthermore, IMUP-1 and IMUP-2 transformants proliferated in reduced serum and shortened cell cycle. These results suggest that ectopic overexpression of IMUP-1 and IMUP-2 may play an important role in acquiring a transformed phenotype, tumorigenicity in vivo, and be related to cellular proliferation.

  13. Mouse 3T3-L1 cell variants unable to respond to mitogenic stimulation of dihydroteleocidin B: genetic evidence for the synergism of tumor promoters with growth factors

    SciTech Connect

    Shimizu, Y.; Fujiki, H.; Sugimura, T.; Shimizu, N.

    1986-08-01

    Dihydroteleocidin B, an indole alkaloid tumor promoter, stimulates confluent, quiescent mouse 3T3-L1 fibroblasts to initiate DNA synthesis and undergo cell division. Using a mitotic shakeoff technique, we have isolated 12 clones of genetic variants which are unable to respond to the mitogenic stimulation of dihydroteleocidin B from a total of 12 million cells. Biochemical characterization of these nonresponsive variants to dihydroteleocidin B revealed that there is no change in the ability to bind (/sup 3/H)phorbol dibutyrate, the activity of protein kinase C, and the turnover of phosphatidylinositol. The evidence indicates that nonresponsiveness to dihydroteleocidin B is caused by several different lesions, including defects in receptors for insulin or epidermal growth factor and in the postreceptor mechanisms. The evidence also suggests that mitogenic signal transfer via the epidermal growth factor receptor system appears to share a common step with dihydroteleocidin B whereas the signal transfer for insulin seems separate from these. These results suggest that phosphatidylinositol turnover followed by protein kinase C activation alone is not sufficient for mitogenic stimulation and that the coordination of the protein kinase C system with the receptor systems for growth factors may be necessary for ''full'' mitogenic response.

  14. Myosin IIA participates in docking of Glut4 storage vesicles with the plasma membrane in 3T3-L1 adipocytes

    SciTech Connect

    Chung, Le Thi Kim, E-mail: ngocanh@nutr.med.tokushima-u.ac.jp [Department of Nutrition and Metabolism, Institute of Health Biosciences, The University of Tokushima Graduate School, 3-18-15 Kuramoto-cho, Tokushima 770-8503 (Japan); Hosaka, Toshio [Department of Public Health and Applied Nutrition, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima (Japan)] [Department of Public Health and Applied Nutrition, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima (Japan); Harada, Nagakatsu; Jambaldorj, Bayasgalan; Fukunaga, Keiko; Nishiwaki, Yuka [Department of Nutrition and Metabolism, Institute of Health Biosciences, The University of Tokushima Graduate School, 3-18-15 Kuramoto-cho, Tokushima 770-8503 (Japan)] [Department of Nutrition and Metabolism, Institute of Health Biosciences, The University of Tokushima Graduate School, 3-18-15 Kuramoto-cho, Tokushima 770-8503 (Japan); Teshigawara, Kiyoshi [Clinical Research Center for Diabetes, Tokushima University Hospital, 2-50-1 Kuramoto-cho, Tokushima 770-8503 (Japan)] [Clinical Research Center for Diabetes, Tokushima University Hospital, 2-50-1 Kuramoto-cho, Tokushima 770-8503 (Japan); Sakai, Tohru [Department of Public Health and Applied Nutrition, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima (Japan)] [Department of Public Health and Applied Nutrition, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima (Japan); Nakaya, Yutaka [Department of Nutrition and Metabolism, Institute of Health Biosciences, The University of Tokushima Graduate School, 3-18-15 Kuramoto-cho, Tokushima 770-8503 (Japan)] [Department of Nutrition and Metabolism, Institute of Health Biosciences, The University of Tokushima Graduate School, 3-18-15 Kuramoto-cho, Tokushima 770-8503 (Japan); Funaki, Makoto, E-mail: m-funaki@clin.med.tokushima-u.ac.jp [Clinical Research Center for Diabetes, Tokushima University Hospital, 2-50-1 Kuramoto-cho, Tokushima 770-8503 (Japan)] [Clinical Research Center for Diabetes, Tokushima University Hospital, 2-50-1 Kuramoto-cho, Tokushima 770-8503 (Japan)

    2010-01-01

    In adipocytes and myocytes, insulin stimulation translocates glucose transporter 4 (Glut4) storage vesicles (GSVs) from their intracellular storage sites to the plasma membrane (PM) where they dock with the PM. Then, Glut4 is inserted into the PM and initiates glucose uptake into these cells. Previous studies using chemical inhibitors demonstrated that myosin II participates in fusion of GSVs and the PM and increase in the intrinsic activity of Glut4. In this study, the effect of myosin IIA on GSV trafficking was examined by knocking down myosin IIA expression. Myosin IIA knockdown decreased both glucose uptake and exposures of myc-tagged Glut4 to the cell surface in insulin-stimulated cells, but did not affect insulin signal transduction. Interestingly, myosin IIA knockdown failed to decrease insulin-dependent trafficking of Glut4 to the PM. Moreover, in myosin IIA knockdown cells, insulin-stimulated binding of GSV SNARE protein, vesicle-associated membrane protein 2 (VAMP2) to PM SNARE protein, syntaxin 4 was inhibited. These data suggest that myosin IIA plays a role in insulin-stimulated docking of GSVs to the PM in 3T3-L1 adipocytes through SNARE complex formation.

  15. Purification and Characterization of Aporphine Alkaloids from Leaves of Nelumbo nucifera Gaertn and Their Effects on Glucose Consumption in 3T3-L1 Adipocytes

    PubMed Central

    Ma, Chengjun; Wang, Jinjun; Chu, Hongmei; Zhang, Xiaoxiao; Wang, Zhenhua; Wang, Honglun; Li, Gang

    2014-01-01

    Aporphine alkaloids from the leaves of Nelumbo nucifera Gaertn are substances of great interest because of their important pharmacological activities, particularly anti-diabetic, anti-obesity, anti-hyperlipidemic, anti-oxidant, and anti-HIV’s activities. In order to produce large amounts of pure alkaloid for research purposes, a novel method using high-speed counter-current chromatography (HSCCC) was developed. Without any initial cleanup steps, four main aporphine alkaloids, including 2-hydroxy-1-methoxyaporphine, pronuciferine, nuciferine and roemerine were successfully purified from the crude extract by HSCCC in one step. The separation was performed with a simple two-phase solvent system composed of n-hexane-ethyl acetate-methanol-acetonitrile-water (5:3:3:2.5:5, v/v/v/v/v). In each operation, 100 mg crude extracts was separated and yielded 6.3 mg of 2-hydroxy-1-methoxyaporphine (95.1% purity), 1.1 mg of pronuciferine (96.8% purity), 8.5 mg of nuciferine (98.9% purity), and 2.7 mg of roemerine (97.4%) respectively. The chemical structure of four aporphine alkaloids are identified by means of electrospray ionization MS (ESI-MS) and nuclear magnetic resonance (NMR) analysis. Moreover, the effects of four separated aporphine alkaloids on insulin-stimulated glucose consumption were examined in 3T3-L1 adipocytes. The results showed that 2-hydroxy-1-methoxyaporphine and pronuciferine increased the glucose consumption significantly as rosiglitazone did. PMID:24577311

  16. Vitronectin Absorbed on Nanoparticles Mediate Cell Viability/Proliferation and Uptake by 3T3 Swiss Albino Mouse Fibroblasts: In Vitro Study

    PubMed Central

    Rosso, F.; Marino, G.; Grimaldi, A.; Cafiero, G.; Chiellini, E.; Chiellini, F.; Barbarisi, M.; Barbarisi, A.

    2013-01-01

    We study the interaction of 3T3 Swiss albino mouse fibroblasts with polymeric nanoparticles (NPs) and investigate cellular behaviour in terms of viability/cytotoxicity, cell cycle, NPs uptake, MAP kinase (ERK1/2), and focal adhesion kinase (FAK) activation. After incubation of NPs with cell culture media, western blot analysis showed that Vitronectin is retained by NPs, while Fibronectin is not detected. From cytotoxicity studies (MTT and BrdU methods) an LD50 of about 1.5?mg/mL results for NPs. However, NPs in the range 0.01–0.30?mg/mL are able to trigger a statistically significant increase in proliferation and cell cycle progression in dose and time depending manner. Also, biochemical evaluation of ERK1/2 and FAK clearly shows an increasing phosphorylation in a dose and time depending manner. Finally, we found by transmission electron microscopy that NPs are internalised by cells. Competitively blocking VN-integrin receptors with echistatin (1??g/mL) results in a decrease of viability/proliferation, cell cycle progression, cellular uptake, and FAK/ERK activation showing the involvement of Vitronectin receptors in signal transduction. In conclusion, our results show that cell surface NPs interactions are mediated by absorbed plasma proteins (i.e., Vitronectin) that represent an external stimuli, switched to the nucleus by FAK enzyme, which in turn modulate fibroblasts viability/proliferation. PMID:23710450

  17. Reduced induction of c-fos but not of c-myc expressions in a nontumorigenic revertant R1 of EJ-ras-transformed NIH/3T3 cells treated with 12-O-tetradecanoylphorbol-13-acetate (TPA)

    SciTech Connect

    Suzuki, Hiroaki; Fujita, Hisakazu; Ogiso, Yoshifumi; Oda, Atsushi; Kuzumaki, Noboru; Uchino, Junichi (Hokkaido Univ., Sapporo (Japan))

    1989-10-01

    It has been reported that both c-fos and c-myc mRNAs are induced in NIH/3T3 cells after 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment. The authors have studied the effect of TPA on the expression of c-fos and c-myc in EJ-ras-transformed NIH/3T3 and its nontumorigenic flat revertant R1 cells. Although TPA treatment induces c-myc mRNA, as in the case of NIH/3T3 cells, the induced level of c-fos mRNA is greatly reduced not only in slow growing EJ-ras-transformed NIH/3T3 but also in quiescent R1 cells. In addition, serum-induced c-fos expression is also reduced in EJ-ras-transformed NIH/3T3 and R1 cells. These observations suggest that the pathway from TPA to c-fos gene is different from that to c-myc gene and that the former pathway is down-regulated in association not with the transformed phenotype, but with EJ-ras expression, and it is possible that this reduced induction of c-fox is not specific to TPA.

  18. Sulfonate groups grafted on Ti6Al4V favor MC3T3-E1 cell performance in serum free medium conditions.

    PubMed

    Felgueiras, Helena; Migonney, Véronique

    2014-06-01

    Ten years ago, we synthesized "bioactive model polymers" bearing sulfonate groups and proposed a mechanism of their modulation effect at different steps of the cell response. Then, we set up the grafting of polymers bearing sulfonate on Ti6Al4V surfaces by a grafting "from" technique making sure of the creation of covalent bonds between the grafted polymer and the Ti6Al4V surface. We have checked and confirmed the positive effect of grafted sulfonate groups on the osteoblastic cell response in vivo and in vitro but we did not elucidate the mechanism. The aim of this basic work consists first in investigating the role of sulfonate groups in the presence and in the absence of proteins at early stages of the osteointegration process on poly(sodium styrene sulfonate) poly(NaSS) grafted and ungrafted Ti6Al4V surfaces, in vitro. To understand the role of poly(NaSS) grafted chains on osteoblast-like cell response and to confirm/elucidate the importance of fetal bovine serum (FBS) proteins in the culture medium, MC3T3-E1 cells were seeded onto poly(NaSS) grafted and non-grafted Ti6Al4V surfaces. Cultures were carried out in a complete (10% FBS) and in a non-complete medium (without FBS). Cell viability assay, cell attachment number and cell adhesion strength were followed up to 3days of culture. The presence of proteins enhanced cell growth and development whatever the surface and the presence of sulfonate groups enhanced the cell attachment even in the absence of proteins, which suggests and confirms that the sulfonate groups can modify the activity of cells such as the secretion of binding proteins. Statistical differences were found in the attachment strength tests on poly(NaSS) grafted and ungrafted surfaces and showed that the sulfonate groups play an important role in the cell resistance to shear stress. PMID:24863216

  19. Isolation of the molecular species of monogalactosyldiacylglycerols from brown edible seaweed Sargassum horneri and their inhibitory effects on triglyceride accumulation in 3T3-L1 adipocytes.

    PubMed

    Ma, Ai-Cui; Chen, Zhen; Wang, Tao; Song, Ni; Yan, Qian; Fang, Yu-Chun; Guan, Hua-Shi; Liu, Hong-Bing

    2014-11-19

    The chemical composition of monogalactosyldiacylglycerols (MGDGs) from brown alga Sargassum horneri and their inhibitory effects on lipid accumulation were investigated in this study. A total of 10 molecular species of MGDGs were identified using nuclear magnetic resonance, alkaline hydrolysis, gas chromatography-flame ionization detector, and high-performance liquid chromatography-tandem mass spectrometry methods. Individual molecular species of MGDGs, including (2S)-1-O-myristoyl-2-O-palmitoleoyl-3-O-?-D-galactopyranosyl-sn-glycerol (1), (2S)-1-O-myristoyl-2-O-linoleyl-3-O-?-D-galactopyranosyl-sn-glycerol (3), (2S)-1-O-palmitoyl-2-O-linolenoyl-3-O-?-D-galactopyranosyl-sn-glycerol (5), (2S)-1-O-myristoyl-2-O-oleyl-3-O-?-D-galactopyranosyl-sn-glycerol (7), (2S)-1-O-palmitoyl-2-O-palmitoleoyl-3-O-?-D-galactopyranosyl-sn-glycerol (8), (2S)-1-O-palmitoyl-2-O-linoleyl-3-O-?-D-galactopyranosyl-sn-glycerol (9), and (2S)-1-O-palmitoyl-2-O-oleyl-3-O-?-D-galactopyranosyl-sn-glycerol (10), were then furnished using semi-preparative high-performance liquid chromatography, and their inhibitory effects on triglyceride (TG) accumulation and free fatty acid (FFA) levels in 3T3-L1 adipocytes were evaluated. Compounds 3 and 9 showed inhibitory effects on TG and FFA accumulation, with TG levels of 1.568 ± 0.2808 and 1.701 ± 0.1460 ?mol/L and FFA levels of 0.149 ± 0.0258 and 0.198 ± 0.0229 mequiv/L, respectively, which were more effective than other compounds. The primary structure-activity relationship suggested that linoleyl [18:2(?-6)] in the sn-2 position played an important role on triglyceride accumulation inhibition. PMID:25363514

  20. Cytotoxicity and morphological transforming potential of cobalt nanoparticles, microparticles and ions in Balb/3T3 mouse fibroblasts: an in vitro model.

    PubMed

    Sabbioni, Enrico; Fortaner, Salvador; Farina, Massimo; Del Torchio, Riccardo; Olivato, Iolanda; Petrarca, Claudia; Bernardini, Giovanni; Mariani-Costantini, Renato; Perconti, Silvia; Di Giampaolo, Luca; Gornati, Rosalba; Di Gioacchino, Mario

    2014-06-01

    We previously described the behaviour of different cobalt forms, i.e., cobalt nanoparticles (CoNP), cobalt microparticles (CoMP) and cobalt ions (Co(2+)), in culture medium (dissolution, interaction with medium components, bioavailability) as well as their uptake and intracellular distribution in Balb/3T3 mouse fibroblasts (Sabbioni, Nanotoxicology, 2012). Here, we assess the cytotoxicity and morphological transformation of CoNP compared not only to Co(2+), but also to CoMP and to released Co products. Cytotoxicity reached maximum at 4-h exposure, with ranking CoMP > CoNP > Co(2+). However, if we consider toxicity as a function of intracellular Co, toxicity of the ionic forms seems to prevail over the particles. Co forms other than Co(2+) released from particles had toxicity intermediate between particles and ions. Alterations in concentrations of essential elements (Cu, Mg, Zn) in cells exposed to Co particles may contribute to toxicity. Both CoMP and CoNP (but not Co(2+) and other released Co forms) induced morphological transformation (CoMP > CoNP). This was dependent on reactive oxygen species production and lipid peroxidation, as indicated by inhibition of type III foci with ascorbic acid. The present results suggest that the previously demonstrated massive mitochondrial and nuclear Co internalisation and DNA adduct formation by CoMP and CoNP (Sabbioni, Nanotoxicology, 2012) induce toxicity and transformation. On the contrary, the role of ions released by particles in culture medium is negligible. Thus, both the chemical and the physical properties of Co particles contribute to cytotoxicity and morphological transformation. PMID:23586465

  1. Long Non-Coding RNA NEAT1 Associates with SRp40 to Temporally Regulate PPAR?2 Splicing during Adipogenesis in 3T3-L1 Cells.

    PubMed

    Cooper, Denise R; Carter, Gay; Li, Pengfei; Patel, Rehka; Watson, James E; Patel, Niketa A

    2014-01-01

    Long non-coding (lnc) RNAs serve a multitude of functions in cells. NEAT1 RNA is a highly abundant 4 kb lncRNA in nuclei, and coincides with paraspeckles, nuclear domains that control sequestration of paraspeckle proteins. We examined NEAT1 RNA levels and its function in 3T3-L1 cells during differentiation to adipocytes. Levels of NEAT1 transcript, measured by RT-PCR, fluctuated in a temporal manner over the course of differentiation that suggested its role in alternative splicing of PPAR? mRNA, the major transcription factor driving adipogenesis. When cells were induced to differentiate by a media cocktail of insulin, dexamethasone, and isobutylmethyxanthine (IBMX) on Day 0, NEAT1 levels dropped on Day 4, when the PPAR?2 variant was spliced and when terminal differentiation occurs The appearance of PPAR?2 coordinates with the PPAR?1 variant to drive differentiation of adipocytes. SiRNA used to deplete NEAT1 resulted in the inability of cells to phosphorylate the serine/arginine-rich splicing protein, SRp40. SiRNA treatment for SRp40 resulted in dysregulation of PPAR?1 and, primarily, PPAR?2 mRNA levels. SRp40 associated with NEAT1, as shown by RNA-IP on days 0 and 8, but decreased on day 4, and concentrations increased over that of IgG control. Overexpression of SRp40 increased PPAR?2, but not ?1. Although lncRNA MALAT1 has been investigated in SR protein function, NEAT1 has not been shown to bind SR proteins for phosphorylation such that alternative splicing results. The ability of cells to increase phosphorylated SR proteins for PPAR?2 splicing suggests that fluxes in NEAT1 levels during adipogenesis regulate alternative splicing events. PMID:25437750

  2. Sodium alginate-cross-linked polymyxin B sulphate-loaded solid lipid nanoparticles: Antibiotic resistance tests and HaCat and NIH/3T3 cell viability studies.

    PubMed

    Severino, Patrícia; Chaud, Marco V; Shimojo, Andrea; Antonini, Danilo; Lancelloti, Marcelo; Santana, Maria Helena A; Souto, Eliana B

    2015-05-01

    Polymyxins are a group of antibiotics with a common structure of a cyclic peptide with a long hydrophobic tail. Polymyxin B sulphate (PLX) has cationic charge, which is an obstacle for the efficient loading into Solid Lipid Nanoparticles (SLN). In the present paper, we describe an innovative method to load PLX into SLN to achieve the sustained release of the drug. PLX was firstly cross-linked with sodium alginate (SA) at different ratios (1:1, 1:2 and 1:3 SA/PLX), and loaded into SLN produced by high pressure homogenization (HPH). Optimized SLN were produced applying 500bar pressure and 5 homogenization cycles. The best results were obtained with SA/PLX (1:1), recording 99.08±1.2% for the association efficiency of the drug with SA, 0.99±10g for the loading capacity and 212.07±5.84% degree of swelling. The rheological profile of aqueous SA solution followed the typical behaviour of concentrated polymeric solutions, whereas aqueous SA/PLX solution exhibited a gel-like dynamic behaviour. Micrographs show that SA/PLX depicted a porous and discontinuous amorphous phase in different ratios. The encapsulation efficiency of SA/PLX (1:1) in SLN, the mean particle diameter, polydispersity index and zeta potential were, respectively, 82.7±5.5%; 439.5±20.42nm, 0.241±0.050 and -34.8±0.55mV. The effect of SLN on cell viability was checked in HaCat and NIH/3T3 cell lines, and the minimal inhibitory concentrations (MIC) were determined in Pseudomonas aeruginosa strains. SA/PLX-loaded SLN were shown to be less toxic than free PLX. Minimal inhibitory concentrations (MIC) showed the presence of the cross-linker polymer-drug complex, and SLN were shown to enhance MIC in the evaluated strains. PMID:25863712

  3. Activation of the Ras/mitogen-activated protein kinase signaling pathway alone is not sufficient to induce glucose uptake in 3T3-L1 adipocytes.

    PubMed Central

    van den Berghe, N; Ouwens, D M; Maassen, J A; van Mackelenbergh, M G; Sips, H C; Krans, H M

    1994-01-01

    The signal transduction pathway by which insulin stimulates glucose transport is largely unknown, but a role for tyrosine and serine/threonine kinases has been proposed. Since mitogen-activated protein (MAP) kinase is activated by insulin through phosphorylation on both tyrosine and threonine residues, we investigated whether MAP kinase and its upstream regulator, p21ras, are involved in insulin-mediated glucose transport. We did this by examining the time- and dose-dependent stimulation of glucose uptake in relation to the activation of Ras-GTP formation and MAP kinase by thrombin, epidermal growth factor (EGF), and insulin in 3T3-L1 adipocytes. Ras-GTP formation was stimulated transiently by all three agonists, with a peak at 5 to 10 min. Thrombin induced a second peak at approximately 30 min. The activation of p21ras was paralleled by both the phosphorylation and the activation of MAP kinase: transient for insulin and EGF and biphasic for thrombin. However, despite the strong activation of Ras-GTP formation and MAP kinase by EGF and thrombin, glucose uptake was not stimulated by these agonists, in contrast to the eightfold stimulation of 2-deoxy-D-[14C]glucose uptake by insulin. In addition, insulin-mediated glucose transport was not potentiated by thrombin or EGF. Although these results cannot exclude the possibility that p21ras and/or MAP kinase is needed in conjunction with other signaling molecules that are activated by insulin and not by thrombin or EGF, they show that the Ras/MAP kinase signaling pathway alone is not sufficient to induce insulin-mediated glucose transport. Images PMID:7511205

  4. Expression and function of chicken integrin beta 1 subunit and its cytoplasmic domain mutants in mouse NIH 3T3 cells

    PubMed Central

    1990-01-01

    Chicken integrin beta 1 cDNA and its site-directed mutants were cloned into a mammalian expression vector and introduced into mouse NIH 3T3 cells. Stable transfectants expressing the chicken beta 1 subunit or its site-directed mutants were identified by immunostaining with antibodies specific for the chicken integrin beta 1 subunit. The chicken beta 1 proteins were expressed predominately in the endoplasmic reticulum of transfectants and to a lesser degree in the plasma membrane. Immunoblots and immunoprecipitations, using anti-chicken integrin antibodies, revealed three different sizes of the chicken subunit (90, 95, and 120 kD) and a mouse 140-kD alpha subunit. Immunoprecipitations of the cell surface receptors showed only two peptides, an 120-kD beta 1 and an 140-kD alpha subunit. Antibodies perturbing mouse and chicken integrin-specific cell adhesions were used to demonstrate that the chimeric receptors functioned in adhesion to both laminin and fibronectin. Immunofluorescent staining with antibodies specific for either the chicken or mouse receptors showed that both the wild type and the chimeric receptors localized in focal contacts. Several mutations in the cytoplasmic domain were synthesized and used in the transfection experiments. In one mutant the tyrosine (Tyr 788) in the consensus sequence for phosphorylation was replaced by a phenylalanine. In another the lysine (Lys 757) at the end of the membrane spanning region was replaced by a leucine. Both of these mutants formed dimers with mouse alpha subunits, participated in adhesion, localized in focal contacts, and displayed biological properties indistinguishable from the wild-type transfection. In contrast, mutants containing deletions greater than 5-15 amino acids nearest the carboxyl end in the cytoplasmic domain neither promoted adhesion nor localized in focal contacts. They did, however, form heterodimers that were expressed on the cell surface. PMID:2104857

  5. Titanium is not "the most biocompatible metal" under cathodic potential: The relationship between voltage and MC3T3 preosteoblast behavior on electrically polarized cpTi surfaces.

    PubMed

    Ehrensberger, Mark T; Sivan, Shiril; Gilbert, Jeremy L

    2010-06-15

    An electrochemically controlled system has been developed which allows for cell culture directly on electrically polarized metal surfaces with simultaneous control and assessment of the electrochemical current, potential, and impedance of the interface. This system was utilized in this study to assess the interactions between electrochemically polarized commercially pure titanium (cpTi) and MC3T3 preosteoblast cells. Cells were cultured on CpTi for 24 h at static potentials between -1000 mV and +1000 mV vs. Ag/AgCl and cell morphology (SEM and cell area) and viability (MTT and Live-Dead assay) were assessed along with the electrochemical current densities and surface oxide impedance properties. The results indicate that cathodic polarization in the range of -600 mV to -1000 mV markedly reduces the spreading and viability of cells cultured directly on cpTi within 24 h, while anodic polarization (-300 mV to +1000 mV) out to 72 h shows no difference in cell behavior as compared to the OCP condition. Analysis of the relationship between the cell outcomes and the electrochemical current densities and impedance indicated the presence of voltage-dependent electrochemical thresholds (cathodic current density, i(c) > 1.0 microA/cm(2), R(p) < 10(5) Omega cm(2)) which may control the biocompatibility of cpTi. In addition, these outcomes have direct clinical significance for modular orthopedic implants whose potential can shift, via fretting corrosion, down into the range of potentials exhibiting poor cell behavior. PMID:20014293

  6. In vitro characterization of membrane associated phospholipase C from normal and Kirsten sarcoma virus-transformed NIH 3T3 cells

    SciTech Connect

    Talwar, H.S.; Thomas, T.P.; Bassin, R.; Anderson, W.B.

    1986-05-01

    Transformation of NIH 3T3 cells with Kirsten sarcoma virus (Ki-SV) increased phosphatidylinositol (PI) metabolism. This suggests possible alterations in the phospholipase C (PLC) and PIP/sub 2/-phosphodiesterase (PIP/sub 2/-PDE) activities responsible for hydrolysis of PI and PIP/sub 2/ with Ki-SV transformation. An in vitro assay is employed to study the hydrolysis of exogenously added (/sup 3/H)PI and (/sup 3/H)PIP/sub 2/ with membranes prepared from normal and Ki-SV transformed cells. Association of these activities with membranes appears to be differentially mediated by metals (Ca/sup 2 +/) since chelator treatment dissociates PLC from the particulate fraction. Hydrolysis of PIP/sub 2/ is markedly enhanced (10 fold) by introducing (/sup 3/H)PIP/sub 2/ to membrane preparations in vesicles prepared with excess phosphatidylethanolamine. These activities are dependent on Ca/sup 2 +/ and exhibit a progressive increase in activity between 10/sup -7/M and 10/sup -3/M Ca/sup 2 +/. The optimal pH for PIP/sub 2/-PDE is 7.0, whereas PI specific PLC exhibits optimal activity at pH 5.5. With this in vitro assay system it is possible to demonstrate that GTP-..gamma..-S addition to isolated membranes stimulates PIP/sub 2/-PDE to hydrolyze exogenously added (/sup 3/H)PIP/sub 2/. This should allow direct studies to determine possible differences in GTP-dependent regulation of PI and PIP/sub 2/ hydrolysis with membranes prepared from normal and transformed cells.

  7. ROS activate KCl cotransport in nonadherent Ehrlich ascites cells but K+ and Cl- channels in adherent Ehrlich Lettré and NIH3T3 cells.

    PubMed

    Lambert, Ian Henry; Klausen, Thomas Kjaer; Bergdahl, Andreas; Hougaard, Charlotte; Hoffmann, Else Kay

    2009-07-01

    Addition of H(2)O(2) (0.5 mM) to Ehrlich ascites tumor cells under isotonic conditions results in a substantial (22 +/- 1%) reduction in cell volume within 25 min. The cell shrinkage is paralleled by net loss of K(+), which was significant within 8 min, whereas no concomitant increase in the K(+) or Cl(-) conductances could be observed. The H(2)O(2)-induced cell shrinkage was unaffected by the presence of clofilium and clotrimazole, which blocks volume-sensitive and Ca(2+)-activated K(+) channels, respectively, and is unaffected by a raise in extracellular K(+) concentration to a value that eliminates the electrochemical driving force for K(+). On the other hand, the H(2)O(2)-induced cell shrinkage was impaired in the presence of the KCl cotransport inhibitor (dihydro-indenyl)oxyalkanoic acid (DIOA), following substitution of NO(3)(-) for Cl(-), and when the driving force for KCl cotransport was omitted. It is suggested that H(2)O(2) activates electroneutral KCl cotransport in Ehrlich ascites tumor cells and not K(+) and Cl(-) channels. Addition of H(2)O(2) to hypotonically exposed cells accelerates the regulatory volume decrease and the concomitant net loss of K(+), whereas no additional increase in the K(+) and Cl(-) conductance was observed. The effect of H(2)O(2) on cell volume was blocked by the serine-threonine phosphatase inhibitor calyculin A, indicating an important role of serine-threonine phosphorylation in the H(2)O(2)-mediated activation of KCl cotransport in Ehrlich cells. In contrast, addition of H(2)O(2) to adherent cells, e.g., Ehrlich Lettré ascites cells, a subtype of the Ehrlich ascites tumor cells, and NIH3T3 mouse fibroblasts increased the K(+) and Cl(-) conductances after hypotonic cell swelling. Hence, H(2)O(2) induces KCl cotransport or K(+) and Cl(-) channels in nonadherent and adherent cells, respectively. PMID:19419998

  8. Differentiation of 3T3-L1 preadipocytes with 3-isobutyl-1-methylxanthine and dexamethasone stimulates cell-associated and soluble chondroitin 4-sulfate proteoglycans

    SciTech Connect

    Calvo, J.C.; Rodbard, D.; Katki, A.; Chernick, S.; Yanagishita, M. (Laboratory of Theoretical and Physical Biology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland (USA))

    1991-06-15

    The proteoglycans (cell-associated and culture media) in 3T3-L1 preadipocytes in culture were analyzed before and during differentiation into adipocytes. Cells were metabolically labeled with (35S)sulfate and (3H) glucosamine for 24 h and then extracted and analyzed. There was a 1.68 {plus minus} 0.07-fold increase in the 35S in medium proteoglycan during differentiation, whereas cell-associated proteoglycan radioactivity showed no increase. Analyses of radiolabeled molecules using ion-exchange chromatography, gel filtration, and high performance liquid chromatography after enzymatic or alkaline digestion indicated that all of the 35S label was recovered as two major species of chondroitin 4-sulfate proteoglycans (CSPG-I and CSPG-II) and 7% as heparan sulfate proteoglycan. CSPG-I has a mass of {approximately} 970 kDa with multiple chondroitin sulfate chains (average of 50 kDa each) and a core protein of {approximately} 370 kDa including oligosaccharides. CSPG-II has a mass of 140 kDa with one or two chondroitin sulfate chains (average of 68 kDa each) and a core protein of 41 kDa including oligosaccharides. CSPG-I appears to be similar to versican, whereas CSPG-II is similar to decorin and/or biglycan, found in other fibroblastic cells. Cell differentiation was associated with a specific increase in CSPG-I (4.0 {plus minus} 0.2-fold in media and 3.2 {plus minus} 0.5-fold in the cell-associated form). This system should facilitate study of the functional roles of proteoglycans during growth and differentiation.

  9. Purification and characterization of a 40 S ribosomal protein S6 kinase from vanadate-stimulated Swiss 3T3 cells.

    PubMed

    Jenö, P; Jäggi, N; Luther, H; Siegmann, M; Thomas, G

    1989-01-15

    Recently we have identified a mitogen-activated S6 kinase from Swiss 3T3 cells (Jenö, P., Ballou, L. M., Novak-Hofer, I., and Thomas, G. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 406-410). Here we describe the detailed purification of this enzyme from high-speed supernatants (400,000 x g) of vanadate-treated cell extracts. The enzyme is purified through six sequential steps including cation- and anion-exchange, sizing, and affinity chromatography. At each step, the enzyme behaves as one entity and, on the final step of purification, is revealed on silver-stained sodium dodecyl sulfate-polyacrylamide gels as a single protein of Mr 70,000. As reported earlier, the overall purification factor is 3,000-fold, and the specific activity of the homogeneously purified enzyme is 0.6 mumol/min/mg of protein. However, recovery of total activity is only 0.2%. This large loss of activity appears to be due to freeze-thawing the enzyme between each step of purification. The purified kinase does not phosphorylate casein, histones 2A and 3S, or phosvitin. It has a Km for ATP of 28 microM and a broad optimum for Mg2+ between 5 and 20 mM. Mn2+ does not affect the basal level of kinase activity, and at concentrations as low as 1 mM, it completely suppresses the effect of 20 mM Mg2+ on kinase activity. The relationship of this enzyme to two other purified S6 kinases is discussed. PMID:2910854

  10. Effects of Coating a Titanium Alloy with Fibronectin on the Expression of Osteoblast Gene Markers in the MC3T3 Osteoprogenitor Cell Line

    PubMed Central

    Rapuano, Bruce E.; Hackshaw, Kyle M.; Schniepp, Hannes C.; MacDonald, Daniel E.

    2013-01-01

    Purpose A number of environmental and patient-related factors contribute to implant failure. A significant fraction of these failures can be attributed to limited osseointegration resulting from poor bone healing responses. The overall goal of this study was to determine whether surface treatment of a titanium-aluminum-vanadium alloy (Ti-6Al-4V) implant material with a biomimetic protein coating could promote the differentiation of attached osteoblastic cells. The specific aims of the study were to investigate whether osteoprogenitor cells cultured on a rigorously cleaned implant specimen showed a normal pattern of differentiation and whether preadsorbed fibronectin accelerated or enhanced osteoblast differentiation. Materials and Methods Ti-6Al-4V disks were rigorously cleaned, passivated in nitric acid, and dry heat–sterilized; some of the disks were then coated with 1 nmol/L fibronectin. MC3T3 osteoprogenitor cells were then cultured on the pretreated disks for several weeks. Quantitative real-time polymerase chain reaction was performed to measure changes over time in the mRNA levels of osteoblast genes. Results Fibronectin increased the peak expression of all analyzed osteoblast gene markers. “Early” genes that normally mark the proliferative phase (0 to 10 days) of osteoblastic development showed peak expression within the first 10 days after cell attachment to the titanium alloy. In contrast, “late” genes that normally mark the differentiation (10 to 20 days) and mineralization (20 to 36 days) phases of osteoblastogenesis achieved peak expression only after approximately 3 to 4 weeks of culture. Conclusions Osteoprogenitors cultured on a rigorously cleaned Ti-6Al-4V alloy were found to demonstrate a normal pattern of osteoblast differentiation. Preadsorbed fibronectin was observed to stimulate osteoblast differentiation during the mineralization phase of osteoblastogenesis. PMID:23057020

  11. Individual trans 18:1 isomers are metabolised differently and have distinct effects on lipogenesis in 3T3-L1 adipocytes.

    PubMed

    Vahmani, P; Meadus, W J; Turner, T D; Duff, P; Rolland, D C; Mapiye, C; Dugan, M E R

    2015-02-01

    The objective of this research was to study the metabolism of individual trans fatty acids (FAs) that can be found in ruminant fat or partially hydrogenated vegetable oils (PHVO) and determine their effects on FA composition and lipogenic gene expression in adipocytes. Differentiated 3T3-L1 adipocytes were treated with 200 µM of either trans-9-18:1, trans-11-18:1, trans-13-18:1, cis-9-18:1 or BSA vehicle control for 120 h. Trans-9-18:1 increased total cell FA content (µmole/well) compared to other FA treatments, which was mainly related to the accumulation of trans-9-18:1 in the cells. Adipocytes were able to desaturate a significant proportion of absorbed trans-11-18:1 and trans-13-18:1 (~20 and 30% respectively) to cis-9,trans-11-18:2 and cis-9,trans-13-18:2, whereas trans-9-18:1 was mostly incorporated intact resulting in a greater lipophilic index (i.e. decreased mean FA fluidity) of adipocytes. Trans-9-18:1 up-regulated (P < 0.05) the expression of lipogenic genes including acetyl-CoA carboxylase (1.65 fold), FA synthase (1.45 fold), FA elongase-5 (1.52 fold) and stearoyl-CoA desaturase-1 (1.49 fold), compared to the control, whereas trans-11-18:1 and trans-13-18:1 did not affect the expression of these genes compared to control. Our results suggest that the metabolism and lipogenic properties of trans-11-18:1 and trans-13-18:1, typically the most abundant trans FA in beef from cattle fed forage-based diets, are similar and are different from those of trans-9-18:1, the predominant trans FA in PHVO. PMID:25544125

  12. PPARgamma induces the insulin-dependent glucose transporter GLUT4 in the absence of C/EBPalpha during the conversion of 3T3 fibroblasts into adipocytes.

    PubMed Central

    Wu, Z; Xie, Y; Morrison, R F; Bucher, N L; Farmer, S R

    1998-01-01

    To define the molecular mechanisms that control GLUT4 expression during adipogenesis, NIH-3T3 fibroblasts ectopically expressing different adipogenic transcription factors (C/EBPbeta, C/EBPdelta, C/EBPalpha, and PPARgamma) under the control of a tetracycline-responsive inducible (C/EBPs) or a constitutive retroviral (PPARgamma) expression system were used. Enhanced production of C/EBPbeta (beta2 cell line), C/EBPbeta together with C/EBPdelta (beta/delta39 cell line), C/EBPalpha (alpha1 cell line), or PPARgamma (Pgamma2 cell line) in cells exposed to dexamethasone and the PPARgamma ligand ciglitazone (a thiazolidinedione) resulted in expression of GLUT4 mRNA as well as other members of the adipogenic gene program, including aP2 and adipsin. Focusing our studies on the beta/delta39 cells, we have demonstrated that C/EBPbeta along with C/EBPdelta in the presence of dexamethasone induces PPARgamma, adipsin, and aP2 mRNA production; however, GLUT4 mRNA is only expressed in cells exposed to ciglitazone. In addition, enhanced expression of a ligand-activated form of PPARgamma in the beta/delta39 fibroblasts stimulates synthesis of GLUT4 protein and gives rise to a population of adipocytic cells that take up glucose in direct response to insulin. C/EBPalpha is not expressed in the beta/delta39 cells under conditions that stimulate the adipogenic program. This observation suggests that PPARgamma alone or in combination with C/EBPbeta and C/EBPdelta is capable of activating GLUT4 gene expression. PMID:9421462

  13. [Overexpression of the nucleolar protein SURF-6 in mouse fibroblasts NIH/3T3 leads to stabilisation of intragenic transcribed spacers of the pre-rRNA].

    PubMed

    Polzikov, M A; Ve?ko, N N; Zharskaia, O O; Magoulas, Kh B; Zatsepina, O V

    2010-01-01

    SURF-6 is an evolutionary conserved nucleolar protein that is required for maintenance of cell viability, but its functional significance in mammals still remains illusive. In the present work we examined effects of SURF-6 overexpression in mouse NIH/3T3 fibroblasts transfected with two plasmids. The plasmid pUHrT62-1 encodes a tetracycline-dependant trans-activator, the protein rtTA, the plasmid pBI-SURF6--the genes of EGFP (enhanced green fluorescent protein) and of mouse SURF-6 which expression was controlled by the rtTA-responsive bi-directorial promoter. Western blot analysis showed that the SURF-6 level was severely augmented in cells transfected with pUHrT62-1 and pBI-SURF6 and incubated with the inducer--doxycycline opposed to the transfected but not-induced cells. The increase of SURF-6 was observed in 24 and 48 h after adding the inducer doxycycline. Dot-hybridization of isolated RNA with biotinilated oligonucleotide probes to various regions of mouse primarily pre-rRNA transcripts showed that overexpression of SURF-6 enhanced levels of the second intragenic transcribed spacer ITS2 in about seven folds and of the 5' external transcribed spacer 5'ETS in two folds. Amounts of fragments corresponding to 18S, 5.8S and 28S rRNA remained almost unchanged. These observations for the first time demonstrated that mammalian SURF-6 helps to stabilize or prevents premature cleavage of the pre-rRNA intragenic transcribed spacers, particularly of ITS2, similar to its homologue in S. cerevisiae the protein Rrp14. Today metazoan proteins that play a similar role in ribosome biogenesis, are not described. PMID:21063453

  14. HIF-1 or HIF-2 induction is sufficient to achieve cell cycle arrest in NIH3T3 mouse fibroblasts independent from hypoxia.

    PubMed

    Hackenbeck, Thomas; Knaup, Karl Xaver; Schietke, Ruth; Schödel, Johannes; Willam, Carsten; Wu, Xiaoqing; Warnecke, Christina; Eckardt, Kai-Uwe; Wiesener, Michael Sean

    2009-05-01

    Hypoxia is a severe stress which induces physiological and molecular adaptations, where the latter is dominated by the Hypoxia-inducible transcription Factor (HIF). A well described response on cellular level upon exposure to hypoxia is a reversible cell cycle arrest, which probably renders the cells more resistant to the difficult environment. The individual roles of hypoxia itself and of the isoforms HIF-1alpha and HIF-2alpha in cell cycle regulation are poorly understood and discussed controversially. In order to characterize the isolated effect of both HIFalpha isoforms on the cell cycle we generated tetracycline inducible, HIF-1alpha and -2alpha expressing NIH3T3 cells. The cDNAs for HIFalpha were mutated to generate stable and active HIF under normoxia. Upon activation of both HIFalpha subunits, the total number of living cells was reduced and long-term stimulation of HIF led to complete loss of transgene expression, implicating a strong negative selection pressure. Equally, colony forming activity was reduced by activation of both HIFalpha subunits. Cell cycle analyses showed that HIF activation resulted in a prominent cell cycle arrest in G(1)-phase, similarly to the hypoxic effect. Both, HIF-1alpha and HIF-2alpha were able to induce the expression of the cyclin-dependent kinase inhibitor p27 on reporter gene and protein level. Our study shows that HIF-1 and HIF-2 can individually arrest the cell cycle independent from hypoxia. These findings have implications for the resistance of tumor cells to the environment and treatment, but also for physiological cells. Importantly, recent approaches to stabilize HIFalpha in normoxia could have deleterious effects on proliferating tissues. PMID:19342889

  15. Derivation and maintenance of human embryonic stem cell line on human adult skin fibroblast feeder cells in serum replacement medium

    Microsoft Academic Search

    R. Tayfur Tecirlioglu; Linh Nguyen; Karen Koh; Alan O. Trounson; Anna E. Michalska

    2010-01-01

    Human embryonic stem (hES) cells were originally isolated and maintained on mouse embryonic fibroblast (MEF) feeder layers\\u000a in the presence of fetal bovine serum (FBS). However, if the hES cells are to be used for therapeutic applications, it is\\u000a preferable to regulatory authorities that they be derived and cultured in animal-free conditions to prevent mouse antigen\\u000a contamination that would exacerbate

  16. Senescence occurs with hTERT repression and limited telomere shortening in human oral keratinocytes cultured with feeder cells.

    PubMed

    Kang, Mo K; Kameta, Ayako; Shin, Ki-Hyuk; Baluda, Marcel A; Park, No-Hee

    2004-06-01

    We investigated the phenotypic and molecular alterations in normal human oral keratinocytes (NHOK) during in vitro replication in two different culture conditions. The cells were cultured either in chemically defined Keratinocyte Growth Medium (KGM) without feeder layers or in serum-containing flavin-adenine dinucleotide (FAD) medium with feeder layers. Primary NHOK underwent 22 +/- 3 population doublings (PDs) in KGM and 42 +/- 4 PDs in FAD medium, reflecting 52% increase in replication capacity with feeder layers. In both culture conditions, exponentially replicating NHOK demonstrated telomerase activity and expression of human telomerase reverse transcriptase (hTERT) gene. Telomerase activity and hTERT expression were rapidly diminished in senescing NHOK, which exhibited small decrease of telomere length for the remaining limited cellular replications until the complete arrest of cell division. However, telomere length in senescent NHOK was 6.7 +/- 0.5 kilobase pairs (kbps), significantly longer than that (5.12 kbps) of senescent human fibroblasts. The onset of senescence was accompanied with marked induction of p16(INK4A), and this occurred in both culture systems using either KGM or FAD medium. These results indicate that replicative senescence of NHOK is associated with loss of telomerase activity followed by limited telomere shortening. PMID:15095283

  17. Micropatterned co-culture of hepatocyte spheroids layered on non-parenchymal cells to understand heterotypic cellular interactions

    NASA Astrophysics Data System (ADS)

    Otsuka, Hidenori; Sasaki, Kohei; Okimura, Saya; Nagamura, Masako; Nakasone, Yuichi

    2013-12-01

    Microfabrication and micropatterning techniques in tissue engineering offer great potential for creating and controlling cellular microenvironments including cell-matrix interactions, soluble stimuli and cell-cell interactions. Here, we present a novel approach to generate layered patterning of hepatocyte spheroids on micropatterned non-parenchymal feeder cells using microfabricated poly(ethylene glycol) (PEG) hydrogels. Micropatterned PEG-hydrogel-treated substrates with two-dimensional arrays of gelatin circular domains (? = 100 ?m) were prepared by photolithographic method. Only on the critical structure of PEG hydrogel with perfect protein rejection, hepatocytes were co-cultured with non-parenchymal cells to be led to enhanced hepatocyte functions. Then, we investigated the mechanism of the functional enhancement in co-culture with respect to the contributions of soluble factors and direct cell-cell interactions. In particular, to elucidate the influence of soluble factors on hepatocyte function, hepatocyte spheroids underlaid with fibroblasts (NIH/3T3 mouse fibroblasts) or endothelial cells (BAECs: bovine aortic endothelial cells) were compared with physically separated co-culture of hepatocyte monospheroids with NIH3T3 or BAEC using trans-well culture systems. Our results suggested that direct heterotypic cell-to-cell contact and soluble factors, both of these between hepatocytes and fibroblasts, significantly enhanced hepatocyte functions. In contrast, direct heterotypic cell-to-cell contact between hepatocytes and endothelial cells only contributed to enhance hepatocyte functions. This patterning technique can be a useful experimental tool for applications in basic science, drug screening and tissue engineering, as well as in the design of artificial liver devices.

  18. 3T3 Cell Lines Stably Expressing Pax6 or Pax6(5a) – A New Tool Used for Identification of Common and Isoform Specific Target Genes

    PubMed Central

    Forsdahl, Siri; Nguyen, Lan Huong Thi; Mikkola, Ingvild

    2012-01-01

    Pax6 and Pax6(5a) are two isoforms of the evolutionary conserved Pax6 gene often co-expressed in specific stochiometric relationship in the brain and the eye during development. The Pax6(5a) protein differs from Pax6 by having a 14 amino acid insert in the paired domain, causing the two proteins to have different DNA binding specificities. Difference in functions during development is proven by the fact that mutations in the 14 amino acid insertion for Pax6(5a) give a slightly different eye phenotype than the one described for Pax6. Whereas quite many Pax6 target genes have been published during the last years, few Pax6(5a) specific target genes have been reported on. However, target genes identified by Pax6 knockout studies can probably be Pax6(5a) targets as well, since this isoform also will be affected by the knockout. In order to identify new Pax6 target genes, and to try to distinguish between genes regulated by Pax6 and Pax6(5a), we generated FlpIn-3T3 cell lines stably expressing Pax6 or Pax6(5a). RNA was harvested from these cell lines and used in gene expression microarrays where we identified a number of genes differentially regulated by Pax6 and Pax6(5a). A majority of these were associated with the extracellular region. By qPCR we verified that Ncam1, Ngef, Sphk1, Dkk3 and Crtap are Pax6(5a) specific target genes, while Tgfbi, Vegfa, EphB2, Klk8 and Edn1 were confirmed as Pax6 specific target genes. Nbl1, Ngfb and seven genes encoding different glycosyl transferases appeared to be regulated by both. Direct binding to the promoters of Crtap, Ctgf, Edn1, Dkk3, Pdgfb and Ngef was verified by ChIP. Furthermore, a change in morphology of the stably transfected Pax6 and Pax6(5a) cells was observed, and the Pax6 expressing cells were shown to have increased proliferation and migration capacities. PMID:22384097

  19. Transformation of BALB/c-3T3 cells: IV. Rank-ordered potency of 24 chemical responses detected in a sensitive new assay procedure.

    PubMed Central

    Matthews, E J; Spalding, J W; Tennant, R W

    1993-01-01

    This report introduces an improved method of detecting chemical-induced morphological transformation of A-31-1-13 BALB/c-3T3 cells. The new procedure uses an increased target cell population to assess chemical-induced damage by increasing the initial seeding density and by delaying the initiation time of chemical treatment. Furthermore, a newly developed co-culture clonal survival assay was used to select chemical doses for the transformation assay. This assay measured the relative cloning efficiency (RCE) of chemical treatments in high-density cell cultures. In addition, transformation assay sensitivity was enhanced through the use of improved methods to solubilize many chemicals. From a group of 24 chemicals tested in at least two trials, clear evidence of chemical-induced transformation was detected for 12 chemicals (aphidicolin, barium chloride-2H2O, 5-bromo-2'-deoxyuridine, C.I. direct blue 15, trans-cinnamaldehyde, cytosine arabinoside, diphenylnitrosamine, manganese sulfate-H2O, 2-mercaptobenzimidazole, mezerein, riddelliine, and 2,6-xylidine); 2 chemicals had equivocal activity [C.I. direct blue 218 and mono(2-ethylhexyl)phthalate], 9 chemicals were inactive [carisoprodol, chloramphenicol sodium succinate, 4-chloro-2-nitroaniline, C.I. acid red 114, isobutyraldehyde, mono(2-ethylhexyl)adipate, sodium fluoride, and 12-O-tetradecanoylphorbol-13-acetate), and 1 chemical had an indeterminate response (2,6-dinitrotoluene). All positive responses were detected in the absence of an exogenous activation system and exhibited significant activity at two or more consecutive doses. This report also presents a mathematical method that uses t-statistics for rank-ordering the potency of chemical-induced transformation responses. This model detects sensitivity differences in experiments used to evaluate chemical-induced transformation. Furthermore, it provides a method to estimate a chemical's transformation response in terms of the historical behavior of the assay, as well as its future activity. The most active of the 24 chemicals was mezerein, and the least active chemical was diphenylnitrosamine. PMID:8243401

  20. Cellular Zn depletion by metal ion chelators (TPEN, DTPA and chelex resin) and its application to osteoblastic MC3T3-E1 cells

    PubMed Central

    Cho, Young-Eun; Lomeda, Ria-Ann R.; Ryu, Sang-Hoon; Lee, Jong-Hwa; Beattie, John H.

    2007-01-01

    Trace mineral studies involving metal ion chelators have been conducted in investigating the response of gene and protein expressions of certain cell lines but a few had really focused on how these metal ion chelators could affect the availability of important trace minerals such as Zn, Mn, Fe and Cu. The aim of the present study was to investigate the availability of Zn for the treatment of MC3T3-E1 osteoblast-like cells and the availability of some trace minerals in the cell culture media components after using chelexing resin in the FBS and the addition of N,N,N',N'-tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN, membrane-permeable chelator) and diethylenetriaminepentaacetic acid (DTPA, membrane-impermeable chelator) in the treatment medium. Components for the preparation of cell culture medium and Zn-treated medium have been tested for Zn, Mn, Fe and Cu contents by atomic absorption spectrophotometer or inductively coupled plasma spectrophotometer. Also, the expression of bone-related genes (ALP, Runx2, PTH-R, ProCOL I, OPN and OC) was measured on the cellular Zn depletion such as chelexing or TPEN treatment. Results have shown that using the chelexing resin in FBS would significantly decrease the available Zn (p<0.05) (39.4 ± 1.5 µM vs 0.61 ± 10.15 µM) and Mn (p<0.05) (0.74 ± 0.01 µM vs 0.12 ± 0.04 µM). However, levels of Fe and Cu in FBS were not changed by chelexing FBS. The use of TPEN and DTPA as Zn-chelators did not show significant difference on the final concentration of Zn in the treatment medium (0, 3, 6, 9, 12 µM) except for in the addition of higher 15 µM ZnCl2 which showed a significant increase of Zn level in DTPA-chelated treatment medium. Results have shown that both chelators gave the same pattern for the expression of the five bone-related genes between Zn- and Zn+, and TPEN-treated experiments, compared to chelex-treated experiment, showed lower bone-related gene expression, which may imply that TPEN would be a stronger chelator than chelex resin. This study showed that TPEN would be a stronger chelator compared to DTPA or chelex resin and TPEN and chelex resin exerted cellular zinc depletion to be enough for cell study for Zn depletion. PMID:20535382

  1. A Phosphatidylinositol 3Kinase Docking Site in the Cytoplasmic Tail of the Jaagsiekte Sheep Retrovirus Transmembrane Protein Is Essential for Envelope-Induced Transformation of NIH 3T3 Cells

    Microsoft Academic Search

    MASSIMO PALMARINI; NAOYOSHI MAEDA; CLAUDIO MURGIA; CLAUDIO DE-FRAJA; ANDREW HOFACRE; HUNG FAN

    2001-01-01

    Jaagsiekte sheep retrovirus (JSRV) is the causative agent of a transmissible lung cancer of sheep known as ovine pulmonary carcinoma. Recently, we have found that the expression of the JSRV envelope (Env) is sufficient to transform mouse NIH 3T3 cells in classical transformation assays. To further investigate the mechanisms of JSRV oncogenesis, we generated a series of envelope chimeras between

  2. Comparative neoplastic transformation responses of Balb\\/3T3 cells, Syrian hamster embryo cells, and Rauscher murine leukemia virus-infected Fischer 344 rat embryo cells to chemical carcinogens

    Microsoft Academic Search

    V. C. Dunkel; R. J. Pienta; A. Sivak; K. A. Traul

    1981-01-01

    This study provides a preliminary comparative evaluation of the responses to a series of 49 chemicals, in in vitro transformation assays, of Balb\\/3T3 cells, Syrian hamster embryo cells, and Fischer 344 rat embryo cells infected with Rauscher murine leukemia virus. The chemicals assayed included aromatic amines; polycylic aromatic hydrocarbons; alkylating agents; nitrosamines, hydrazines, and related compounds; heterocyclic compounds, amides, ureas,

  3. Primary Design and Analysis of Feeder for ITER Poloidal Field

    NASA Astrophysics Data System (ADS)

    Lei, Mingzhun; Song, Yuntao; Liu, Sumei; Lu, Kun; Wang, Zhongwei

    2011-10-01

    An electromagnetic (EM) analytic model for the PF feeder, applied to ITER and needed to convey the cryogenic supply and electrical power to the PF magnets, was built up. The magnetic flux density and the EM force under the worst conditions with the maximum working current in each coil were then calculated. Based on the EM analysis and theoretical calculation, the relationship between the busbar stress and the distance of neighbouring busbar supports was obtained, which provides an approach to optimize the design of the busbar supports. In order to check the feasibility of the PF feeder structure, a finite element model was built up and the ANSYS code was applied to analyze the stress and displacement. The numerical results show that the stress of the PF feeder is within the allowable limits and the structure is feasible.

  4. A magnetic flux leakage NDE system for CANDU feeder pipes

    NASA Astrophysics Data System (ADS)

    Mak, Thomas Don

    This work examines the application of different magnetic flux leakage (MFL) inspection concepts to the non destructive evaluation (NDE) of residual (elastic) stresses in CANDURTM reactor feeder pipes. The stress sensitivity of three MFL inspection techniques was examined with flat plate samples, with stress-induced magnetic anisotropy (SMA) demonstrating the greatest stress sensitivity. A prototype SMA testing system was developed to apply magnetic NDE to feeders. The system consists of a flux controller that incorporates feedback from a wire coil and a Hall sensor (FCV2), and a magnetic anisotropy prototype (MAP) probe. The combination of FCV2 and the MAP probe was shown to provide SMA measurements on feeder pipe samples and predict stresses from SMA measurements with a mean accuracy of +/-38MPa.

  5. The anti-obesity effects of a tuna peptide on 3T3-L1 adipocytes are mediated by the inhibition of the expression of lipogenic and adipogenic genes and by the activation of the Wnt/?-catenin signaling pathway.

    PubMed

    Kim, Young-Min; Kim, In-Hye; Choi, Jeong-Wook; Lee, Min-Kyeong; Nam, Taek-Jeong

    2015-08-01

    The differentiation of 3T3-L1 cells into adipocytes involves the activation of an organized system of obesity-related genes, of which those encoding CCAAT/enhancer?binding proteins (C/EBPs) and the Wnt-10b protein may play integral roles. In a previous study of ours, we found that a specific peptide found in tuna (sequence D?I?V?D?K?I?E?I; termed TP?D) inhibited 3T3?L1 cell differentiation. In the present study, we observed that the expression of expression of C/EBPs and Wnt?10b was associated with obesity. The initial step of 3T3?L1 cell differentiation involved the upregulation of C/EBP?? expression, which in turn activated various subfactors. An upstream effector of glycogen synthase kinase-3? (GSK?3?) inhibited Wnt?10b expression in 3T3?L1 adipocytes. In a previous study of ours, we sequenced the tuna peptide via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS?PAGE) and quadrupole time-of-flight mass spectrometry (Q-TOF MS/MS) and confirmed the anti?obesity effects thereof in 3T3?L1 adipocytes. In the present study, we demonstrate that TP?D inhibits C/EBP and promotes Wnt?10b mRNA expression, thus activating the Wnt pathway. The inhibition of lipid accumulation was measured using a glucose and triglyceride (TG) assay. Our results confirmed that TP?D altered the expression levels of C/EBP?related genes in a dose?dependent manner and activated the Wnt signaling pathway. In addition, we confirmed that total adiponectin and high?molecular weight (HMW) adiponectin levels were reduced by treatment with TP?D. These data indicate that TP?D inhibits adipocyte differentiation through the inhibition of C/EBP genes and the subsequent activation of the Wnt/?-catenin signaling pathway. PMID:26046125

  6. 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 ?g of recombinant construct and 6 ?l 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

  7. Vitamin A Studies in Fattening Feeder Calves and Yearlings.

    E-print Network

    Marion, Paul T.; Ellis, N. R.; Black, W. H.; Howe, P. E.; Kemmerer, A. R.; Riggs, J. K.; Jones, J. M.; Fraps, G. S.; Dickson, R. E.; Schmidt, H.; Jones, John H.

    1943-01-01

    and from other work at Spur from 1934 to 1937 VITAMIN A STCDY I10 FATTENIXG FEEDER CALVES AKD YEAR,,,,,, ~d Table 3. Performance of steers on 450 microgram carotene level, Experiment 3 ivky" 1 Total I Daily 145 . 421 1 .11 1,54 Z26 1.24 472 49s . E...- TEXAS AGRICULTURAL EXPERIMENT STATION A. B. CONNER, DIRECTOR College Station. Texas BULLETIPI' NO. 630 APRIL 1943 VITAMIN A STUDIES IN FATTENING FEEDER CALVES AND YEARLINGS . H.,JONES, H. SCHMIDT, R. E. DIICKSON, G. S. FRAPS, J. M. JONE 3...

  8. Dynamic voltage compensation on distribution feeders using flywheel energy storage

    SciTech Connect

    Weissbach, R.S.; Karady, G.G.; Farmer, R.G. [Arizona State Univ., Tempe, AZ (United States)] [Arizona State Univ., Tempe, AZ (United States)

    1999-04-01

    Advancements in power electronics bearings and materials have made flywheel energy storage systems a viable alternative to electrochemical batteries. A future application of such a device is as an uninterruptible power supply for critical loads on a distribution feeder. However, the same power electronics and flywheel system could also be used for dynamic voltage compensation. A comparison is made between series and parallel connection of such dynamic compensation techniques used to maintain rated load voltage on distribution feeders when there are momentary dips in the supply voltage. For each case a mathematical model is presented and analyzed. The two cases are compared and the series compensation technique is more effective.

  9. Trafficking of glucose transporters in 3T3-L1 cells. Inhibition of trafficking by phenylarsine oxide implicates a slow dissociation of transporters from trafficking proteins.

    PubMed Central

    Yang, J; Clark, A E; Harrison, R; Kozka, I J; Holman, G D

    1992-01-01

    We have compared the rates of insulin stimulation of cell-surface availability of glucose-transporter isoforms (GLUT1 and GLUT4) and the stimulation of 2-deoxy-D-glucose transport in 3T3-L1 cells. The levels of cell-surface transporters have been assessed by using the bismannose compound 2-N-[4-(1-azi-2,2,2-trifluoroethyl)benzoyl]-1,3-bis(D-mannos -4-yloxy) propyl-2-amine (ATB-BMPA). At 27 degrees C the half-times for the appearance of GLUT1 and GLUT4 at the cell surface were 5.7 and 5.4 min respectively and were slightly shorter than that for the observed stimulation of transport activity (t 1/2 8.6 min). This lag may be due to a slow dissociation of surface transporters from trafficking proteins responsible for translocation. When fully-insulin-stimulated cells were subjected to a low-pH washing procedure to remove insulin at 37 degrees C, the cell-surface levels of GLUT1 and GLUT4 decreased, with half-times of 9.2 and 6.8 min respectively. These times correlated well with decrease in 2-deoxy-D-glucose transport activity that occurred during this washing procedure (t1/2 6.5 min). When fully-insulin-stimulated cells were treated with phenylarsine oxide (PAO), a similar decrease in transport activity occurred (t1/2 9.8 min). However, surface labelling showed that this corresponded with a decrease in GLUT4 only (t1/2 7.8 min). The cell-surface level of GLUT1 remained high throughout the PAO treatment. Light-microsome membranes were isolated from cells which had been cell-surface-labelled with ATB-BMPA. Internalization of both transporter isoforms to this pool occurred when cells were maintained in the presence of insulin for 60 min. In contrast with the surface-labelling results, we have shown that the transfer to the light-microsome pool of both transporters occurred in cells treated with insulin and PAO. These results suggest that both transporters are recycled by fluid-phase endocytosis and exocytosis. PAO may inhibit this recycling at a stage which involves the re-emergence of internalized transporters at the plasma membrane. The GLUT1 transporters that are recycled to the surface in insulin- and PAO-treated cells appear to have low transport activity. This may be because of a failure to dissociate fully from trafficking proteins at the cell surface. GLUT4 transporters appear to have a greater tendency to remain internalized if the normal mechanisms that commit transporters to the cell surface, such as dissociation from trafficking proteins, are uncoupled. PMID:1536656

  10. Novel design and development of an active feeder

    Microsoft Academic Search

    Patrick S. K. Chua

    2007-01-01

    Purpose – This paper aims to focus on the novel design and development of an automatic feeding system which is capable of feeding cylindrical parts which are fragile and powdery in nature and possess asymmetrical features such as a groove near to one end. Design\\/methodology\\/approach – It is an active feeder, performing its task without having to reject any feeding

  11. LOADING REEL AND PERFECTO STRIP STOCK FEEDER FOR #84 WATERBURYFARREL ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    LOADING REEL AND PERFECTO STRIP STOCK FEEDER FOR #84 WATERBURY-FARREL (U.S. GOVERNMENT) PRESS. THIS CONTINUOUS-FEED, 2-DRAW, 100 TON PRESS IS ONE OF TWO IN THE U.S. UNDER CONTRACT WITH THE DEPARTMENT OF DEFENSE FOR PRODUCTION OF BULLET JACKETS AND CARTRIDGE CASINGS. - American Brass Foundry, 70 Sayre Street, Buffalo, Erie County, NY

  12. Optimal Placement of Distributed Generation on a Radial Feeder

    Microsoft Academic Search

    Du Yun-Feng; Huang Qi

    2010-01-01

    Abstract-Distributed generation (DG) can be used to generate a customer's entire electricity supply; for peak shaving; for standby or emergency generation; as a green power source; or for increased reliability. In this paper, analytical approaches for optimal placement of DG with unity power factor in power systems are presented. Placement of DG in a radial feeder is analyzed and the

  13. The Effects of High School Feeder Networks on College Enrollment

    ERIC Educational Resources Information Center

    Wolniak, Gregory C.; Engberg, Mark E.

    2007-01-01

    This study examines the influence of networks established among high schools and colleges. Focusing on the college choice process, the authors analyzed data from the 2006 admissions and financial aid records at eight private, four-year colleges representing nearly 18,000 students. Results show that high school feeder networks have an important…

  14. Distribution feeder one-line diagram generation: a visibility representation

    Microsoft Academic Search

    P. S. Nagendra Rao; Ravishankar Deekshit

    2004-01-01

    An algorithm to automatically generate an aesthetically ‘pleasing’ and ‘readable’ one-line diagram of a radial distribution feeder is presented. Readability is sought to be achieved by using the visibility representation where buses and transmission lines are drawn as axis-parallel horizontal and vertical lines, respectively. In addition to this choice, a set of layout specifications for enhancing the readability of the

  15. Marginal sinus fistula supplied exclusively by vertebral artery feeders.

    PubMed

    Tekle, Wondwossen G; Grigoryan, Mikayel; Tummala, Ramachandra P

    2013-12-01

    A 54-year-old woman is reported with severe pulsatile tinnitus. Digital subtraction angiography demonstrated dural arteriovenous fistula of the marginal sinus with feeders arising exclusively from bilateral vertebral arteries. Patient underwent successful transarterial Onyx embolization with complete angiographic and clinical cure. PMID:24358414

  16. Marginal sinus fistula supplied exclusively by vertebral artery feeders

    PubMed Central

    Tekle, Wondwossen G; Grigoryan, Mikayel; Tummala, Ramachandra P

    2013-01-01

    A 54-year-old woman is reported with severe pulsatile tinnitus. Digital subtraction angiography demonstrated dural arteriovenous fistula of the marginal sinus with feeders arising exclusively from bilateral vertebral arteries. Patient underwent successful transarterial Onyx embolization with complete angiographic and clinical cure. PMID:24358414

  17. Development of a Feeder for Uninterrupted Centrifugation Studies

    NASA Technical Reports Server (NTRS)

    Mulenburg, Gerald M.; Vasques, Marilyn F.; Gundo, Daniel P.; Griffith, Jon B.; Wade, Charles E. (Technical Monitor)

    1994-01-01

    A specialized paste diet feeder was developed in support of a hypergravity (2G) centrifuge study. The centrifuge study was to be compared to a previously flown Russian Cosmos spaceflight so experimental parameters of the 14 day spaceflight had to be duplicated. In order to duplicate at hyper G an experiment that took place in weightlessness, all other conditions must be as identical as possible. Stopping the centrifuge to provide maintenance for the animals causes unacceptable changes in experimental research results. Thus the experimental protocol required the delivery of a designated amount of paste diet at regular intervals for a two week period without stopping the centrifuge. A centrifuge and a stationary control cage, each containing 10 laboratory rats, were fitted with feeders that were calibrated to provide 140 plus or minus 2g of paste diet every 6 hours. This paper describes development of the feeder design and results of its operation over the two week experiment. The design philosophy and details of the feeder system are provided with recommendations for future such devices.

  18. Implementation of heuristic search strategies for distribution feeder reconfiguration

    Microsoft Academic Search

    T. Taylor; D. Lubkeman

    1990-01-01

    This paper describes a method for feeder reconfiguration with the potential for handling realistic operating constraints. The approach taken is to set up a decision tree to represent the various switching operations available. A best-first tree searching strategy, based on heuristics, is used to evaluate the various alternatives. Switching options which could result in overloads, voltage problems, or other operating

  19. Optimal Operation of Distribution Feeders in Smart Grids

    Microsoft Academic Search

    Sumit Paudyal; Claudio A. Canizares; Kankar Bhattacharya

    2011-01-01

    This paper presents a generic and comprehensive distribution optimal power flow (DOPF) model that can be used by local distribution companies (LDCs) to integrate their distribution system feeders into a Smart Grid. The proposed three-phase DOPF framework incorporates detailed modeling of distribution system components and considers various operating objectives. Phase specific and voltage dependent modeling of customer loads in the

  20. Some aspects of water filtering activity of filter-feeders

    Microsoft Academic Search

    S. A. Ostroumov

    2005-01-01

    On the basis of the previous publications, our new data and the existing scientific literature, we have formulated some fundamental principles that characterize the pivotal roles of the biodiversity of filter-feeders in ecosystems. Among those roles are: (1) the role of ecological repair of water quality, (2) the role of contributing to reliability and stability of the functioning of the

  1. Dynamic voltage compensation on distribution feeders using flywheel energy storage

    Microsoft Academic Search

    R. S. Weissbach; G. G. Karady; R. G. Farmer

    1999-01-01

    Advancements in power electronics bearings and materials have made flywheel energy storage systems a viable alternative to electrochemical batteries. A future application of such a device is as an uninterruptible power supply for critical loads on a distribution feeder. However, the same power electronics and flywheel system could also be used for dynamic voltage compensation. A comparison is made between

  2. The forces exerted by aquatic suction feeders on their prey

    E-print Network

    Wainwright, Peter C.

    magnitude under conditions that are common in aquatic predator­prey interactions. We focus on three forces), the nature of the interaction of this flow with the prey has received less attention (Lauder & Clark 1984; deThe forces exerted by aquatic suction feeders on their prey Peter C. Wainwright1,* and Steven W

  3. Dry coal feeder development program at Ingersoll-Rand Research, Incorporated. [for coal gasification systems

    NASA Technical Reports Server (NTRS)

    Mistry, D. K.; Chen, T. N.

    1977-01-01

    A dry coal screw feeder for feeding coal into coal gasification reactors operating at pressures up to 1500 psig is described. Results on the feeder under several different modes of operation are presented. In addition, three piston feeder concepts and their technical and economical merits are discussed.

  4. Development and Evaluation of Improved Feeders for Goats Suitable to Stall-fed Management System

    Microsoft Academic Search

    Chet R Upreti; Bahadur S Kuwar; Shambhu B Panday

    2010-01-01

    Five different types of feeders were designed and tested on goats to find out their effectiveness in reducing feed wastage and cost fabrication. Experiment was conducted at Agriculture Research Station (ARS)-Bandipur for two years. Tested feeders were hexagonal, rectangular, hay rack, chain barrel and conventional wooden Tatnu. Feeders were fabricated using iron bars and woods. They were tested with the

  5. Cyanidin 3-glucoside protects 3T3-L1 adipocytes against H 2O 2- or TNF-?-induced insulin resistance by inhibiting c-Jun NH 2-terminal kinase activation

    Microsoft Academic Search

    Honghui Guo; Wenhua Ling; Qing Wang; Chi Liu; Yan Hu; Min Xia

    2008-01-01

    Anthocyanins are naturally occurring plant pigments and exhibit an array of pharmacological properties. Our previous study showed that black rice pigment extract rich in anthocyanin prevents and ameliorates high-fructose-induced insulin resistance in rats. In present study, cyanidin 3-glucoside (Cy-3-G), a typical anthocyanin most abundant in black rice was used to examine its protective effect on insulin sensitivity in 3T3-L1 adipocytes

  6. An early transient increase of intracellular Na + may be one of the first components of the mitogenic signal. Direct detection by 23Na-NMR spectroscopy in quiescent 3T3 mouse fibroblasts stimulated by growth factors

    Microsoft Academic Search

    Elisha Berman; Ilana Sharon; Henri Atlan

    1995-01-01

    23Na-NMR spectroscopy was designed to allow for continuous recording of intracellular Na+ in 3T3 fibroblasts stimulated by serum growth-factors in the presence of ion transport inhibitors. The metabolic state of cells at rest and following stimulation was monitored by 31P-NMR spectra of ATP and related high-energy phosphates. The study demonstrates that early activation of ion transporters by addition of serum

  7. Modulation and Lack of Cross-Talk between Signal Transducer and Activator of Transcription 5 and Suppressor of Cytokine Signaling3 in Insulin and Growth Hormone Signaling in 3T3-L1 Adipocytes

    Microsoft Academic Search

    David J. Story; Jacqueline M. Stephens

    2006-01-01

    Objective: To examine the role of signal transducer and activator of transcription (STAT) 5 and suppressor of cytokine signaling (SOCS)-3 in the cross-talk between growth hormone and insulin (INS) signaling in fat cells.Research Methods and Procedures: Fully differentiated 3T3-L1 adipocytes were exposed to INS, growth hormone (GH), or both of these growth factors, and the activation of STAT5 proteins and

  8. Inhibition of insulin-stimulated glucose transport in 3T3-L1 cells by Clostridium difficile toxin B, Clostridium sordellii lethal toxin, and Clostridium botulinum C2 toxin

    Microsoft Academic Search

    Gudrun Schmid; Annette Schürmann; Christine Huppertz; Fred Hofmann; Klaus Aktories; Hans-Georg Joost

    1998-01-01

    The role of the actin cytoskeleton and\\/or GTPases of the Rho\\/Rac-family in glucose transport regulation was investigated in\\u000a 3T3-L1 cells with clostridial toxins which depolymerize actin by inactivation of Rho\\/Rac (Clostridium difficile toxin B and Clostridium sordellii lethal toxin (LT)) or by direct ADP-ribosylation (Clostridium botulinum C2 toxin). Toxin B and C2 reduced insulin-stimulated, but not basal, 2-deoxyglucose (2-DOG) uptake

  9. Effects of BMP2, BMP4, and BMP6 on Osteoblastic Differentiation of Bone Marrow-Derived Stromal Cell Lines, ST2 and MC3T3-G2\\/PA6

    Microsoft Academic Search

    Akira Yamaguchi; Toshinori Ishizuya; Naoki Kintou; Yasuhiro Wada; Takenobu Katagiri; John M. Wozney; Vicki Rosen; Shusaku Yoshiki

    1996-01-01

    The effects of bone morphogenetic protein-2 (BMP-2) on osteoblastic differentiation of bone marrow stromal cells were investigated using two bone marrow stromal cell lines, ST2 and MC3T3-G2\\/PA6 (PA6). BMP-2 stimulated ALP activity and induced parathyroid hormone (PTH)-dependent production of cAMP in both ST2 and PA6 cells, but these effects were more apparent in ST2 cells than in PA6 cells. BMP-2

  10. Cytogenetic detection of a trans-species bystander effect: induction of sister chromatid exchanges in murine 3T3 cells by ganciclovir metabolized in HSV thymidine kinase gene-transfected Chinese hamster ovary cells.

    PubMed

    Thust, R; Tomicic, M T; Gräbner, R; Friedrichs, C; Wutzler, P; Kaina, B

    2004-01-01

    Due to the very limited transduction capacity of hitherto available vectors, the success of gene therapy of tumours by means of suicide genes has so far essentially depended on the transfer of toxic drug metabolites from transduced (metabolizing) cells to adjacent non-transduced cells via gap junctions (bystander effect). Most experimental systems for the detection of a bystander effect yield net data of cell losses and cannot differentiate between killed transduced versus killed bystander cells. Here we report on metabolic cooperation in vitro between CHO cells stably transfected with the thymidine kinase gene of herpes simplex virus type-1 (HSVtk) (metabolizing cells) and Swiss albino 3T3 cells (bystander cells). Both cell lines are readily distinguishable by single cell and colony morphology and by their chromosomal constitution. While 3T3 cells cultured alone were refractory to the antiviral drug ganciclovir (GCV), co-culture with CHO-HSVtk(+) cells led to a dramatic reduction in plating efficiency as well as to a 4-fold increase in sister chromatid exchange rates induced by very low GCV concentrations in the 3T3 bystander cells. The modulator of gap junctional cooperation, all-trans retinoic acid, caused a strong augmentation of the bystander effect, while 18alpha-glycyrrhetinic acid, an inhibitor of gap junctional communication, drastically diminished the toxicity of GCV in the bystander cells. Whereas CHO-HSVtk(+) cells showed a distinct immunoreactivity for connexin43 in the cell membranes, 3T3 cells were almost negative. The co-culture system described here allows unequivocal distinction between metabolizing and bystander cells. In this way, mechanistic aspects of the transfer of genotoxic/cytotoxic metabolites to cells, which per se are unable to form them, become accessible to investigation. PMID:14681310

  11. Activation of PKA and phosphorylation of sodium-dependent vitamin C transporter 2 by prostaglandin E2 promote osteoblast-like differentiation in MC3T3-E1 cells

    Microsoft Academic Search

    X Wu; L-H Zeng; T Taniguchi; Q-M Xie

    2007-01-01

    Sodium-dependent vitamin C transporter (SVCT) 2-mediated L-ascorbic acid (AA) uptake is required in osteoblast-like differentiation of MC3T3-E1 cells, and prostaglandin E2 (PGE2) is among the most important local factors in bone formation, but the detailed mechanism by which PGE2 induces osteoblast differentiation remains obscure. We revealed that PGE2 induced AA uptake and osteoblast-like differential markers including alkaline phosphatase, collagen, osteocalcin

  12. Ras Activity Late in G 1 Phase Required for p27 kip1 Downregulation, Passage through the Restriction Point, and Entry into S Phase in Growth Factor-Stimulated NIH 3T3 Fibroblasts

    Microsoft Academic Search

    NORIKO TAKUWA; YOH TAKUWA

    1997-01-01

    It is well documented that Ras functions as a molecular switch for reentry into the cell cycle at the border between G0 and G1 by transducing extracellular growth stimuli into early G1 mitogenic signals. In the present study, we investigated the role of Ras during the late stage of the G1 phase by using NIH 3T3 (M17) fibroblasts in which

  13. Effect of Nd:YAG laser-nitriding-treated titanium nitride surface over Ti6Al4V substrate on the activity of MC3T3-E1 cells.

    PubMed

    Wang, Min; Ning, Yingyuan; Zou, Haixiao; Chen, Si; Bai, Yi; Wang, Aihua; Xia, Haibin

    2014-01-01

    Ti6Al4V discs with a thickness of 2.5 mm and dimensions of 15 × 15 mm2 were fabricated. The titanium nitride (TiN) surface was formed via Nd:YAG laser-nitriding. A sandblast acid-etched (SA) surface was used as a control. Scanning electron microscopy (SEM), X-ray diffraction analysis (XRD), and surface roughness tests were conducted to study the surface and cross-section morphologies as well as the properties of TiN and SA surfaces. MC3T3-E1 osteoblast-like cells were cultured on the TiN and SA surfaces to evaluate the effect of TiN surface on cellular behaviors, including attachment, proliferation and differentiation. Morphological testing results revealed that the cross-section of TiN exhibited dendritic crystallization without cracking. The proliferation and differentiation of MC3T3-E1 cells on the laser-nitriding TiN surface were significantly increased compared to those cultured on SA surface. These findings suggested that the TiN surface generated from Nd:YAG laser-nitriding were favorable for the proliferation and differentiation of MC3T3-E1 cells, which is significant for implant surface modification. PMID:24211949

  14. Eicosapentaenoic acid increases lipolysis through up-regulation of the lipolytic gene expression and down-regulation of the adipogenic gene expression in 3T3-L1 adipocytes

    PubMed Central

    Lee, Mak-Soon; Kwun, In-Sook

    2007-01-01

    In this study, we investigated the lipolytic effects of eicosapentaenoic acid (EPA) in 3T3-L1 adipocytes. The differentiated 3T3-L1 adipocytes were treated in a serum-free medium with 300 ?M of EPA for 3, 6, 12, and 24 h. In comparison with the control, intracellular lipid accumulation was significantly decreased by 24% at 24 h following the addition of EPA (P < 0.05). Under the same experimental conditions, there was an increase of glycerol and free fatty acids (FFAs). The mRNA level of carnitine palmitoyltransferase I-a, a component of the fatty-acid shuttle system involved in the mitochondrial oxidation of long-chain fatty acids, was also significantly elevated by EPA (P < 0.05). However, the expression of peroxisome proliferator-activated receptor-? and acetyl-CoA carboxylase (ACC), which are involved in adipogenesis, was significantly down-regulated by EPA (P < 0.05). These results suggest that EPA may modulate lipid metabolism by stimulation of lipolysis, which was associated with induction of lipolytic gene expression and suppression of adipogenic gene expression in 3T3-L1 adipocytes. PMID:18850226

  15. Sida rhomboidea. Roxb Leaf Extract Down-Regulates Expression of PPAR?2 and Leptin Genes in High Fat Diet Fed C57BL/6J Mice and Retards in Vitro 3T3L1 Pre-Adipocyte Differentiation

    PubMed Central

    Thounaojam, Menaka C.; Jadeja, Ravirajsinh N.; Ramani, Umed V.; Devkar, Ranjitsinh V.; Ramachandran, A. V.

    2011-01-01

    Sida rhomboidea. Roxb leaf extract (SRLE) is being used by the populace of North-East India to alleviate symptoms of diabetes and obesity. We have previously reported its hypolipidemic and anti-diabetic properties. In this study, we report the effect of SRLE on (i) in vivo modulation of genes controlling high fat diet (HFD) induced obesity and (ii) in vitro 3T3L1 pre-adipocyte differentiation and leptin release. Supplementation with SRLE significantly prevented HFD induced increment in bodyweight, plasma lipids and leptin, visceral adiposity and adipocyte hypertrophy. Also, SRLE supplementation reduced food intake, down regulated PPAR?2, SREBP1c, FAS and LEP expressions and up-regulated CPT-1 in epididymal adipose tissue compared to obese mice. In vitro adipogenesis of 3T3L1 pre-adipocytes was significantly retarded in the presence of SRLE extract. Also decreased triglyceride accumulation, leptin release and glyceraldehyde-3-Phosphate dehydrogenase activity along with higher glycerol release without significant alteration of viability of 3T3L1 pre-adipocytes, was recorded. Our findings suggest that prevention of HFD induced visceral adiposity is primarily by down regulation of PPAR?2 and leptin gene expression coupled with attenuation of food intake in C57BL/6J mice. SRLE induced prevention of pre-adipocytes differentiation, and leptin release further substantiated these findings and scientifically validates the potential application of SRLE as a therapeutic agent against obesity. PMID:21845103

  16. Cellular regulation of poly ADP-ribosylation of proteins: II. Augmentation of poly(ADP-ribose) polymerase in SV40 3T3 cells following methotrexate-induced G1/S inhibition of cell cycle progression

    SciTech Connect

    Sooki-Toth, A.; Asghari, F.; Kirsten, E.; Kun, E. (Univ. of California, San Francisco (USA))

    1987-05-01

    SV40-3T3 cells were exposed in monolayer cultures to 5{times}10{sup {minus}7} M methotrexate (MTX), that inhibited thymidylate synthetase, arrested cell growth without cell killing in 24 h and did not induce single- (ss) or double-strand (ds) breaks in DNA. Following 24, up to 72 h, the poly(ADP-ribose) polymerase content of attached cells was induced by 5{times}10{sup {minus}7} MTX and the augmentation of the enzyme increased with the time of exposure to the drug. Inhibition of protein or RNA synthesis abolished augmentation of enzymatic activity; so too did the initiation of maximal cell growth by thymidine + hypoxanthine, by-passing the inhibitory site of MTX. Isolation of the ADP-ribosylated enzyme protein by gel electrophoresis identified poly(ADP-ribose) polymerase protein as the molecule that was induced by 5{times}10{sup {minus}7} M MTX. Under identical conditions, the poly(ADP-ribose) polymerase induction in 3T3 cells could not be demonstrated. A possible cell-cycle dependent biosynthesis of the enzyme protein is proposed in SV40 3T3 cells.

  17. Two chalcones, 4-hydroxyderricin and xanthoangelol, stimulate GLUT4-dependent glucose uptake through the LKB1/AMP-activated protein kinase signaling pathway in 3T3-L1 adipocytes.

    PubMed

    Ohta, Mitsuhiro; Fujinami, Aya; Kobayashi, Norihiro; Amano, Akiko; Ishigami, Akihito; Tokuda, Harukuni; Suzuki, Nobutaka; Ito, Fumitake; Mori, Taisuke; Sawada, Morio; Iwasa, Koichi; Kitawaki, Jo; Ohnishi, Katsunori; Tsujikawa, Muneo; Obayashi, Hiroshi

    2015-07-01

    4-Hydroxyderricin (4HD) and xanthoangelol (XAG) are major components of n-hexane/ethyl acetate (5:1) extract of the yellow-colored stem juice of Angelica keiskei. 4-Hydroxyderricin and XAG have been reported to increase glucose transporter 4 (GLUT4)-dependent glucose uptake in 3T3-L1 adipocytes, but the detailed mechanism of this phenomenon remains unknown. This present study was aimed at clarifying the detailed mechanism by which 4HD and XAG increase GLUT4-dependent glucose uptake in 3T3-L1 adipocytes. Both 4HD and XAG increased glucose uptake and GLUT4 translocation to the plasma membrane. 4-Hydroxyderricin and XAG also stimulated the phosphorylation of 5' adenosine monophosphate-activated protein kinase (AMPK) and its downstream target acetyl-CoA carboxylase. In addition, phosphorylation of liver kinase B1 (LKB1), which acts upstream of AMPK, was also increased by 4HD and XAG treatment. Small interfering RNA knockdown of LKB1 attenuated 4HD- and XAG-stimulated AMPK phosphorylation and suppressed glucose uptake. These findings demonstrate that 4HD and XAG can increase GLUT4-dependent glucose uptake through the LKB1/AMPK signaling pathway in 3T3-L1 adipocytes. PMID:26077869

  18. Derivation of embryonic stem cells from Kunming mice IVF blastocyst in feeder- and serum-free condition.

    PubMed

    Liu, Xiaokun; Wei, Qiang; Zhang, Junhong; Yang, Wanli; Zhao, Xiaoe; Ma, Baohua

    2015-06-01

    Kunming mice are widely used in China; however, it is difficult to isolate embryonic stem cells (ESCs) in conventional derivation condition containing feeder cells and serum. 6-Bromoindirubin-3'-oxime (BIO), a glycogen synthase kinase 3 (GSK3) inhibitor, could facilitate the maintenance of pluripotency of ESCs. Therefore, BIO could be considered as a candidate to replace feeder cells and serum. On the other hand, in vitro fertilization (IVF) is an important technology in assisted reproduction. It is reported that there was some difference in gene expression between IVF and in vivo developed blastocyst. ESCs derived from IVF blastocyst could provide a valuable tool to research the effect of IVF on differentiation and development. In the present study, we established two novel ESC lines from IVF blastocyst of Kunming mice in a feeder- and serum-free condition containing 2.5 ?M BIO. In this condition, expanded IVF blastocyst could spontaneously hatch from zonae pellucidae and attached to the gelatin-coated bottom of dishes. ESC-like outgrowth could be observed without overfull trophoblast cells. After further propagation, two Kunming mice ESC lines, designated as KMES1 and KMES2, were obtained. These two novel ESCs shared common morphological characteristics with other rodent ESCs, showed strong alkaline phosphatase activity, and expressed pluripotent markers, including Oct-4, Nanog, and SSEA-1. Embryoid body (EB) and teratoma test indicated that these ESCs could spontaneously differentiate into cells representative of all three embryonic germ layers. PMID:25592083

  19. Feederism: an exaggeration of a normative mate selection preference?

    PubMed

    Terry, Lesley L; Suschinsky, Kelly D; Lalumičre, Martin L; Vasey, Paul L

    2012-02-01

    Quinsey and Lalumičre (1995) suggested that some, if not most, paraphilias are exaggerated manifestations of more normative and functional mate selection preferences. The present study tested whether Feederism, a fat fetish focused on erotic eating, feeding, and gaining weight, is an exaggeration of a sexual arousal pattern commonly seen in the general population. Thirty participants (15 men and 15 women) recruited from the general population were assessed using penile plethysmography and vaginal photoplethysmography, respectively. None of the participants were self-identified Feeders or Feedees. Participants were shown sexual, neutral, and feeding still images while listening to audio recordings of sexual, neutral, and feeding stories. Participants did not genitally respond to feeding stimuli. However, both men and women subjectively rated feeding stimuli as more sexually arousing than neutral stimuli. We discuss the discordance between physiological and self-reported sexual arousal in the context of sex differences in sexual concordance and implications for future research. PMID:22392517

  20. POPULATION DYNAMICS OF BLUE JAYS AT A BIRD FEEDER

    Microsoft Academic Search

    MARGARET B. HICKEY; MARGARET CLARK BRITTINGHAM

    1991-01-01

    &sraxr.-Hickey color banded and monitored 2373 Blue Jays (Cyanocitta cristata) from 1953 until 1976 at her bird-feeder in Madison, Wisconsin. The mean annual survival rate of Blue Jays calculated from reobservation and recapture data by the Jolly-Seber method was 0.54. The annual survival rates ofjuvenile and adult jays calculated from band returns by the life table approach were 0.45 and

  1. Can Bivalve Suspension-Feeders Affect Pelagic Food Web Structure?

    Microsoft Academic Search

    Theo Prins; Vincent Escaravage; V. L. Escaravage

    2005-01-01

    Bivalve suspension-feeders are considered to be keystone herbivores in many estuarine ecosystems. However, bivalves can also\\u000a feed upon organisms that belong to the microzooplankton and on mesozooplankton. Laboratory experiments showed that nauplii\\u000a of the copepod Temora longicornis were filtered by mussels at the same rate as algae. Adult T. longicornis were also susceptible to filtration by mussels and oysters, but

  2. Advanced coal feeder tests at Sunbury Station. Final report

    SciTech Connect

    Brat, J. [CQ, Inc., Homer City, PA (United States)

    1995-06-01

    As part of an effort to develop equipment that will assure uninterrupted delivery of fuel with poor handleability characteristics, the Stamet Solids Pump system, a new but simple technology for moving solid materials, was field tested at Pennsylvania Power & Light`s Sunbury Steam Electric Stations Unit 1A. The Stamet pump consists of two discs mounted on a shaft to form a spool. The spool is enclosed within a housing, which forms a duct through the feeder. Solid material enters from above, is carried around by the spool, and exits after rotating between 200{degrees} and 300{degrees} of arc. The Stamet pump delivered significantly more coal than the existing vibratory feeders. As shown by EPRI`s Coal Quality Impact Model (CQIM), the Stamet pump reduces plant costs by $111,000 per year and would allow an increase in silt bum of approximately 6% at the same performance level. The pump also reduced excess air from 24 to 16%, resulting in a calculated further reduction in costs of approximately $71,000 per year. Excess air reduction resulted in a boiler efficiency increase of 0.93%. Reductions in feeder pluggages brought about by use of the new pump enhanced safety while minimizing pulverizer and boiler swings.

  3. Automated fault location and diagnosis on electric power distribution feeders

    SciTech Connect

    Zhu, J. [Advanced Control Systems, Inc., Norcross, GA (United States); Lubkeman, D.L.; Girgis, A.A. [Clemson Univ., SC (United States). Dept. of Electrical and Computer Engineering

    1997-04-01

    This paper presents new techniques for locating and diagnosing faults on electric power distribution feeders. The proposed fault location and diagnosis scheme is capable of accurately identifying the location of a fault upon its occurrence, based on the integration of information available from disturbance recording devices with knowledge contained in a distribution feeder database. The developed fault location and diagnosis system can also be applied to the investigation of temporary faults that may not result in a blown fuse. The proposed fault location algorithm is based on the steady-state analysis of the faulted distribution network. To deal with the uncertainties inherent in the system modeling and the phasor estimation, the fault location algorithm has been adapted to estimate fault regions based on probabilistic modeling and analysis. Since the distribution feeder is a radial network, multiple possibilities of fault locations could be computed with measurements available only at the substation. To identify the actual fault location, a fault diagnosis algorithm has been developed to prune down and rank the possible fault locations by integrating the available pieces of evidence. Testing of the developed fault location and diagnosis system using field data has demonstrated its potential for practical use.

  4. Mechanical Analysis and Optimization of ITER Upper ELM Coil & Feeder

    NASA Astrophysics Data System (ADS)

    Zhang, Shanwen; Song, Yuntao; Wang, Zhongwei; Lu, Su; Ji, Xiang; Du, Shuangsong; Liu, Xufeng; Feng, Changle; Yang, Hong; Wang, Songke; Luo, Zhiren

    2014-08-01

    International thermonuclear experimental reactor (ITER) edge localized mode (ELM) coils are used to mitigate or suppress ELMs. The location of the coils in the vacuum vessel and behind the blankets exposes them to high radiation levels and high temperatures. The feeders provide the power and cooling water for ELM coils. They are located in the chimney ports and experience lower radiation and temperature levels. These coils and feeders work in a high magnetic field environment and are subjected to alternating electromagnetic force due to the interaction between high magnetic field and alternating current (AC) current in the coils. They are also subjected to thermal stresses due to thermal expansion. Using the ITER upper ELM coil and feeder as an example, mechanical analyses are performed to verify and optimize the updated design to enhance their structural performance. The results show that the conductor, jacket and bracket can meet the static, fatigue and crack threshold criteria. The optimization indicates that adding chamfers to the bracket can reduce the high stress of the bracket, and removing two rails can reduce the peak reaction force on the two rails arising from thermal expansion.

  5. A novel coal feeder for production of low sulfur fuel

    SciTech Connect

    Khang, S.J.; Lin, L.; Keener, T.C.; Yeh, P.

    1991-01-01

    A dual-screw feeder was designed for desulfurization of coal. This reactor contains two screw tubes, the inner tube acting as a coal pyrolizer and the outer tube acting as a desulfurizer with hot calcined lime pellets or other renewable sorbent pellets. The objectives of this project is to study the feasibility of an advanced concept of desulfurization and possibly some denitrification in this coal feeder. In this year, two basic studies have been performed: (1) the desulfurization and (2) the denitrification due to mild pyrolysis. Specifically, the following tasks have been performed: (1) Setting up the Dual-Screw reactor, (2) Determination of the pyrolysis product and the sulfur distribution in char, tar and gas based on experimental data, (3) Study of the devolatilization, the desulfurization kinetics and the denitrification kinetics and obtaining the basic kinetic parameters, (4) Study of the sulfur removal efficiency of lime pellets fed into the outer tube of the dual-feeder reactor, (5) Study of the effect of the coal particle size on pyrolysis and desulfurization, (6) Study of the coal pyrolysis and desulfurization using a TGA(Thermal Gravimetric Analyzer).

  6. Effect of broiler breeder feeding programme and feeder space change at photostimulation using maize- or wheat-based diets on broiler progeny growth performance and leg health.

    PubMed

    Eusebio-Balcazar, P; Oviedo-Rondón, E O; Wineland, M J; Osborne, J; Brake, J

    2015-06-01

    1. The aim of this study was to evaluate the effects of diet type, maternal feeding programme at 29 weeks of age and breeder feeder space change at photostimulation on broiler progeny performance and leg health at 6 weeks of age. 2. Fast-feathering Cobb 500 broiler breeders were fed on either maize- or wheat-based diets that had been formulated to have similar nutrient composition during growing and layer phases. Two feeding programmes, fast or flow, were used from 14 to 29 weeks of age. At 22 weeks, 69 females from each pen were placed in a layer house where feeder space was either similar to that in rearing (6.3 to 6.5 cm/female) or was increased from 6.3 to 8.4 cm/female. Eggs produced at 32 and 44 weeks of age were collected and incubated for two broiler experiments. A total of 16 male and 16 female one-d-old chicks were placed in floor pens in two experiments, respectively, with 6 and 4 replicate pens. Broiler gait scores and leg problem prevalence were evaluated at 6 weeks of age. 3. Data were analysed as a 2 × 2 × 2 factorial design with diet type, feeding programme and feeder space change as main factors. 4. The wheat diet increased the probability of observing crooked toes in broiler progeny compared to the use of maize, but only when breeders were fed according to the fast feeding programme and given similar feeder space as during rearing. 5. Breeders given more feeder space in the laying period produced progeny with more locomotion problems compared with those provided similar feeder space, but only when maize was used and the slow feeding programme was applied to the breeders. 6. The maternal feeding programme interacted with other factors to influence progeny leg health, but it did not solely influence walking ability or leg problems of progeny. 7. In conclusion, an increased probability of observing walking impairment of broiler progeny was detected when breeders were given greater feeder space at photostimulation rather than no change and fed according to the slow feeding programme using maize diets in breeders and progeny. PMID:25811235

  7. Construction of Plasmid Insulin Gene Vector Containing Metallothionein IIA (pcDNAMTChIns) and Carbohydrate Response Element (ChoRE), and Its Expression in NIH3T3 Cell Line

    PubMed Central

    Piri, Hossein; Kazemi, Bahram; Rezaei, Mohsen; Bandehpour, Mojgan; Khodadadi, Iraj; Hassanzadeh, Taghi; Karimi, Jamshid; Yarian, Fatemeh; Peirovi, Habibollah; Tavakoli, Amir Hossein; Goodarzi, Mohammad Taghi

    2012-01-01

    Background Type 1 diabetes mellitus is one of the metabolic diseases that cause insulin-producing pancreatic ß cells be destroyed by immune system self-reactive T cells. Recent­ly, new treatment methods have been developed including use of the stem cells, ß islet cells transplantation and gene therapy by viral and non-viral gene constructs. Objectives The aim of this project was preparing the non-viral vector containing the glucose inducible insulin gene and using it in the NIH3T3 cell line. Materials and Methods Cloning was carried out by standard methods. Total RNA was extracted from pancreatic tissue, RNA was converted to cDNA using RT-PCR reaction and preproinsulin gene was amplified using specific primers. PNMTCH plasmid was extract­ed and digested by NotI, HindIII, and MTIIA and ChoRE genes were purified and cloned into pcDNA3.1 (-) plasmid and named pcDNAMTCh. Finally, the preproinsulin genes were cloned into pcDNA3.1 (-) plasmid and pcDNAMTChIns was built. Results The cloned gene constructs were evaluated by restriction enzyme digestion and RT-PCR. The NIH3T3 cells were transfected by plasmid naked DNA containing preproinsu­lin gene and expression was confirmed by Reverse Transcriptase PCR and Western Blot­ting Techniques. Conclusions Gel electrophoresis of PCR products confirmed that cloning was per­formed correctly. The expression of preproinsulin gene in recombinant plasmid in NI­H3T3 cell line was observed for the first time. The findings in this study can be the basis of further research on diabetes mellitus type 1 gene therapy on animals. PMID:23843817

  8. Extracellular calcium-sensing-receptor (CaR)-mediated opening of an outward K(+) channel in murine MC3T3-E1 osteoblastic cells: evidence for expression of a functional CaR

    NASA Technical Reports Server (NTRS)

    Ye, C. P.; Yamaguchi, T.; Chattopadhyay, N.; Sanders, J. L.; Vassilev, P. M.; Brown, E. M.; O'Malley, B. W. (Principal Investigator)

    2000-01-01

    The existence in osteoblasts of the G-protein-coupled extracellular calcium (Ca(o)(2+))-sensing receptor (CaR) that was originally cloned from parathyroid and kidney remains controversial. In our recent studies, we utilized multiple detection methods to demonstrate the expression of CaR transcripts and protein in several osteoblastic cell lines, including murine MC3T3-E1 cells. Although we and others have shown that high Ca(o)(2+) and other polycationic CaR agonists modulate the function of MC3T3-E1 cells, none of these actions has been unequivocally shown to be mediated by the CaR. Previous investigations using neurons and lens epithelial cells have shown that activation of the CaR stimulates Ca(2+)-activated K(+) channels. Because osteoblastic cells express a similar type of channel, we have examined the effects of specific "calcimimetic" CaR activators on the activity of a Ca(2+)-activated K(+) channel in MC3T3-E1 cells as a way of showing that the CaR is not only expressed in those cells but is functionally active. Patch-clamp analysis in the cell-attached mode showed that raising Ca(o)(2+) from 0.75 to 2.75 mmol/L elicited about a fourfold increase in the open state probability (P(o)) of an outward K(+) channel with a conductance of approximately 92 pS. The selective calcimimetic CaR activator, NPS R-467 (0.5 micromol/L), evoked a similar activation of the channel, while its less active stereoisomer, NPSS-467 (0.5 micromol/L), did not. Thus, the CaR is not only expressed in MC3T3-E1 cells, but is also functionally coupled to the activity of a Ca(2+)-activated K(+) channel. This receptor, therefore, could transduce local or systemic changes in Ca(o)(2+) into changes in the activity of this ion channel and related physiological processes in these and perhaps other osteoblastic cells.

  9. SP600125 reduces lipopolysaccharide-induced apoptosis and restores the early-stage differentiation of osteoblasts inhibited by LPS through the MAPK pathway in MC3T3-E1 cells.

    PubMed

    Guo, Chun; Wang, Sheng-Li; Xu, Song-Tao; Wang, Jian-Guo; Song, Guo-Hua

    2015-05-01

    Bone degradation is a serious complication of chronic inflammatory diseases such as septic arthritis, osteomyelitis, and infected orthopedic implant failure. Effective therapeutic treatments for bacteria-caused bone destruction are limited. In a previous study, we found that lipopolysaccharide (LPS) induced osteoblast apoptosis and inhibited early and late-stage differentiation of osteoblasts via activation of the C-Jun N-terminal kinase (JNK) pathway. This study aimed to investigate the effect of JNK inhibition by SP600125 on the apoptosis and differentiation of MC3T3-E1 osteoblasts suppressed by LPS. Following pretreatment with SP600125 for 2 h, MC3T3-E1 cells were treated LPS. Following this treatment, cell viability, activity of alkaline phosphatase (ALP) and caspase-3 were measured. mRNA and protein expression of osteoblast-specific genes, mitogen-activated protein kinases (MAPKs), Bax, Bcl-2 and caspase-3 were determined by quantitative polymerase chain reaction (qPCR) and western blot analysis. The results showed that SP600125 significantly restored LPS-inhibited cell metabolism and ALP activity and reduced the upregulated caspase-3 activity of MC3T3-E1 cells induced by LPS. SP600125 also significantly restored the LPS-suppressed mRNA and protein expression levels of early-stage osteoblast-associated genes in a dose-dependent manner. SP600125 significantly downregulated expression of Bax and caspase-3 but upregulated Bcl-2 expression in MC3T3-E1 cells stimulated by LPS. Furthermore, SP600125 selectively triggered the MAPK pathway by reducing the expression of JNK1, while enhancing the expression of extracellular signal-regulated kinase 1 (ERK1). Our results suggested that SP600125 reduced LPS-induced osteoblast apoptosis and restored early-stage differentiation of osteoblasts inhibited by LPS through MAPK signaling. These findings suggest that the therapeutic agent that inhibited JNK1 is of potential use for the restoration of osteoblast function in bacteria-induced bone diseases. PMID:25760015

  10. Comparison of defined culture systems for feeder cell free propagation of human embryonic stem cells

    PubMed Central

    Akopian, Veronika; Beil, Stephen; Benvenisty, Nissim; Brehm, Jennifer; Christie, Megan; Ford, Angela; Fox, Victoria; Gokhale, Paul J.; Healy, Lyn; Holm, Frida; Hovatta, Outi; Knowles, Barbara B.; Ludwig, Tenneille E.; McKay, Ronald D. G.; Miyazaki, Takamichi; Nakatsuji, Norio; Oh, Steve K. W.; Pera, Martin F.; Rossant, Janet; Stacey, Glyn N.; Suemori, Hirofumi

    2010-01-01

    There are many reports of defined culture systems for the propagation of human embryonic stem cells in the absence of feeder cell support, but no previous study has undertaken a multi-laboratory comparison of these diverse methodologies. In this study, five separate laboratories, each with experience in human embryonic stem cell culture, used a panel of ten embryonic stem cell lines (including WA09 as an index cell line common to all laboratories) to assess eight cell culture methods, with propagation in the presence of Knockout Serum Replacer, FGF-2, and mouse embryonic fibroblast feeder cell layers serving as a positive control. The cultures were assessed for up to ten passages for attachment, death, and differentiated morphology by phase contrast microscopy, for growth by serial cell counts, and for maintenance of stem cell surface marker expression by flow cytometry. Of the eight culture systems, only the control and those based on two commercial media, mTeSR1 and STEMPRO, supported maintenance of most cell lines for ten passages. Cultures grown in the remaining media failed before this point due to lack of attachment, cell death, or overt cell differentiation. Possible explanations for relative success of the commercial formulations in this study, and the lack of success with other formulations from academic groups compared to previously published results, include: the complex combination of growth factors present in the commercial preparations; improved development, manufacture, and quality control in the commercial products; differences in epigenetic adaptation to culture in vitro between different ES cell lines grown in different laboratories. PMID:20186512

  11. Zanthoxylum schinifolium leaf ethanol extract inhibits adipocyte differentiation through inactivation of the extracellular signal regulated kinase and phosphoinositide 3-kinase/Akt signaling pathways in 3T3-L1 pre-adipocytes.

    PubMed

    Choi, Eun-Ok; Park, Cheol; Shin, Soon Shik; Cho, Eun-Ju; Kim, Byung Woo; Hwang, Jin Ah; Hwang, Hye-Jin; Choi, Yung Hyun

    2015-07-01

    Zanthoxylum schinifolium is widely used as a food flavoring in east Asia. Although this plant has also been used in traditional oriental medicine for the treatment of the common cold, toothache, stomach ache, diarrhea and jaundice, its anti?obesity activity remains to be elucidated. The present study investigated the effects of ethanol extract from the leaves of Z. schinifolium (EEZS) on adipocyte differentiation, and its underlying mechanism, in 3T3?L1 pre?adipocytes. The results demonstrated that EEZS effectively suppressed intracellular lipid accumulation at non?toxic concentrations, and was associated with the downregulation of several adipocyte?specific transcription factors, including peroxisome proliferation?activity receptor ? (PPAR?), CCAAT/enhancer binding protein (C/EBP)? and C/EBP?, in a concentration?dependent manner. Furthermore, it was observed that EEZS markedly inactivated the extracellular signal?regulated protein kinase (ERK) and phosphatidylinositide 3?kinase (PI3K)/Akt pathways, which act upstream of PPAR? and C/EBPs in adipogenesis. These results suggested that EEZS inhibited lipid accumulation by downregulating the major transcription factors involved in the pathway of adipogenesis, including PPAR?, C/EBP? and C/EBP?, via regulation of the ERK and PI3K/Akt signaling pathways in 3T3?L1 adipocyte differentiation. This indicated the potential use of EEZS as an anti?obesity agent. PMID:25760758

  12. Nucleocytoplasmic transport is enhanced concomitant with nuclear accumulation of epidermal growth factor (EGF) binding activity in both 3T3-1 and EGF receptor reconstituted NR-6 fibroblasts

    PubMed Central

    1990-01-01

    Measurements of nucleocytoplasmic transport of fluorescent-labeled macromolecules were performed in both an EGF-nonresponsive mutant fibroblast line (3T3-NR6) and in the same cell line reconstituted with active EGF receptors derived from rat hepatic membrane fraction. Immunolocalization studies of exogenously incorporated EGF receptors in reconstituted 3T3-NR6 fibroblasts demonstrated predominantly intracellular localization. The EGF receptor constructs also showed EGF- stimulated incorporation of [3H]thymidine, providing biochemical evidence for functional integration of the exogenously supplied EGF receptors into the reconstituted fibroblasts. Additional support for the functional incorporation of receptor may be inferred from the enhanced cellular accumulation of 125I-EGF in cells treated with chloroquine and leupeptin. 125I-EGF binding and transnuclear macromolecular transport measurements in mutant and reconstituted cells, in conjunction with such measurements on nuclei isolated from these cells, provide data consistent with a growth factor/nuclear signaling mechanism dependent on the nuclear acquisition of EGF binding activity from the plasma membrane. PMID:2307699

  13. Desensitization of prostaglandin F2 alpha-stimulated inositol phosphate generation in NIH-3T3 fibroblasts transformed by overexpression of normal c-Ha-ras-1, c-Ki-ras-2 and c-N-ras genes.

    PubMed Central

    Black, F M; Wakelam, M J

    1990-01-01

    The stimulation of inositol phosphate generation in control and ras-gene-transformed NIH-3T3 cells by prostaglandin F2 alpha (PGF2 alpha) was investigated. Compared with the control cells, a desensitization of the response was observed in cells transformed by the overexpression of N-, Ha-, or Ki-ras genes. This desensitization was without effect upon the concentration causing half-maximal effect (EC50), dissociation constant (Kd) or number of PGF2 alpha receptors. Inhibition of PG synthesis was without effect upon desensitization, demonstrating that the effect was not agonist-induced. Desensitization could be induced in NIH-3T3 cells by culturing under conditions where the cells were all in the exponential growth phase, or by a 12 h exposure to a C-kinase-activating phorbol ester. These results suggest that desensitization of certain agonist-induced inositol phospholipid responses in ras-transformed cells is a consequence of increased cell proliferation and associated amplification in C-kinase activity and is an indirect consequence of transformation by ras. PMID:2187437

  14. Inhibition of Reactive Oxygen Species (ROS) and Nitric Oxide (NO) by Gelidium elegans Using Alternative Drying and Extraction Conditions in 3T3-L1 and RAW 264.7 Cells.

    PubMed

    Jeon, Hui-Jeon; Choi, Hyeon-Son; Lee, Ok-Hwan; Jeon, You-Jin; Lee, Boo-Yong

    2012-06-01

    Gelidium (G.) elegans is a red alga inhabiting intertidal areas of North East Asia. We examined anti-oxidative and anti-inflammatory effects of G. elegans, depending on drying and extraction conditions, by determining reactive oxygen species (ROS) and nitric oxide (NO) in 3T3-L1 and RAW 264.7 cells. Extraction yields of samples using hot air drying (HD) and far-infrared ray drying (FID) were significantly higher than those using natural air drying (ND). The 70% ethanol extracts showed the highest total phenol and flavonoid contents compared to other extracts (0, 30, and 50% ethanol) under tested drying conditions. The scavenging activity on 2,2-diphenyl-1-picrylhydrazyl (DPPH) and nitrite correlated with total phenol or flavonoid content in the extracts. The greatest DPPH scavenging effect was observed in 70% ethanol extract from FID and HD conditions. The production of ROS and NO in 3T3-L1 and macrophage cells greatly decreased with the 70% ethanol extraction derived from FID. This study suggests that 70% ethanol extraction of G. elegans dried by FID is the most optimal condition to obtain efficiently antioxidant compounds of G. elegans. PMID:24471073

  15. Derivation and maintenance of human embryonic stem cell line on human adult skin fibroblast feeder cells in serum replacement medium.

    PubMed

    Tecirlioglu, R Tayfur; Nguyen, Linh; Koh, Karen; Trounson, Alan O; Michalska, Anna E

    2010-04-01

    Human embryonic stem (hES) cells were originally isolated and maintained on mouse embryonic fibroblast (MEF) feeder layers in the presence of fetal bovine serum (FBS). However, if the hES cells are to be used for therapeutic applications, it is preferable to regulatory authorities that they be derived and cultured in animal-free conditions to prevent mouse antigen contamination that would exacerbate an immune response to foreign proteins, and the potential risk of transmission of retroviral and other zoonotic pathogens to humans. As a step towards this goal, we derived a new hES cell line (MISCES-01) on human adult skin fibroblasts as feeder cells using serum replacement (SR) medium. The MISCES-01 cells have a normal diploid karyotype (46XX), express markers of pluripotency (OCT4, GCTM-2, TRA-1-60, TRA-1-81, SSEA-3, SSEA-4, and alkaline phosphatase) and following in vitro and in vivo differentiation, give rise to derivatives of the three primary germ layers. This cell line can be obtained for research purposes from the Australian Stem Cell Centre (http://www.stemcellcentre.edu.au). PMID:20178002

  16. Feeder-Cell Ingestion of Seeding Aerosol from Cloud Base Determined by Tracking Radar Chaff.

    NASA Astrophysics Data System (ADS)

    Reinking, Roger F.; Martner, Brooks E.

    1996-09-01

    Questions of delivery, transport, and dispersion of cloud seeding aerosol in a convective feeder cloud are addressed by using radar chaff as a surrogate for aerosol and tracking it with circular-polarization radar. In a case study, a line source of chaff was released by an aircraft at the roots of a growing cloud flanking and feeding into a thunderstorm line. The chaff was tracked as it dispersed in the boundary layer and rose more than 3 km from the cloud base at +14°C to levels cold enough to nucleate ice-forming seeding aerosols. Quantitative measures of the rates of loft and dispersion, and the volume filling and dilution were obtained. The measurements permit examination of the hypotheses and potential efficacy of cloud-base seeding to increase rain and suppress hail. Notably, the problem of delivery, transport, and dispersion of cloud seeding aerosol is much the same as the air quality question of the nature and effect of cloud venting of the boundary layer, and the findings here apply in that context as well.

  17. Benthic suspension feeders: their paramount role in littoral marine food webs

    Microsoft Academic Search

    Josep-Maria Gili; Rafel Coma

    1998-01-01

    In recent years, particular attention has been paid to coupling and energy transfer between benthos and plankton. Because of their abundance, certain benthic suspension feeders have been shown to have a major impact in marine ecosystems. They capture large quantities of particles and might directly regulate primary production and indirectly regulate secondary production in littoral food chains. Suspension feeders develop

  18. Artificial neural-network based feeder reconfiguration for loss reduction in distribution systems

    Microsoft Academic Search

    Hoyong Kim; Yunseok Ko; Kyunghee Jung

    1993-01-01

    Neural networks have the capability to map the complex and extremely non-linear relationship between the load levels of zone and system topologies, which is required for feeder reconfiguration in distribution systems. This study is intended to propose the strategies to reconfigure the feeder, by using artificial neural networks with mapping ability. Artificial neural networks determine the appropriate system topology that

  19. A New Angle on Microscopic Suspension Feeders near Boundaries Rachel E. Pepper,

    E-print Network

    Koehl, Mimi

    and calculations, we show that living suspension feeders (Vorticella) likely actively regulate the angle attached to underwater surfaces and form an essential link in the aquatic carbon chain by consuming). Such estimates of sea water filtration enhance our under- standing of the contributions of sessile filter feeders

  20. Optimization issues in automated production of printed circuit boards: operations sequencing and feeder configuration problems

    Microsoft Academic Search

    E. Demirkol

    1995-01-01

    In the manufacturing of PCB, the determination of component insertion sequence and the assignment of components to the feeder carriage cells, in a way to minimize the overall time losses due to carrier board and feeder carriage movements and component leg spread adjustments are important factors directly contributing to the overall productivity of the production system. In the light of

  1. 30 CFR 75.1003-1 - Other requirements for guarding of trolley wires and trolley feeder wires.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ...2010-07-01 false Other requirements for guarding of trolley wires and trolley feeder wires. 75.1003-1 Section 75.1003-1...MANDATORY SAFETY STANDARDS-UNDERGROUND COAL MINES Trolley Wires and Trolley Feeder Wires §...

  2. 30 CFR 75.1003-1 - Other requirements for guarding of trolley wires and trolley feeder wires.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ...2011-07-01 false Other requirements for guarding of trolley wires and trolley feeder wires. 75.1003-1 Section 75.1003-1...MANDATORY SAFETY STANDARDS-UNDERGROUND COAL MINES Trolley Wires and Trolley Feeder Wires §...

  3. 30 CFR 75.1003 - Insulation of trolley wires, trolley feeder wires and bare signal wires; guarding of trolley...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... Mineral Resources 1 2011-07-01 2011-07-01 false Insulation of trolley wires, trolley feeder wires and bare signal wires; guarding of trolley wires and trolley feeder wires. 75.1003 Section 75.1003 Mineral...

  4. 30 CFR 75.1003 - Insulation of trolley wires, trolley feeder wires and bare signal wires; guarding of trolley...

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... Mineral Resources 1 2010-07-01 2010-07-01 false Insulation of trolley wires, trolley feeder wires and bare signal wires; guarding of trolley wires and trolley feeder wires. 75.1003 Section 75.1003 Mineral...

  5. 30 CFR 77.1802 - Insulation of trolley wires, trolley feeder wires and bare signal wires; guarding of trolley...

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... Mineral Resources 1 2011-07-01 2011-07-01 false Insulation of trolley wires, trolley feeder wires and bare signal wires; guarding of trolley wires and trolley feeder wires. 77.1802 Section 77.1802 Mineral...

  6. 30 CFR 77.1802 - Insulation of trolley wires, trolley feeder wires and bare signal wires; guarding of trolley...

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... Mineral Resources 1 2010-07-01 2010-07-01 false Insulation of trolley wires, trolley feeder wires and bare signal wires; guarding of trolley wires and trolley feeder wires. 77.1802 Section 77.1802 Mineral...

  7. Production of the monoterpene limonene and modulation of apoptosis-related proteins in embryonic-mouse NIH 3T3 fibroblast cells by introduction of the limonene synthase gene isolated from Japanese catnip (Schizonepeta tenuifolia).

    PubMed

    Satomi, Yoshiko; Ohara, Kazuaki; Yazaki, Kazufumi; Ito, Michiho; Honda, Gisho; Nishino, Hoyoku

    2009-03-01

    The monoterpene D-limonene shows cancer preventative and cancer therapeutic activities in vitro and in vivo. Unlike plants, animals are unable to synthesize limonene de novo and obtain limonene through dietary sources. In the present study we established transgenic mouse embryonic NIH 3T3 fibroblast cells that produce limonene by introducing the D-limonene synthase gene obtained from Japanese catnip (Schizonepeta tenuifolia). Apoptosis was not observed in the limonene-producing cells. A concomitant increase in the level of apoptosis-related protein Bcl-2 (B-cell lymphoma protein 2) and decreases in the levels of Bad (Bcl-2 antagonist of cell death) and phosphorylated JNK (c-Jun N-terminal kinase) were observed in limonene-producing cells. Limonene-producing cells may provide a useful new system to investigate the in vivo function of this monoterpene. PMID:18547169

  8. Ginsenosides Rb1 and Rg1 suppress triglyceride accumulation in 3T3-L1 adipocytes and enhance beta-cell insulin secretion and viability in Min6 cells via PKA-dependent pathways.

    PubMed

    Park, Sunmin; Ahn, Il Sung; Kwon, Dae Young; Ko, Byoung Seob; Jun, Won Kyung

    2008-11-01

    Ginseng root is known to induce anti-diabetic activity, but the key components involved are unknown. We investigated which major ginsenosides in ginseng enhanced glucose homeostasis by in vitro studies. Rb1 and Rg1 reduced the triglyceride accumulation in 3T3-L1 adipocytes by activating PKA with increased intracellular cAMP. However, the insulin-stimulated glucose uptake was enhanced by Rb1 and Rg1 via activation of phosphatidylinositol-3 kinase. Rb1 and Rg1 promoted glucose-stimulated insulin secretion and cell viability in Min6 cells through PKA which augmented IRS2 expression to enhance insulin/IGF-1 signaling. These results suggest that Rb1 and Rg1 improved glucose homeostasis through the activation of a PKA like glucagon-like peptide-1 receptor agonist. PMID:18997435

  9. OM2, a Novel Oligomannuronate-Chromium(III) Complex, Promotes Mitochondrial Biogenesis and Lipid Metabolism in 3T3-L1 Adipocytes via the AMPK-PGC1? Pathway

    PubMed Central

    Hao, Jiejie; Hao, Cui; Zhang, Lijuan; Liu, Xin; Zhou, Xiaolin; Dun, Yunlou; Li, Haihua; Li, Guangsheng; Zhao, Xiaoliang; An, Yuanyuan; Liu, Jiankang; Yu, Guangli

    2015-01-01

    Background In our previous studies, we prepared novel oligomannuronate-chromium(III) complexes (OM2, OM4) from marine alginate, and found that these compounds sensitize insulin action better than oligomannuronate(OM), chromium, and metformin in C2C12 skeletal muscle cells. In the present study, we studied their effects on mitochondrial biogenesis, lipid metabolism, and the underlying molecular mechanisms in differentiated 3T3-L1 adipocytes. Methodology/Principal Findings We firstly used the pGL3-PGC1? and pGL3-ATGL promoter plasmids to compare their effects on PGC1? and ATGL transcription activities. Then mitochondrial biogenesis was quantified by transmission electron microscopy and MitoTracker staining. Mitochondrial oxygen consumption and fatty acid oxidation were measured by an oxygen biosensor system and łH-labelled water scintillation. The mitochondrial DNA and mRNA involved in mitochondrial biogenesis and lipid oxidation were evaluated by real-time PCR. AMPK together with other protein expression levels were measured by western blotting. The inhibitor compound C and siRNA of PGC1? were used to inhibit the OM2-induced AMPK-PGC1? signaling pathway. And we found that OM2 stimulated AMPK-PGC1? pathway in the 3T3-L1 adipocytes, which were correlated with induced mitochondrial biogenesis, improved mitochondrial function, and reduced lipid accumulation by enhanced fatty acid ?-oxidation and augmented ATGL protein expression. Conclusions/Significance Our data indicated that the marine oligosaccharide-derived OM2 might represent a novel class of molecules that could be useful for type 2 diabetes prevention and treatment by up-regulating AMPK-PGC1? signaling pathway. PMID:26176781

  10. Tumor necrosis factor (TNF)-?-induced repression of GKAP42 protein levels through cGMP-dependent kinase (cGK)-I? causes insulin resistance in 3T3-L1 adipocytes.

    PubMed

    Ando, Yasutoshi; Shinozawa, Yusuke; Iijima, Yumi; Yu, Bu-Chin; Sone, Meri; Ooi, Yuko; Watanaka, Yusuke; Chida, Kazuhiro; Hakuno, Fumihiko; Takahashi, Shin-Ichiro

    2015-02-27

    Insulin receptor substrates (IRSs) have been shown to be major mediators of insulin signaling. Recently, we found that IRSs form high-molecular weight complexes, and here, we identify by yeast two-hybrid screening a novel IRS-1-associated protein: a 42-kDa cGMP-dependent protein kinase-anchoring protein (GKAP42). GKAP42 knockdown in 3T3-L1 adipocytes suppressed insulin-dependent IRS-1 tyrosine phosphorylation and downstream signaling, resulting in suppression of GLUT4 translocation to plasma membrane induced by insulin. In addition, GLUT4 translocation was also suppressed in cells overexpressing GKAP42-N (the IRS-1 binding region of GKAP42), which competed with GKAP42 for IRS-1, indicating that GKAP42 binding to IRS-1 is required for insulin-induced GLUT4 translocation. Long term treatment of 3T3-L1 adipocytes with TNF-?, which induced insulin resistance, significantly decreased the GKAP42 protein level. We then investigated the roles of cGMP-dependent kinase (cGK)-I?, which bound to GKAP42, in these changes. cGK-I? knockdown partially rescued TNF-?-induced decrease in GKAP42 and impairment of insulin signals. These data indicated that TNF-?-induced repression of GKAP42 via cGK-I? caused reduction of insulin-induced IRS-1 tyrosine phosphorylation at least in part. The present study describes analysis of the novel TNF-?-induced pathway, cGK-I?-GKAP42, which regulates insulin-dependent signals and GLUT4 translocation. PMID:25586176

  11. Prostaglandin E2-induced up-regulation of c-fos messenger ribonucleic acid is primarily mediated by 3',5'-cyclic adenosine monophosphate in MC3T3-E1 osteoblasts

    NASA Technical Reports Server (NTRS)

    Fitzgerald, J.; Dietz, T. J.; Hughes-Fulford, M.

    2000-01-01

    The mechanism by which the proto-oncogene, c-fos, is up-regulated in response to PGE2 in the mouse osteoblastic (MC3T3-E1) cell line was investigated using RT-PCR. c-fos messenger RNA up-regulation by dmPGE2 is rapid, starting 10 min post stimulation, and transient. The specific protein kinase A (PKA) inhibitor, H89, inhibited c-fos induction. Moreover, down-regulation of protein kinase C (PKC) activity by chronic TPA treatment had no effect on the induction of c-fos by dmPGE2. We conclude that up-regulation of c-fos by dmPGE2 is primarily dependent on PKA in MC3T3-E1 osteoblasts. In S49 lymphoma wild-type but not S49 cyc- cells, which are deficient in cAMP signaling, dmPGE2 up-regulates c-fos and increases cell growth compared with unstimulated cells. Thus in S49 lymphoma cells, c-fos induction by PGE2 is also dependent on cAMP signaling. The minimal c-fos promoter region required for dmPGE2-induced expression was identified by transfecting c-fos promoter deletion constructs coupled to the chloramphenicol acetyltransferase (CAT) reporter gene into Vero cells. Transfection of a plasmid containing 99 bp c-fos proximal promoter was sufficient to direct c-fos/CAT expression following stimulation with dmPGE2. Because induction of c-fos is mediated by cAMP, these data are consistent with activation of c-fos via the CRE/ATF cis element.

  12. Testosterone enhances lipopolysaccharide-induced interleukin-6 and macrophage chemotactic protein-1 expression by activating the extracellular signal-regulated kinase 1/2/nuclear factor-?B signalling pathways in 3T3-L1 adipocytes.

    PubMed

    Su, Chunlin; Chen, Min; Huang, Haiyan; Lin, Jinfang

    2015-07-01

    Low-grade chronic inflammation is commonly found in patients with polycystic ovary syndrome (PCOS) who exhibit hyperandrogenism or hyperandrogenemia. Clinical studies have shown that hyperandrogenemia is closely correlated with low-grade chronic inflammation. However, the mechanism underlying this correlation remains unclear. Recent studies have suggested that adipocytes increase the production of proinflammatory mediators such as interleukin-6 (IL-6) and macrophage chemotactic protein-1 (MCP-1) when the inflammatory signal transduction cascade system is activated by external stimuli. The present study aimed to evaluate the effects of testosterone on the innate signalling and expression of proinflammatory mediators in 3T3-L1 adipocytes, which were or were not induced by lipopolysaccharide (LPS). The effects of testosterone on the expression of proinflammatory mediators, nuclear factor-?B (NF-?B), and extracellular signal-regulated kinase 1/2 (ERK1/2) signalling pathways were investigated using an enzyme-linked immunosorbent assay, reverse transcriptase-polymerase chain reaction, western blot analysis and an electrophoresis mobility shift assay. Testosterone induces IL-6 and MCP-1, and enhances LPS-induction of IL-6 and MCP-1. However, the effects are not simply additive, testosterone significantly enhanced the effects of LPS-induced inflammation factors. Testosterone induces the phosphorylation of ERK1/2 and NF-?B. The effect of testosterone on the expression of IL-6 and MCP-1 is inhibited by PD98059 , an ERK1/2 inhibitor, and PDTC, an NF-?B inhibitor. The results indicate that testosterone enhances LPS-induced IL-6 and MCP-1 expression by activating the ERK1/2/NF-?B signalling pathways in 3T3-L1 adipocytes. PMID:25738264

  13. 30 CFR 77.1802 - Insulation of trolley wires, trolley feeder wires and bare signal wires; guarding of trolley...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ...Resources 1 2013-07-01 2013-07-01 false Insulation of trolley wires, trolley feeder wires and bare signal wires...MINES Trolley Wires and Trolley Feeder Wires § 77.1802 Insulation of trolley wires, trolley feeder wires and bare signal...

  14. 30 CFR 75.1003 - Insulation of trolley wires, trolley feeder wires and bare signal wires; guarding of trolley...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ...Resources 1 2014-07-01 2014-07-01 false Insulation of trolley wires, trolley feeder wires and bare signal wires...MINES Trolley Wires and Trolley Feeder Wires § 75.1003 Insulation of trolley wires, trolley feeder wires and bare signal...

  15. 30 CFR 77.1802 - Insulation of trolley wires, trolley feeder wires and bare signal wires; guarding of trolley...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ...Resources 1 2012-07-01 2012-07-01 false Insulation of trolley wires, trolley feeder wires and bare signal wires...MINES Trolley Wires and Trolley Feeder Wires § 77.1802 Insulation of trolley wires, trolley feeder wires and bare signal...

  16. 30 CFR 77.1802 - Insulation of trolley wires, trolley feeder wires and bare signal wires; guarding of trolley...

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ...Resources 1 2014-07-01 2014-07-01 false Insulation of trolley wires, trolley feeder wires and bare signal wires...MINES Trolley Wires and Trolley Feeder Wires § 77.1802 Insulation of trolley wires, trolley feeder wires and bare signal...

  17. 30 CFR 75.1003 - Insulation of trolley wires, trolley feeder wires and bare signal wires; guarding of trolley...

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ...Resources 1 2012-07-01 2012-07-01 false Insulation of trolley wires, trolley feeder wires and bare signal wires...MINES Trolley Wires and Trolley Feeder Wires § 75.1003 Insulation of trolley wires, trolley feeder wires and bare signal...

  18. 30 CFR 75.1003 - Insulation of trolley wires, trolley feeder wires and bare signal wires; guarding of trolley...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ...Resources 1 2013-07-01 2013-07-01 false Insulation of trolley wires, trolley feeder wires and bare signal wires...MINES Trolley Wires and Trolley Feeder Wires § 75.1003 Insulation of trolley wires, trolley feeder wires and bare signal...

  19. 7 CFR 1230.113 - Collection and remittance of assessments for the sale of feeder pigs and market hogs.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ...the sale of feeder pigs and market hogs. 1230.113 Section...AGRICULTURE PORK PROMOTION, RESEARCH, AND CONSUMER INFORMATION...the sale of feeder pigs and market hogs. Pursuant to the provisions...purchasers of feeder pigs or market hogs shall collect...

  20. 7 CFR 1230.113 - Collection and remittance of assessments for the sale of feeder pigs and market hogs.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ...the sale of feeder pigs and market hogs. 1230.113 Section...AGRICULTURE PORK PROMOTION, RESEARCH, AND CONSUMER INFORMATION...the sale of feeder pigs and market hogs. Pursuant to the provisions...purchasers of feeder pigs or market hogs shall collect...

  1. Minimal incorporation of Deepwater Horizon oil by estuarine filter feeders.

    PubMed

    Fry, Brian; Anderson, Laurie C

    2014-03-15

    Natural abundance carbon isotope analyses are sensitive tracers for fates and use of oil in aquatic environments. Use of oil carbon in estuarine food webs should lead to isotope values approaching those of oil itself, -27‰ for stable carbon isotopes reflecting oil origins and -1000‰ for carbon-14 reflecting oil age. To test for transfer of oil from the 2010 Deepwater Horizon spill into estuarine food webs, filter-feeding barnacles (Balanus sp.) and marsh mussels (Geukensia demissa) were collected from Louisiana estuaries near the site of the oil spill. Carbon-14 analyses of these animals from open waters and oiled marshes showed that oil use was <1% and near detection limits estimated at 0.3% oil incorporation. Respiration studies showed no evidence for enhanced microbial activity in bay waters. Results are consistent with low dietary impacts of oil for filter feeders and little overall impact on respiration in the productive Louisiana estuarine systems. PMID:24461698

  2. Microplastic in a macro filter feeder: Humpback whale Megaptera novaeangliae.

    PubMed

    Besseling, E; Foekema, E M; Van Franeker, J A; Leopold, M F; Kühn, S; Bravo Rebolledo, E L; Heße, E; Mielke, L; IJzer, J; Kamminga, P; Koelmans, A A

    2015-06-15

    Marine filter feeders are exposed to microplastic because of their selection of small particles as food source. Baleen whales feed by filtering small particles from large water volumes. Macroplastic was found in baleen whales before. This study is the first to show the presence of microplastic in intestines of a baleen whale (Megaptera novaeangliae). Contents of its gastrointestinal tract were sieved, dissolved in 10% potassium hydroxide and washed. From the remaining dried material, potential synthetic polymer particles were selected based on density and appearance, and analysed by Fourier transform infrared (FTIR) spectroscopy. Several polymer types (polyethylene, polypropylene, polyvinylchloride, polyethylene terephthalate, nylon) were found, in varying particle shapes: sheets, fragments and threads with a size of 1mm to 17cm. This diversity in polymer types and particle shapes, can be interpreted as a representation of the varying characteristics of marine plastic and the unselective way of ingestion by M. novaeangliae. PMID:25916197

  3. Development of an extruder-feeder biomass direct liquefaction process

    SciTech Connect

    White, D.H.; Wolf, D. (Arizona Univ., Tucson, AZ (United States). Dept. of Chemical Engineering)

    1991-10-01

    As an abundant, renewable, domestic energy resource, biomass could help the United States reduce its dependence on imported oil. Biomass is the only renewable energy technology capable of addressing the national need for liquid transportation fuels. Thus, there is an incentive to develop economic conversion processes for converting biomass, including wood, into liquid fuels. Through research sponsored by the US DOE's Biomass Thermochemical Conversion Program, the University of Arizona has developed a unique biomass direct liquefaction system. The system features a modified single-screw extruder capable of pumping solid slurries containing as high as 60 wt% wood flour in wood oil derived vacuum bottoms at pressures up to 3000 psi. The extruder-feeder has been integrated with a unique reactor by the University to form a system which offers potential for improving high pressure biomass direct liquefaction technology. The extruder-feeder acts simultaneously as both a feed preheater and a pumping device for injecting wood slurries into a high pressure reactor in the biomass liquefaction process. An experimental facility was constructed and following shakedown operations, wood crude oil was produced by mid-1985. By July 1988, a total of 57 experimental continuous biomass liquefaction runs were made using White Birch wood feedstock. Good operability was achieved at slurry feed rates up to 30 lb/hr, reactor pressures from 800 to 3000 psi and temperatures from 350{degree}C to 430{degree}C under conditions covering a range of carbon monoxide feed rates and sodium carbonate catalyst addition. Crude wood oils containing as little as 6--10 wt% residual oxygen were produced. 38 refs., 82 figs., 26 tabs.

  4. [Developing of a new feeder-free system and characterization of human embryonic stem cell sublines derived in this system under autogenic and allogenic culturing].

    PubMed

    Kol'tsova, A M; Voronkina, I V; Gordeeva, O F; Zenin, V V; Lifantseva, N V; Musorina, A S; Smagina, L V; Iakovleva, T K; Polianskaia, G G

    2012-01-01

    A new feeder-free culture system for human embryonic stem cells (hESC) was developed. It consist of extracellular matrix proteins synthesized by feeder cells--mesenchymal stem cell line SC5-MSC, which was derived from initial hESC line SC5. The major ECM proteins--fibronectin and laminin--that maintain hESC growth in feeder-free system were identified. An essential component of this system is a SC5-MSC-conditioned medium. Two hESC sublines were derived. The subline SC5-FF was cultured in autogenic and subline SC7-FF in allogenic system. Sublines SC5-FF and SC7-FF passed through more than 300 and 115 cell population doublings, retained normal diploid karyotype and an ability of in vitro differentiation into derivates of three germ layers. These sublines express markers of undifferentiated hESC: alkaline phosphatase, Oct-4, SSEA-4, TRA-1-81 and multidrug resistance transporter--ABCG2. The RT-PCR analysis revealed that undifferentiated cells SC5-FF subline, like cells of initial feeder-maintained hESC line SC5, expressed genes OCT4 and NANOG, and germ line specific genes such as DPPA3/STELLA and DAZL. An expression of OCT4, NANOG, DPPA3/STELLA ans DAZL was down-regulated during embryonic bodies differentiation, whereas expression of somatic lineages specific genes like GATA4 and AFP (extra embryonic and embryonic endoderm), PAX6 (neuroectoderm) and BRY (mesoderm) was up-regulated. The comparative analysis of some typical features (karyotype structure, the average population doubling time and the number of undifferentiated cells in populations) did not reveal essential differences between initial SC5 and SC7 lines and their sublines SC5-FF and SC7-FF. This shows that feeder-free culture systems, which are much more stable than any feeder systems, do not break main hESC features during long cultivation and can be recommended for fundamental, biomedicine and pharmacological investigations, using hESCs. PMID:23074854

  5. Aflatoxin production in supplemental feeders provided for northern bobwhite in Texas and Oklahoma.

    PubMed

    Oberheu, D G; Dabbert, C B

    2001-07-01

    Mycotoxins are toxic metabolites produced by varous species of fungi. Aflatoxin (AF), a particular type of mycotoxin, can negatively impact many wildlife species in the laboratory; however, the magnitude of the problem in the field environment is unclear. Wild birds generally consume a combination of native foods and agricultural grains. A common practice in which birds, such as northern bobwhite (Colinus virginianus), contact stored agricultural grain is through supplemental feeding. This feeding practice may promote the production of AF. The objectives of this study were to (1) examine AF production in supplemental feeders and (2) examine the relationship between weather and AF production in supplemental feeders. Samples were collected from supplemental feeders from November through February of 1996-97 and 1997-98. Mean monthly AF concentration of samples from feeders ranged from 0.57+/-2.86 to 15.47+/-14.69 ppb. Aflatoxin concentration in supplemental feeders increased from pre-sample to one month after filling the feeders each year. AF production in supplemental feeders was highly variable among months with no real temporal pattern between years. Instead, AF production was related to the highly variable relative humidity of the study area which influences moisture content of grain. Average relative humidity can be used to predict AF production. PMID:11504221

  6. Prolonged insulin stimulation down-regulates GLUT4 through oxidative stress-mediated retromer inhibition by a protein kinase CK2-dependent mechanism in 3T3-L1 adipocytes.

    PubMed

    Ma, Jinhui; Nakagawa, Yuko; Kojima, Itaru; Shibata, Hiroshi

    2014-01-01

    Although insulin acutely stimulates glucose uptake by promotion of GLUT4 translocation from intracellular compartments to the plasma membrane in adipocytes and muscles, long term insulin stimulation causes GLUT4 depletion that is particularly prominent in the insulin-responsive GLUT4 storage compartment. This effect is caused mainly by accelerated lysosomal degradation of GLUT4, although the mechanism is not fully defined. Here we show that insulin acutely induced dissociation of retromer components from the low density microsomal membranes of 3T3-L1 adipocytes that was accompanied by disruption of the interaction of Vps35 with sortilin. This insulin effect was dependent on the activity of protein kinase CK2 but not phosphatidylinositol 3-kinase or extracellular signal-regulated kinase 1/2. Knockdown of Vps26 decreased GLUT4 to a level comparable with that with insulin stimulation for 4 h. Vps35 with a mutation in the CK2 phosphorylation motif (Vps35-S7A) was resistant to insulin-induced dissociation from the low density microsomal membrane, and its overexpression attenuated GLUT4 down-regulation with insulin. Furthermore, insulin-generated hydrogen peroxide was an upstream mediator of the insulin action on retromer and GLUT4. These results suggested that insulin-generated oxidative stress switches the GLUT4 sorting direction to lysosomes through inhibition of the retromer function in a CK2-dependent manner. PMID:24240093

  7. Design, synthesis and characterization of novel binary V(V)-Schiff base materials linked with insulin-mimetic vanadium-induced differentiation of 3T3-L1 fibroblasts to adipocytes. Structure-function correlations at the molecular level.

    PubMed

    Halevas, E; Tsave, O; Yavropoulou, M P; Hatzidimitriou, A; Yovos, J G; Psycharis, V; Gabriel, C; Salifoglou, A

    2015-06-01

    Among the various roles of vanadium in the regulation of intracellular signaling, energy metabolism and insulin mimesis, its exogenous activity stands as a contemporary challenge currently under investigation and a goal to pursue as a metallodrug against Diabetes mellitus II. In this regard, the lipogenic activity of vanadium linked to the development of well-defined anti-diabetic vanadodrugs has been investigated through: a) specifically designing and synthesizing Schiff base organic ligands L, bearing a variable number of terminal alcohols, b) a series of well-defined soluble binary V(V)-L compounds synthesized and physicochemically characterized, c) a study of their cytotoxic effect and establishment of adipogenic activity in 3T3-L1 fibroblasts toward mature adipocytes, and d) biomarker examination of a closely-linked molecular target involving or influenced by the specific V(V) forms, cumulatively delineating factors involved in potential pathways linked to V(V)-induced insulin-like activity. Collectively, the results a) project the importance of specific structural features in Schiff ligands bound to V(V), thereby influencing the emergence of its (a)toxicity and for the first time its insulin-like activity in pre-adipocyte differentiation, b) contribute to the discovery of molecular targets influenced by the specific vanadoforms seeking to induce glucose uptake, and c) indicate an interplay of V(V) structural speciation and cell-differentiation biological activity, thereby gaining insight into vanadium's potential as a future metallodrug in Diabetes mellitus. PMID:25920352

  8. Changes in protein kinase C epsilon phosphorylation status and intracellular localization as 3T3 and 3T6 fibroblasts grow to confluency and quiescence: a role for phosphorylation at ser-729?

    PubMed Central

    England, K; Rumsby, M G

    2000-01-01

    Protein kinase C (PKC) epsilon in 3T3 and 3T6 fibroblasts and in C6 glioma cells migrated on SDS/PAGE predominantly as a doublet with molecular masses of 87 and 95 kDa (PKC epsilon(87) and PKC epsilon(95) respectively). PKC epsilon(95) predominates when cells reach confluency but PKC epsilon(87) was the main form detected within 15 min when confluent cells were passaged at low cell density into fresh medium containing serum and allowed to adhere. Matrix-assisted laser-desorption ionization-time-of-flight MS analysis and experiments with phosphospecific antibodies revealed that PKC epsilon(87) is phosphorylated at Thr-566 and Ser-703, and PKC epsilon(95) is additionally phosphorylated at Ser-729. Cell fractionation studies revealed that PKC epsilon(95) is associated with the nuclear fraction, whereas PKC epsilon(87) was found in the 100,000 g cytosol fraction. Immunofluorescence studies confirmed these findings and showed that PKC epsilon(95) had a perinuclear, probably Golgi, localization and PKC epsilon(87) was distributed in the cytosol. It is proposed that phosphorylation at Ser-729 may be important for determining the intracellular localization of PKC epsilon, and that a specific Ser-729 phosphatase may be activated on cell passage to convert PKC epsilon(95) to PKC epsilon(87). PMID:11062054

  9. Insulin-Mimetic Action of Rhoifolin and Cosmosiin Isolated from Citrus grandis (L.) Osbeck Leaves: Enhanced Adiponectin Secretion and Insulin Receptor Phosphorylation in 3T3-L1 Cells

    PubMed Central

    Rao, Yerra Koteswara; Lee, Meng-Jen; Chen, Keru; Lee, Yi-Ching; Wu, Wen-Shi; Tzeng, Yew-Min

    2011-01-01

    Citrus grandis (L.) Osbeck (red wendun) leaves have been used in traditional Chinese medicine to treat several illnesses including diabetes. However, there is no scientific evidence supporting these actions and its active compounds. Two flavone glycosides, rhoifolin and cosmosiin were isolated for the first time from red wendun leaves and, identified these leaves are rich source for rhoifolin (1.1%, w/w). In differentiated 3T3-L1 adipocytes, rhoifolin and cosmosiin showed dose-dependent response in concentration range of o.oo1–5??M and 1–20??M, respectively, in biological studies beneficial to diabetes. Particularly, rhoifolin and cosmosiin at 0.5 and 20??M, respectively showed nearly similar response to that 10?nM of insulin, on adiponectin secretion level. Furthermore, 5??M of rhoifolin and 20??M of cosmosiin showed equal potential with 10?nM of insulin to increase the phosphorylation of insulin receptor-?, in addition to their positive effect on GLUT4 translocation. These findings indicate that rhoifolin and cosmosiin from red wendun leaves may be beneficial for diabetic complications through their enhanced adiponectin secretion, tyrosine phosphorylation of insulin receptor-? and GLUT4 translocation. PMID:20008903

  10. Gelidium elegans, an edible red seaweed, and hesperidin inhibit lipid accumulation and production of reactive oxygen species and reactive nitrogen species in 3T3-L1 and RAW264.7 cells.

    PubMed

    Jeon, Hui-Jeon; Seo, Min-Jung; Choi, Hyeon-Son; Lee, Ok-Hwan; Lee, Boo-Yong

    2014-11-01

    Gelidium elegans is an edible red alga native to the intertidal area of northeastern Asia. We investigated the effect of G.?elegans extract and its main flavonoids, rutin and hesperidin, on lipid accumulation and the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in 3T3-L1 and RAW264.7 cells. Our data show that G.?elegans extract decreased lipid accumulation and ROS/RNS production in a dose-dependent manner. The extract also inhibited the mRNA expression of adipogenic transcription factors, such as peroxisome proliferator-activated receptor gamma and CCAAT/enhancer-binding protein alpha, while enhancing the protein expression of the antioxidant enzymes superoxide dismutases 1 and 2, glutathione peroxidase, and glutathione reductase compared with controls. In addition, lipopolysaccharide-induced nitric oxide production was significantly reduced in G.?elegans extract-treated RAW264.7 cells. In analysis of the effects of G.?elegans flavonoids on lipid accumulation and ROS/RNS production, only hesperidin showed an inhibitory effect on lipid accumulation and ROS production; rutin did not affect adipogenesis and ROS status. The antiadipogenic effect of hesperidin was evidenced by the downregulation of peroxisome proliferator-activated receptor gamma, CCAAT/enhancer-binding protein alpha, and fatty acid binding protein 4 gene expression. Collectively, our data suggest that G.?elegans is a potential food source containing antiobesity and antioxidant constituents. PMID:24930594

  11. SOIL DISTURBANCE REDUCES FEEDER ROOT MASS UNDER IRRIGATED OR NON-IRRIGATED 'HASS' AVOCADO TREES

    Microsoft Academic Search

    J. Dixon; D. B. Smith; A. C. Greenwood

    Alternate bearing of 'Hass' avocado trees is common in New Zealand and leads to reduced grower income. The causes of alternate bearing may be related to the reduced growth of new feeder roots in an \\

  12. An algorithm for adaptive phase overcurrent protection of power distribution feeders

    E-print Network

    Flournoy, Caroll Lynn

    1989-01-01

    AN ALGORITHM FOR ADAPTIVE PHASE OVERCURRENT PROTECTION OF POWER DISTRIBUTION FEEDERS A Thesis by CAROLL LYNN FLOURNOY Submitted to the Office of Graduate Studies of Texas ARM University in partial fulfillment of the requirements... for the degree oi' MASTER OF SCIENCE December 1989 a~or Subject: Electrical Engineering AN ALGORITHM FOR ADAPTIVE PHASE OVERCURRENT PROTECTION OF POWER DISTRIBUTION FEEDERS A Thesis by CAROLL LYNN FLOL RNOY Approved as to style and content by: B. Don...

  13. Method of detecting the direction of arcing faults on power distribution feeders

    E-print Network

    Fernando, W. Anand Krisantha

    1992-01-01

    METHOD OF DETECTING THE DIRECTION OF ARCING FAULTS ON POWER DISTRIBUTION FEEDERS A Thesis by W. ANAND KRISANTHA FERNANDO Submitted to the Office of Graduate Studies of Texas A&M University in partial fulfillment of the requirements... for the degree of MASTER OF SCIENCE May 1992 Major Subject: Electrical Engineering METHOD OF DETECTING THE DIRECTION OF ARCING FAULTS ON POWER DISTRIBUTION FEEDERS A Thesis by W. ANAND KRISANTHA FERNANDO Approved as to style and content by: B. Don...

  14. A probabilistic model of wall thinning in CANDU feeders due to flow-accelerated corrosion

    Microsoft Academic Search

    X.-X. Yuan; M. D. Pandey; G. A. Bickel

    2008-01-01

    Wall thinning due to flow-accelerated corrosion (FAC) is a pervasive form of degradation in feeder pipes of the primary heat transport system of CANDU reactors. Prediction of the end-of-life of a feeder from wall thickness measurement data is confounded by the sampling and temporal uncertainties associated with the FAC degradation phenomenon. This paper presents a probabilistic model of wall thinning

  15. Method of detecting the direction of arcing faults on power distribution feeders 

    E-print Network

    Fernando, W. Anand Krisantha

    1992-01-01

    METHOD OF DETECTING THE DIRECTION OF ARCING FAULTS ON POWER DISTRIBUTION FEEDERS A Thesis by W. ANAND KRISANTHA FERNANDO Submitted to the Office of Graduate Studies of Texas A&M University in partial fulfillment of the requirements... for the degree of MASTER OF SCIENCE May 1992 Major Subject: Electrical Engineering METHOD OF DETECTING THE DIRECTION OF ARCING FAULTS ON POWER DISTRIBUTION FEEDERS A Thesis by W. ANAND KRISANTHA FERNANDO Approved as to style and content by: B. Don...

  16. Coplanar phased array antenna with optical feeder and photonic bandgap structure

    Microsoft Academic Search

    Guy-Aymar Chakam; Wolfgang Freude

    1999-01-01

    A novel coplanar broadband dipole phased array sending antenna with optical feeder network and photonic bandgap structure has been developed. Hybridly integrated opto-electronic receivers and high-gain MMIC power amplifiers detect the modulated optical input power and deliver a 20-GHz microwave signal to the antenna elements. A planar photonic bandgap structure surrounding each dipole and its electric feeder line suppresses parallel

  17. The analysis of the performance of porous electrodes with two equipotential current feeders

    Microsoft Academic Search

    A. I. Masliy; N. P. Poddubny

    1997-01-01

    On the basis of a one-dimensional model of the porous electrode (pe) and the total polarization curve involving the main and side electrode reactions, the dependence of performance efficiency of the two-section electrode with two short-circuited current feeder on the solid and liquid phase conductivity relation and the position of an additional current feeder is analysed. The introduction of an

  18. The Stamet Posimetric{reg_sign} high-pressure solids feeder

    SciTech Connect

    Hay, A.; Crowley, M. [Stamet, Inc., Gardena, CA (United States); Maronde, C. [Dept. of Energy, Pittsburgh, PA (United States). Federal Energy Technology Center

    1998-12-31

    In the late 1980`s, Stamet, Inc. started development of a device for pumping granular solids utilizing particle interlocking and internal friction. Developmental work resulted in a novel rotary mechanical feeder capable of feeding granular solids at very consistent rates at ambient or elevated pressures. In 1995, Stamet received an R and D 100 Award from Power Magazine for the design of the Posimetric feeder. The Posimetric feeder is presently operating in approximately 20 commercial installations feeding various materials under ambient conditions. The Posimetric feeder is also a very significant development for advanced combustion systems, including PFBC and IGCC applications, where it is expected to substantially be more reliable and less expensive to install and operate than the lock-hopper and paste systems currently used for feeding coal into high gas pressure environments. To date, a 600 lb/hr unit has been successfully tested for pumping granular coal mixtures into a gas pressure of 250 psig. Stamet currently is continuing development of the feeder for operating at gas discharge pressures of up to 450 psig and for pressure letdown applications. This paper will detail the unique design and operational characteristics of the Posimetric feeder and provide an update on Stamet`s continuing developmental activities on the unit.

  19. Flow accelerated corrosion of carbon steel feeder pipes from pressurized heavy water reactors

    NASA Astrophysics Data System (ADS)

    Singh, J. L.; Kumar, Umesh; Kumawat, N.; Kumar, Sunil; Kain, Vivekanand; Anantharaman, S.; Sinha, A. K.

    2012-10-01

    Detailed investigation of a number of feeder pipes received from Rajasthan Atomic Power Station Unit 2 (RAPS#2) after en-masse feeder pipe replacement after 15.67 Effective Full Power Years (EFPYs) was carried out. Investigations included ultrasonic thickness measurement by ultrasonic testing, optical microscopy, scanning electron microscopy, chemical analysis and X-ray Diffraction (XRD). Results showed that maximum thickness reduction of the feeder had occurred downstream and close to the weld in 32 NB (1.25?/32.75 mm ID) elbows. Rate of Flow Accelerated Corrosion (FAC) was measured to be higher in the lower diameter feeder pipes due to high flow velocity and turbulence. Weld regions had thinned to a lower extent than the parent material due to higher chromium content in the weld. A weld protrusion has been shown to add to the thinning due to FAC and lead to faster thinning rate at localized regions. Surface morphology of inner surface of feeder had shown different size scallop pattern over the weld and parent material. Inter-granular cracks were also observed along the weld fusion line and in the parent material in 32 NB outlet feeder elbow.

  20. The relative cytotoxicity of personal care preservative systems in Balb/C 3T3 clone A31 embryonic mouse cells and the effect of selected preservative systems upon the toxicity of a standard rinse-off formulation.

    PubMed

    Smith, C N; Alexander, B R

    2005-10-01

    Biocide chemicals are commonly used as preservatives for cosmetic and personal care products and the conditions for their use are stipulated in Annex VI of the Cosmetics Directive. In these studies the cytotoxicity (EC50 and EC90) of a range of preservatives including the isothiazolinone family, formaldehyde donors, parabens mixtures and organic acids have been established in the Balb/C 3T3 clone A31 fibroblast cell-line following a 1h exposure. Cell viability was established using the neutral red uptake assay 24h after exposure. The potency of the preservatives spanned several orders of magnitude from the isothiazolinones (EC50<10ppm) to the organic acids (EC50>10,000ppm). Although these values are directly proportional to the anti-microbial efficacy of the actives, they do not reflect the addition levels commonly used to preserve formulations, which are intended to provide prolonged protection against a wide spectrum of spoilage organisms. In a further study, the cytotoxic profile of an unpreserved standard rinse-off body wash formulation was assessed. Two concentrations of the formulation were selected: 0.1% v/v (EC98) and 0.15% v/v (EC82) to study the effects of selected preservative chemicals at recommended addition levels upon the cytotoxicity of the formulation. At 0.1%, only preservation with benzoate/sorbate at the highest addition level increased the toxicity, whereas at 0.15%, preservation with 2-bromo-2-nitro-propane-1,3-diol increased the cytotoxicity of the formulation. No other preservatives, including isothiazolinones and formaldehyde donors affected the basal cytotoxicity of the formulation. Theses studies have provided a standardised assessment of the cytotoxicity of cosmetic preservatives and demonstrated that preservation of a rinse-off formulation at recommended addition levels is unlikely to affect the cytotoxic profile. PMID:16055304

  1. Lipolytic and antiadipogenic effects of (3,3-dimethylallyl) halfordinol on 3T3-L1 adipocytes and high fat and fructose diet induced obese C57/BL6J mice.

    PubMed

    Saravanan, Munisankar; Pandikumar, Perumal; Saravanan, Subramaniam; Toppo, Erenius; Pazhanivel, Natesan; Ignacimuthu, Savarimuthu

    2014-10-01

    Aegle marmelos Correa., (Rutaceae) is a medium sized tree distributed in South East Asia and used traditionally for the management of obestiy and diabetes. In this study the lipolytic and antiadipogenic effects of (3,3-dimethylallyl) halfordinol (Hfn) isolated from leaves of A. marmelos have been investigated. Intracellular lipid accumulation was measured by oil red O staining and glycerol secretion. The expression of genes related to adipocyte differentiation was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR). Hfn decreased intracellular triglyceride accumulation and increased glycerol release in a dose dependent manner (5-20 ?g/ml) in differentiated 3T3-L1 adipocytes. In high fat diet fed C57/BL 6J mice, treatment with Hfn for four weeks reduced plasma glucose, insulin and triglyceride levels and showed a significant reduction in total adipose tissue mass by 37.85% and visceral adipose tissue mass by 62.99% at 50mg/kg b.w. concentration. RT-PCR analyses indicated that Hfn decreased the expression of peroxisome proliferator-activated receptor ? (PPAR?) and CCAAT enhancer binding protein ? (CEBP?) and increased the expression of sterol regulatory enzyme binding protein (SREBP-1c), peroxisome proliferator-activated receptor ? (PPAR?), Adiponectin and Glucose transporter protein 4 (GLUT4) compared to the high fat diet group. These results suggested that Hfn decreased adipocyte differentiation and stimulated lipolysis of adipocytes. This study justifies the folklore medicinal uses and claims about the therapeutic values of this plant for the management of insulin resistance and obesity. PMID:24952133

  2. Quantification of Hormone Sensitive Lipase Phosphorylation and Colocalization with Lipid Droplets in Murine 3T3L1 and Human Subcutaneous Adipocytes via Automated Digital Microscopy and High-Content Analysis

    PubMed Central

    Ingermanson, Randall S.; Loy, Patricia A.; Koon, Erick D.; Whittaker, Ross; Laris, Casey A.; Hilton, Jeffrey M.; Nicoll, James B.; Buehrer, Benjamin M.; Price, Jeffrey H.

    2011-01-01

    Abstract Lipolysis in adipocytes is associated with phosphorylation of hormone sensitive lipase (HSL) and translocation of HSL to lipid droplets. In this study, adipocytes were cultured in a high-throughput format (96-well dishes), exposed to lipolytic agents, and then fixed and labeled for nuclei, lipid droplets, and HSL (or HSL phosphorylated on serine 660 [pHSLser660]). The cells were imaged via automated digital fluorescence microscopy, and high-content analysis (HCA) methods were used to quantify HSL phosphorylation and the degree to which HSL (or pHSLser660) colocalizes with the lipid droplets. HSL:lipid droplet colocalization was quantified through use of Pearson's correlation, Mander's M1 Colocalization, and the Tanimoto coefficient. For murine 3T3L1 adipocytes, isoproterenol, Lys-?3-melanocyte stimulating hormone, and forskolin elicited the appearance and colocalization of pHSLser660, whereas atrial natriuretic peptide (ANP) did not. For human subcutaneous adipocytes, isoproterenol, forskolin, and ANP activated HSL phosphorylation/colocalization, but Lys-?3-melanocyte stimulating hormone had little or no effect. Since ANP activates guanosine 3?,5?-cyclic monophosphate (cGMP)-dependent protein kinase, HSL serine 660 is likely a substrate for cGMP-dependent protein kinase in human adipocytes. For both adipocyte model systems, adipocytes with the greatest lipid content displayed the greatest lipolytic responses. The results for pHSLser660 were consistent with release of glycerol by the cells, a well-established assay of lipolysis, and the HCA methods yielded Z? values >0.50. The results illustrate several key differences between human and murine adipocytes and demonstrate advantages of utilizing HCA techniques to study lipolysis in cultured adipocytes. PMID:21186937

  3. Control of insulin receptor level in 3T3 cells: effect of insulin-induced down-regulation and dexamethasone-induced up-regulation on rate of receptor inactivation.

    PubMed Central

    Knutson, V P; Ronnett, G V; Lane, M D

    1982-01-01

    Chronic exposure of 3T3 mouse fibroblasts to insulin or to the glucocorticoid dexamethasone induces down-regulation and up-regulation, respectively, of cell-surface and total cellular insulin binding capacity. Both processes are reversed upon withdrawal of the inducer. Scatchard analysis of insulin binding for receptors in the down- and up-regulated states indicates that the changes in binding capacity result primarily from alterations in insulin receptor level. That these alterations in total receptor level are due to changes in cell-surface receptor level is indicated by the fact that the level of trypsin-insensitive, presumably intracellular, insulin binding sites does not change appreciably upon down- and up-regulation. The effects of insulin-induced down-regulation and dexamethasone-induced up-regulation on the rates of insulin receptor synthesis and decay were assessed by the heavy-isotope density-shift technique. Cells were shifted to medium containing heavy (2H, 13C, 15N) amino acids and, at various times after the shift, light and heavy receptors solubilized from total cellular membranes were resolved by isopycnic banding on density gradients and then quantitated. It was demonstrated that the insulin- and dexamethasone-induced alterations in insulin receptor level were due entirely to changes in the rate constant for receptor inactivation. The decrease in the first-order rate constant for receptor decay caused by dexamethasone is unexpected in view of the known action of steroid hormones in the induction of the synthesis of specific proteins. PMID:7045873

  4. Combined potentiating action of phytochemical(s) from Cinnamomum tamala and Aloe vera for their anti-diabetic and insulinomimetic effect using in vivo rat and in vitro NIH/3T3 cell culture system.

    PubMed

    Singh, Vineeta; Singh, S P; Singh, Manoj; Gupta, Atul Kumar; Kumar, Anil

    2015-03-01

    The present investigation was undertaken to analyze the ethanolic extracts of leaves of Cinnamomum tamala and Aloe vera for their anti-diabetic and insulinomimitic effect by determining the levels of blood sugar, glycosylated hemoglobin, and serum lipid profile (total cholesterol, triglycerides, high density lipoprotein (HDL), and low density lipoprotein (LDL)) after daily administration of each alone and in combined at 250 mg/kg in alloxan (ALX)-induced diabetic rats. Treatment of diabetic rats with the extracts restored the elevated biochemical parameters significantly. The anti-diabetic effect further potentiated the insulin signaling pathway by co-administration of both extracts. The molecular mechanisms of modulating gene expression and cellular signaling through the insulin receptor were also evaluated on specific targets of the insulin signaling pathway, including insulin receptor substrate (IRS), phosphatidylinositol 3-kinase (PI3-K), AKT, and the glucose transporter (GLUT4) on NIH/3T3 cell line by western blotting, ELISA, semiquantitative RT-PCR, and real-time PCR. The active principle of both extracts revealed insulin mimicking effect as indicated by increased expression of pIRS1 and pAKT in time-dependent manner. There was no significant difference in PI3-K content between unchallenged and challenged groups. Enhanced expression of GLUT-4 transcript further suggested that the Cinnamomum and Aloe phytochemicals could serve as a good adjuvant in the present armamentarium of anti-diabetic drugs by either mimicking or improving insulin action. This study reveals that ethanolic extracts of C. tamala and A. vera have potent therapeutic efficacy and prospect for the development of phytomedicine for diabetes mellitus. PMID:25536877

  5. The Fate of the Nucleolus during Mitosis: Comparative Analysis of Localization of Some Forms of Pre-rRNA by Fluorescent in Situ Hybridization in NIH/3T3 Mouse Fibroblasts

    PubMed Central

    Shishova, K.V.; Zharskaya, ?.?.; Zatsepina, ?.V.

    2011-01-01

    Nucleolus is the major structural domain of the cell nucleus, which in addition to proteins contains ribosomal RNA (rRNA) at different stages of maturation (or pre-rRNA). In mammals, the onset of mitosis is accompanied by the inhibition of rRNA synthesis, nucleolus disassembly, and the migration of pre-rRNA to the cytoplasm. However, the precise role of cytoplasmic pre-rRNA in mitosis remains unclear, and no comparative analysis of its different forms at consequent mitotic stages has thus far been performed. The focus of this research was the study of the localization of pre-rRNA in mitotic NIH/3T3 mouse fibroblasts by fluorescentin situhybridization (FISH) with probes to several regions of mouse primary 47S pre-rRNA transcripts and by confocal laser microscopy. The results reveal that all types of pre-rRNA appear in the cytoplasm at the beginning of mitosis, following the breakdown of the nucleolus and nuclear envelope. However, not all pre-rRNA are transported by chromosomes from maternal cells into daughter cells. At the end of mitosis, all types of pre-rRNA and 28S rRNA can be visualized in nucleolus-derived foci (NDF), structures containing many proteins of mature nucleoli the appearance of which indicates the commencement of nucleologenesis. However, early NDF are enriched in less processed pre-RNA, whereas late NDF contain predominantly 28S rRNA. Altogether, the results of this study strengthen the hypotheses that postulate that different forms of pre-rRNA may play various roles in mitosis, and that NDF can be involved in the maturation of pre-rRNA, remaining preserved in the cytoplasm of dividing cells. PMID:22649709

  6. Hydrogels as feeder-free scaffolds for long-term self-renewal of mouse induced pluripotent stem cells.

    PubMed

    Yang, Jing Jing; Liu, Jian Fang; Kurokawa, Takayuki; Kitada, Kazuhiro; Gong, Jian Ping

    2015-04-01

    Expanding undifferentiated induced pluripotent stem (iPS) cells in vitro is a basic requirement for application of iPS cells in both fundamental research and clinical regeneration. In this study, we intended to establish a simple, low cost and efficient method for the long-term self-renewal of mouse induced pluripotent stem (miPS) cells without using feeder-cells and adhesive proteins. Three scaffolds were selected for the long-term subculture of miPS cells over two months starting from passages 14 to 29: 1) a gelatin coated polystyrene (Gelatin-PS) that is a widely used scaffold for self-renewal of mouse embryonic stem (mES) cells; 2) a neutral hydrogel poly(N,N-dimethylacrylamide) (PDMAAm); and 3) a negatively charged hydrogel poly(2-acrylamido-2-methyl-propane sulfonic acid sodium salt) (PNaAMPS). Each passaged miPS cells on these scaffolds were cryopreserved successfully and the revived cells showed high viability and proliferation. The passaged miPS cells maintained a high undifferentiated state on all three scaffolds and a high level of pluripotency by expressing differentiation markers in vitro and forming teratomas in SCID mice with derivatives of all three germ layers. Compared to Gelatin-PS, the two hydrogels exhibited much better self-renewal performance in terms of high proliferation rate and level of expression of undifferentiated gene markers as well as efficiency in pluripotent teratoma formation. Furthermore, the PNaAMPS hydrogel demonstrated a slightly higher efficiency and simpler operation of cell expansion than the PDMAAm hydrogel. To conclude, PNaAMPS hydrogel is an excellent feeder-free scaffold because of its simplicity, low cost and high efficiency in expanding a large number of miPS cells in vitro. PMID:23166055

  7. ANALYSIS OF DISTRIBUTION FEEDER LOSSES DUE TO ADDITION OF DISTRIBUTED PHOTOVOLTAIC GENERATORS

    SciTech Connect

    Tuffner, Francis K.; Singh, Ruchi

    2011-08-09

    Distributed generators (DG) are small scale power supplying sources owned by customers or utilities and scattered throughout the power system distribution network. Distributed generation can be both renewable and non-renewable. Addition of distributed generation is primarily to increase feeder capacity and to provide peak load reduction. However, this addition comes with several impacts on the distribution feeder. Several studies have shown that addition of DG leads to reduction of feeder loss. However, most of these studies have considered lumped load and distributed load models to analyze the effects on system losses, where the dynamic variation of load due to seasonal changes is ignored. It is very important for utilities to minimize the losses under all scenarios to decrease revenue losses, promote efficient asset utilization, and therefore, increase feeder capacity. This paper will investigate an IEEE 13-node feeder populated with photovoltaic generators on detailed residential houses with water heater, Heating Ventilation and Air conditioning (HVAC) units, lights, and other plug and convenience loads. An analysis of losses for different power system components, such as transformers, underground and overhead lines, and triplex lines, will be performed. The analysis will utilize different seasons and different solar penetration levels (15%, 30%).

  8. Calculation and Optimization of ITER Upper VS Feeder Under an Electromagnetic Load

    NASA Astrophysics Data System (ADS)

    Wang, Xianwei; Xie, Fei; Jin, Huan

    2014-11-01

    The upper vertical stability (VS) feeder is a part connected to the upper VS coil by a welding joint. The function of the feeder is to transfer current and coolant water to the VS coil. A giant electromagnetic force will be generated during normal operation by the current flowing in the VS coils, interacting with the external background field. The Lorentz force will induce Tresca stress in the feeder. The amplitudes of the magnetic field and Lorentz force along the conductor running direction have been calculated based on Maxwell's equations. To extract the Tresca stress in the feeder, a finite element model was created using the software ANSYS and an electromagnetic load was applied on the model. According to the analytical design, the stresses were classified and evaluated based on ASME. In order to reduce the Tresca stress, some optimization works have been done and the Tresca stress has had a significant reduction in the optimized model. This analytical work figured out the stress distribution in the feeder and checked the feasibility of the prototype design model. The ANSYS analysis results will provide a guidance for later improvement and fabrication.

  9. Dynamic feeder dyke systems in basaltic volcanoes: the exceptional example of the 1809 Etna eruption (Italy)

    NASA Astrophysics Data System (ADS)

    Geshi, Nobuo; Neri, Marco

    2014-07-01

    In this paper, we describe the 1809 eruption of Mt. Etna, Italy, which represents one historical and rare case in which it is possible to closely observe the internal structure of the feeder system. This is possible thanks to the presence of two large pit craters located in the middle of the eruptive fracture field that allow studying a section of the shallow feeder system. Along the walls of one of these craters, we analysed well-exposed cross sections of the uppermost 15–20 m of the feeder system and related volcanic products. Here, we describe the structure, morphology and lithology of this portion of the 1809 feeder system, including the host rock which conditioned the propagation of the dyke, and compare the results with other recent eruptions. Finally, we propose a dynamic model of the magma behaviour inside a laterally–propagating feeder dyke, demonstrating how this dynamic triggered important changes in the eruptive style (from effusive/Strombolian to phreatomagmatic) during the same eruption. This is therefore an exceptional case to understand how basaltic magmas move during the propagation of an eruptive fissure. Our results are also useful for hazard assessment related to the development of flank eruptions, potentially the most hazardous type of eruption from basaltic volcanoes in densely urbanized areas like those at Mt. Etna.

  10. Epigallocathechin-3 Gallate Selectively Inhibits the PDGF-BB–induced Intracellular Signaling Transduction Pathway in Vascular Smooth Muscle Cells and Inhibits Transformation of sis-transfected NIH 3T3 Fibroblasts and Human Glioblastoma Cells (A172)

    PubMed Central

    Ahn, Hee-Yul; Hadizadeh, Kourosch Reza; Seul, Claudia; Yun, Yeo-Pyo; Vetter, Hans; Sachinidis, Agapios

    1999-01-01

    Enhanced activity of receptor tyrosine kinases such as the PDGF ?-receptor and EGF receptor has been implicated as a contributing factor in the development of malignant and nonmalignant proliferative diseases such as cancer and atherosclerosis. Several epidemiological studies suggest that green tea may prevent the development of cancer and atherosclerosis. One of the major constituents of green tea is the polyphenol epigallocathechin-3 gallate (EGCG). In an attempt to offer a possible explanation for the anti-cancer and anti-atherosclerotic activity of EGCG, we examined the effect of EGCG on the PDGF-BB–, EGF-, angiotensin II-, and FCS-induced activation of the 44 kDa and 42 kDa mitogen-activated protein (MAP) kinase isoforms (p44mapk/p42mapk) in cultured vascular smooth muscle cells (VSMCs) from rat aorta. VSMCs were treated with EGCG (1–100 ?M) for 24 h and stimulated with the above mentioned agonists for different time periods. Stimulation of the p44mapk/p42mapk was detected by the enhanced Western blotting method using phospho-specific MAP kinase antibodies that recognized the Tyr204-phosphorylated (active) isoforms. Treatment of VSMCs with 10 and 50 ?M EGCG resulted in an 80% and a complete inhibition of the PDGF-BB–induced activation of MAP kinase isoforms, respectively. In striking contrast, EGCG (1–100 ?M) did not influence MAP kinase activation by EGF, angiotensin II, and FCS. Similarly, the maximal effect of PDGF-BB on the c-fos and egr-1 mRNA expression as well as on intracellular free Ca2+ concentration was completely inhibited in EGCG-treated VSMCs, whereas the effect of EGF was not affected. Quantification of the immunoprecipitated tyrosine-phosphorylated PDGF-R?, phosphatidylinositol 3?-kinase, and phospholipase C-?1 by the enhanced Western blotting method revealed that EGCG treatment effectively inhibits tyrosine phosphorylation of these kinases in VSMCs. Furthermore, we show that spheroid formation of human glioblastoma cells (A172) and colony formation of sis-transfected NIH 3T3 cells in semisolid agar are completely inhibited by 20–50 ?M EGCG. Our findings demonstrate that EGCG is a selective inhibitor of the tyrosine phosphorylation of PDGF-R? and its downstream signaling pathway. The present findings may partly explain the anti-cancer and anti-atherosclerotic activity of green tea. PMID:10198059

  11. Layered granodiorites at Chebucto Head, South Mountain batholith, Nova Scotia

    Microsoft Academic Search

    D. B. Clarke; G. K. C. Clarke

    1998-01-01

    A sequence of more than 40 distinct, rhythmically-banded layers (typically 10–30cm thick), occurs in the contact-zone granodiorite of the South Mountain batholith, Nova Scotia. The layered sequence shows well-developed modal mineralogical variations, grain-size variations, layer bifurcations, scour-and-fill structures, cross-bedding, slump structures, and feeder dykes. Various processes (granitization of sediments, closed-system fractionation, double-diffusion, shear flow) are incapable of explaining the field

  12. Effect of artificial feeders on pollen loads of the hummingbirds of Cerro de la Muerte, Costa Rica.

    PubMed

    Avalos, Gerardo; Soto, Alejandra; Alfaro, Willy

    2012-03-01

    Although sugar-water feeders are commonly used by enthusiasts to attract hummingbirds, little is known about how they affect hummingbird behavior and flower use. We studied the highland hummingbird assemblage of Cerro de La Muerte, Costa Rica, both at a site with permanent feeders (La Georgina Restaurant) and further from it. We examined how feeder use and monopolization affected seasonal changes in pollen loads during four sampling periods, including dry and wet seasons, from 2003-2005. We expected that species monopolizing the feeders would carry little or no pollen whatsoever, and would have pollen loads characterized by low floral diversity, in contrast with species less dependent on feeders. We obtained pollen samples from 183 individuals of four hummingbird species captured around the feeders using mist nets, which were compared with a pollen reference collection of plants with a pollination syndrome by hummingbirds. The same methods were implemented at a site 3km away from the feeders. Feeder usage was quantified by counting the number of times hummingbirds drank from the feeders in periods of 4min separated by 1min. The effects of hummingbird species and season on pollen load categories were assessed using a nominal logistic regression. The alpha species at the site, the Fiery-throated Hummingbird (Panterpe insignis), dominated the feeders during the dry season. Meanwhile, in the wet season, feeder usage was more evenly distributed across species, with the exception of the Volcano Hummingbird, Selasphorus flammula, which occupies the last place in the dominance hierarchy. Pollen loads of hummingbirds captured near feeders were low in abundance (more than 50% of captured individuals had zero or low pollen loads), and low in species richness (96% of the hummingbirds with pollen from only one plant genus, Centropogon). Overall pollen loads increased during the dry season coinciding with peaks in flower availability, although the majority of captured hummingbirds carried no pollen. Mist nets located 3km from La Georgina returned few captures (one-to-three specimens) per sampling date, contrasting with observations made before feeders were present. These results suggest that sugar-water feeders gather hummingbirds in over considerable distances drawing them away from flowers. The competitive and antagonistic pattern shown between feeders and flowers indicate that natural pollination system could be significantly altered. Supplementing hummingbirds with food seems likely to interfere with pollination networks already stressed by many anthropogenic effects. PMID:22458209

  13. Conspray dynamic sleeve piston coal feeder. Phase II. Verification tests. Final technical report

    SciTech Connect

    Not Available

    1984-01-26

    This report details the performance of Phase II: Verification Tests of the Conspray dynamic sleeve piston coal feeder. The machine performed for 200 hours at 700 psi backpressure, utilizing a 70% to 200 mesh Utah bituminous coal as feedstock. All test work was satisfactorily completed. A post-test inspection was performed. A report of component wear and failures incurred in testing is included as well as suggestions for machine upgrades. The overall conclusion is that the dynamic sleeve piston feeder has proven its ability to operate safely and reliably. When problems have occurred, the machine has demonstrated inherent safety by shutting down without endangering process or personnel. With the recommended improvements incorporated into the feeder, the unit will be ready for installation on a pilot scale coal gasifier. 9 figures, 11 tables.

  14. Factors affecting the selling price of feeder cattle sold at Arkansas livestock auctions in 2005.

    PubMed

    Barham, B L; Troxel, T R

    2007-12-01

    Data were collected from 15 Arkansas livestock auctions to determine factors affecting selling price. Data included how calves were sold (single or groups), sex, breed or breed type, color, muscle thickness, horn status, frame score, fill, body condition, age, health, BW, and price. Data were randomly collected on 52,401 lots consisting of 105,542 calves. Selling prices for steers ($124.20 +/- 0.07), bulls ($117.93 +/- 0.12), and heifers ($112.81 +/- 0.07) were different from each other (P <0.001). Hereford x Charolais feeder calves sold for the highest price ($122.66 +/- 0.14) and Longhorns sold for the lowest price ($74.52 +/- 0.46). Yellow feeder cattle received the highest selling price ($96.47 +/- 0.12), and spotted or striped feeder cattle received the lowest price ($83.84 +/- 0.23). The selling price of singles was lower than the price for calves sold in groups of 6 or more ($117.26 +/- 0.06 vs. $122.61 +/- 0.21; P <0.001). For cattle classified as having muscle scores of 1, 2, 3, and 4, selling prices were $120.45 +/- 0.05, $111.31 +/- 0.09, $96.28 +/- 0.44, and $82.21 +/- 1.87, respectively. Polled feeder cattle sold for $118.57 +/- 0.05, and horned feeder cattle sold for $114.87 +/- 0.14 (P <0.001). Interactions (P <0.001) were detected between frame score and BW groups, and muscle score and BW groups on the selling price of cattle. A number of management and genetic factors affected the selling price of feeder cattle. PMID:17709785

  15. Optimization of multi-feeder (depot) printed circuit board manufacturing with error guarantees

    Microsoft Academic Search

    Burak Kazaz; Kemal Altinkemer

    2003-01-01

    Abstract This paper considers an integrated optimization,problem,enhancing,productivity in printed circuit board,(PCB) manufacturing.,The problems,of assigning component,types to feeder locations and sequencing,component,placements on the PCB are simultaneously,formulated,in a mathematical,model. Our model,differs from earlier studies by allowing component types to be placed in multiple feeders. Although such flexibility adds complexity to the original problem, we develop,an integrated solution that has promising,results. We develop,an

  16. Feed intake and growth of fast and slow growing strains of rainbow trout ( Oncorhynchus mykiss) fed by automatic feeders or by self-feeders

    Microsoft Academic Search

    L. M. P. Valente; B. Fauconneau; E. F. S. Gomes; T. Boujard

    2001-01-01

    Rainbow trout (8.5–9.5 g) of two strains (C and M) differing in growth potential were compared with respect to feeding motivation and feeding rhythms, over a 65-day experimental period, employing self-feeding or automatic feeding. Growth rate, feed gain ratio, feed intake and pattern of feeding activity of fish fed with self-feeders, were recorded, as was body composition of both strains.

  17. Wave Propagation Regime to Point to Faulty Feeder in a Mixed Cable-and-Lines Distribution Systems

    E-print Network

    Paris-Sud XI, Université de

    by multiple refractions and reflections from busbar, fault and loads. The initial propagation zone presents or on the soil resistivity, and if scrutinized on all feeders it can point out from busbar to fault in systems occurring on feeder a. The analyzed data are residual current waves as recorded by sensors on the busbar

  18. Production, Manufacturing and Logistics Optimization of multi-feeder (depot) printed circuit board manufacturing with error guarantees

    Microsoft Academic Search

    Burak Kazaz; Kemal Altinkemer

    This paper considers an integrated optimization problem enhancing productivity in printed circuit board (PCB) manufacturing. The problems of assigning component types to feeder locations and sequencing component placements on the PCB are simultaneously formulated in a mathematical model. Our model differs from earlier studies by allowing component types to be placed in multiple feeders. Although such flexibility adds complexity to

  19. Optimization issues in automated production of printed circuit boards: operations sequencing, feeder configuration and load balancing problems

    Microsoft Academic Search

    Ekrem Duman

    1996-01-01

    In electronics industry, the widely used automatic placement machines insert or amount electronic components, supplied from sequential or random access feeders, to predefined locations on printed circuit boards. The sequencing of placement operations, the assignment of component types to feeder cells and load balancing in these machines directly influence productivity. In this research, such sequencing, assignment and balancing problems, arising

  20. Effects of bale feeder type and supplementation of monensin on hay waste, intake, and performance of beef cattle

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The effects of feeder type and supplemental monensin on hay utilization in beef cows was investigated using 56 crossbred beef cows (BW= 494 ± 50 kg; BCS= 5.2 ± 0.5) in a split-plot treatment arrangement with a completely randomized design. Supplement treatment served as the main plot and feeder desi...

  1. Statistic Characteristic Estimations of Harmonic And Flicker on Electric Arc Furnace Feeders

    Microsoft Academic Search

    Ming-Tang Chen; Chen-Wen Lu

    2006-01-01

    This paper proposes the estimation results of the stochastic characteristics of harmonic and flicker for an AC and a DC electric arc furnace feeders. These characteristics include stationarity, normality and correlation. Besides, the statistic probability was assessed, too. The estimation and the assessment are implemented by a PC-based virtual instrumentation system. The results show that (1) most harmonic and flicker

  2. A heuristic method for feeder reconfiguration and service restoration in distribution networks

    Microsoft Academic Search

    S. P. Singh; G. S. Raju; G. K. Rao; M. Afsari

    2009-01-01

    A sequential switch opening method is proposed for minimum loss feeder reconfiguration in this paper. The algorithm is further extended for service restoration. The method is based on the branch power flow rather than the current flow as reported in earlier methods. The final algorithm arrives at opening of a branch in a loop carrying minimum resistive power flow to

  3. Species Visitation at Quail Feeders and Guzzlers in Southern New Mexico

    Microsoft Academic Search

    Dale Rollins; Ben D. Taylor; Troy D. Sparks; Tom E. Wadell; George Richards

    Providing supplemental feed and water are sometimes used to manage scaled quail (Callipepla squamata) in the Chihuahuan Desert even though their biological and economical efficacies are questionable. Seasonal visitation rates of scaled quail and various nontarget species are important parameters affecting the efficacy of feeding and watering practices. However, empirical data on visitation by scaled quail at feeders and guzzlers

  4. A Novel Algorithm for Automatic Generation of One-Line Diagrams of Distribution Feeders

    Microsoft Academic Search

    P. S. NAGENDRA RAO; RAVISHANKAR DEEKSHIT

    2004-01-01

    This article presents an algorithm for automatically generating readable one-line diagrams of radial distribution feeders. As readability is a subjective view, we propose a set of specifications for the drawing (aesthetic criteria) that ensures a good visualization of distribution systems. The network data of distribution systems that is generally available does not contain any information regarding node locations. Therefore, the

  5. Estimating the Value of Source Verification in Iowa Feeder Cattle Markets

    Microsoft Academic Search

    Godfred Yeboah; John D. Lawrence; Gary J. May

    2002-01-01

    Source verification and pooling of feeder cattle into larger lots resulted in higher selling prices compared to more typical sales at a southern Iowa auction market. After higher prices due to larger lot sizes were accounted for, cattle that received a specified management program and were source verified as to origin received additional price premiums. The data do not distinguish

  6. A fault location approach for fuzzy fault section estimation on radial distribution feeders 

    E-print Network

    Andoh, Kwame Sarpong

    2000-01-01

    for the uncertainty use fuzzy logic techniques to estimate bounds of possibility of the input data and calculated quantities, or probabilistic modeling of the input data to estimate the likelihood of the location of the fault on a particular section of the feeder...

  7. Robotic assembly of printed circuit boards with component feeder location consideration

    Microsoft Academic Search

    PIUS J. EGBELU; CHUNG-TE WU; RAJIV PILGAONKAR

    1996-01-01

    The use of industrial robots for the assembly of printed circuit boards (PCB) is fast gaining popularity. Minimization of the time required for board assembly while maintaining production quality is the process planner's main task. This task involves the specification of the insertion sequence of components in their respective locations and the assignment of component types to feeders or dispensing

  8. Optimum Number, Location, and Size of Shunt Capacitors in Radial Distribution Feeders A Dynamic Programming Approach

    Microsoft Academic Search

    HERNANDO DURAN

    1968-01-01

    A new method is described which determines the optimum number, location, and size of shunt capacitors in a radial distribution feeder with discrete lumped loads so as to maximize overall savings, including the cost of capacitors. The method also determines when capacitors are not economically justified. Dynamic programming techniques are used and several algorithms developed to obtain the optimal solution

  9. Materials for the manufacture of the cutting blades of gob feeders (a review)

    Microsoft Academic Search

    A. M. Naerman; B. M. Zuev; I. M. Divinskii

    1984-01-01

    The problem of increasing the resistance of the cutting tool of the feeder of glass-molding machines is considered from different points of view. At the present time work is being carried out with the aim of improving the resistance of the blades and reducing the traces left by the cut on the finished article. These studies go in three main

  10. [Liquid embolization for arteriovenous malformation with temporary balloon occlusion of another feeders].

    PubMed

    Abe, H; Koike, T; Minakawa, T; Tanaka, R

    1989-06-01

    A case of frontal arteriovenous malformation, embolized using a new balloon catheter technique, was reported. During liquid embolization of the main feeder from anterior cerebral artery, middle cerebral artery was temporary occluded with another balloon catheter, to decrease blood flow from branches of middle cerebral artery. About 90% of the AVM could be obliterated by this technique. PMID:2614983

  11. Feeder-independent continuous culture of the PICM-19 pig liver stem cell line

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The PICM-19 pig liver stem cell line is a bipotent cell line, i.e., capable of forming either bile ductules or hepatocyte monolayers in vitro, that was derived from the primary culture of pig embryonic stem cells. The cell line has been strictly feeder-dependent in that cell replication morphology,...

  12. Nursing Homes for the Birds: A Control-Relevant Intervention with Bird Feeders.

    ERIC Educational Resources Information Center

    Banziger, George; Roush, Sharon

    Many gerontologists have noted the tendency of nursing homes to nurture dependency and learned helplessness in residents. To test the effectiveness of a control-relevant intervention strategy, nursing home residents (N=40) were given the opportunity to care for wild birds by tending individually placed bird feeders. Residents were assigned to one…

  13. Co-ordination of directional overcurrent protection with load current for parallel feeders

    SciTech Connect

    Wright, J.W.; Lloyd, G.; Hindle, P.J. [Alstom, Inc., Stafford (United Kingdom). T and D Protection and Control

    1999-11-01

    Directional phase overcurrent relays are commonly applied at the receiving ends of parallel feeders or transformer feeders. Their purpose is to ensure full discrimination of main or back-up power system overcurrent protection for a fault near the receiving end of one feeder. This paper reviews this type of relay application and highlights load current setting constraints for directional protection. Such constraints have not previously been publicized in well-known text books. A directional relay current setting constraint that is suggested in some text books is based purely on thermal rating considerations for older technology relays. This constraint may not exist with modern numerical relays. In the absence of any apparent constraint, there is a temptation to adopt lower current settings with modern directional relays in relation to reverse load current at the receiving ends of parallel feeders. This paper identifies the danger of adopting very low current settings without any special relay feature to ensure protection security with load current during power system faults. A system incident recorded by numerical relays is also offered to highlight this danger. In cases where there is a need to infringe the identified constraints an implemented and testing relaying technique is proposed.

  14. SHIPPING SUPPRESSES LYMPHOCYTE BLASTOGENIC RESPONSES IN ANGUS AND BRAHMAN Ť ANGUS FEEDER CALVES 1,2

    Microsoft Academic Search

    Frank Blecha; Stephen L. Boyles; Jack G. Riley

    2010-01-01

    Summary An experiment using 40 Angus or Brahman x Angus preconditioned feeder calves was conducted to evaluate the influence of shipping on cellular immune reactivity. Steers were allotted on the basis of weight and breed to a control or shipped group. Shipped steers were trucked 700 km to a feedlot; control steers remained at the ranch of origin. Total and

  15. Efficient Reprogramming of Naďve-Like Induced Pluripotent Stem Cells from Porcine Adipose-Derived Stem Cells with a Feeder-Independent and Serum-Free System

    PubMed Central

    Zhang, Pengfei; Li, Xia; Liu, Tong; Pu, Yong; Li, Yunsheng; Cao, Zubing; Cao, Hongguo; Liu, Ya; Zhang, Xiaorong; Zhang, Yunhai

    2014-01-01

    Induced pluripotent stem cells (iPSCs) are somatic cells reprogrammed by ectopic expression of transcription factors or small molecule treatment, which resemble embryonic stem cells (ESCs). They hold great promise for improving the generation of genetically modified large animals. However, few porcine iPSCs (piPSCs) lines obtained currently can support development of cloned embryos. Here, we generated iPSCs from porcine adipose-derived stem cells (pADSCs), using drug-inducible expression of defined human factors (Oct4, Sox2, c-Myc and Klf4). Reprogramming of iPSCs from pADSCs was more efficient than from fibroblasts, regardless of using feeder-independent or feeder-dependent manners. By addition of Lif-2i medium containing mouse Lif, CHIR99021 and PD0325901 (Lif-2i), naďve-like piPSCs were obtained under feeder-independent and serum-free conditions. These successfully reprogrammed piPSCs were characterized by short cell cycle intervals, alkaline phosphatase (AP) staining, expression of Oct4, Sox2, Nanog, SSEA3 and SSEA4, and normal karyotypes. The resemblance of piPSCs to naďve ESCs was confirmed by their packed dome morphology, growth after single-cell dissociation, Lif-dependency, up-regulation of Stella and Eras, low expression levels of TRA-1-60, TRA-1-81 and MHC I and activation of both X chromosomes. Full reprogramming of naďve-like piPSCs was evaluated by the significant up-regulation of Lin28, Esrrb, Utf1 and Dppa5, differentiating into cell types of all three germ layers in vitro and in vivo. Furthermore, nuclear transfer embryos from naďve-like piPSCs could develop to blastocysts with improved quality. Thus, we provided an efficient protocol for generating naďve-like piPSCs from pADSCs in a feeder-independent and serum-free system with controlled regulation of exogenous genes, which may facilitate optimization of culture media and the production of transgenic pigs. PMID:24465482

  16. A novel dual-screw coal feeder for production of low sulfur fuel

    SciTech Connect

    Lin, L.; Khang, S.J.; Keener, T.C.

    1993-06-15

    In this project, the following tasks have been performed: (1) Setting up the Dual-Screw feeder reactor. (2) Determination of the pyrolysis product and the sulfur distribution in char, tar and gas based on experimental data. (3) Study of the devolatilization and the desulfurization kinetics and obtaining the basic kinetic parameters. (4) Study of the sulfur removal efficiency of lime pellets fed into the outer tube of the dual-screw feeder reactor. (5) Study of the effect of the coal particle size on pyrolysis and desulfurization. (6) Study of the coal pyrolysis using a TGA (Thermal Gravimetric Analyzer). (7) Study of the coal desulfurization using a tube oven. (8) Setting up a combustor. (9) Study of the combustion characteristics of the pyrolysis products from the dual-screw feeder reactor. (10) Process simulation of the dual-screw feeder reactor. The experimental results of devolatilization and desulfurization of an Ohio {number_sign}8 coal demonstrate that an increasing the temperature in mild coal pyrolysis leads to the increase of both the devolatilization yield and the desulfurization yield. Under the experimental conditions, mainly the organic sulfur releases in the form of H{sub 2}S. Both the devolatilization and the desulfurization processes can be described by using the first-order-reaction model which gives the activation energy values for pyrolysis and desulfurization of 170,021 kJ/mol and 78,783 kJ/mol, indicating the sulfur is easier to release than volatiles. The outer screw region of CaO pellets also demonstrated almost a complete removal of hydrogen sulfide from volatiles. At a temperature of 475{degree}C and a residence time of 6 minutes, 73.1% of the organic sulfur was removed in the screw feeder reactor. The investigation of the combustion characteristics of the pyrolysis products showed a negligible reduction of the total heating value of the char and volatile products.

  17. Polymeric nanofibrous substrates stimulate pluripotent stem cells to form three-dimensional multilayered patty-like spheroids in feeder-free culture and maintain their pluripotency.

    PubMed

    Alamein, Mohammad A; Wolvetang, Ernst J; Ovchinnikov, Dmitry A; Stephens, Sebastien; Sanders, Katherine; Warnke, Patrick H

    2014-11-25

    Expansion of pluripotent stem cells in defined media devoid of animal-derived feeder cells to generate multilayered three-dimensional (3D) bulk preparations or spheroids, rather than two-dimensional (2D) monolayers, is advantageous for many regenerative, biological or disease-modelling studies. Here we show that electrospun polymer matrices comprised of nanofibres that mimic the architecture of the natural fibrous extracellular matrix allow for feeder-free expansion of pluripotent human induced pluripotent stem cells (IPSCs) and human embryonic stem cells (HESCs) into multilayered 3D 'patty-like' spheroid structures in defined xeno-free culture medium. The observation that IPSCs and HESCs readily revert to 2D growth in the absence of the synthetic nanofibre membranes suggests that this 3D expansion behaviour is mediated by the physical microenvironment and artificial niche provided by the nanofibres only. Importantly, we could show that such 3D growth as patties maintained the pluripotency of cells as long as they were kept on nanofibres. The generation of complex multilayered 3D structures consisting of only pluripotent cells on biodegradable nanofibre matrices of the desired shape and size will enable both industrial-scale expansion and intricate organ-tissue engineering applications with human pluripotent stem cells, where simultaneous coupling of differentiation pathways of all germ layers from one stem cell source may be required for organ formation. Copyright © 2014 John Wiley & Sons, Ltd. PMID:25423911