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Sample records for model t-3 gem-t3

  1. T3_MM: a Markov model effectively classifies bacterial type III secretion signals.

    Science.gov (United States)

    Wang, Yejun; Sun, Ming'an; Bao, Hongxia; White, Aaron P

    2013-01-01

    Type III Secretion Systems (T3SSs) play important roles in the interaction between gram-negative bacteria and their hosts. T3SSs function by translocating a group of bacterial effector proteins into the host cytoplasm. The details of specific type III secretion process are yet to be clarified. This research focused on comparing the amino acid composition within the N-terminal 100 amino acids from type III secretion (T3S) signal sequences or non-T3S proteins, specifically whether each residue exerts a constraint on residues found in adjacent positions. We used these comparisons to set up a statistic model to quantitatively model and effectively distinguish T3S effectors. In this study, the amino acid composition (Aac) probability profiles conditional on its sequentially preceding position and corresponding amino acids were compared between N-terminal sequences of T3S and non-T3S proteins. The profiles are generally different. A Markov model, namely T3_MM, was consequently designed to calculate the total Aac conditional probability difference, i.e., the likelihood ratio of a sequence being a T3S or a non-T3S protein. With T3_MM, known T3S and non-T3S proteins were found to well approximate two distinct normal distributions. The model could distinguish validated T3S and non-T3S proteins with a 5-fold cross-validation sensitivity of 83.9% at a specificity of 90.3%. T3_MM was also shown to be more robust, accurate, simple, and statistically quantitative, when compared with other T3S protein prediction models. The high effectiveness of T3_MM also indicated the overall Aac difference between N-termini of T3S and non-T3S proteins, and the constraint of Aac exerted by its preceding position and corresponding Aac. An R package for T3_MM is freely downloadable from: http://biocomputer.bio.cuhk.edu.hk/softwares/T3_MM. T3_MM web server: http://biocomputer.bio.cuhk.edu.hk/T3DB/T3_MM.php.

  2. An evaluation of the Cray T3D programming paradigms in atmospheric chemistry/transport models

    NARCIS (Netherlands)

    J.G. Blom (Joke); C. Keßler (Carsten); J.G. Verwer (Jan)

    1996-01-01

    textabstractIn this paper we compare the different programming paradigms available on the Cray T3D for the implementation of a 3D prototype of an Atmospheric Chemistry/Transport Model. We discuss the amount of work needed to convert existing codes to the T3D and the portability of the resulting

  3. Finite element modelling of aluminum alloy 2024-T3 under transverse impact loading

    Science.gov (United States)

    Abdullah, Ahmad Sufian; Kuntjoro, Wahyu; Yamin, A. F. M.

    2017-12-01

    Fiber metal laminate named GLARE is a new aerospace material which has great potential to be widely used in future lightweight aircraft. It consists of aluminum alloy 2024-T3 and glass-fiber reinforced laminate. In order to produce reliable finite element model of impact response or crashworthiness of structure made of GLARE, one can initially model and validate the finite element model of the impact response of its constituents separately. The objective of this study was to develop a reliable finite element model of aluminum alloy 2024-T3 under low velocity transverse impact loading using commercial software ABAQUS. Johnson-Cook plasticity and damage models were used to predict the alloy's material properties and impact behavior. The results of the finite element analysis were compared to the experiment that has similar material and impact conditions. Results showed good correlations in terms of impact forces, deformation and failure progressions which concluded that the finite element model of 2024-T3 aluminum alloy under low velocity transverse impact condition using Johnson-Cook plastic and damage models was reliable.

  4. Review and study of physics driven pitting corrosion modeling in 2024-T3 aluminum alloys

    Science.gov (United States)

    Yu, Lingyu; Jata, Kumar V.

    2015-04-01

    Material degradation due to corrosion and corrosion fatigue has been recognized to significantly affect the airworthiness of civilian and military aircraft, especially for the current fleet of airplanes that have served beyond their initial design life. The ability to predict the corrosion damage development in aircraft components and structures, therefore, is of great importance in managing timely maintenance for the aging aircraft vehicles and in assisting the design of new ones. The assessment of aircraft corrosion and its influence on fatigue life relies on appropriate quantitative models that can evaluate the initiation of the corrosion as well as the accumulation during the period of operation. Beyond the aircraft regime, corrosion has also affected the maintenance, safety and reliability of other systems such as nuclear power systems, steam and gas turbines, marine structures and so on. In the work presented in this paper, we reviewed and studied several physics based pitting corrosion models that have been reported in the literature. The classic work of particle induced pitting corrosion by Wei and Harlow is reviewed in detail. Two types of modeling, a power law based simplified model and a microstructure based model, are compared for 2024-T3 alloy. Data from literatures are used as model inputs. The paper ends with conclusions and recommendations for future work.

  5. Numerical modeling of AA2024-T3 friction stir welding process for residual stress evaluation, including softening effects

    DEFF Research Database (Denmark)

    Sonne, Mads Rostgaard; Carlone, Pierpaolo; Palazzo, Gaetano S.

    2014-01-01

    In the present paper, a numerical finite element model of the precipitation hardenable AA2024-T3 aluminum alloy, consisting of a heat transfer analysis based on the Thermal Pseudo Mechanical model for heat generation, and a sequentially coupled quasi-static stress analysis is proposed. Metallurgi......In the present paper, a numerical finite element model of the precipitation hardenable AA2024-T3 aluminum alloy, consisting of a heat transfer analysis based on the Thermal Pseudo Mechanical model for heat generation, and a sequentially coupled quasi-static stress analysis is proposed...

  6. 3D Finite Element Modelling of Drilling Process of Al2024-T3 Alloy with solid tooling and Experimental Validation

    DEFF Research Database (Denmark)

    Davoudinejad, Ali; Tosello, Guido

    2017-01-01

    Drilling is an indispensable process for many manufacturing industries due to its importance for assembling components. This study presents a 3D finite element modelling (3D FEM) approach for drilling process of aluminium 2024-T3. The 3D model of drilling tools for two facet HSSCo and four facet...... area were determined numerically. The results confirm the ability and advantage of 3D FE modelling of the drilling process....

  7. Statistical coronary motion models for 2D + t/3D registration of X-ray coronary angiography and CTA

    DEFF Research Database (Denmark)

    Baka, N.; Metz, C. T.; Schultz, C.

    2013-01-01

    Accurate alignment of intra-operative X-ray coronary angiography (XA) and pre-operative cardiac CT angiography (CTA) may improve procedural success rates of minimally invasive coronary interventions for patients with chronic total occlusions. It was previously shown that incorporating patient...... motion models to provide constraints for the 2D+t/3D registration. We propose a methodology for building statistical motion models of the coronary arteries from a training population of 4D CTA datasets. We compare the 2D+t/3D registration performance of the proposed statistical models with other motion...... estimates, including the patient specific motion extracted from 4D CTA, the mean motion of a population, the predicted motion based on the cardiac shape. The coronary motion models, constructed on a training set of 150 patients, had a generalization accuracy of 1mm root mean square point-to-point distance...

  8. The effect of hardening laws and thermal softening on modeling residual stresses in FSW of aluminum alloy 2024-T3

    DEFF Research Database (Denmark)

    Sonne, Mads Rostgaard; Tutum, Cem Celal; Hattel, Jesper Henri

    2013-01-01

    In the present paper, a numerical model consisting of a heat transfer analysis based on the Thermal Pseudo Mechanical (TPM) model for heat generation, and a sequentially coupled quasi-static stress analysis with a built-in metallurgical softening model was implemented in ABAQUS. Both isotropic...... or kinematic hardening together with the metallurgical softening model were applied in order to give a first impression of the tendencies in residual stresses in friction stir welds when choosing different hardening and softening behaviors. Secondly, real friction stir butt welding of aluminum alloy 2024-T3...

  9. Anti-osteoporosis activity of Sanguinarine in preosteoblast MC3T3-E1 cells and an ovariectomized rat model.

    Science.gov (United States)

    Zhang, Fuzhan; Xie, Jile; Wang, Genlin; Zhang, Ge; Yang, Huilin

    2018-06-01

    Sanguinarine, a benzophenanthridine alkaloid, has been previously demonstrated to exert antimicrobial, anti-inflammatory, and anti-tumor activities. A previous study has identified Sanguinarine as a potential drug candidate for osteoporosis treatment by computational bioinformatics analysis. This study further evaluated the effects of Sanguinarine on the differentiation of murine preosteoblast MC3T3-E1 cells and its anti-osteoporosis activity in an ovarietomized rat model. Sanguinarine treatment (0.25, 0.5, 1, and 2 µm) of MC3T3-E1 cells significantly increased alkaline phosphatase (ALP) activity and the phoshporalyation of AMP-activated protein kinase α subunit (AMPKα), but did not affect cell proliferation. The induction effects of Sanguinarine treatment (2 µm) on ALP activity, AMPKα phosphorylation, Smad1 phosphorylation, and the expression of three osteoblast differentiation-regulators (bone morphogenetic protein 2 [BMP2], osterix [OSX], and osteoprotegerin [OPG]) were partially reversed by Compound C treatment. More importantly, Sanguinarine treatment promoted bone tissue growth in an ovariectomized (OVX) osteoporosis rat model as evaluated by histological examination, micro-CT analysis, and serum parameter detection. In conclusion, these results indicate that Sanguinarine induces the differentiation of MC3T3-E1 cells through the activation of the AMPK/Smad1 signaling pathway. Sanguinarine can stimulate bone growth in vivo and may be an effective drug for osteoporosis treatment. © 2017 Wiley Periodicals, Inc.

  10. Loop Quantization of Polarized Gowdy Model on $T^3$: Kinematical States and Constraint Operators

    OpenAIRE

    Banerjee, Kinjal; Date, Ghanashyam

    2007-01-01

    In this second paper on loop quantization of Gowdy model, we introduce the kinematical Hilbert space on which appropriate holonomies and fluxes are well represented. The quantization of the volume operator and the Gauss constraint is straightforward. Imposition of the Gauss constraint can be done on the kinematical Hilbert space to select subspace of gauge invariant states. We carry out the quantization of the Hamiltonian constraint making specific choices. Alternative choices are briefly dis...

  11. Trichosanthes cucumerina extracts enhance glucose uptake and regulate adiponectin and leptin concentrations in 3T3-L1 adipocytes model

    Directory of Open Access Journals (Sweden)

    Sassi, A.,

    2017-10-01

    Full Text Available Trichosanthes cucumerina (Cucurbitaceae commonly known as Snake gourd or Labu Ular is considered the largest genre in the Cucurbitaceae family and is mainly found in the southeast areas of Asia. It has been used in Ayurvedic medicine as a treatment for certain diseases such as Diabetes mellitus, but these acclaims lack scientific-based evidence. In this study, water and ethanol extracts of three parts of Trichosanthes cucumerina namely; whole vegetable, peels, and seeds, were assessed for toxicity through a cell viability assay using 3T3-L1 pre-adipocytes model which revealed a maximum toleration concentration of 0.063 mg/mL. The extracts were further tested on adipocytes’ differentiation and positively showed a stimulation of lipid droplets formation during adipogenesis and significantly (p<0.001 increased glycerol release levels (75.34±3.69 μg/ml during adipolysis. The extracts also significantly (p<0.001 promoted the uptake of glucose into the cells (2636.22±91.33 Bq in an action similar to that of insulin. Similar results were observed during ELISA assay with a significant increase (p<0.001 in adiponectin concentrations (3593.1±225.25 ng/mL and a decrease in leptin concentrations (23870±5066.07 pg/mL. The present study results indicate a beneficial effect of Trichosanthes cucumerina extracts on adipogenesis, adipolysis and glucose uptake, in addition to a regulation of adiponectin and leptin concentrations in 3T3-L1 adipocytes which can be of clinical importance in energy regulation which is a key factor in treating diabetes, obesity, and metabolic syndrome.

  12. Modelling the residual stresses and microstructural evolution in Friction Stir Welding of AA2024-T3 including the Wagner-Kampmann precipitation model

    DEFF Research Database (Denmark)

    Sonne, Mads Rostgaard; Hattel, Jesper Henri

    In this work, a numerical finite element model for friction stir welding of 2024-T3 aluminum alloy, consisting of a heat transfer analysis and a sequentially coupled quasi-static stress analysis is proposed. Metallurgical softening of the material is properly considered and included...

  13. Mathematical Modeling of the Pituitary-Thyroid Feedback Loop: Role of a TSH-T3-Shunt and Sensitivity Analysis.

    Science.gov (United States)

    Berberich, Julian; Dietrich, Johannes W; Hoermann, Rudolf; Müller, Matthias A

    2018-01-01

    Despite significant progress in assay technology, diagnosis of functional thyroid disorders may still be a challenge, as illustrated by the vague upper limit of the reference range for serum thyrotropin ( TSH ). Diagnostical problems also apply to subjects affected by syndrome T, i.e., those 10% of hypothyroid patients who continue to suffer from poor quality of life despite normal TSH concentrations under substitution therapy with levothyroxine ( L - T 4 ). In this paper, we extend a mathematical model of the pituitary-thyroid feedback loop in order to improve the understanding of thyroid hormone homeostasis. In particular, we incorporate a TSH - T 3 -shunt inside the thyroid, whose existence has recently been demonstrated in several clinical studies. The resulting extended model shows good accordance with various clinical observations, such as a circadian rhythm in free peripheral triiodothyronine ( FT 3 ). Furthermore, we perform a sensitivity analysis of the derived model, revealing the dependence of TSH and hormone concentrations on different system parameters. The results have implications for clinical interpretation of thyroid tests, e.g., in the differential diagnosis of subclinical hypothyroidism.

  14. Characterisation of Shigella Spa33 and Thermotoga FliM/N reveals a new model for C-ring assembly in T3SS.

    Science.gov (United States)

    McDowell, Melanie A; Marcoux, Julien; McVicker, Gareth; Johnson, Steven; Fong, Yu Hang; Stevens, Rebecca; Bowman, Lesley A H; Degiacomi, Matteo T; Yan, Jun; Wise, Adam; Friede, Miriam E; Benesch, Justin L P; Deane, Janet E; Tang, Christoph M; Robinson, Carol V; Lea, Susan M

    2016-02-01

    Flagellar type III secretion systems (T3SS) contain an essential cytoplasmic-ring (C-ring) largely composed of two proteins FliM and FliN, whereas an analogous substructure for the closely related non-flagellar (NF) T3SS has not been observed in situ. We show that the spa33 gene encoding the putative NF-T3SS C-ring component in Shigella flexneri is alternatively translated to produce both full-length (Spa33-FL) and a short variant (Spa33-C), with both required for secretion. They associate in a 1:2 complex (Spa33-FL/C2) that further oligomerises into elongated arrays in vitro. The structure of Spa33-C2 and identification of an unexpected intramolecular pseudodimer in Spa33-FL reveal a molecular model for their higher order assembly within NF-T3SS. Spa33-FL and Spa33-C are identified as functional counterparts of a FliM-FliN fusion and free FliN respectively. Furthermore, we show that Thermotoga maritima FliM and FliN form a 1:3 complex structurally equivalent to Spa33-FL/C2 , allowing us to propose a unified model for C-ring assembly by NF-T3SS and flagellar-T3SS. © 2015 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.

  15. Characterisation of Shigella Spa33 and Thermotoga FliM/N reveals a new model for C‐ring assembly in T3SS

    Science.gov (United States)

    McDowell, Melanie A.; Marcoux, Julien; McVicker, Gareth; Johnson, Steven; Fong, Yu Hang; Stevens, Rebecca; Bowman, Lesley A. H.; Degiacomi, Matteo T.; Yan, Jun; Wise, Adam; Friede, Miriam E.; Benesch, Justin L. P.; Deane, Janet E.; Tang, Christoph M.; Robinson, Carol V.

    2015-01-01

    Summary Flagellar type III secretion systems (T3SS) contain an essential cytoplasmic‐ring (C‐ring) largely composed of two proteins FliM and FliN, whereas an analogous substructure for the closely related non‐flagellar (NF) T3SS has not been observed in situ. We show that the spa33 gene encoding the putative NF‐T3SS C‐ring component in S higella flexneri is alternatively translated to produce both full‐length (Spa33‐FL) and a short variant (Spa33‐C), with both required for secretion. They associate in a 1:2 complex (Spa33‐FL/C2) that further oligomerises into elongated arrays in vitro. The structure of Spa33‐C 2 and identification of an unexpected intramolecular pseudodimer in Spa33‐FL reveal a molecular model for their higher order assembly within NF‐T3SS. Spa33‐FL and Spa33‐C are identified as functional counterparts of a FliM–FliN fusion and free FliN respectively. Furthermore, we show that T hermotoga maritima  FliM and FliN form a 1:3 complex structurally equivalent to Spa33‐FL/C2, allowing us to propose a unified model for C‐ring assembly by NF‐T3SS and flagellar‐T3SS. PMID:26538516

  16. Stochastic modeling of adipogenesis in 3T3-L1 cultures to determine probabilities of events in the cell's life cycle.

    Science.gov (United States)

    Shoham, Naama; Gefen, Amit

    2011-10-01

    3T3-L1 preadipocytes are often being used in research of adipose-related diseases such as obesity, insulin resistance, and hyperlipidemia. We developed a stochastic model that simulated differentiation of four 3T3-L1 culture conditions distinct by the insulin concentration in the differentiation medium (2.5, 5, 7.5, or 10 μg/mL). The model simulated culture behavior and the accumulation of lipid droplets in the maturing cells from the day of induction of differentiation through 28 days after that. The cellular processes including cell adhesion, mitosis, growing after undergoing mitosis, commitment to the adipocyte lineage, and apoptosis were referred to as stochastic events in the modeling. By minimizing the error between our model and experimental results, we found that the probability for becoming committed to the adipocyte lineage in a single division and the probability for growing after undergoing mitosis were 0.02 and 0.8, respectively, regardless of the insulin concentration. The probability for undergoing mitosis was equal to 0.2 and 0.4 in cultures that had insulin concentrations of 2.5 and 5-10 μg/mL in the differentiation medium, respectively; hence the insulin concentration affected the probability for mitosis in the 3T3-L1 cells. The model and resulted probabilities now allow quantitative and visual predictions of adipogenesis in 3T3-L1 cultures, toward computational design of cell culturing protocols.

  17. T3RU test

    Science.gov (United States)

    ... the Test is Performed" for more about TBG): Male hormones (androgens) Serious illness Kidney disease Other medicines that ... is done to check your thyroid function. Thyroid function depends on the ... including thyroid-stimulating hormone (TSH), T3, and T4. ...

  18. An Atmospheric General Circulation Model with Chemistry for the CRAY T3E: Design, Performance Optimization and Coupling to an Ocean Model

    Science.gov (United States)

    Farrara, John D.; Drummond, Leroy A.; Mechoso, Carlos R.; Spahr, Joseph A.

    1998-01-01

    The design, implementation and performance optimization on the CRAY T3E of an atmospheric general circulation model (AGCM) which includes the transport of, and chemical reactions among, an arbitrary number of constituents is reviewed. The parallel implementation is based on a two-dimensional (longitude and latitude) data domain decomposition. Initial optimization efforts centered on minimizing the impact of substantial static and weakly-dynamic load imbalances among processors through load redistribution schemes. Recent optimization efforts have centered on single-node optimization. Strategies employed include loop unrolling, both manually and through the compiler, the use of an optimized assembler-code library for special function calls, and restructuring of parts of the code to improve data locality. Data exchanges and synchronizations involved in coupling different data-distributed models can account for a significant fraction of the running time. Therefore, the required scattering and gathering of data must be optimized. In systems such as the T3E, there is much more aggregate bandwidth in the total system than in any particular processor. This suggests a distributed design. The design and implementation of a such distributed 'Data Broker' as a means to efficiently couple the components of our climate system model is described.

  19. Chlamydia induces anchorage independence in 3T3 cells and detrimental cytological defects in an infection model.

    Directory of Open Access Journals (Sweden)

    Andrea E Knowlton

    Full Text Available Chlamydia are gram negative, obligate intracellular bacterial organisms with different species causing a multitude of infections in both humans and animals. Chlamydia trachomatis is the causative agent of the sexually transmitted infection (STI Chlamydia, the most commonly acquired bacterial STI in the United States. Chlamydial infections have also been epidemiologically linked to cervical cancer in women co-infected with the human papillomavirus (HPV. We have previously shown chlamydial infection results in centrosome amplification and multipolar spindle formation leading to chromosomal instability. Many studies indicate that centrosome abnormalities, spindle defects, and chromosome segregation errors can lead to cell transformation. We hypothesize that the presence of these defects within infected dividing cells identifies a possible mechanism for Chlamydia as a cofactor in cervical cancer formation. Here we demonstrate that infection with Chlamydia trachomatis is able to transform 3T3 cells in soft agar resulting in anchorage independence and increased colony formation. Additionally, we show for the first time Chlamydia infects actively replicating cells in vivo. Infection of mice with Chlamydia results in significantly increased cell proliferation within the cervix, and in evidence of cervical dysplasia. Confocal examination of these infected tissues also revealed elements of chlamydial induced chromosome instability. These results contribute to a growing body of data implicating a role for Chlamydia in cervical cancer development and suggest a possible molecular mechanism for this effect.

  20. Insulin-IGF signaling affects cell transformation in the BALB/c 3T3 cell model.

    Science.gov (United States)

    Poburski, Doerte; Leovsky, Christiane; Boerner, Josefine Barbara; Szimmtenings, Luisa; Ristow, Michael; Glei, Michael; Thierbach, René

    2016-11-16

    The increased cancer mortality of diabetes type 2 patients is most likely an evidence of the tight connection between tumor development and energy metabolism. A major focus of today's research is still the identification of key proteins of both diseases and the development of corresponding inhibitors. In this study we combined the two-stage BALB/c-3T3 cell transformation assay (BALB-CTA) with the IR/IGF-1R inhibitor OSI-906 (linsitinib) and analyzed alterations in protein activity and energy parameters in non-transformed as well as transformed cells. OSI-906 successfully inhibited the phosphorylation of IR/IGF-1R and decreased cell growth in non-transformed cells. In the BALB-CTA, a permanent treatment with OSI-906 reduced cellular transformation dose-dependently, whereas a temporary treatment gave evidence for a preventive effect in the promotion phase. Furthermore, even though several key proteins were affected, it was possible to show that the phosphorylation of GSK3, Erk 1/2 and the S6 protein are not crucial for the cell foci reducing effect of OSI-906. Taken together, the BALB-CTA confirmed results of OSI-906 from animal studies and enhanced the knowledge of its mode of action. Therefore, the BALB-CTA offers the opportunity to analyze alterations in the transformation process more precisely and will be helpful to identify effective cancer treatments.

  1. AICAR inhibits adipocyte differentiation in 3T3L1 and restores metabolic alterations in diet-induced obesity mice model

    Directory of Open Access Journals (Sweden)

    Key Lyndon

    2006-08-01

    Full Text Available Abstract Background Obesity is one of the principal causative factors involved in the development of metabolic syndrome. AMP-activated protein kinase (AMPK is an energy sensor that regulates cellular metabolism. The role of AMP-activated protein kinase in adipocyte differentiation is not completely understood, therefore, we examined the effect of 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR, a pharmacological activator of AMP-activated protein kinase (AMPK on adipocyte differentiation in 3T3L1 cells and in a mouse Diet induced obesity (DIO model. Methods To examine the effect of AICAR on adipocyte differentiation in 3T3L1 cells and in a mouse Diet induced obesity (DIO model, 3T3L1 cells were differentiatied in the presence or absence of different concentration of AICAR and neutral lipid content and expression of various adipocyte-specific transcription factors were examined. In vivo study, treated and untreated mice with AICAR (0.1–0.5 mg/g body weight were fed high-fat diet (60% kcal% fat to induce DIO and several parameters were studied. Results AICAR blocked adipogenic conversion in 3T3L1 cells along with significant decrease in the neutral lipid content by downregulating several adipocyte-specific transcription factors including peroxisome proliferators-activated receptor γ (PPARγ, C/EBPα and ADD1/SREBP1, which are critical for adipogenesis in vitro. Moreover, intraperitoneal administration of AICAR (0.5 mg g/body weight to mice fed with high-fat diet (60% kcal% fat to induce DIO, significantly blocked the body weight gain and total content of epididymal fat in these mice over a period of 6 weeks. AICAR treatment also restored normal adipokine levels and resulted in significant improvement in glucose tolerance and insulin sensitivity. The reduction in adipose tissue content in AICAR treated DIO mice was due to reduction in lipid accumulation in the pre-existing adipocytes. However, no change was observed in the expression

  2. A refined model of the prototypical Salmonella SPI-1 T3SS basal body reveals the molecular basis for its assembly.

    Science.gov (United States)

    Bergeron, Julien R C; Worrall, Liam J; Sgourakis, Nikolaos G; DiMaio, Frank; Pfuetzner, Richard A; Felise, Heather B; Vuckovic, Marija; Yu, Angel C; Miller, Samuel I; Baker, David; Strynadka, Natalie C J

    2013-01-01

    The T3SS injectisome is a syringe-shaped macromolecular assembly found in pathogenic Gram-negative bacteria that allows for the direct delivery of virulence effectors into host cells. It is composed of a "basal body", a lock-nut structure spanning both bacterial membranes, and a "needle" that protrudes away from the bacterial surface. A hollow channel spans throughout the apparatus, permitting the translocation of effector proteins from the bacterial cytosol to the host plasma membrane. The basal body is composed largely of three membrane-embedded proteins that form oligomerized concentric rings. Here, we report the crystal structures of three domains of the prototypical Salmonella SPI-1 basal body, and use a new approach incorporating symmetric flexible backbone docking and EM data to produce a model for their oligomeric assembly. The obtained models, validated by biochemical and in vivo assays, reveal the molecular details of the interactions driving basal body assembly, and notably demonstrate a conserved oligomerization mechanism.

  3. Dynamics of a quasiparticle in the α-T3 model: Role of pseudospin polarization and transverse magnetic field on zitterbewegung.

    Science.gov (United States)

    Biswas, Tutul; Ghosh, Tarun Kanti

    2018-01-09

    We consider the $\\alpha$-$T_3$ model which provides a smooth crossover between the honeycomb lattice with pseudospin $1/2$ and the dice lattice with pseudospin $1$ through the variation of a parameter $\\alpha$. We study the dynamics of a wave packet representing a quasiparticle in the $\\alpha$-T$_3$ model with zero and finite transverse magnetic field. For zero field, it is shown that the wave packet undergoes a transient $zitterbewegung$ (ZB). Various features of ZB depending on the initial pseudospin polarization of the wave packet have been revealed. For an intermediate value of the parameter $\\alpha$ i.e. for $0<\\alpha<1$ the resulting ZB consists of two distinct frequencies when the wave packet was located initially in $rim$ site. However, the wave packet exhibits single frequency ZB for $\\alpha=0$ and $\\alpha=1$. It is also unveiled that the frequency of ZB corresponding to $\\alpha=1$ gets exactly half of that corresponding to the $\\alpha=0$ case. On the other hand, when the initial wave packet was in $hub$ site, the ZB consists of only one frequency for all values of $\\alpha$. Using stationary phase approximation we find analytical expression of velocity average which can be used to extract the associated timescale over which the transient nature of ZB persists. On the contrary the wave packet undergoes permanent ZB in presence of a transverse magnetic field. Due to the presence of large number of Landau energy levels the oscillations in ZB appear to be much more complicated. The oscillation pattern depends significantly on the initial pseudospin polarization of the wave packet. Furthermore, it is revealed that the number of the frequency components involved in ZB depends on the parameter $\\alpha$. © 2018 IOP Publishing Ltd.

  4. In vitro and in vivo study of microporous ceramics using MC3T3 cells, CAM assay and a pig animal model.

    Science.gov (United States)

    Tomco, Marek; Petrovova, Eva; Giretova, Maria; Almasiova, Viera; Holovska, Katarina; Cigankova, Viera; Jenca, Andrej; Jencova, Janka; Jenca, Andrej; Boldizar, Martin; Balazs, Kosa; Medvecky, Lubomir

    2017-09-01

    Bone tissue engineering combines biomaterials with biologically active factors and cells to hold promise for reconstructing craniofacial defects. In this study the biological activity of biphasic hydroxyapatite ceramics (HA; a bone substitute that is a mixture of hydroxyapatite and β-tricalcium phosphate in fixed ratios) was characterized (1) in vitro by assessing the growth of MC3T3 mouse osteoblast lineage cells, (2) in ovo by using the chick chorioallantoic membrane (CAM) assay and (3) in an in vivo pig animal model. Biocompatibility, bioactivity, bone formation and biomaterial degradation were detected microscopically and by radiology and histology. HA ceramics alone demonstrated great biocompatibility on the CAM as well as bioactivity by increased proliferation and alkaline phosphatase secretion of mouse osteoblasts. The in vivo implantation of HA ceramics with bone marrow mesenchymal stem cells (MMSCs) showed de novo intramembranous bone healing of critical-size bone defects in the right lateral side of pig mandibular bodies after 3 and 9 weeks post-implantation. Compared with the HA ceramics without MMSCs, the progress of bone formation was slower with less-developed features. This article highlights the clinical use of microporous biphasic HA ceramics despite the unusually shaped elongated micropores with a high length/width aspect ratio (up to 20) and absence of preferable macropores (>100 µm) in bone regenerative medicine.

  5. Improved Brain Insulin/IGF Signaling and Reduced Neuroinflammation with T3D-959 in an Experimental Model of Sporadic Alzheimer's Disease.

    Science.gov (United States)

    de la Monte, Suzanne M; Tong, Ming; Schiano, Irio; Didsbury, John

    2017-01-01

    Alzheimer's disease (AD) is associated with progressive impairments in brain insulin, insulin-like growth factor (IGF), and insulin receptor substrate (IRS) signaling through Akt pathways that regulate neuronal growth, survival, metabolism, and plasticity. The intracerebral streptozotocin (i.c. STZ) model replicates the full range of abnormalities in sporadic AD. T3D-959, an orally active PPAR-delta/gamma agonist remediates neurocognitive deficits and AD neuropathology in the i.c. STZ model. This study characterizes the effects of T3D-959 on AD biomarkers, insulin/IGF/IRS signaling through Akt pathways, and neuroinflammation in an i.c. STZ model. Long Evans rats were treated with i.c. STZ or saline, followed by daily oral doses of T3D-959 (1 mg/kg) or saline initiated 1 day (T3D-959-E) or 7 days (T3D-959-L) later through Experimental Day 28. Protein and phospho-protein expression and pro-inflammatory cytokine activation were measured in temporal lobe homogenates by duplex or multiplex bead-based ELISAs. i.c. STZ treatments caused neurodegeneration with increased pTau, AβPP, Aβ42, ubiquitin, and SNAP-25, and reduced levels of synaptophysin, IGF-1 receptor (R), IRS-1, Akt, p70S6K, mTOR, and S9-GSK-3β. i.c. STZ also broadly increased neuroinflammation. T3D-959 abrogated or reduced most of the AD neuropathological and biomarker abnormalities, increased/normalized IGF-1R, IRS-1, Akt, p70S6K, and S9-GSK-3β, and decreased expression of multiple pro-inflammatory cytokines. T3D-959-E or -L effectively restored insulin/IGF signaling, whereas T3D-959-L more broadly resolved neuroinflammation. AD remediating effects of T3D-959 are potentially due to enhanced expression of key insulin/IGF signaling proteins and inhibition of GSK-3β and neuroinflammation. These effects lead to reduced neurodegeneration, cognitive impairment, and AD biomarker levels in the brain.

  6. Toxicological evaluation of the topoisomerase inhibitor, etoposide, in the model animal Caenorhabditis elegans and 3T3-L1 normal murine cells.

    Science.gov (United States)

    Lee, So Young; Kim, Joo Yeon; Jung, Yu-Jin; Kang, Kyungsu

    2017-06-01

    Etoposide, a topoisomerase II inhibitor, has been widely used as a clinical anticancer drug to treat diverse cancer patients. Since not only rapidly dividing cancer cells but also the cells of normal human tissues and every living organism in environmental ecosystems have topoisomerases, it is crucial to study the toxicity of etoposide in other organisms in addition to cancer cells. In this study, we evaluated the toxicity of etoposide in both a soil nematode, Caenorhabditis elegans, and 3T3-L1 normal murine cells. Etoposide significantly retarded the growth, egg laying, and hatching in C. elegans. Etoposide also affected the reproductive gonad tissue, decreased the number of germ cells and induced abnormally enlarged nuclei in C. elegans. In addition, etoposide inhibited 3T3-L1 cell proliferation, with IC 50 values of 37.8 ± 7.3 and 9.8 ± 1.8 μM after 24 and 48 hours of treatment, respectively, via the induction of cell cycle arrest at the G2/M phase and apoptotic cell death. Etoposide also induced nuclear enlargement in 3T3-L1 normal murine cells. The reproductive toxicity and abnormal nuclear morphological changes seemed to correlate with the adverse effects of etoposide. We suggest that these experimental platforms, i.e., the toxicological evaluation of both nematodes and 3T3-L1 cells, may be useful to study the mechanisms underlying the side effects of chemicals, including topoisomerase inhibitors. © 2017 Wiley Periodicals, Inc.

  7. Identification of chlamydial T3SS inhibitors through virtual screening against T3SS ATPase.

    Science.gov (United States)

    Grishin, Alexander V; Luyksaar, Sergey I; Kapotina, Lidiya N; Kirsanov, Dmitry D; Zayakin, Egor S; Karyagina, Anna S; Zigangirova, Naylia A

    2018-03-01

    Chlamydia trachomatis is a widespread sexually transmitted pathogen that resides within a special vacuole inside host cells. Although acute infection can be treated with antibiotics, chlamydia can enter persistent state, leading to chronic infection that is difficult to cure. Thus, novel anti-chlamydial compounds active against persistent chlamydia are required. Chlamydiae rely upon type III secretion system (T3SS) to inject effector proteins into host cell cytoplasm, and T3SS inhibitors are viewed as promising compounds for treatment of chlamydial infections. C. trachomatis ATPase SctN is an important T3SS component and has not been targeted before. We thus used virtual screening against homology modeled SctN structure to search for SctN inhibitors. Selected compounds were tested for their ability to inhibit chlamydial survival and development within eukaryotic cells, and for the ability to suppress normal T3SS functioning. We identified two compounds that were able to block normal protein translocation through T3SS and inhibit chlamydial survival within eukaryotic cells in 50-100 μm concentrations. These two novel T3SS inhibitors also possessed relatively low toxicity toward eukaryotic cells. A small series of derivatives was further synthesized for the most active of two inhibitors to probe SAR properties. © 2017 John Wiley & Sons A/S.

  8. Radical radiotherapy for T3 laryngeal cancers

    International Nuclear Information System (INIS)

    Uno, T.; Itami, J.; Kotaka, K.; Toriyama, M.

    1996-01-01

    From 1974 through 1992, 37 previously untreated patients with T3 laryngeal cancer (supraglottic 15, glottic 22) were treated with initial radical radiotherapy and surgery for salvage. Two-year local control rate with radiotherapy alone, ultimate voice preservation rate, and ultimate local control rate for T3 supraglottic cancer were 33%, 33%, and 60%, respectively. Corresponding figures for T3 glottic cancer were 32%, 23%, and 77%, respecitvely. Five-year cause-specific survival rate for T3 supraglottic cancer and glottic cancer were 47% and 77%, respectively. In T3 supraglottic cancer, none of the 4 patients with subglottic tumor extension attained local control by radiotherapy alone, and local-regional recurrence-free time were significantly shorter in patients with subglottic tumor extension or tracheostomy before radiotherapy. There were no serious late complications such as chondronecrosis, rupture of carotid artery attributed to radical radiotherapy, while 3 patients had severe laryngeal edema requiring total laryngectomy. (orig.) [de

  9. Radical radiotherapy for T3 laryngeal cancers

    Energy Technology Data Exchange (ETDEWEB)

    Uno, T. [International Medical Center of Japan, Tokyo (Japan). Dept. of Radiation Therapy; Itami, J. [International Medical Center of Japan, Tokyo (Japan). Dept. of Radiation Therapy; Kotaka, K. [International Medical Center of Japan, Tokyo (Japan). Dept. of Radiation Therapy; Toriyama, M. [International Medical Center of Japan, Tokyo (Japan). Dept. of Otolaryngology

    1996-08-01

    From 1974 through 1992, 37 previously untreated patients with T3 laryngeal cancer (supraglottic 15, glottic 22) were treated with initial radical radiotherapy and surgery for salvage. Two-year local control rate with radiotherapy alone, ultimate voice preservation rate, and ultimate local control rate for T3 supraglottic cancer were 33%, 33%, and 60%, respectively. Corresponding figures for T3 glottic cancer were 32%, 23%, and 77%, respecitvely. Five-year cause-specific survival rate for T3 supraglottic cancer and glottic cancer were 47% and 77%, respectively. In T3 supraglottic cancer, none of the 4 patients with subglottic tumor extension attained local control by radiotherapy alone, and local-regional recurrence-free time were significantly shorter in patients with subglottic tumor extension or tracheostomy before radiotherapy. There were no serious late complications such as chondronecrosis, rupture of carotid artery attributed to radical radiotherapy, while 3 patients had severe laryngeal edema requiring total laryngectomy. (orig.) [Deutsch] Von 1974 bis 1992 wurden 37 zuvor nicht behandelte Patienten mit T3-Larynxkarzinomen (15 supraglottisch, 22 glottisch) primaer kurativ bestrahlt und, wenn erforderlich, einer Salvage-Operation unterzogen. Die Zwei-Jahres-Kontrollrate bei alleiniger Strahlentherapie, die Rate der Stimmerhaltung sowie die unter Einschluss der Operation erreichbare lokale Kontrollrate bei supraglottischen T3-Larynxkarzinomen betrugen 33%, 33% und 60%. Bei glottischen T3-Karzinomen wurden jeweils 32%, 23% und 77% erreicht. Die Fuenf-Jahres-Ueberlebensrate betrug 47% bei supraglottischen T3-Karzinomen und 77% bei den glottischen Karzinomen. Im Fall von supraglottischen Karzinomen erreichte keiner der vier Patienten mit subglottischer Tumorausdehnung eine lokale Kontrolle durch alleinige Strahlentherapie. Die lokoregionale rezidivfreie Zeit war bei den Patienten mit subglottischer Tumorausdehnung oder Tracheostomie vor Einleitung der

  10. Polynucleotide phosphorylase and the T3SS.

    Science.gov (United States)

    Rosenzweig, Jason A; Schesser, Kurt

    2007-01-01

    Low temperatures as well as encounters with host phagocytes are two stresses that have been relatively well studied in many species of bacteria. The exoribonuclease polynucleotide phosphorylase (PNPase) has previously been shown to be required by several species of bacteria, including Yersinia, for low-temperature growth. We have shown that PNPase also enhances the ability of Yersinia to withstand the killing activities of murine macrophages. We have gone on to show that PNPase is required for the optimal functioning of Yersinia's type three secretion system (T3SS), an organelle that injects effector proteins directly into host cells. Surprisingly, the PNPase-mediated effect on T3SS activity is independent of PNPase's ribonuclease activity and instead requires only its S1 RNA-binding domain. In stark contrast, the catalytic activity of PNPase is strictly required for enhanced growth at low temperature. Preliminary experiments suggest that the RNA-binding interface of the S1 domain is critical for its T3SS-enhancing activity. Our findings indicate that PNPase plays versatile roles in promoting Yersinia's survival in response to stressful conditions.

  11. Enterohepatic circulation of triiodothyronine (T3) in rats: Importance of the microflora for the liberation and reabsorption of T3 from biliary T3 conjugates

    Energy Technology Data Exchange (ETDEWEB)

    Rutgers, M.; Heusdens, F.A.; Bonthuis, F.; de Herder, W.W.; Hazenberg, M.P.; Visser, T.J. (Erasmus Univ. Medical School, Rotterdam (Netherlands))

    1989-12-01

    In normal rats, T3 glucuronide (T3G) is the major biliary T3 metabolite, but excretion of T3 sulfate (T3S) is greatly increased after inhibition of type I deiodinase, e.g. with 6-propyl-2-thiouracil (PTU). In this study, the fate of the T3 conjugates excreted with bile was studied to assess the significance of a putative enterohepatic circulation of T3 in rats. Conventional (CV) or intestine-decontaminated (ID) rats received iv (125I)T3G or (125I)T3S, the latter usually after pretreatment with PTU (1 mg/100 g BW). Radioactivity in plasma and bile or feces was analyzed by Sephadex LH-20 chromatography and HPLC. Within 1 h, 88% of injected T3G was excreted in bile of CV or ID rats, independent of PTU. About 75% of the injected T3S was excreted within 4 h in PTU-treated rats, in contrast to only 20% in controls. Up to 13 h after iv administration of T3G or T3S (+PTU) to intact ID and CV rats, fecal radioactivity consisted of more than 90% T3 in all CV rats, 95% of T3S in T3S-injected ID rats, and 30% T3 and 67% T3G in T3G-injected ID rats. In overnight-fasted CV rats injected with T3G, total plasma radioactivity rapidly declined until a nadir of 0.10% dose/ml at about 2.5 h, but radioactivity reappeared with a broad maximum of 0.12% dose/ml between 5.5-10 h. In the latter phase, plasma radioactivity consisted of predominantly I- and T3 in a ratio of 2:1. Reabsorption was diminished in fed CV rats and prevented in ID rats. Plasma T3 4-10 h after iv T3G injection to overnight-fasted CV rats was 12, 2, and 3 times higher than that in bile-diverted rats, fed CV rats, and ID rats, respectively, and similar to that 4 h after the injection of T3 itself. Total plasma radioactivity as well as plasma T3 6-13 h after iv administration T3S in PTU-treated rats were significantly increased in CV vs. ID rats, e.g. T3 0.016% vs. 0.005% dose/ml.

  12. The SGI/Cray T3E: Experiences and Insights

    Science.gov (United States)

    Bernard, Lisa Hamet

    1998-01-01

    The NASA Goddard Space Flight Center is home to the fifth most powerful supercomputer in the world, a 1024 processor SGI/Cray T3E-600. The original 512 processor system was placed at Goddard in March, 1997 as part of a cooperative agreement between the High Performance Computing and Communications Program's Earth and Space Sciences Project (ESS) and SGI/Cray Research. The goal of this system is to facilitate achievement of the Project milestones of 10, 50 and 100 GFLOPS sustained performance on selected Earth and space science application codes. The additional 512 processors were purchased in March, 1998 by the NASA Earth Science Enterprise for the NASA Seasonal to Interannual Prediction Project (NSIPP). These two "halves" still operate as a single system, and must satisfy the unique requirements of both aforementioned groups, as well as guest researchers from the Earth, space, microgravity, manned space flight and aeronautics communities. Few large scalable parallel systems are configured for capability computing, so models are hard to find. This unique environment has created a challenging system administration task, and has yielded some insights into the supercomputing needs of the various NASA Enterprises, as well as insights into the strengths and weaknesses of the T3E architecture and software. The T3E is a distributed memory system in which the processing elements (PE's) are connected by a low latency, high bandwidth bidirectional 3-D torus. Due to the focus on high speed communication between PE's, the T3E requires PE's to be allocated contiguously per job. Further, jobs will only execute on the user specified number of PE's and PE timesharing is possible but impractical. With a highly varied job mix in both size and runtime of jobs, the resulting scenario is PE fragmentation and an inability to achieve near 100% utilization. SGI/Cray has provided several scheduling and configuration tools to minimize the impact of fragmentation. These tools include PSche

  13. Production of sheep anti triiodothyronine T3 antisera for development of T3 RIA kit

    International Nuclear Information System (INIS)

    Elsadig, M. G.; Abdalla, O. M.; Eltayed, E. A.

    2009-01-01

    This study was a trial to produce antisera against triiodothyronine T 3 . Small amounts of antibodies were detected due to a poor response of sheep to immunization. Consequently the amount of T 3 tracer bound by antibodies was found to be less than 10% with weak discrimination between zero and high standard doses in all bleeds which dose not satisfy radioimmunoassay (RIA) technique requirements. In this study, two local male Sudanese sheep (ovis aries) were immunized with T 3 -immuno gen intramuscularly and subcutaneously in different sites along their backs. Sheep (I) was immunized with 25μg of antigen per kg body weight in first and boosting injections, while the dose was only 10μg per kg body weight for sheep (II). T 3 -immunogen was emulsified in FCA for the first injection and in FIA for the boosting injections. The sera obtained from both sheep after each injection were subjected to evaluation for the levels of circulating anti T 3 antibodies through both qualitative and quantitative tests. The tests were performed for both purified and non-purified forms of antibodies with different separation techniques. Theses me methods include, precipitation by second antibody assisted by polyethylene glycol (PEG), polystyrene beads, and liquid phase separation techniques.(Author)

  14. Production of sheep anti triiodothyronine (T3) antisera for development of T3 RIA kit

    International Nuclear Information System (INIS)

    Karar, M. G. E.

    2006-11-01

    This study is a trial to produce antisera against triiodothyronine (T 3 ) which resulted in a poor response of sheep to immunization, since small amount of antibodies were detected, hence the amount of T 3 tracer bound by the antibodies in terms of percentage was found to be less than 10% with weak discrimination between zero and high standard doses in all bleeds which dose not satisfy radioimmunoassay (RIA) technique requirements. In this study, two local males Sudanese sheep (ovis aries) were immunized with T 3 -immuno gen intramuscularly and subcutaneously in different sites along their backs. Sheep (I) was immunized with 25μg of antigen per kg body weight in first and boosting injections, while the dose was only 10μg per kg body weight for sheep(II). T 3 -immuno gen was emulsified in FCA for the first injection and in FIA for the boosting injections. The sera obtained from both sheep after each injection were subjected to evaluation for the levels of circulating anti-T 3 antibodies through both qualitative and quantitative tests (titration test). The tests were performed for both purified and non-purified from with different separation methods, these methods included, precipitation by second antibody assisted by polyethylene glycol (PEG), polystyrene beads, and liquid phase separation technique. Tests for the quality and success of polystyrene beads coating process for the titration were done using pre characterized antibodies, namely anti progesterone antibodies and anti thyroxine antibodies. These tests revels that, the coating process including the activation of polystyrene beads, was of good quality, and the results obtained was due to weak response to T 3 -immuno gen. The results obtained from qualitative tests of the two sheep sera did not show clear precipitate, in spite of the positive result obtained in a neat sera, which was an indication for weak antibody formation. The result of the titration tests for all bleeds (third, forth, fifth, sixth, and

  15. Inhibition of nuclear T3 binding by fatty acids

    NARCIS (Netherlands)

    Wiersinga, W. M.; Chopra, I. J.; Teco, G. N.

    1988-01-01

    Studies were performed to evaluate a possible modulatory role of lipids on the binding of T3 to rat liver nuclear receptors in vitro. Unsaturated fatty acids were potent inhibitors of the binding of [125I] T3 to isolated rat liver nuclei. Doses (in mumol/L) causing a 50% inhibition of nuclear T3

  16. Corrosion of AA2024-T3 Part III: Propagation

    International Nuclear Information System (INIS)

    Glenn, A.M.; Muster, T.H.; Luo, C.; Zhou, X.; Thompson, G.E.; Boag, A.; Hughes, A.E.

    2011-01-01

    Research highlights: → Corrosion of AA2024 in 0.1 M NaCl was examined for immersion times up to 120 min. → Rings of corrosion product with H 2 evolution developed after 5 min immersion. → Intergranular attack penetrated up to 60 μm below the rings within 120 min. → After 240 min mixed intergranular attack and grain etchout were observed. - Abstract: Optical and electron microscopies and EBSD were used to study the early stages of corrosion propagation during stable pit formation on AA2024-T3. Polished AA2024-T3 developed large scale rings of corrosion product, typically a few hundred microns in diameter, within 2 h of exposure to 0.1 M NaCl at room temperature. These features were sectioned using diamond ultramicrotomy and substantial subsurface attack, in the form of intergranular corrosion was observed beneath these sites with virtually no grain etchout. A model is proposed for the mechanism of stable pit progression which involves extensive grain boundary attack, followed by grain etchout leading to open pit formation.

  17. Development and characterization of radioimmunoassay methods for the measurement of iodothyronines (T4, T3 and rT3)

    International Nuclear Information System (INIS)

    Russo, E.M.K.; Vieira, J.G.H.; Barros Maciel, R.M. de; Fonseca, R.M.G.

    1982-01-01

    The experience acquired in the development of radioimmunoassay for T 4 , T 3 and rT 3 in unextrated serum is described. Antisera were produced in rabbits using iodothyronines conjugated to bovine serum albumin: the antisera selected provided the development of sensitive and specific radioassay methods. Stable high activity T 3 , T 4 and rT 3 tracers were prepared by iodination of 3,5 T 2 , T 3 and 3,3' T 2 by the chloramine-T method, and purified by column chromatography on Sephadex G25. Binding of those iodothyronines to endogenous serum proteins was blocked by including 8-aniline-1-naphtalene sulphonic acid (ANSA) in the T 4 and T 3 assays and thymerosal in the rT 3 assay. Normal values were defined in 46 healthy euthyroid adults of both sexes: T 4 = 7,1 +- 1,3μg/dl; T 3 = 139 +- 35ng/dl and rT 3 = 18,0 +- 7,9ng/dl. (Author) [pt

  18. Modulation of nuclear T3 binding by T3 in a human hepatocyte cell-line (Chang-liver) - T3 stimulation of cell growth but not of malic enzyme, glucose-6-phosphatdehydrogenase or 6-phosphogluconate-dehydrogenase

    DEFF Research Database (Denmark)

    Matzen, L E; Kristensen, S R; Kvetny, J

    1991-01-01

    The T3 modulation of nuclear T3 binding (NBT3), the T3 effect on cell growth, and the T3 and insulin effects on malic enzyme (ME), glucose-6-phosphat-dehydrogenase (G6PD) and 6-phosphogluconat-dehydrogenase (G6PD) were studied in a human hepatocyte cell-line (Chang-liver). T3 was bound to a high...

  19. Comparison of only T3 and T3-T4 sympathectomy for axillary hyperhidrosis regarding treatment effect and compensatory sweating.

    Science.gov (United States)

    Yuncu, Gökhan; Turk, Figen; Ozturk, Gökhan; Atinkaya, Cansel

    2013-08-01

    Patients diagnosed with axillary hyperhidrosis can face psychosocial issues that can ultimately hinder their quality of life both privately and socially. The routine treatment for axillary hyperhidrosis is T3-T4 sympathectomy, but compensatory sweating is a serious side effect that is commonly seen with this approach. This study was designed to evaluate whether a T3 sympathectomy was effective for the treatment of axillary hyperhidrosis and whether this treatment led to less compensatory sweating than T3-T4 sympathectomies among our 60-patient population. One hundred and twenty endoscopic thoracic sympathectomies were performed on 60 patients who had axillary hyperhidrosis. The sympathectomies were accomplished by means of a single-lumen endotracheal tube and a single port. The axillary hyperhidrosis patients were randomly divided into two groups with 17 patients in Group 1 undergoing T3-T4 sympathectomies and 43 in Group 2 undergoing only T3 sympathectomies. We analysed the data associated with the resolution of axillary hyperhidrosis, the degree of patient satisfaction with the surgical outcome and the quality of life in parallel with compensatory sweating after the procedure as reported by the patient and confirmed by the examiner. Moreover, the results were compared statistically. No statistically significant difference was observed between the groups based on age (P=0.56), gender (P=0.81), duration of the surgery (P=0.35) or postoperative satisfaction levels (P=0.45). However, the incidence and degree of compensatory sweating were lower in the T3 group than the T3-T4 group at the 1-year follow-up (P=0.008). T3 sympathectomy was as effective as T3-T4 sympathectomy for the treatment of axillary hyperhidrosis based on the patients' reported postoperative satisfaction, and the T3 group demonstrated lower compensatory sweating at the 1-year follow-up.

  20. Increased reverse T3 concentration in patients with anorexia nerrosa

    International Nuclear Information System (INIS)

    Baranowska, B.; Kaniewski, M.; Zgliczynski, S.

    1980-01-01

    In 20 female patients with anorexia nervosa, aging 16 - 26 years, the thyroid function was estimated by +- determining TSH secretion in response to TRH, and serum thyroxine (T 4 ), 3,5,3'L-triiodothyronine (T 3 ) and 3,3',5'L-triiodothyronine (reverse T 3 ) concentrations. 14 healthy women of the same age were included into the control group. If compared with control group, a marked supression of TRH stimulated TSH secretion and a lowering of serum T 3 concentration was found in patients with anorexia nervosa. On the other hand, serum reverse T 3 concentration was markedly higher in patients with anorexia nervosa than in control ones. Gain of body weight leads to normalization of thyroid hormones level in the serum. Obtained results show for peripheral mechanism of described hormonal disorders. (author)

  1. Triple3 Redundant Spacecraft Subsystems (T3RSS) Project

    Data.gov (United States)

    National Aeronautics and Space Administration — T3RSS is the system engineer's tool that allows a systematic approach to ensuring that even if one or more failures occur in a single component or subsystem, then...

  2. Basic and clinical investigation of T3 immunoassay kit

    International Nuclear Information System (INIS)

    Konishi, Junji; Nakajima, Akiko; Morita, Rikushi; Endo, Keigo; Ikekubo, Katsuji

    1976-01-01

    T 3 immunoassay kit was investigated basically and clinically. A good result was obtained at the prescribed incubation temperature and for 16 hours of incubation time. Moreover, it was thought to be possible that incubation time could be shortened to 1 - 4 hours at 37 0 C. Specificity of antibody was good. Recovery of added T 3 was 100+-5 (S.D.) % on an average and parallel of dilution curve of high T 3 serum was also good. Variation coefficient of accuracy of this kit was 1.5 - 2.1 % and that of reproducibility was 1.3 - 6.6 %. Mild hemolysis did not affect measurement value. Serum T 3 level in normals, untreated patients with Basedow's disease and patients with primary hypothyroidism was 142+-21 ng/100 ml, 452+-156 ng/100 ml and 67+-17 ng/100 ml, respectively. Serum T 3 level in patients with Hashimoto's disease was distributed to a wide extent, but that of patients with goiter and simple goiter ranged within normal range. On the other side, serum T 3 level of normal pregnant woman was high and that of patients with anorexia nervosa showed low level. From the above mentioned results, it was concluded that this kit was simple in method and good in sensitivity, specificity and reproducibility and it was also useful for clinical applications. (M. Tsunoda)

  3. Mediator subunit MED1 is a T3-dependent and T3-independent coactivator on the thyrotropin β gene promoter

    Energy Technology Data Exchange (ETDEWEB)

    Matsui, Keiji; Oda, Kasumi; Mizuta, Shumpei; Ishino, Ruri; Urahama, Norinaga; Hasegawa, Natsumi [Laboratory of Hematology, Division of Medical Biophysics, Kobe University Graduate School of Health Sciences, 7-10-2 Tomogaoka, Suma-ku, Kobe 654-0142 (Japan); Roeder, Robert G. [Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10065 (United States); Ito, Mitsuhiro, E-mail: itomi@med.kobe-u.ac.jp [Laboratory of Hematology, Division of Medical Biophysics, Kobe University Graduate School of Health Sciences, 7-10-2 Tomogaoka, Suma-ku, Kobe 654-0142 (Japan); Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10065 (United States); Department of Family and Community Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 654-0142 (Japan); Consolidated Research Institute for Advanced Science and Medical Care, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 159-8555 (Japan)

    2013-10-11

    Highlights: •MED1 is a bona fide T3-dependent coactivator on TSHB promoter. •Mice with LxxLL-mutant MED1 have attenuated TSHβ mRNA and thyroid hormone levels. •MED1 activates TSHB promoter T3-dependently in cultured cells. •T3-dependent MED1 action is enhanced when SRC1/SRC2 or HDAC2 is downregulated. •MED1 is also a T3-independent GATA2/Pit1 coactivator on TSHB promoter. -- Abstract: The MED1 subunit of the Mediator transcriptional coregulator complex is a nuclear receptor-specific coactivator. A negative feedback mechanism of thyroid-stimulating hormone (TSH, or thyrotropin) expression in the thyrotroph in the presence of triiodothyronine (T3) is employed by liganded thyroid hormone receptor β (TRβ) on the TSHβ gene promoter, where conventional histone-modifying coactivators act as corepressors. We now provide evidence that MED1 is a ligand-dependent positive cofactor on this promoter. TSHβ gene transcription was attenuated in MED1 mutant mice in which the nuclear receptor-binding ability of MED1 was specifically disrupted. MED1 stimulated GATA2- and Pit1-mediated TSHβ gene promoter activity in a ligand-independent manner in cultured cells. MED1 also stimulated transcription from the TSHβ gene promoter in a T3-dependent manner. The transcription was further enhanced when the T3-dependent corepressors SRC1, SRC2, and HDAC2 were downregulated. Hence, MED1 is a T3-dependent and -independent coactivator on the TSHβ gene promoter.

  4. Measurement of weak rates for stellar evolution via the (t,3He) reaction

    International Nuclear Information System (INIS)

    Zegers, R.G.T.

    2007-01-01

    The (t, 3 He) charge-exchange reaction has been developed as a tool to extract Gamow-Teller strengths on nuclei of importance for stellar evolution. A secondary triton beam of 115 MeV/nucleon is used, either produced from a primary α beam, or, since recently, from a primary 16 O beam. Here, the (t, 3 He) reaction is used to study the Gamow-Teller strength distribution in 58 Co via the 58 Ni(t, 3 He) reaction. The experimental results are compared with calculations in large-scale shell models using the kb3g and gxpf1 interactions, as well as existing data from 58 Ni(n, p) and 58 Ni(d, 2 He) experiments. The differences between the data and theoretical models are studied in terms of electron-capture rates in the pre-collapse stages of core-collapse supernovae

  5. Assembly and structure of the T3SS.

    Science.gov (United States)

    Burkinshaw, Brianne J; Strynadka, Natalie C J

    2014-08-01

    The Type III Secretion System (T3SS) is a multi-mega Dalton apparatus assembled from more than twenty components and is found in many species of animal and plant bacterial pathogens. The T3SS creates a contiguous channel through the bacterial and host membranes, allowing injection of specialized bacterial effector proteins directly to the host cell. In this review, we discuss our current understanding of T3SS assembly and structure, as well as highlight structurally characterized Salmonella effectors. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey. Copyright © 2014 Elsevier B.V. All rights reserved.

  6. Host Cell Targeting by Enteropathogenic Bacteria T3SS Effectors.

    Science.gov (United States)

    Pinaud, Laurie; Sansonetti, Philippe J; Phalipon, Armelle

    2018-04-01

    Microbial pathogens possess a diversity of weapons that disrupt host homeostasis and immune defenses, thus resulting in the establishment of infection. The best-characterized system mediating bacterial protein delivery into target eukaryotic cells is the type III secretion system (T3SS) expressed by Gram-negative bacteria, including the human enteric pathogens Shigella, Salmonella, Yersinia, and enteropathogenic/enterohemorragic Escherichia coli (EPEC/EHEC). The emerging global view is that these T3SS-bearing pathogens share similarities in their ability to target key cellular pathways such as the cell cytoskeleton, trafficking, cell death/survival, and the NF-κB and MAPK signaling pathways. In particular, multiple host proteins are targeted in a given pathway, and different T3SS effectors from various pathogens share functional similarities. Copyright © 2018 Elsevier Ltd. All rights reserved.

  7. Isoproterenol Increases Uncoupling, Glycolysis, and Markers of Beiging in Mature 3T3-L1 Adipocytes.

    Directory of Open Access Journals (Sweden)

    Colette N Miller

    Full Text Available Beta-adrenergic activation stimulates uncoupling protein 1 (UCP1, enhancing metabolic rate. In vitro, most work has studied brown adipocytes, however, few have investigated more established adipocyte lines such as the murine 3T3-L1 line. To assess the effect of beta-adrenergic activation, mature 3T3-L1s were treated for 6 or 48 hours with or without isoproterenol (10 and 100 μM following standard differentiation supplemented with thyroid hormone (T3; 1 nM. The highest dose of isoproterenol increased lipid content following 48 hours of treatment. This concentration enhanced UCP1 mRNA and protein expression. The increase in UCP1 following 48 hours of isoproterenol increased oxygen consumption rate. Further, coupling efficiency of the electron transport chain was disturbed and an enhancement of glycolytic rate was measured alongside this, indicating an attempt to meet the energy demands of the cell. Lastly, markers of beige adipocytes (protein content of CD137 and gene transcript of CITED1 were also found to be upregulated at 48 hours of isoproterenol treatment. This data indicates that mature 3T3-L1 adipocytes are responsive to isoproterenol and induce UCP1 expression and activity. Further, this finding provides a model for further pharmaceutical and nutraceutical investigation of UCP1 in 3T3-L1s.

  8. Study of the charge-exchange reaction 24 Mg(t,3 He)

    International Nuclear Information System (INIS)

    Gareev, F.A.; Semchenkov, A.G.

    1994-01-01

    The charge-exchange reaction 24 Mg(t, 3 He) in the region of Δ-isobar excitation at initial momentum 9.15 GeV/c is analyzed using (π + ρ + g')-model and effective number formalism. Contribution from different topologies and their influence on Δ-isobar inclusive peak are considered. (author). 15 refs., 9 figs., 3 tabs

  9. Using T3, an improved decision tree classifier, for mining stroke-related medical data.

    Science.gov (United States)

    Tjortjis, C; Saraee, M; Theodoulidis, B; Keane, J A

    2007-01-01

    Medical data are a valuable resource from which novel and potentially useful knowledge can be discovered by using data mining. Data mining can assist and support medical decision making and enhance clinical management and investigative research. The objective of this work is to propose a method for building accurate descriptive and predictive models based on classification of past medical data. We also aim to compare this method with other well established data mining methods and identify strengths and weaknesses. We propose T3, a decision tree classifier which builds predictive models based on known classes, by allowing for a certain amount of misclassification error in training in order to achieve better descriptive and predictive accuracy. We then experiment with a real medical data set on stroke, and various subsets, in order to identify strengths and weaknesses. We also compare performance with a very successful and well established decision tree classifier. T3 demonstrated impressive performance when predicting unseen cases of stroke resulting in as little as 0.4% classification error while the state of the art decision tree classifier resulted in 33.6% classification error respectively. This paper presents and evaluates T3, a classification algorithm that builds decision trees of depth at most three, and results in high accuracy whilst keeping the tree size reasonably small. T3 demonstrates strong descriptive and predictive power without compromising simplicity and clarity. We evaluate T3 based on real stroke register data and compare it with C4.5, a well-known classification algorithm, showing that T3 produces significantly more accurate and readable classifiers.

  10. Operation and maintenance of the T-3 cask system

    International Nuclear Information System (INIS)

    Hussey, M.W.; Berger, J.D.; Peterson, J.M.

    1983-01-01

    The T-3 cask system consists of three lead-shielded casks and the associated payload containers, internal fixturing, tiedowns, transportation trailers and handling devices. The three casks were designed to meet the requirements of Title 10 of the Code of Federal Regulations, Part 71. The Nuclear Regulatory Commission cask licensing activities for original design and for licensing revisions have required significant analytical support. Commercial transportation contractors can provide needed services including provisions of suitable equipment, compliances with security requirements, and safe movement of the shipment at a potential savings over DOE-owned transportation systems. Proper periodic inspection/maintenance activities supported by adequate decontamination facilities are a must in keeping the T-3 casks available for service

  11. Ames T-3 fire test facility - Aircraft crash fire simulation

    Science.gov (United States)

    Fish, R. H.

    1976-01-01

    There is a need to characterize the thermal response of materials exposed to aircraft fuel fires. Large scale open fire tests are costly and pollute the local environment. This paper describes the construction and operation of a subscale fire test that simulates the heat flux levels and thermochemistry of typical open pool fires. It has been termed the Ames T-3 Test and has been used extensively by NASA since 1969 to observe the behavior of materials exposed to JP-4 fuel fires.

  12. Rapid Identification of Sporothrix Species by T3B Fingerprinting

    Science.gov (United States)

    de Oliveira, Manoel Marques Evangelista; Sampaio, Paula; Almeida-Paes, Rodrigo; Pais, Célia; Gutierrez-Galhardo, Maria Clara

    2012-01-01

    This article describes PCR fingerprinting using the universal primer T3B to distinguish among species of the Sporothrix complex, S. brasiliensis, S. globosa, S. mexicana, and S. schenckii. This methodology generated distinct banding patterns, allowing the correct identification of all 35 clinical isolates at the species level, confirmed by partial calmodulin (CAL) gene sequence analyses. This methodology is simple, reliable, rapid, and cheap, making it an ideal routine identification system for clinical mycology laboratories. PMID:22403427

  13. Modulation of nuclear T3 binding by T3 in a human hepatocyte cell-line (Chang-liver) - T3 stimulation of cell growth but not of malic enzyme, glucose-6-phosphatdehydrogenase or 6-phosphogluconate-dehydrogenase

    DEFF Research Database (Denmark)

    Matzen, L E; Kristensen, S R; Kvetny, J

    1991-01-01

    The T3 modulation of nuclear T3 binding (NBT3), the T3 effect on cell growth, and the T3 and insulin effects on malic enzyme (ME), glucose-6-phosphat-dehydrogenase (G6PD) and 6-phosphogluconat-dehydrogenase (G6PD) were studied in a human hepatocyte cell-line (Chang-liver). T3 was bound to a high...... modulation of NBT3 associated to receptor saturation; 2) stimulation of cell growth; 3) contrary to the findings in rat hepatocytes no stimulation of ME, G6PD or 6PGD. Insulin enhanced ME and 6PGD....

  14. Topiramate effects lipolysis in 3T3-L1 adipocytes.

    Science.gov (United States)

    Martins, Gabriela Poltronieri Campagnaro; Souza, Camila Oliveira; Marques, Scherolin; Luciano, Thais Fernandes; DA Silva Pieri, Bruno Luiz; Rosa, José César; DA Silva, Adelino Sanchez Ramos; Pauli, José Rodrigo; Cintra, Dennys Esper; Ropelle, Eduardo Rochete; Rodrigues, Bruno; DE Lira, Fabio Santos; DE Souza, Claudio Teodoro

    2015-11-01

    Studies have shown that topiramate (TPM)-induced weight loss can be dependent on the central nervous system (CNS). However, the direct action of TPM on adipose tissue has not been tested previously. Thus, the present study aimed to examine whether TPM modulates lipolysis in 3T3-L1. The 3T3-L1 cells were incubated in 50 µM TPM for 30 min. The β-adrenergic stimulator, isoproterenol, was used as a positive control. The release of lactate dehydrogenase, non-esterified fatty acid, glycerol and incorporation of 14 C-palmitate to lipid were analyzed. The phosphorylation of protein kinase A (PKA), hormone-sensitive lipase (HSL), adipocyte triglyceride lipase (ATGL) and perilipin A, as well as the protein levels of comparative genetic identification 58 (CGI-58) were assessed. The levels of glycerol and non-esterified fatty acid increased markedly when the cells were treated with TPM. The TPM effects were similar to the isoproterenol positive control. Additionally, TPM reduced lipogenesis. These results were observed without any change in cell viability. Finally, the phosphorylation of PKA, HSL, ATGL and perilipin A, as well as the protein levels of CGI-58 were increased compared to the control cells. These results were similar to those observed in the cells treated with isoproterenol. The present results show that TPM increased the phosphorylation of pivotal lipolytic enzymes, which induced lipolysis in 3T3-L1 adipocytes, suggesting that this drug may act directly in the adipose tissue independent from its effect on the CNS.

  15. Sympathectomy versus Sympathicotomy in Palmar Hyperhidrosis Comparing T3 Ablation.

    Science.gov (United States)

    Aydemir, Bülent; Imamoglu, Oya; Okay, Tamer; Celik, Muharrem

    2015-12-01

    The objective of this study was to evaluate and compare thoracoscopic sympathectomy and sympathicotomy at the third ganglia (T3) level for the treatment of primary palmar hyperhidrosis in terms of initial surgery results, complications, and patient satisfaction. Two groups of patient underwent T3 thoracoscopic sympathectomy and thoracoscopic sympathicotomy under general anesthesia using single-lung ventilation via a double-lumen endotracheal tube by the same surgical team for the treatment of severe primary palmar hyperhidrosis or a combination of levels for multiarea between 2008 and 2013. The groups were homogeneous for relevant demographic, physiological, and clinical data. All patients were examined preoperatively and were followed up at 6 months postoperatively. In both groups, patient's satisfaction was evaluated 6 months after surgery by a detailed interview and scored into three grades (1 = very satisfied, 2 = satisfied, and 3 = dissatisfied). No operative mortality, major intraoperative complication, infections, and Horner syndrome were recorded. There was no treatment failure. The average time of operation was 50 minutes for Group A (sympathectomy) and 36 minutes for Group B (sympathicotomy). Compensatory sweating occurred in 40 patients (89% for Group A and 85.11% for Group B) with a different accumulation of the severity degree. The satisfaction rate was 91.11% for Group A and 93.61% for Group B. There was no significant difference between thoracoscopic sympathectomy and sympathicotomy at the third ganglia (T3) level for the treatment of primary palmar hyperhidrosis in terms of initial surgery results, complications, and patient satisfaction. Neither surgical technique is better than the other one for palmar hyperhidrosis treatment. Development of severe compensatory sweating and postoperative pain are major determinant factors of patient dissatisfaction. Sympathicotomy should be preferred for palmar hyperhidrosis treatment, as it is much

  16. Cannabidiol promotes browning in 3T3-L1 adipocytes.

    Science.gov (United States)

    Parray, Hilal Ahmad; Yun, Jong Won

    2016-05-01

    Recruitment of the brown-like phenotype in white adipocytes (browning) and activation of existing brown adipocytes are currently being investigated as a means to combat obesity. Thus, a wide variety of dietary agents that contribute to browning of white adipocytes have been identified. The present study was designed to investigate the effects of cannabidiol (CBD), a major nonpsychotropic phytocannabinoid of Cannabis sativa, on induction of browning in 3T3-L1 adipocytes. CBD enhanced expression of a core set of brown fat-specific marker genes (Ucp1, Cited1, Tmem26, Prdm16, Cidea, Tbx1, Fgf21, and Pgc-1α) and proteins (UCP1, PRDM16, and PGC-1α). Increased expression of UCP1 and other brown fat-specific markers contributed to the browning of 3T3-L1 adipocytes possibly via activation of PPARγ and PI3K. In addition, CBD increased protein expression levels of CPT1, ACSL, SIRT1, and PLIN while down-regulating JNK2, SREBP1, and LPL. These data suggest possible roles for CBD in browning of white adipocytes, augmentation of lipolysis, thermogenesis, and reduction of lipogenesis. In conclusion, the current data suggest that CBD plays dual modulatory roles in the form of inducing the brown-like phenotype as well as promoting lipid metabolism. Thus, CBD may be explored as a potentially promising therapeutic agent for the prevention of obesity.

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

    Energy Technology Data Exchange (ETDEWEB)

    Lai, Jing [The First Affiliated Hospital, Jinan University, Guangzhou, Guangdong (China); Liu, Gexiu [Institute of Hematology, School of Medicine, Jinan University, Guangzhou, Guangdong (China); Yan, Guoyao [The First Affiliated Hospital, Jinan University, Guangzhou, Guangdong (China); He, Dongmei [Institute of Hematology, School of Medicine, Jinan University, Guangzhou, Guangdong (China); Zhou, Ying [The First Affiliated Hospital, Jinan University, Guangzhou, Guangdong (China); Chen, Shengting, E-mail: shengtingchen@sina.cn [The First Affiliated Hospital, Jinan University, Guangzhou, Guangdong (China)

    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 PPARγ, C/EBPα, 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, β-catenin, were both upregulated compared with the control group. siRNA-mediated knockdown of ROCKII and β-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 β-catenin are involved in this process. - Highlights: • WEHI-3, an acute myelomonocytic leukemia cell line, inhibited 3T3-L1 preadipocyte from differentiating into adipocyte. • WEHI-3 cells can arrest 3T3-L1 cells in G0/G1 phase by secreting soluble factors and thus inhibit their proliferation. • WEHI-3 cells reduced bone marrow pimelosis in the murine model. • Both ROCKII and β-catenin were involved in the WEHI-3-mediated anti-adipogenic effects.

  18. Preoperative infusional chemoradiation therapy for stage T3 rectal cancer

    Energy Technology Data Exchange (ETDEWEB)

    Rich, T.A.; Skibber, J.M.; Ajani, J.A. [Univ. of Texas M. D. Anderson Cancer Center, Houston, TX (United States)] [and others

    1995-07-15

    To evaluate preoperative infusional chemoradiation for patients with operable rectal cancer. Preoperative chemoradiation therapy using infusional 5-fluorouracil (5-FU), (300 mg/m{sup 2}/day) together with daily irradiation (45 Gy/25 fractions/5 weeks) was administered to 77 patients with clinically Stage T3 rectal cancer. Endoscopic ultrasound confirmed the digital rectal exam in 63 patients. Surgery was performed approximately 6 weeks after the completion of chemoradiation therapy and included 25 abdominoperineal resections and 52 anal-sphincter-preserving procedures. Posttreatment tumor stages were T1-2, N0 in 35%, T3, N0 in 25%, and T1-3, N1 in 11%; 29% had no evidence of tumor. Local tumor control after chemoradiation was seen in 96% (74 out of 77); 2 patients had recurrent disease at the anastomosis site and were treated successfully with abdominoperineal resection. Overall, pelvic control was obtained in 99% (76 out of 77). The survival after chemoradiation was higher in patients without node involvement than in those having node involvement (p = n.s.). More patients with pathologic complete responses or only microscopic foci survived than did patients who had gross residual tumor (p = 0.07). The actuarial survival rate was 83% at 3 years; the median follow-up was 27 months, with a range of 3 to 68 months. Acute, perioperative, and late complications were not more numerous or more severe with chemoradiation therapy than with traditional radiation therapy (XRT) alone. Excellent treatment response allowed two-thirds of the patients to have an anal-sphincter-sparing procedure. Gross residual disease in the resected specimen indicates a poor prognosis, and therapies specifically targeting these patients may improve survival further. 22 refs., 2 figs., 3 tabs.

  19. Effects of shugan jieyu panacea on behavior and levels of ACTH in plasma and T3, T4, TSH and rT3 in serum in depression rats

    International Nuclear Information System (INIS)

    Li Huijing; Li Yang; Yao Jinghui; Li Youtian

    2010-01-01

    Objective: To investigate the effects of Shugan Jieyu Panacea (SJP) on behavior and the levels of ACTH in plasma and T 3 , T 4 , TSH, rT 3 in serum in depression model rats and explore the mechanism. Methods: The model rats were lonely fed and received chronic moderate intensive unpredictable stimulation. Normal control group, depressed model group, high dosage SJP group, middle dosage SJP group, low dosage SJP group and fluoxetine group were set up. Different drugs were used in various groups for 21 d, then the body mass, sugar consumption and the behavior changes of the rats were determined and the levels of ACTH in plasma and T 3 , T 4 , TSH, rT 3 in serum were detected with radioimmunoassay. Results: Compared with normal group,the body mass was decreased (P 4 , rT 3 markedly decreased (P 3 was increased (P<0.05) in high, middle, low dosage SJP groups after treatment. At the same time, there was no obvious difference between SJP groups and fluoxetine groups. Conclusion: SJP can significantly improve the depression in rats, its mechanism may be connected with adjusting the function of HPAA and HPTA. (authors)

  20. Ultrasound associated uptake of chitosan nanoparticles in MC3T3-E1 cells

    Science.gov (United States)

    Wu, Junyi

    Chitosan is a natural linear polysaccharide that has been well known for its applications in drug delivery system due to its unique physicochemical and biological properties. However, challenges still remain for it to become a fully realized therapeutic agent. In this study, we investigated the uptake of chitosan nanoparticles (CNP) under the ultrasound stimulation, using a model cell culture system (MC3T3-E1 mouse pre-osteoblasts). The CNP were fabricated by an ionic gelation method and were lyophilized prior to characterization and delivery to cells. Particle size and zeta potential were measured using Dynamic Light Scattering (DLS); the efficiency of chitosan complexation was measured using the ninhydrin assay. Cytotoxicity was examined by neutral red assay within 48 hours after delivery. The effect of ultrasound (US) on the efficiency of nanoparticle delivery to the MC3T3-E1 cells was examined at 1MHz and at either 1 or 2 W/cm2. Fluorescein isothiocyanate (FITC)-conjugated-CNP were used to visualize the internalized particles within the cytosol. The uptake of FITC-CNP exhibits a dose and time dependent effect, a strong FITC fluorescence was detected at the concentration of 500microg/mL under fluorescence microscope. Ultrasound assisted uptake of FITC-CNP performed a significant positive effect at 2W/cm2 with 60s of ultrasound exposure time. CNP displayed a slightly decrease in cell viability from 25microg/mL to 100microg/mL, while higher concentration of CNP facilitates the proliferation of MC3T3-E1 cells. Less than 10% of reduction in cell viability was observed for US at 1W/cm2 and 2W/cm2 with 30s and 60s of exposure time, which suggest a mild effect of US to MC3T3-E1 cell line.

  1. Pore formation by T3SS translocators: liposome leakage assay.

    Science.gov (United States)

    Faudry, Eric; Perdu, Caroline; Attrée, Ina

    2013-01-01

    Gram-negative bacteria utilize a dedicated membrane-embedded apparatus, the type III secretion system (T3SS), to inject proteins into host cells. The passage of the proteins across the target membrane is accomplished by a proteinaceous pore-the translocon-formed within the host-cell cytoplasmic membrane. Translocators bound to their chaperones can be expressed in Escherichia coli and subsequently dissociated from the chaperone by guanidine treatment. The pore formation properties of the translocators can then be studied by an in-vitro liposome leakage assay. Sulforhodamine-B is encapsulated within lipid vesicles during liposome preparation. At high concentration, this fluorochrome exhibits self-quenching limiting fluorescence emission. Upon pore formation, liposome leakage leads to the dilution of Sulforhodamine-B in the medium and fluorescence emission increases. Alternatively, fluorochromes coupled to large dextran molecules can be encapsulated in order to estimate pore dimensions. Here we describe protein expression and purification, dye-liposome preparation, and leakage assay conditions.

  2. T3DB: an integrated database for bacterial type III secretion system

    OpenAIRE

    Wang, Yejun; Huang, He; Sun, Ming’an; Zhang, Qing; Guo, Dianjing

    2012-01-01

    Abstract Background Type III Secretion System (T3SS), which plays important roles in pathogenesis or symbiosis, is widely expressed in a variety of gram negative bacteria. However, lack of unique nomenclature for T3SS genes has hindered T3SS related research. It is necessary to set up a knowledgebase integrating T3SS-related research data to facilitate the communication between different research groups interested in different bacteria. Description A T3SS-related Database (T3DB) was developed...

  3. Cold exposure rapidly induces virtual saturation of brown adipose tissue nuclear T3 receptors

    International Nuclear Information System (INIS)

    Bianco, A.C.; Silva, J.E.

    1988-01-01

    Cold exposure induces a rapid increase in uncoupling protein (UCP) concentration in the brown adipose tissue (BAT) of euthyroid, but not hypothyroid, rats. To normalize this response with exogenous 3,5,3'-triiodothyronine (T 3 ), it is necessary to cause systemic hyperthyroidism. In contrast, the same result can be obtained with just replacement doses of thyroxine (T 4 ) and, in euthyroid rats, the normal response of UCP to cold occurs without hyperthyroid plasma T 3 levels. Consequently, the authors explored the possibility that the cold-induced activation of the type II 5'-deiodinase resulted in high levels of nuclear T 3 receptor occupancy in euthyroid rats. Studies were performed with pulse injections of tracer T 3 or T 4 in rats exposed to 4 degree C for different lengths of time (1 h-3 wk). Within 4 h of cold exposure, they observed a significant increase in the nuclear [ 125 I]T 3 derived from the tracer [ 125 I]T 4 injections (T 3 [T 4 ]) and a significant reduction in the nuclear [ 125 I]T 3 derived from [ 125 I]T 3 injections (T 3 [T 3 ]). The number of BAT nuclear T 3 receptors did not increase for up to 3 wk of observation at 4 degree C. The mass of nuclear-bound T 3 was calculated from the nuclear tracer [ 125 I]T 3 [T 3 ] and [ 125 I]T 3 [T 4 ] at equilibrium and the specific activity of serum T 3 and T 4 , respectively. By 4 h after the initiation of the cold exposure, the receptors were >95% occupied and remained so for the 3 weeks of observation. They conclude that the simultaneous activation of the deiodinase with adrenergic BAT stimulation serves the purpose of nearly saturating the nuclear T 3 receptors. This makes possible the realization of the full thermogenic potential of the tissue without causing systemic hyperthyroidism

  4. ExsE Is a Negative Regulator for T3SS Gene Expression inVibrio alginolyticus.

    Science.gov (United States)

    Liu, Jinxin; Lu, Shao-Yeh; Orfe, Lisa H; Ren, Chun-Hua; Hu, Chao-Qun; Call, Douglas R; Avillan, Johannetsy J; Zhao, Zhe

    2016-01-01

    Type III secretion systems (T3SSs) contribute to microbial pathogenesis of Vibrio species, but the regulatory mechanisms are complex. We determined if the classic ExsACDE protein-protein regulatory model from Pseudomonas aeruginosa applies to Vibrio alginolyticus . Deletion mutants in V. alginolyticus demonstrated that, as expected, the T3SS is positively regulated by ExsA and ExsC and negatively regulated by ExsD and ExsE. Interestingly, deletion of exsE enhanced the ability of V. alginolyticus to induce host-cell death while cytotoxicity was inhibited by in trans complementation of this gene in a wild-type strain, a result that differs from a similar experiment with Vibrio parahaemolyticus ExsE. We further showed that ExsE is a secreted protein that does not contribute to adhesion to Fathead minnow epithelial cells. An in vitro co-immunoprecipitation assay confirmed that ExsE binds to ExsC to exert negative regulatory effect on T3SS genes. T3SS in V. alginolyticus can be activated in the absence of physical contact with host cells and a separate regulatory pathway appears to contribute to the regulation of ExsA. Consequently, like ExsE from P. aeruginosa , ExsE is a negative regulator for T3SS gene expression in V. alginolyticus . Unlike the V. parahaemolyticus orthologue, however, deletion of exsE from V. alginolyticus enhanced in vitro cytotoxicity.

  5. [Importance of 3T3 feeder layer to establish epithelial cultures from cell suspension obtained from corneo-scleral rims].

    Science.gov (United States)

    Cristovam, Priscila Cardoso; Glória, Maria Aparecida da; Melo, Gustavo Barreto; Gomes, José Alvaro Pereira

    2008-01-01

    To evaluate the importance of the presence of 3T3 fibroblasts for establishing limbal epithelial cultures from cell suspension obtained from corneo-scleral rims (CSR). Corneo-scleral rims from different donors (n=6) had their posterior stroma and endothelium stripped away. Each corneo-scleral rim was divided into three equal segments that were set up in tissue culture in three different conditions: one of the segments was placed with the epithelial side up on the bottom of a 6-well culture plate (Group A). The other two fragments were trypsinized and the obtained cell suspension was cultured with (Group B) or without (Group C) irradiaded 3T3 cells. The cells were cultured in supplemental hormonal epithelial medium (SHEM), the epithelial migration and clone formation in groups A, B and C were evaluated with phase contrast microscopy and rodamine B staining. Epithelial cell growth was observed in 4/6 rims (Group A). All epithelial cell suspensions that were cultured with 3T3 cells (Group B) formed clones. No adhesion or true clone formation (holo- or meroclones) was observed in the cell suspensions that were cultivated without 3T3 (Group C) (p=0.009). Epithelial cell suspension obtained from corneo-scleral rims in this model needs to be cultivated with 3T3 cells in order to form clones and establish limbal epithelial cell colonies with the potential to be used for ocular surface reconstruction.

  6. Comparison of only T3 and T3–T4 sympathectomy for axillary hyperhidrosis regarding treatment effect and compensatory sweating

    Science.gov (United States)

    Yuncu, Gökhan; Turk, Figen; Ozturk, Gökhan; Atinkaya, Cansel

    2013-01-01

    OBJECTIVES Patients diagnosed with axillary hyperhidrosis can face psychosocial issues that can ultimately hinder their quality of life both privately and socially. The routine treatment for axillary hyperhidrosis is T3–T4 sympathectomy, but compensatory sweating is a serious side effect that is commonly seen with this approach. This study was designed to evaluate whether a T3 sympathectomy was effective for the treatment of axillary hyperhidrosis and whether this treatment led to less compensatory sweating than T3–T4 sympathectomies among our 60-patient population. METHODS One hundred and twenty endoscopic thoracic sympathectomies were performed on 60 patients who had axillary hyperhidrosis. The sympathectomies were accomplished by means of a single-lumen endotracheal tube and a single port. The axillary hyperhidrosis patients were randomly divided into two groups with 17 patients in Group 1 undergoing T3–T4 sympathectomies and 43 in Group 2 undergoing only T3 sympathectomies. We analysed the data associated with the resolution of axillary hyperhidrosis, the degree of patient satisfaction with the surgical outcome and the quality of life in parallel with compensatory sweating after the procedure as reported by the patient and confirmed by the examiner. Moreover, the results were compared statistically. RESULTS No statistically significant difference was observed between the groups based on age (P = 0.56), gender (P = 0.81), duration of the surgery (P = 0.35) or postoperative satisfaction levels (P = 0.45). However, the incidence and degree of compensatory sweating were lower in the T3 group than the T3–T4 group at the 1-year follow-up (P = 0.008). CONCLUSIONS T3 sympathectomy was as effective as T3–T4 sympathectomy for the treatment of axillary hyperhidrosis based on the patients’ reported postoperative satisfaction, and the T3 group demonstrated lower compensatory sweating at the 1-year follow-up. PMID:23644731

  7. Early life trauma is associated with decreased peripheral levels of thyroid-hormone T3 in adolescents.

    Science.gov (United States)

    Machado, T D; Salum, G A; Bosa, V L; Goldani, M Z; Meaney, M J; Agranonik, M; Manfro, G G; Silveira, P P

    2015-12-01

    An adverse early life environment can induce changes on behavioral and metabolic responses later in life. Recent studies in rats showed that the quality of maternal care as measured by high levels of pup licking and grooming (LG) was associated with changes in the relationship between the precursor thyroid-hormone T4 and the more active T3. Here we investigated if early exposure to childhood abuse is associated with thyroid-hormone levels in human adolescents. Given the empirical evidence from animal models showing that good maternal care was associated with increased conversion of T4 to T3, we hypothesized that early adversity would be associated with a decreased peripheral conversion of T4 to T3. A sample of 80 adolescents (10-18 years) participated in this study. We used the Childhood Trauma Questionnaire to investigate early life stress. We calculate the body mass index (BMI) assessing weight and height and sexual maturation stage was determined by self-assessment. Blood samples were collected to measure T3 and T4 levels. ANCOVA were used to evaluate the influence of the Physical Abuse domain of the Childhood Trauma Questionnaire as the early life stress variable in T3 and T4 separately, adjusted for potential confounders such as pubertal status, gender, socioeconomic status and BMI. Early life trauma was associated with reduced T3 levels in adolescents, when adjusted for potential confounders (p=0.013), but not with peripheral T4 levels (p=0.625). We extended findings from animal models showing that adverse early experience persistently impacts on the individual's responses to stress, which is marked by an abnormal metabolism of thyroid hormones. Further studies are needed to further investigate the nature of such associations. Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. The Structure of a Type 3 Secretion System (T3SS) Ruler Protein Suggests a Molecular Mechanism for Needle Length Sensing*

    Science.gov (United States)

    Bergeron, Julien R. C.; Fernández, Lucia; Wasney, Gregory A.; Vuckovic, Marija; Reffuveille, Fany; Hancock, Robert E. W.; Strynadka, Natalie C. J.

    2016-01-01

    The type 3 secretion system (T3SS) and the bacterial flagellum are related pathogenicity-associated appendages found at the surface of many disease-causing bacteria. These appendages consist of long tubular structures that protrude away from the bacterial surface to interact with the host cell and/or promote motility. A proposed “ruler” protein tightly regulates the length of both the T3SS and the flagellum, but the molecular basis for this length control has remained poorly characterized and controversial. Using the Pseudomonas aeruginosa T3SS as a model system, we report the first structure of a T3SS ruler protein, revealing a “ball-and-chain” architecture, with a globular C-terminal domain (the ball) preceded by a long intrinsically disordered N-terminal polypeptide chain. The dimensions and stability of the globular domain do not support its potential passage through the inner lumen of the T3SS needle. We further demonstrate that a conserved motif at the N terminus of the ruler protein interacts with the T3SS autoprotease in the cytosolic side. Collectively, these data suggest a potential mechanism for needle length sensing by ruler proteins, whereby upon T3SS needle assembly, the ruler protein's N-terminal end is anchored on the cytosolic side, with the globular domain located on the extracellular end of the growing needle. Sequence analysis of T3SS and flagellar ruler proteins shows that this mechanism is probably conserved across systems. PMID:26589798

  9. Length quantization of DNA partially expelled from heads of a bacteriophage T3 mutant

    Energy Technology Data Exchange (ETDEWEB)

    Serwer, Philip, E-mail: serwer@uthscsa.edu [Department of Biochemistry, The University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900 (United States); Wright, Elena T. [Department of Biochemistry, The University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900 (United States); Liu, Zheng; Jiang, Wen [Markey Center for Structural Biology, Department of Biological Sciences, Purdue University, West Lafayette, IN 47907 (United States)

    2014-05-15

    DNA packaging of phages phi29, T3 and T7 sometimes produces incompletely packaged DNA with quantized lengths, based on gel electrophoretic band formation. We discover here a packaging ATPase-free, in vitro model for packaged DNA length quantization. We use directed evolution to isolate a five-site T3 point mutant that hyper-produces tail-free capsids with mature DNA (heads). Three tail gene mutations, but no head gene mutations, are present. A variable-length DNA segment leaks from some mutant heads, based on DNase I-protection assay and electron microscopy. The protected DNA segment has quantized lengths, based on restriction endonuclease analysis: six sharp bands of DNA missing 3.7–12.3% of the last end packaged. Native gel electrophoresis confirms quantized DNA expulsion and, after removal of external DNA, provides evidence that capsid radius is the quantization-ruler. Capsid-based DNA length quantization possibly evolved via selection for stalling that provides time for feedback control during DNA packaging and injection. - Graphical abstract: Highlights: • We implement directed evolution- and DNA-sequencing-based phage assembly genetics. • We purify stable, mutant phage heads with a partially leaked mature DNA molecule. • Native gels and DNase-protection show leaked DNA segments to have quantized lengths. • Native gels after DNase I-removal of leaked DNA reveal the capsids to vary in radius. • Thus, we hypothesize leaked DNA quantization via variably quantized capsid radius.

  10. Analysis of the tool plunge in friction stir welding - comparison of aluminium alloys 2024 T3 and 2024 T351

    Directory of Open Access Journals (Sweden)

    Veljić Darko

    2016-01-01

    Full Text Available Temperature, plastic strain and heat generation during the plunge stage of the friction stir welding (FSW of high-strength aluminium alloys 2024 T3 and 2024 T351 are considered in this work. The plunging of the tool into the material is done at different rotating speeds. A three-dimensional finite element (FE model for thermomechanical simulation is developed. It is based on arbitrary Lagrangian-Eulerian formulation, and Johnson-Cook material law is used for modelling of material behaviour. From comparison of the numerical results for alloys 2024 T3 and 2024 T351, it can be seen that the former has more intensive heat generation from the plastic deformation, due to its higher strength. Friction heat generation is only slightly different for the two alloys. Therefore, temperatures in the working plate are higher in the alloy 2024 T3 for the same parameters of the plunge stage. Equivalent plastic strain is higher for 2024 T351 alloy, and the highest values are determined under the tool shoulder and around the tool pin. For the alloy 2024 T3, equivalent plastic strain is the highest in the influence zone of the tool pin. [Projekat Ministarstva nauke Republike Srbije, br. TR 34016 i br. TR 35006

  11. T3SS-dependent microvascular thrombosis and ischemic enteritis in human gut xenografts infected with enteropathogenicEscherichia coli.

    Science.gov (United States)

    Nissim-Eliraz, Einat; Nir, Eilam; Shoval, Irit; Marsiano, Noga; Nissan, Israel; Shemesh, Hadar; Nagy, Nandor; Goldstein, Allan M; Gutnick, Michael; Rosenshine, Ilan; Yagel, Simcha; Shpigel, Nahum Y

    2017-08-07

    Enteropathogenic Escherichia coli (EPEC) is a leading cause of severe intestinal disease and infant mortality in developing countries. Virulence is mediated by a type three secretion system (T3SS) causing the hallmark lesions of attaching and effacing (AE) and actin-rich pedestal formation beneath the infecting bacteria on the apical surface of enterocytes. EPEC is a human-specific pathogen whose pathogenesis cannot be studied in animal models. We therefore established an EPEC infection model in human gut xenografts in SCID mice and used it to study the role of T3SS in the pathogenesis of the disease. Following EPEC O127:H6 strain E2348/69 infection, T3SS-dependent AE lesions and pedestals were demonstrated in all infected xenografts. We report here the development of T3SS-dependent intestinal thrombotic microangiopathy (iTMA) and ischemic enteritis in ∼50% of infected human gut xenografts. Using species-specific CD31 immunostaining, we showed that iTMA was limited to the larger human-mouse chimeric blood vessels which are located between the muscularis mucosa and circular muscular layer of the human gut. These blood vessels were massively invaded by bacteria which adhered to and formed pedestals on endothelial cells and aggregated with mouse neutrophils in the lumen. We conclude that endothelial infection, iTMA and ischemic enteritis might be central mechanisms underlying severe EPEC-mediated disease. Copyright © 2017 American Society for Microbiology.

  12. Calpain inhibition rescues troponin T3 fragmentation, increases Cav1.1, and enhances skeletal muscle force in aging sedentary mice.

    Science.gov (United States)

    Zhang, Tan; Pereyra, Andrea S; Wang, Zhong-Min; Birbrair, Alexander; Reisz, Julie A; Files, Daniel Clark; Purcell, Lina; Feng, Xin; Messi, Maria L; Feng, Hanzhong; Chalovich, Joseph; Jin, Jian-Ping; Furdui, Cristina; Delbono, Osvaldo

    2016-06-01

    Loss of strength in human and animal models of aging can be partially attributed to a well-recognized decrease in muscle mass; however, starting at middle-age, the normalized force (force/muscle cross-sectional area) in the knee extensors and single muscle fibers declines in a curvilinear manner. Strength is lost faster than muscle mass and is a more consistent risk factor for disability and death. Reduced expression of the voltage sensor Ca(2+) channel α1 subunit (Cav1.1) with aging leads to excitation-contraction uncoupling, which accounts for a significant fraction of the decrease in skeletal muscle function. We recently reported that in addition to its classical cytoplasmic location, fast skeletal muscle troponin T3 (TnT3) is fragmented in aging mice, and both full-length TnT3 (FL-TnT3) and its carboxyl-terminal (CT-TnT3) fragment shuttle to the nucleus. Here, we demonstrate that it regulates transcription of Cacna1s, the gene encoding Cav1.1. Knocking down TnT3 in vivo downregulated Cav1.1. TnT3 downregulation or overexpression decreased or increased, respectively, Cacna1s promoter activity, and the effect was ablated by truncating the TnT3 nuclear localization sequence. Further, we mapped the Cacna1s promoter region and established the consensus sequence for TnT3 binding to Cacna1s promoter. Systemic administration of BDA-410, a specific calpain inhibitor, prevented TnT3 fragmentation, and Cacna1s and Cav1.1 downregulation and improved muscle force generation in sedentary old mice. © 2016 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

  13. Lysophosphatidic acid receptor-5 negatively regulates cellular responses in mouse fibroblast 3T3 cells

    Energy Technology Data Exchange (ETDEWEB)

    Dong, Yan; Hirane, Miku; Araki, Mutsumi [Division of Cancer Biology and Bioinformatics, Department of Life Science, Faculty of Science and Engineering, Kinki University, 3-4-1, Kowakae, Higashiosaka, Osaka 577-8502 (Japan); Fukushima, Nobuyuki [Division of Molecular Neurobiology, Department of Life Science, Faculty of Science and Engineering, Kinki University, 3-4-1, Kowakae, Higashiosaka, Osaka 577-8502 (Japan); Tsujiuchi, Toshifumi, E-mail: ttujiuch@life.kindai.ac.jp [Division of Cancer Biology and Bioinformatics, Department of Life Science, Faculty of Science and Engineering, Kinki University, 3-4-1, Kowakae, Higashiosaka, Osaka 577-8502 (Japan)

    2014-04-04

    Highlights: • LPA{sub 5} inhibits the cell growth and motile activities of 3T3 cells. • LPA{sub 5} suppresses the cell motile activities stimulated by hydrogen peroxide in 3T3 cells. • Enhancement of LPA{sub 5} on the cell motile activities inhibited by LPA{sub 1} in 3T3 cells. • The expression and activation of Mmp-9 were inhibited by LPA{sub 5} in 3T3 cells. • LPA signaling via LPA{sub 5} acts as a negative regulator of cellular responses in 3T3 cells. - Abstract: Lysophosphatidic acid (LPA) signaling via G protein-coupled LPA receptors (LPA{sub 1}–LPA{sub 6}) mediates a variety of biological functions, including cell migration. Recently, we have reported that LPA{sub 1} inhibited the cell motile activities of mouse fibroblast 3T3 cells. In the present study, to evaluate a role of LPA{sub 5} in cellular responses, Lpar5 knockdown (3T3-L5) cells were generated from 3T3 cells. In cell proliferation assays, LPA markedly stimulated the cell proliferation activities of 3T3-L5 cells, compared with control cells. In cell motility assays with Cell Culture Inserts, the cell motile activities of 3T3-L5 cells were significantly higher than those of control cells. The activity levels of matrix metalloproteinases (MMPs) were measured by gelatin zymography. 3T3-L5 cells stimulated the activation of Mmp-2, correlating with the expression levels of Mmp-2 gene. Moreover, to assess the co-effects of LPA{sub 1} and LPA{sub 5} on cell motile activities, Lpar5 knockdown (3T3a1-L5) cells were also established from Lpar1 over-expressing (3T3a1) cells. 3T3a1-L5 cells increased the cell motile activities of 3T3a1 cells, while the cell motile activities of 3T3a1 cells were significantly lower than those of control cells. These results suggest that LPA{sub 5} may act as a negative regulator of cellular responses in mouse fibroblast 3T3 cells, similar to the case for LPA{sub 1}.

  14. Lysophosphatidic acid receptor-5 negatively regulates cellular responses in mouse fibroblast 3T3 cells

    International Nuclear Information System (INIS)

    Dong, Yan; Hirane, Miku; Araki, Mutsumi; Fukushima, Nobuyuki; Tsujiuchi, Toshifumi

    2014-01-01

    Highlights: • LPA 5 inhibits the cell growth and motile activities of 3T3 cells. • LPA 5 suppresses the cell motile activities stimulated by hydrogen peroxide in 3T3 cells. • Enhancement of LPA 5 on the cell motile activities inhibited by LPA 1 in 3T3 cells. • The expression and activation of Mmp-9 were inhibited by LPA 5 in 3T3 cells. • LPA signaling via LPA 5 acts as a negative regulator of cellular responses in 3T3 cells. - Abstract: Lysophosphatidic acid (LPA) signaling via G protein-coupled LPA receptors (LPA 1 –LPA 6 ) mediates a variety of biological functions, including cell migration. Recently, we have reported that LPA 1 inhibited the cell motile activities of mouse fibroblast 3T3 cells. In the present study, to evaluate a role of LPA 5 in cellular responses, Lpar5 knockdown (3T3-L5) cells were generated from 3T3 cells. In cell proliferation assays, LPA markedly stimulated the cell proliferation activities of 3T3-L5 cells, compared with control cells. In cell motility assays with Cell Culture Inserts, the cell motile activities of 3T3-L5 cells were significantly higher than those of control cells. The activity levels of matrix metalloproteinases (MMPs) were measured by gelatin zymography. 3T3-L5 cells stimulated the activation of Mmp-2, correlating with the expression levels of Mmp-2 gene. Moreover, to assess the co-effects of LPA 1 and LPA 5 on cell motile activities, Lpar5 knockdown (3T3a1-L5) cells were also established from Lpar1 over-expressing (3T3a1) cells. 3T3a1-L5 cells increased the cell motile activities of 3T3a1 cells, while the cell motile activities of 3T3a1 cells were significantly lower than those of control cells. These results suggest that LPA 5 may act as a negative regulator of cellular responses in mouse fibroblast 3T3 cells, similar to the case for LPA 1

  15. Long-term L-Triiodothyronine (T3) treatment in stable systolic heart failure patients

    DEFF Research Database (Denmark)

    Holmager, Pernille; Schmidt, Ulla; Mark, Peter

    2015-01-01

    BACKGROUND: Chronic heart failure (HF) is characterized by reduced serum T3 levels and increased activity of the T3 degrading enzyme deiodinase D3. This may result in an intracellular composition of the cardiomyocyte mimicking that of hypothyroidism. Short-term T3-administration to systolic HF...... during T3-treatment and neither did the neurohormonal profile. There were no side-effects in terms of cardiac arrhythmias and no change in resting heart rate. CONCLUSIONS: This study does not support the hypothesis that oral T3 treatment might be beneficial to patients with chronic, stable systolic HF...... with a modest degree of reduced LVEF and low-normal serum T3 concentrations. The study included both functional studies of heart contractility as well as measures of the neurohormonal activation....

  16. Building a secreting nanomachine: a structural overview of the T3SS.

    Science.gov (United States)

    Abrusci, Patrizia; McDowell, Melanie A; Lea, Susan M; Johnson, Steven

    2014-04-01

    To fulfill complex biological tasks, such as locomotion and protein translocation, bacteria assemble macromolecular nanomachines. One such nanodevice, the type III secretion system (T3SS), has evolved to provide a means of transporting proteins from the bacterial cytoplasm across the periplasmic and extracellular spaces. T3SS can be broadly classified into two highly homologous families: the flagellar T3SS which drive cell motility, and the non-flagellar T3SS (NF-T3SS) that inject effector proteins into eukaryotic host cells, a trait frequently associated with virulence. Although the structures and symmetries of ancillary components of the T3SS have diversified to match requirements of different species adapted to different niches, recent genetic, molecular and structural studies demonstrate that these systems are built by arranging homologous modular protein assemblies. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

  17. Initial experience with 3T 3D-TOF MRA in the diagnosis of intracranial aneurysms

    International Nuclear Information System (INIS)

    Senba, Yoshiki; Takahashi, Shizue; Matsubara, Ichiro; Sadamoto, Kazuhiko; Miki, Hitoshi; Mochizuki, Teruhito

    2006-01-01

    We assessed the value of 3T 3D-time of flight (TOF) MR angiography (MRA) in the diagnosis of intracranial aneurysms compared with 1.5T 3D-TOF MRA. Twenty-one patients with 22 aneurysms underwent MRA at 1.5T and 3T. Images were interpreted by two radiologists. Each of nine aneurysms that had been considered ''definite'' at 1.5T 3D-TOF MRA were considered ''definite'' at 3T 3D-TOF MRA. Seven aneurysms that had been considered ''suspicious'' at 1.5T MRA were considered ''definite'' at 3T. And four aneurysms that had been considered ''suspicious'' at 1.5T were considered ''negative'' at 3T. We concluded that 3T 3D-TOF MRA is superior to 1.5T 3D-TOF MRA in the diagnosis of intracranial aneurysms. (author)

  18. Numerical Simulation of Tensile Behavior of Corroded Aluminum Alloy 2024 T3 Considering the Hydrogen Embrittlement

    Directory of Open Access Journals (Sweden)

    Marina C. Vasco

    2018-01-01

    Full Text Available A multi-scale modeling approach for simulating the tensile behavior of the corroded aluminum alloy 2024 T3 was developed, accounting for both the geometrical features of corrosion damage and the effect of corrosion-induced hydrogen embrittlement (HE. The approach combines two Finite Element (FE models: a model of a three-dimensional Representative Unit Cell (RUC, representing an exfoliated area and its correspondent hydrogen embrittled zone (HEZ, and a model of the tensile specimen. The models lie at the micro- and macro-scales, respectively. The characteristics of the HEZ are determined from measurements of nanoindentation hardness, conducted on pre-corroded specimens. Using the model of the RUC, the local homogenized mechanical behavior of the corroded material is simulated. Then, the behavior of the exfoliated areas is assigned into different areas (elements of the tensile specimen and final analyses are performed to simulate the tensile behavior of the corroded material. The approach was applied to model specimens after 8, 16 and 24 h exposure periods of the Exfoliation Corrosion (EXCO test. For validation of the approach, tensile tests were used. The numerical results show that this approach is suitable for accurately simulating the tensile behavior of pre-corroded experimental specimens, accounting for both geometrical features of corrosion damage and corrosion-induced HE.

  19. The Shigella T3SS needle transmits a signal for MxiC release, which controls secretion of effectors.

    Science.gov (United States)

    Martinez-Argudo, Isabel; Blocker, Ariel J

    2010-12-01

    Type III secretion systems (T3SSs) are key determinants of virulence in many Gram-negative bacteria, including animal and plant pathogens. They inject 'effector' proteins through a 'needle' protruding from the bacterial surface directly into eukaryotic cells after assembly of a 'translocator' pore in the host plasma membrane. Secretion is a tightly regulated process, which is blocked until physical contact with a host cell takes place. Host cell sensing occurs through a distal needle 'tip complex' and translocators are secreted before effectors. MxiC, a Shigella T3SS substrate, prevents premature effector secretion. Here, we examine how the different parts of T3SSs work together to allow orderly secretion. We show that T3SS assembly and needle tip composition are not altered in an mxiC mutant. We find that MxiC not only represses effector secretion but that it is also required for translocator release. We provide genetic evidence that MxiC acts downstream of the tip complex and then the needle during secretion activation. Finally, we show that the needle controls MxiC release. Therefore, for the first time, our data allow us to propose a model of secretion activation that goes from the tip complex to cytoplasmic MxiC via the needle. © 2010 Blackwell Publishing Ltd.

  20. bFGF rescues imatinib/STI571-induced apoptosis of sis-NIH3T3 fibroblasts.

    Science.gov (United States)

    Ohshima, Mitsuhiro; Yamaguchi, Yoko; Kappert, Kai; Micke, Patrick; Otsuka, Kichibee

    2009-04-03

    PDGF-B-transfected, sis-NIH3T3 fibroblasts serve as a model system for examining the role of PDGF signaling in tumors. We have found that imatinib/STI571, a tyrosine kinase inhibitor targeting PDGF receptors, induces apoptosis of sis-NIH3T3 fibroblasts cultured under serum free conditions, which was rescued by the addition of 10% newborn calf serum (NCS). Therefore, growth factors included in serum were tested with regard to their ability to rescue imatinib-induced apoptosis. While PDGF-AB, EGF, and IGF-I failed to protect imatinib-induced sis-NIH3T3 cell apoptosis, bFGF rescued it. The effects of bFGF were confirmed by both cell viability assays and Bax/Bcl-2 gene expression ratio. An FGF receptor inhibitor, PD166866, invalidated the protective effect of bFGF. However, combination of imatinib and PD166866 failed to induce cell death of sis-NIH3T3 cells when cultured in 10% NCS. These results indicate that synergistic administration of some types of tyrosine kinase inhibitors need to be tested under in vivo-like conditions to establish novel strategies in anti-cancer therapy.

  1. TRH Stimulation Tests Compared with T3 Suppression Tests in Patients With Marginal Hyperthyroidism

    International Nuclear Information System (INIS)

    Choi, Sung Jae; Koh, Chang Soon

    1980-01-01

    TRH stimulation tests and T 3 suppression tests were done in 13 patients with clinically suspected mild or early hyperthyroidism who were all conventional thyroid function tests gave results within the accepted normal range. The results were as follows 1) 6 patients with normal T 3 suppression test revealed normal TRH stimulation test and could be easily diagnosed as euthyroidism 2) 7 patients with abnormal T 3 suppression test exhibited no TSH response to TRH stimulation test and could be easily diagnosed as hyperthyroidism. The TRH stimulation test is a single, sensitive and reliable test of thyroid function and can well replace T 3 suppression test in the diagnosis of marginal hyperthyroidism

  2. Effects of Maillard reaction on allergenicity of buckwheat allergen Fag t 3 during thermal processing.

    Science.gov (United States)

    Yang, Zhen-Huang; Li, Chen; Li, Yu-Ying; Wang, Zhuan-Hua

    2013-04-01

    Fag t 3 is a major allergenic protein in tartary buckwheat. The Maillard reaction commonly occurs in food processing, but few studies have been conducted on the influence of thermal processing on the allergenic potential of buckwheat allergen. The aim of the present study was to investigate the effects of autologous plant polysaccharides on the immunoreactivity of buckwheat Fag t 3 (11S globulin) following the Maillard reaction. Fag t 3 and crude polysaccharides were prepared from tartary buckwheat (Fagopyrum tataricum) flour. After heating, the polysaccharides were covalently linked to Fag t 3 via a Maillard reaction, and the IgE/IgG-binding properties of Fag t 3 decreased dramatically, with significant changes also being observed in the electrophoretic mobility, secondary structure and solubility of the glycated Fag t 3. The great influence of glycation on IgE/IgG binding to Fag t 3 was correlated with a significant change in the structure and epitopes of the allergenic protein. These data indicated that conjugation of polysaccharides to Fag t 3 markedly reduced the allergen's immunoreactivity. Glycation that occurs via the Maillard reaction during the processing of buckwheat food may be an efficient method to reduce Fag t 3 allergenicity. © 2012 Society of Chemical Industry.

  3. Duality symmetry of N=4 Yang-Mills theory on T3

    International Nuclear Information System (INIS)

    Hacquebord, F.; Verlinde, H.

    1997-01-01

    We study the spectrum of BPS states in N=4 supersymmetric U(N) Yang-Mills theory. This theory has been proposed to describe M-theory on T 3 in the discrete light-cone formalism. We find that the degeneracy of irreducible BPS bound states in this model exhibits a (partially hidden) SL(5,Z) duality symmetry. Besides the electro-magnetic symmetry, this duality group also contains Nahm-like transformations that interchange the rank N of the gauge group with some of the magnetic or electric fluxes. In the M-theory interpretation, this mapping amounts to a reflection that interchanges the longitudinal direction with one of the transverse directions. (orig.)

  4. Multiple Crack Growth Prediction in AA2024-T3 Friction Stir Welded Joints, Including Manufacturing Effects

    DEFF Research Database (Denmark)

    Carlone, Pierpaolo; Citarella, Roberto; Sonne, Mads Rostgaard

    2016-01-01

    boundary element method (FEM-DBEM) procedure, coupling the welding process simulation to the subsequent crack growth assessment, is proposed and applied to simulate multiple crack propagation, with allowance for manufacturing effects. The friction stir butt welding process of the precipitation hardened AA......2024-T3 alloy was simulated using a thermo-mechanical FEM model to predict the process induced residual stress field and material softening. The computed stress field was transferred to a DBEM environment and superimposed to the stress field produced by a remote fatigue traction load applied...... on a notched specimen. The whole procedure was finally tested comparing simulation outcomes with experimental data. The good agreement obtained highlights the predictive capability of the method. The influence of the residual stress distribution on crack growth and the mutual interaction between propagating...

  5. Triiodothyronine (T3)-associated upregulation and downregulation of nuclear T3 binding in the human fibroblast cell (MRC-5)--stimulation of malic enzyme, glucose-6-phosphate-dehydrogenase, and 6-phosphogluconate-dehydrogenase by insulin, but not by T3

    DEFF Research Database (Denmark)

    Matzen, L E; Kristensen, S R; Kvetny, J

    1991-01-01

    The specific nuclear binding of triiodothyronine (T3) (NBT3) and the activity of malic enzyme (ME), glucose-6-phosphate-dehydrogenase (G6PD), and 6-phosphogluconate-dehydrogenase (6PGD) were studied in the human fibroblast cell (MRC-5). The overall apparent binding affinity (Ka) was 2.7 x 10(9) L...... and a low-affinity binding site with Ka2 2.9 +/- 1.1 x 10(8) L.mol-1 and MBC2 124.7 +/- 22.1 fmol/mg DNA (n = 6). Incubation of cells with 6 nmol/L T3 for 20 hours reduced NBT3 to 62.2% +/- 15.7% (P less than .01, n = 11). The Ka estimated from kinetic studies was reduced to 6.7 x 10(7) L.mol-1......, and the scatchard plots were linear, with Ka 4.5 +/- 1.6 x 10(8) L.mol-1 and MBC 137.0 +/- 44.6 fmol/mg DNA (n = 3) of the same magnitude as the low-affinity binding site in cells incubated without T3 (NS). The reduction in NBT3 was reversible and maximal at T3 concentrations saturating the high-affinity binding...

  6. Triiodothyronine (T3)-associated upregulation and downregulation of nuclear T3 binding in the human fibroblast cell (MRC-5)--stimulation of malic enzyme, glucose-6-phosphate-dehydrogenase, and 6-phosphogluconate-dehydrogenase by insulin, but not by T3

    DEFF Research Database (Denmark)

    Matzen, L E; Kristensen, S R; Kvetny, J

    1991-01-01

    The specific nuclear binding of triiodothyronine (T3) (NBT3) and the activity of malic enzyme (ME), glucose-6-phosphate-dehydrogenase (G6PD), and 6-phosphogluconate-dehydrogenase (6PGD) were studied in the human fibroblast cell (MRC-5). The overall apparent binding affinity (Ka) was 2.7 x 10(9) L...

  7. The time development of hadronic showers and the T3B experiment

    International Nuclear Information System (INIS)

    Soldner, Christian

    2013-01-01

    photons on the light sensor could be determined with sub-nanosecond precision. Embedded in a custom calibration and analysis framework, this allows for a precise study of shower timing on the nanosecond level. The T3B experiment could prove an increased contribution of the delayed shower component in tungsten with respect to steel via a detailed study of the time distribution of energy depositions. In addition, it is observed that the relative importance of late energy depositions increases with radial distance from the shower axis. This increase is substantially more pronounced in tungsten with respect to steel. It could be shown that the standard hadronic shower model QGSPBERT, used for shower simulations at the LHC as well as for most CLIC physics studies, overestimates the delayed shower evolution systematically, while high precision extensions using precise neutron tracking models can reproduce the shower timing adequately. No significant difference in the delayed shower contribution was observed for different particle energies in a range between 60 GeV and 180 GeV.

  8. The time development of hadronic showers and the T3B experiment

    Energy Technology Data Exchange (ETDEWEB)

    Soldner, Christian

    2013-06-06

    , the time of arrival of photons on the light sensor could be determined with sub-nanosecond precision. Embedded in a custom calibration and analysis framework, this allows for a precise study of shower timing on the nanosecond level. The T3B experiment could prove an increased contribution of the delayed shower component in tungsten with respect to steel via a detailed study of the time distribution of energy depositions. In addition, it is observed that the relative importance of late energy depositions increases with radial distance from the shower axis. This increase is substantially more pronounced in tungsten with respect to steel. It could be shown that the standard hadronic shower model QGSPBERT, used for shower simulations at the LHC as well as for most CLIC physics studies, overestimates the delayed shower evolution systematically, while high precision extensions using precise neutron tracking models can reproduce the shower timing adequately. No significant difference in the delayed shower contribution was observed for different particle energies in a range between 60 GeV and 180 GeV.

  9. T3i: A Tool for Generating and Querying Test Suites for Java

    NARCIS (Netherlands)

    Prasetya, I.S.W.B.

    2015-01-01

    T3i is an automated unit-testing tool to test Java classes. To expose interactions T3i generates test-cases in the form of sequences of calls to the methods of the target class. What separates it from other testing tools is that it treats test suites as first class objects and allows users to e.g.

  10. Different sensitivity of BALB/c 3T3 cell clones in the response to carcinogens.

    Science.gov (United States)

    Colacci, Annamaria; Mascolo, Maria Grazia; Perdichizzi, Stefania; Quercioli, Daniele; Gazzilli, Antonio; Rotondo, Francesca; Morandi, Elena; Guerrini, Angela; Silingardi, Paola; Grilli, Sandro; Vaccari, Monica

    2011-09-01

    Cell transformation assays (CTAs) are currently regarded as the only possible in vitro alternative to animal testing for carcinogenesis studies. CTAs have been proposed as screening tests for the carcinogenic potential of compounds that have no evidence of genotoxicity but present structural alerts for carcinogenicity. We have extensively used the BALB/c 3T3 model based on the A31 cell clone to test single chemicals, complex mixtures and environmental pollutants. In the prevalidation study carried out by ECVAM, the improved protocol is based on BALB/c 3T3 A31-1-1 cells, a clone derived by A31 cells, that is very sensitive to PAH-induced transformation. The present study was performed in the aim to compare the results obtained with the two different clones exposed to different classes of carcinogens. Cells were treated with PAHs (3-methylcholanthrene, benzo(a)pyrene), alkylating agents (melphalan) and aloethanes (1,2-dibromoethane). The induction of cytotoxicity and the onset of chemically transformed foci were evaluated by two experimental protocols, differing for cell seeding density and chemical treatment duration. The A31-1-1 cells showed higher inherent transformation rate after PAHs treatment, but they were insensitive to 1,2-dibromoethane at concentrations that usually induced transformation in A31 cells. As 1,2-dibromoethane is bioactivated to reactive forms able to bind DNA mainly through the conjugation with intracellular glutathione, these results suggested a reduced activity of phase-2 enzymes involved in glutathione conjugation in A31-1-1 cells. Our results give evidence that inherent metabolic capacity of cells may play a critical role in in vitro cell transformation, cautioning against possible misclassification of chemicals. Copyright © 2011 Elsevier Ltd. All rights reserved.

  11. Comparison of the methods for tissue triiodothyronine T(3) extraction and subsequent radioimmunoassay

    International Nuclear Information System (INIS)

    Takaishi, M.; Miyachi, Y.; Aoki, M.; Shishiba, Y.; Asahi Life Foundation, Tokyo

    1978-01-01

    Although there have been various reports on tissue T 3 concentration, the examination of the quality of radioimmunoassay has not been available. In the present study, we tried to determine whether the available methods for T 3 extraction are adequate for the various methods of T 3 radioimmunoassays used. T 3 was extracted from liver by ethanol extraction or by acid butanol extraction (Flock's method) and the extract was applied to radioimmunoassay either by Seralute T 3 column, ANS-double antibody or the ANS-charcoal method. The values of T 3 were compared with those obtained by isotope-equilibration method. The dilution curve of ethanol extract was not parallel with that of the standard in ANS-charcoal or ANS-double antibody technique. When the extract was tested by Seralate method, the dilution curve was parallel to the standard, whereas the T 3 value obtained with this method was two-fold higher than that with the isotope equilibration technique. The analysis of the ethanol extract suggested that the lipid extracted by ethanol interfered with the assay. The acid butanol extract when tested either by the ANS-double antibody or Seralate method, showed parallelism to the standard curve and gave T 3 values almost identical with those by the isotope-equilibration method. When tested by ANS-charcoal method, the dilution curve of the acid butanol extract was not parallel to the standard. Thus, to obtain reliable results, tissue extraction by Flock's method and subsequent T 3 radioimmunoassay by either ANS-double antibody or Seralate T 3 method are recommended. (author)

  12. Kinetic Analysis of Drug Release from Compounded Slow-release Capsules of Liothyronine Sodium (T3).

    Science.gov (United States)

    Bakhteyar, Hamid; Cassone, Clayton; Kohan, Hamed Gilzad; Sani, Shabnam N

    2017-01-01

    The purpose of this study was to formulate extemporaneously compounded Liothyronine Sodium (T3) slow-release capsules and to evaluate their in vitro drug release performance. Twenty-one formulations containing T3 (7.5 µg) with various compositions of two different grades of Methocel E4M and K100M premium (30% to 90%), and/or SimpleCap/Lactose (10% to 70%) were examined. Quality assessment of the capsules was conducted by standard quality control criteria of the United States Pharmacopeia (i.e., weight variation, content uniformity) to ensure their compliance. The dissolution release profile of the formulations was evaluated using United States Pharmacopeia Apparatus type II (paddle method) at a speed of 50 rpm and temperature of 37°C in phosphate buffered saline media ( pH = 7.2 to 7.4). Aliquots from the media were taken periodically up to 24 hours and analyzed using a validated enzyme-linked immunosorbent assay method. The cumulative percentage of drug release for each formulation was fitted to eleven major release kinetic equations to determine the best-fit model of drug release, as well as the mechanism of release. Assay sensitivity was as low as 1 ng/mL and the optimal calibration range was found to be between 0 ng/mL and 7.5 ng/mL, which corresponded well with the average physiological plasma concentrations of T3. Liothyronine sodium with either SimpleCap (100%) or Methocel E4M (100%) exhibited slowrelease kinetic patterns of Peppas and Zero Order, respectively. The formulation with SimpleCap (100%) had a higher percentage of drug release (as compared to 100% Methocel E4M) within the first four hours; this formulation released 80% of the drug within 12 hours when the release was plateaued thereafter. The formulation with 30% Methocel E4M and 70% SimpleCap released 100% of the drug within the initial 12 hours and exhibited a Zero Order slow-release kinetic pattern. In general, the release kinetic rate of the formulations containing Methocel K100M appeared to

  13. T24 HRAS transformed NIH/3T3 mouse cells (GhrasT-NIH/3T3) in serial tumorigenic in vitro/in vivo passages give rise to increasingly aggressive tumorigenic cell lines T1-A and T2-A and metastatic cell lines T3-HA and T4-PA.

    Science.gov (United States)

    Ray, Durwood B; Merrill, Gerald A; Brenner, Frederic J; Lytle, Laurie S; Lam, Tan; McElhinney, Aaron; Anders, Joel; Rock, Tara Tauber; Lyker, Jennifer Kier; Barcus, Scott; Leslie, Kara Hust; Kramer, Jill M; Rubenstein, Eric M; Pryor Schanz, Karen; Parkhurst, Amy J; Peck, Michelle; Good, Kimberly; Granath, Kristi Lemke; Cifra, Nicole; Detweiler, Jessalee Wantz; Stevens, Laura; Albertson, Richard; Deir, Rachael; Stewart, Elisabeth; Wingard, Katherine; Richardson, Micah Rose; Blizard, Sarah B; Gillespie, Lauren E; Kriley, Charles E; Rzewnicki, Daniel I; Jones, David H

    2016-01-01

    Cancer cells often arise progressively from "normal" to "pre-cancer" to "transformed" to "local metastasis" to "metastatic disease" to "aggressive metastatic disease". Recent whole genome sequencing (WGS) and spectral karyotyping (SKY) of cancer cells and tumorigenic models have shown this progression involves three major types of genome rearrangements: ordered small step-wise changes, more dramatic "punctuated evolution" (chromoplexy), and large catastrophic steps (chromothripsis) which all occur in random combinations to generate near infinite numbers of stochastically rearranged metastatic cancer cell genomes. This paper describes a series of mouse cell lines developed sequentially to mimic this type of progression. This starts with the new GhrasT-NIH/Swiss cell line that was produced from the NIH/3T3 cell line that had been transformed by transfection with HRAS oncogene DNA from the T24 human bladder carcinoma. These GhrasT-NIH/Swiss cells were injected s.c. into NIH/Swiss mice to produce primary tumors from which one was used to establish the T1-A cell line. T1-A cells injected i.v. into the tail vein of a NIH/Swiss mouse produced a local metastatic tumor near the base of the tail from which the T2-A cell line was established. T2-A cells injected i.v. into the tail vein of a nude NIH/Swiss mouse produced metastases in the liver and one lung from which the T3-HA (H=hepatic) and T3-PA (P=pulmonary) cell lines were developed, respectively. T3-HA cells injected i.v. into a nude mouse produced a metastasis in the lung from which the T4-PA cell line was established. PCR analysis indicated the human T24 HRAS oncogene was carried along with each in vitro/in vivo transfer step and found in the T2-A and T4-PA cell lines. Light photomicrographs indicate that all transformed cells are morphologically similar. GhrasT-NIH/Swiss cells injected s.c. produced tumors in 4% of NIH/Swiss mice in 6-10 weeks; T1-A cells injected s.c. produced tumors in 100% of NIH/Swiss mice in 7

  14. Integrated FEM-DBEM simulation of crack propagation in AA2024-T3 FSW butt joints considering manufacturing effects

    DEFF Research Database (Denmark)

    Sonne, Mads Rostgaard; Carlone, P.; Citarella, R.

    2015-01-01

    This paper deals with a numerical and experimental investigation on the influence of residual stresses on fatigue crack growth in AA2024-T3 friction stir welded butt joints. An integrated FEM-DBEM procedure for the simulation of crack propagation is proposed and discussed. A numerical FEM model...... of the contour method. The computed stress field is transferred to a DBEM environment and superimposed to the stress field produced by a remote fatigue traction load applied on a friction stir welded cracked specimen. Numerical results are compared with experimental data showing good agreement and highlighting...... of the welding process of precipitation hardenable AA2024-T3 aluminum alloy is employed to infer the process induced residual stress field. The reliability of the FEM simulations with respect to the induced residual stresses is assessed comparing numerical outcomes with experimental data obtained by means...

  15. Effect of tumor-conditioned medium on intercellular communication and proliferation of Balb/c 3T3 cells.

    Science.gov (United States)

    Tóth, L; Pásti, G; Sárváry, A; Balázs, M; Adány, R

    2000-04-03

    The possible role of tumor cell-derived factors in the regulation of gap junctional intercellular communication and proliferation of fibroblasts was studied in a model system of Balb/c 3T3 cells growing in tumor conditioned medium by Lucifer Yellow CH dye-transfer and BrdU incorporation assays. Six to 24 h incubation of Balb/c 3T3 cells in a medium conditioned by WiDr adenocarcinoma cells enhanced the gap junctional communication between the cells by 25-40% as revealed by intercellular transfer of a fluorescent dye Lucifer Yellow CH. Simultaneously the cell proliferation rates were examined and found to be reduced by 23% at 24 h treatment. Since adenocarcinoma cells are known to secrete different growth factor-like polypeptides into their conditioned medium, we suppose that tumors that produce these molecules might alter their host environment through the enhancement of cell-cell communication thereby facilitating the exchange of modulatory factors.

  16. Comparative studies on the structural proteins of T3 and T7 phages

    DEFF Research Database (Denmark)

    Issinger, O G; Falk, H

    1976-01-01

    different from that of the wildtype. Treatment of wildtype particles with ammonium nitrate or sodium pyrophosphate led to morphologically abberrant forms which had partially or completely lost the hexagonal head structure. After treatment with ammonium nitrate ball-like structures were obtained, both for T3...... and T7. However, in the case of T3 these abberrant forms contained the proteins specified by genes 7, 8 and 10 whereas those derived from T7 contained only the proteins specified by genes 8 and 10. Sodium pyrophosphate treatment of T3 and T7 wildtype particles led to a release of the phage tails...

  17. The Structure of a Type 3 Secretion System (T3SS) Ruler Protein Suggests a Molecular Mechanism for Needle Length Sensing.

    Science.gov (United States)

    Bergeron, Julien R C; Fernández, Lucia; Wasney, Gregory A; Vuckovic, Marija; Reffuveille, Fany; Hancock, Robert E W; Strynadka, Natalie C J

    2016-01-22

    The type 3 secretion system (T3SS) and the bacterial flagellum are related pathogenicity-associated appendages found at the surface of many disease-causing bacteria. These appendages consist of long tubular structures that protrude away from the bacterial surface to interact with the host cell and/or promote motility. A proposed "ruler" protein tightly regulates the length of both the T3SS and the flagellum, but the molecular basis for this length control has remained poorly characterized and controversial. Using the Pseudomonas aeruginosa T3SS as a model system, we report the first structure of a T3SS ruler protein, revealing a "ball-and-chain" architecture, with a globular C-terminal domain (the ball) preceded by a long intrinsically disordered N-terminal polypeptide chain. The dimensions and stability of the globular domain do not support its potential passage through the inner lumen of the T3SS needle. We further demonstrate that a conserved motif at the N terminus of the ruler protein interacts with the T3SS autoprotease in the cytosolic side. Collectively, these data suggest a potential mechanism for needle length sensing by ruler proteins, whereby upon T3SS needle assembly, the ruler protein's N-terminal end is anchored on the cytosolic side, with the globular domain located on the extracellular end of the growing needle. Sequence analysis of T3SS and flagellar ruler proteins shows that this mechanism is probably conserved across systems. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. C2C12 myotubes inhibit the proliferation and differentiation of 3T3-L1 preadipocytes by reducing the expression of glucocorticoid receptor gene

    Energy Technology Data Exchange (ETDEWEB)

    Chu, Weiwei; Wei, Wei; Yu, Shigang; Han, Haiyin; Shi, Xiaoli [College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095 (China); Sun, Wenxing [College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095 (China); College of Public Health, Nantong University, Nantong 226019 (China); Gao, Ying [College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095 (China); Zhang, Lifan [College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095 (China); Chen, Jie, E-mail: jiechen@njau.edu.cn [College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095 (China)

    2016-03-25

    Obesity is a well-established risk factor to health for its relationship with insulin resistance, diabetes and metabolic syndrome. Myocyte-adipocyte crosstalk model plays a significant role in studying the interaction of muscle and adipose development. Previous related studies mainly focus on the effects of adipocytes on the myocytes activity, however, the influence of myotubes on the preadipocytes development remains unclear. The present study was carried out to settle this issue. Firstly, the co-culture experiment showed that the proliferation, cell cycle, and differentiation of 3T3-L1 preadipocytes were arrested, and the apoptosis was induced, by differentiated C2C12 myotubes. Next, the sensitivity of 3T3-L1 preadipocytes to glucocorticoids (GCs), which was well known as cell proliferation, differentiation, apoptosis factor, was decreased after co-cultured with C2C12 myotubes. What's more, our results showed that C2C12 myotubes suppressed the mRNA and protein expression of glucocorticoid receptor (GR) in 3T3-L1 preadipocytes, indicating the potential mechanism of GCs sensitivity reduction. Taken together, we conclude that C2C12 myotubes inhibited 3T3-L1 preadipocytes proliferation and differentiation by reducing the expression of GR. These data suggest that decreasing GR by administration of myokines may be a promising therapy for treating patients with obesity or diabetes. - Highlights: • C2C12 myotubes inhibited proliferation and differentiation of 3T3-L1 preadipocytes. • C2C12 myotubes arrested cell cycle of 3T3-L1 preadipocytes. • C2C12 myotubes induced apoptosis of 3T3-L1 preadipocytes. • C2C12 inhibit 3T3-L1 cells by reducing the expression of glucocorticoid receptor gene.

  19. T3P as an efficient cyclodehydration reagent for the one-pot ...

    Indian Academy of Sciences (India)

    Abstract. A scalable and environmentally friendly one-pot method for the synthesis of 2-amino-1,3,4-oxadiazoles from acylhydrazides and isocyanates has been achieved with propane phosponic anhydride (T3P) acting as cyclodehydrating reagent.

  20. Cis-2-dodecenoic acid signal modulates virulence of Pseudomonas aeruginosa through interference with quorum sensing systems and T3SS.

    Science.gov (United States)

    Deng, Yinyue; Boon, Calvin; Chen, Shaohua; Lim, Amy; Zhang, Lian-Hui

    2013-10-18

    Cis-2-dodecenoic acid (BDSF) is well known for its important functions in intraspecies signaling in Burkholderia cenocepacia. Previous work has also established an important role of BDSF in interspecies and inter-kingdom communications. It was identified that BDSF modulates virulence of Pseudomonas aeruginosa. However, how BDSF interferes with virulence of P. aeruginosa is still not clear. We report here that BDSF mediates the cross-talk between B. cenocepacia and P. aeruginosa through interference with quorum sensing (QS) systems and type III secretion system (T3SS) of P. aeruginosa. Bioassay results revealed that exogenous addition of BDSF not only reduced the transcriptional expression of the regulator encoding gene of QS systems, i.e., lasR, pqsR, and rhlR, but also simultaneously decreased the production of QS signals including 3-oxo-C12-HSL, Pseudomonas quinolone signal (PQS) and C4-HSL, consequently resulting in the down-regulation of biofilm formation and virulence factor production of P. aeruginosa. Furthermore, BDSF and some of its derivatives are also capable of inhibiting T3SS of P. aeruginosa at a micromolar level. Treatment with BDSF obviously reduced the virulence of P. aeruginosa in both HeLa cell and zebrafish infection models. These results depict that BDSF modulates virulence of P. aeruginosa through interference with QS systems and T3SS.

  1. Low T3 State Is Correlated with Cardiac Mitochondrial Impairments after Ischemia Reperfusion Injury: Evidence from a Proteomic Approach

    Directory of Open Access Journals (Sweden)

    Francesca Forini

    2015-11-01

    Full Text Available Mitochondria are major determinants of cell fate in ischemia/reperfusion injury (IR and common effectors of cardio-protective strategies in cardiac ischemic disease. Thyroid hormone homeostasis critically affects mitochondrial function and energy production. Since a low T3 state (LT3S is frequently observed in the post infarction setting, the study was aimed to investigate the relationship between 72 h post IR T3 levels and both the cardiac function and the mitochondrial proteome in a rat model of IR. The low T3 group exhibits the most compromised cardiac performance along with the worst mitochondrial activity. Accordingly, our results show a different remodeling of the mitochondrial proteome in the presence or absence of a LT3S, with alterations in groups of proteins that play a key role in energy metabolism, quality control and regulation of cell death pathways. Overall, our findings highlight a relationship between LT3S in the early post IR and poor cardiac and mitochondrial outcomes, and suggest a potential implication of thyroid hormone in the cardio-protection and tissue remodeling in ischemic disease.

  2. Role of T3SS-1 SipD Protein in Protecting Mice against Non-typhoidal Salmonella Typhimurium.

    Science.gov (United States)

    Jneid, Bakhos; Moreau, Karine; Plaisance, Marc; Rouaix, Audrey; Dano, Julie; Simon, Stéphanie

    2016-12-01

    Salmonella enterica species are enteric pathogens that cause severe diseases ranging from self-limiting gastroenteritis to enteric fever and sepsis in humans. These infectious diseases are still the major cause of morbidity and mortality in low-income countries, especially in children younger than 5 years and immunocompromised adults. Vaccines targeting typhoidal diseases are already marketed, but none protect against non-typhoidal Salmonella. The existence of multiple non-typhoidal Salmonella serotypes as well as emerging antibiotic resistance highlight the need for development of a broad-spectrum protective vaccine. All Salmonella spp. utilize two type III Secretion Systems (T3SS 1 and 2) to initiate infection, allow replication in phagocytic cells and induce systemic disease. T3SS-1, which is essential to invade epithelial cells and cross the barrier, forms an extracellular needle and syringe necessary to inject effector proteins into the host cell. PrgI and SipD form, respectively, the T3SS-1 needle and the tip complex at the top of the needle. Because they are common and highly conserved in all virulent Salmonella spp., they might be ideal candidate antigens for a subunit-based, broad-spectrum vaccine. We investigated the immunogenicity and protective efficacy of PrgI and SipD administered by subcutaneous, intranasal and oral routes, alone or combined, in a mouse model of Salmonella intestinal challenge. Robust IgG (in all immunization routes) and IgA (in intranasal and oral immunization routes) antibody responses were induced against both proteins, particularly SipD. Mice orally immunized with SipD alone or SipD combined with PrgI were protected against lethal intestinal challenge with Salmonella Typhimurium (100 Lethal Dose 50%) depending on antigen, route and adjuvant. Salmonella T3SS SipD is a promising antigen for the development of a protective Salmonella vaccine, and could be developed for vaccination in tropical endemic areas to control infant

  3. Regulatory mechanisms of exoribonuclease PNPase and regulatory small RNA on T3SS of Dickeya dadantii.

    Science.gov (United States)

    Zeng, Quan; Ibekwe, A Mark; Biddle, Eulandria; Yang, Ching-Hong

    2010-10-01

    The type III secretion system (T3SS) is an essential virulence factor for many bacterial pathogens. Polynucleotide phosphorylase (PNPase) is one of the major exoribonucleases in bacteria and plays important roles in mRNA degradation, tRNA processing, and small RNA (sRNA) turnover. In this study, we showed that PNPase downregulates the transcription of T3SS structural and effector genes of the phytopathogenic bacterium Dickeya dadantii. This negative regulation of T3SS by PNPase occurs by repressing the expression of hrpL, encoding a master regulator of T3SS in D. dadantii. By reducing rpoN mRNA stability, PNPase downregulates the transcription of hrpL, which leads to a reduction in T3SS gene expression. Moreover, we have found that PNPase downregulates T3SS by decreasing hrpL mRNA stability. RsmB, a regulatory sRNA, enhances hrpL mRNA stability in D. dadantii. Our results suggest that PNPase decreases the amount of functional RsmB transcripts that could result in reduction of hrpL mRNA stability. In addition, bistable gene expression (differential expression of a single gene that creates two distinct subpopulations) of hrpA, hrpN, and dspE was observed in D. dadantii under in vitro conditions. Although PNPase regulates the proportion of cells in the high state and the low state of T3SS gene expression, it appears that PNPase is not the key switch that triggers the bistable expression patterns of T3SS genes.

  4. Commonalities and differences of T3SSs in rhizobia and plant pathogenic bacteria

    Science.gov (United States)

    Tampakaki, Anastasia P.

    2014-01-01

    Plant pathogenic bacteria and rhizobia infect higher plants albeit the interactions with their hosts are principally distinct and lead to completely different phenotypic outcomes, either pathogenic or mutualistic, respectively. Bacterial protein delivery to plant host plays an essential role in determining the phenotypic outcome of plant-bacteria interactions. The involvement of type III secretion systems (T3SSs) in mediating animal- and plant-pathogen interactions was discovered in the mid-80's and is now recognized as a multiprotein nanomachine dedicated to trans-kingdom movement of effector proteins. The discovery of T3SS in bacteria with symbiotic lifestyles broadened its role beyond virulence. In most T3SS-positive bacterial pathogens, virulence is largely dependent on functional T3SSs, while in rhizobia the system is dispensable for nodulation and can affect positively or negatively the mutualistic associations with their hosts. This review focuses on recent comparative genome analyses in plant pathogens and rhizobia that uncovered similarities and variations among T3SSs in their genetic organization, regulatory networks and type III secreted proteins and discusses the evolutionary adaptations of T3SSs and type III secreted proteins that might account for the distinguishable phenotypes and host range characteristics of plant pathogens and symbionts. PMID:24723933

  5. Development of an 'In vitro' system for the caption essay of T3

    International Nuclear Information System (INIS)

    Lavalley, C.; Ferro, G.; Zambrano, F.; Lezama, J.

    1990-02-01

    Triiodothyronine uptake (T3U) is a qualitative technique for evaluation of the unsaturation capacity of thyroid binding globulin (TBG). This paper presents results related to a T3 standardized serum and the integration of T3-I-125, and adsorbent for labelled hormone. Labelled hormone were prepared by the chloramine T method and then purified by high performance liquid chromatography. The specific activity was 500 μ Ci/ μ g. Various adsorbents such as: Norit A Charcoal, calcium silicate, talc, bovine serum albumin macroaggregated (BSAM) were used in different buffers as: Tris-HCl, barbital and Michaelis. Standardized serum was prepared by mixing different euthyroid sera. Best conditions for T3U assays were achieved with 15 mg/ml. BSAM at pH 8.6 in presence of Tris-HCl buffer for hypothyroid and hyperthyroid sera, for which we obtained 1.1 ± 0.05 respectively as a T3U index with a 3.0 % of coefficient variation. The reagents so prepared can be conveniently used for T3U assays. (Author)

  6. The effect of control of diabetes mellitus on plasma T4, T3, rT3 levels and half muscle relaxation period

    International Nuclear Information System (INIS)

    Ismail, A.A.; Hafiez, A.A.; Sayed, S.N.; Abbas, E.Z.; Halawa, F.A.; Youssef, M.M.

    1984-01-01

    25 diabetics of the maturity onset type who showed no clinical evidence of either peripheral neruropathy or diabetic amyotrophy were selected for this study. All patients were subjected to the following investigations: estimation of half muscle relaxation period of the quadriceps muscle knee-jerk, measurement of plasma levels of thyroxine (T 4 ), triiodothyronine (T 3 ) and reverse triiodothronine (rT 3 ), determination of fasting and two hours postprandial blood sugar levels. The quadriceps muscle relaxation period in uncontrolled diabetics was significantly longer than in normals. Control of diabetes by glibenclamide or gliclazide did not cause a significant change in muscle relaxation period. There was also no significant difference between the effects of the two drugs. (author)

  7. Effects of Methylmercury exposure in 3T3-L1 Adipocytes

    Directory of Open Access Journals (Sweden)

    Theresa Vertigan

    2017-02-01

    Full Text Available Mercury-containing compounds are environmental pollutants that have become increasingly consequential in the Arctic regions of North America due to processes of climate change increasing their release and availability at northern latitudes. Currently, the form of mercury known to be most detrimental to human health is methylmercury, CH3Hg+, which is found in the environment and accumulates in the tissues of piscivores, including those consumed by Alaska Natives through subsistence gathering. Much is known about the neurotoxicity of methylmercury after exposure to high concentrations, but little is known about toxicity to other tissues and cell types, particularly for long-term exposure and the lower concentrations that would occur through fish consumption. Effects of methylmercury exposure on 3T3-L1 adipocytes in culture were assessed using assays for cytotoxicity and an ELISA assay for vascular endothelial growth factor (VEGF, a signaling molecule shown to be important for maintaining metabolic status in adipose tissue. Results showed that exposure to methylmercury leads to significant toxicity in adipocytes at exposures of 100 ng/mL during later stages of differentiation, but lower methylmercury concentrations produced little to no toxicity. Results also showed that VEGF secretion is elevated in adipocytes exposed to methylmercury after the process of differentiating into mature, fat-storing cells. These results provide a basis for further exploration into metabolic consequences of methylmercury exposure on specific cell types and cell models.

  8. Effective dynamics of the hybrid quantization of the Gowdy T3 universe

    International Nuclear Information System (INIS)

    Brizuela, David; Mena Marugan, Guillermo A.; Pawlowski, Tomasz

    2011-01-01

    The quantum dynamics of the linearly polarized Gowdy T 3 model (compact inhomogeneous universes admitting linearly polarized gravitational waves) is analyzed within loop quantum cosmology by means of an effective dynamics. The analysis, performed via analytical and numerical methods, proves that the behavior found in the evolution of vacuum (homogeneous) Bianchi I universes is preserved qualitatively also in the presence of inhomogeneities. More precisely, the initial singularity is replaced by a big bounce which joins deterministically two large classical universes. In addition, we show that the size of the universe at the bounce is at least of the same order of magnitude (roughly speaking) as the size of the corresponding homogeneous universe obtained in the absence of gravitational waves. In particular, a precise lower bound for the ratio of these two sizes is found. Finally, the comparison of the amplitudes of the gravitational wave modes in the distant future and past shows that, statistically (i.e., for large samples of universes), the difference in amplitude is enhanced for nearly homogeneous universes, whereas this difference vanishes in inhomogeneity-dominated cases. The presented analysis constitutes the first systematic effective study of an inhomogeneous system within loop quantum cosmology, and it proves the robustness of the results obtained for homogeneous cosmologies in this context.

  9. Anodic galvanostatic polarization of AA2024-T3 aircraft alloy in conventional mineral acids

    Energy Technology Data Exchange (ETDEWEB)

    Kozhukharov, S., E-mail: stephko1980@abv.bg [Department of Chemical Sciences, University of Chemical Technology and Metallurgy, 8 “Kliment Okhridski” Blvd, 1756, Sofia (Bulgaria); Girginov, Ch. [Department of Chemical Sciences, University of Chemical Technology and Metallurgy, 8 “Kliment Okhridski” Blvd, 1756, Sofia (Bulgaria); Avramova, I. [Institute of General and Inorganic Chemistry, Bulgarian Academy of Science, 11 “Georgi Bonchev” Str., 1113, Sofia (Bulgaria); Machkova, M. [Department of Chemical Sciences, University of Chemical Technology and Metallurgy, 8 “Kliment Okhridski” Blvd, 1756, Sofia (Bulgaria)

    2016-09-01

    The present study is devoted to the determination of the impact of the anodization of AA2024-T3 alloys in HCl, HNO{sub 3}, H{sub 2}SO{sub 4} or H{sub 3}PO{sub 4} on the samples’ surface morphology and properties. Subsequent systematic assessments were performed by Scanning Electron Microscopy (SEM), Energy Dispersion X-Ray Spectroscopy (EDX) and X-ray Photoelectron Spectroscopy (XPS). These observations were combined with Linear Voltammetry (LVA) and Electrochemical Impedance Spectroscopy (EIS) after 48 and 168 h of exposure to a 3.5% NaCl model corrosive medium. The main result is, that completely different effects were observed in accordance to the acid used. It was established that the monoprotonic acids have a deep destructive effect due to dissolution of the alloy components, whereas the polyprotonic ones possess either indistinguishable influence, or surface film formation. - Highlights: • AA2024 was polarized anodically in 15%{sub wt} acid solutions at 15 mA cm{sup −2} for 2 h. • Four mineral acids were selected for investigation: HCl, HNO{sub 3}, H{sub 2}SO{sub 4} and H{sub 3}PO{sub 4}. • SEM, EDX and XPS were applied for morphological description. • Electrochemical characterizations were performed by EIS and linear voltammetry. • The acid used predetermines completely different interaction with the AA2024 alloy.

  10. SlyA regulates type III secretion system (T3SS) genes in parallel with the T3SS master regulator HrpL in Dickeya dadantii 3937.

    Science.gov (United States)

    Zou, Lifang; Zeng, Quan; Lin, Haiping; Gyaneshwar, Prasad; Chen, Gongyou; Yang, Ching-Hong

    2012-04-01

    The hypersensitive response and pathogenicity (hrp) genes of Dickeya dadantii 3937 encode a type III secretion system (T3SS) which is essential for its full virulence. Previous studies of the T3SS regulation in D. dadantii 3937 revealed that the expression of the hrp genes is regulated by a master regulator, HrpL, through the HrpX-HrpY-HrpS-HrpL and GacS-GacA-rsmB-RsmA pathways. In this work, we identified a novel regulator of the SlyA/MarR family, SlyA, which regulates hrp genes of the HrpL regulon in parallel with HrpL in D. dadantii. SlyA regulates the T3SS in a two-tier manner. It negatively regulates the expression of hrpL by downregulating hrpS and upregulating rsmA. Interestingly, concomitant with its downregulation of the hrpL, SlyA positively regulates the expression of hrpA and hrpN, two hrp genes located in the HrpL regulon. In contrast to Pectobacterium carotovorum, the expression of slyA is not controlled by ExpR and ExpI in D. dadantii 3937. We further show that SlyA is involved in controlling swimming motility and pellicle formation in D. dadantii 3937.

  11. Effects of ethiodol on T3 kinetics in endemic goiter area

    International Nuclear Information System (INIS)

    Watanabe, Tomas; Degrossi, O.J.; Zaninovich, A.A.; Altschuler, Noe; Degrossi, E.B.

    1986-01-01

    T3 kinetics were carried out in 32 children (11-15 years old) of a mild endemic goiter area. The incidence of goiter of the whole school population was 19% (Ia or Ib degree of the WHO). The mean urinary iodine was 84 μ g/d+-3. Six months before this investigation, 10 of the children received 2 ml of ethiodol by mouth and the other 22 (control group-CG) a placebo. The kinetics studies were carried out using less than 0,5 μ Ci of I-125 per Kg b.w. and the single injection method, non compartimental analysis. The CG shows, distribution volumen (V) 20.1+-0.6; serum T3 (sT3) 1.55 ng/ml+-0.03; plasma T3 T1/2 21.6+-0.6 and production rate (PR) 27.4μ g/d+-1.2. The treated group (TG): V, 17.3+-0.6; sT3 1.48 ng/ml+-0.09; T1/2 17.6+-0.6 and PR 25.9μ g/d+-1.4. The TG, after 6 months of ethiodol administration, showed a decrease of the distribution volume and a slight reduction in serum T3 values. As a consequence a significant diminution on the T3 extrathyroidal pool and an increase on the T3 plasma clearance (0.68 l/h+-0.01 in CG versus 0.73+-0.3 in TG) were observed. The production ratewas similar in both groups. These results indicate that the administration of ethiodol to children of a not severe endemic goiter area, produces moderate changes in the different parameters of T3 peripheral metabolism, which compensate each other and lead to similar final values on T3-PR, that were previously normal in these cases. This mechanism reflects the role of thyroid homeostasis in maintaining euthyroidism in these children of an endemic goiter area with decreased iodine supply. (Author) [es

  12. Cytotoxic Vibrio T3SS1 Rewires Host Gene Expression to Subvert Cell Death Signaling and Activate Cell Survival Networks

    Science.gov (United States)

    De Nisco, Nicole J.; Kanchwala, Mohammed; Li, Peng; Fernandez, Jessie; Xing, Chao; Orth, Kim

    2017-01-01

    Bacterial effectors are potent manipulators of host signaling pathways. The marine bacterium Vibrio parahaemolyticus (V. para), delivers effectors into host cells through two type three secretion systems (T3SS). The ubiquitous T3SS1 is vital for V. para survival in the environment, whereas T3SS2 causes acute gastroenteritis in human hosts. Although the natural host is undefined, T3SS1 effectors attack highly conserved cellular processes and pathways to orchestrate non-apoptotic cell death. Much is known about how T3SS1 effectors function in isolation, but we wanted to understand how their concerted action globally affects host cell signaling. To assess the host response to T3SS1, we compared gene expression changes over time in primary fibroblasts infected with V. para that have a functional T3SS1 (T3SS1+) to those in cells infected with V. para lacking T3SS1 (T3SS1−). Overall, the host transcriptional response to both T3SS1+ and T3SS1− V. para was rapid, robust, and temporally dynamic. T3SS1 re-wired host gene expression by specifically altering the expression of 398 genes. Although T3SS1 effectors target host cells at the posttranslational level to cause cytotoxicity, network analysis indicated that V. para T3SS1 also precipitates a host transcriptional response that initially activates cell survival and represses cell death networks. The increased expression of several key pro-survival transcripts mediated by T3SS1 was dependent on a host signaling pathway that is silenced later in infection by the posttranslational action of T3SS1. Taken together, our analysis reveals a complex interplay between roles of T3SS1 as both a transcriptional and posttranslational manipulator of host cell signaling. PMID:28512145

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

    International Nuclear Information System (INIS)

    Boschi, Federico; Rizzatti, Vanni; Zamboni, Mauro; Sbarbati, Andrea

    2014-01-01

    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

  14. ZnT3 Gene Deletion Reduces Colchicine-Induced Dentate Granule Cell Degeneration

    Directory of Open Access Journals (Sweden)

    Bo Young Choi

    2017-10-01

    Full Text Available Our previous study demonstrated that colchicine-induced dentate granule cell death is caused by blocking axonal flow and the accumulation of intracellular zinc. Zinc is concentrated in the synaptic vesicles via zinc transporter 3 (ZnT3, which facilitates zinc transport from the cytosol into the synaptic vesicles. The aim of the present study was to identify the role of ZnT3 gene deletion on colchicine-induced dentate granule cell death. The present study used young (3–5 months mice of the wild-type (WT or the ZnT3−/− genotype. Colchicine (10 µg/kg was injected into the hippocampus, and then brain sections were evaluated 12 or 24 h later. Cell death was evaluated by Fluoro-Jade B; oxidative stress was analyzed by 4-hydroxy-2-nonenal; and dendritic damage was detected by microtubule-associated protein 2. Zinc accumulation was detected by N-(6-methoxy-8-quinolyl-para-toluenesulfonamide (TSQ staining. Here, we found that ZnT3−/− reduced the number of degenerating cells after colchicine injection. The ZnT3−/−-mediated inhibition of cell death was accompanied by suppression of oxidative injury, dendritic damage and zinc accumulation. In addition, ZnT3−/− mice showed more glutathione content than WT mice and inhibited neuronal glutathione depletion by colchicine. These findings suggest that increased neuronal glutathione by ZnT3 gene deletion prevents colchicine-induced dentate granule cell death.

  15. Long term dietary deficiency in steers: vital functions and T3 and IGF-1 relationships

    Directory of Open Access Journals (Sweden)

    Alessandra S. Lima

    2014-09-01

    Full Text Available To evaluate the influence of diets with different degrees of energy deficiency on the hormonal profile and vital functions, 12 steers were randomly distributed into 3 groups of 4 animals. For 140 days, each group received (G1 a diet to promote a weight gain of 900gr/day (17.7 Mcal/d DE and 13% CP, (G2 80% of the maintenance requirements (5.8 Mcal/d DE and 7% CP, or (G3 60% of the maintenance requirements (4.7 Mcal/d DE and 5% CP. In G2 and G3, the energy deficit caused a marked decrease in the heart rate and respiratory rate and a reduction in the blood levels of Insulin like growth factor-1 (IGF-1 and triiodothyronine (T3. The decrease in heart rate, respiratory movement and, to a lesser extent, reduction of the rectal temperature, reflected the low status of energy and was negatively impacted by the low levels of T3. There was a strong correlation between the hormones T3 and IGF-1 (r=0.833. There were also strong correlations between T3 and HR (r=0.701, T3 and RR (r=0.632, IGF-1 and HR (r=0.731, and IGF-1 and RR (r=0.679. There were intermediate correlations between T3 and TºC (r=0.484, T3 and insulin (r=0.506, IGF-1 and insulin (r=0.517, and IGF-1 and TºC (r=0.548. This study showed the influence of a long period of providing an energy-deficient diet on animal performance, correlating hormonal status and vital functions in growing cattle. The results indicated that the evaluated parameters represent an important tool for the early detection of dietary deficiency.

  16. Experimental and Numerical Study of Needle Peening Effects in Aluminium Alloy 2024-T3 Sheets

    Science.gov (United States)

    Mendez Romero, Julio Alberto

    Peening of metallic components is an effective treatment used in aerospace and automotive applications to improve fatigue properties or to blend and repair localized damage. This process is typically carried out using metallic airborne media, called shot. However, different processes make use of different media, such is the case of hard, pneumatically powered needles of needle peening equipment. In order to obtain a better understanding of the effects of needle peening in the same context as shot peening, this research work had as an objective to study in detail the behavior of the needle peening equipment in order to characterize the process, design an experimental campaign to measure the effects of needle peening on AA2024-T3 and to develop and validate a Finite Element (FE) model capable of replicating the results of needle peening. The needle peening equipment prototype, called SPIKERRTM, was developed by Shockform Aeronautique Inc. The equipment was characterized by utilizing high-speed camera recording in order to study its behaviour by varying the operating pressure. The obtained collection of images was ran through a newly developed digital image algorithm, so as to quantify the needles' velocity and frequency. The impact velocity and impact frequency were determined for different equipment operating parameters. It was concluded that both the average impact velocity and the impact frequency increase as the pressure becomes larger. Behaviour anomalies among the different needles, such as frequency and velocity variations, were brought to light; these conclusions could be of interest to the manufacturer. Ideally, all of the needles should behave as similarly as possible so as to produce a more uniform process. The response to needle peening of AA2024-T3 in 1.6 mm thick sheet form was studied by needle peening test specimens with dimensions of an Almen strips using the SPIKERRTM. AA2024-T3 was selected since it was extensively studied at Ecole Polytechnique de

  17. Prognostic impact of perirenal fat or adrenal gland involvement in patients with pT3b renal cell carcinoma.

    Science.gov (United States)

    Fujita, Tetsuo; Iwamura, Masatsugu; Yanagisawa, Nobuyuki; Muramoto, Masatoshi; Hirayama, Takahiro; Okayasu, Isao; Baba, Shiro

    2007-05-01

    To analyze the prognostic impact of the pT3a factors, perirenal fat or adrenal gland involvement, in patients with pT3b renal cell carcinoma (RCC). A total of 43 patients with pT3b RCC who underwent radical nephrectomy, with complete resection of tumor thrombus, at our institution from March 1972 to September 2005 were enrolled in this study. The presence of pT3a factors was reviewed, and the disease-specific survival was compared according to the reclassification as pT3b only or pT3b with pT3a. After review by a single pathologist, 23 patients (53.5%) were identified as having pT3b only and 20 patients (46.5%) as having pT3b with pT3a. The mean disease-specific survival time in those with pT3b only was significantly longer at 70.9 +/- 9.1 (SE) months compared with 25.0 +/- 4.4 (SE) months in those with pT3b with pT3a (P = 0.0032). On univariate analyses, the presence of pT3a factors (P = 0.0065), preoperative metastasis (P = 0.0025), surgical specimens positive for lymph node metastasis (P = 0.0183), and spindle cell factor (P = 0.0233) were recognized as predictors of a poor prognosis. The presence of perirenal fat or adrenal gland involvement in patients with pT3b RCC renders the prognosis significantly worse. Careful postoperative examination should be required, along with reclassification for Stage pT3b with pT3a in RCC.

  18. Protein turnover and cellular autophagy in growing and growth-inhibited 3T3 cells

    Energy Technology Data Exchange (ETDEWEB)

    Papadopoulos, T.; Pfeifer, U. (Univ. of Wuerzburg (West Germany))

    1987-07-01

    The relationship between growth, protein degradation, and cellular autophagy was tested in growing and in growth-inhibited 3T3 cell monolayers. For the biochemical evaluation of DNA and protein metabolism, growth-inhibited 3T3 cell monolayers with high cell density and growing 3T3 cell monolayers with low cell density were labeled simultaneously with ({sup 14}C)thymidine and ({sup 3}H)leucine. The evaluation of the DNA turnover and additional ({sup 3}H)thymidine autoradiography showed that 24 to 5% of 3T3 cells continue to replicate even in the growth-inhibited state, where no accumulation of protein and DNA can be observed. Cell loss, therefore, has to be assumed to compensate for the ongoing cell proliferation. When the data of protein turnover were corrected for cell loss, it was found that the rate constant of protein synthesis in nongrowing monolayers was reduced to half the value found in growing monolayers. Simultaneously, the rate constant of protein degradation in nongrowing monolayers was increased to about 1.5-fold the value of growing monolayers. These data are in agreement with the assumption that cellular autophagy represents a major pathway of regulating protein degradation in 3T3 cells and that the regulation of autophagic protein degradation is of relevance for the transition from a growing to a nongrowing state.

  19. Dual effect of Mesorhizobium loti T3SS functionality on the symbiotic process.

    Science.gov (United States)

    Sánchez, Cintia; Mercante, Virginia; Babuin, María F; Lepek, Viviana C

    2012-05-01

    Mesorhizobium loti MAFF303099 has a functional type III secretory system (T3SS) involved in the nodulation process on Lotus tenuis and Lotus japonicus. Four putative M. loti T3SS effectors (Mlr6358, Mlr6331, Mlr6361, and Mlr6316) have been previously described, and it has been demonstrated that the N-terminal regions of Mlr6361 and Mlr6358 mediate the secretion via a T3SS. Here, we demonstrate the capacity of Mlr6316 and Mlr6331 N-terminal regions to direct the secretion of a translational fusion to a reporter peptide through T3SS. By using single, double, and triple mutants, we demonstrated the positive and negative participation of some of these proteins in the determination of competitiveness on Lotus spp. Low competitiveness values correlated with low nodulation efficiency for a mutant deficient in three of the putative M. loti effectors. Our data suggest that the net effect of M. loti T3SS function on symbiotic process with Lotus results from a balance between positive and negative effects. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  20. Using Inertial Fusion Implosions to Measure the T+^{3}He Fusion Cross Section at Nucleosynthesis-Relevant Energies.

    Science.gov (United States)

    Zylstra, A B; Herrmann, H W; Johnson, M Gatu; Kim, Y H; Frenje, J A; Hale, G; Li, C K; Rubery, M; Paris, M; Bacher, A; Brune, C R; Forrest, C; Glebov, V Yu; Janezic, R; McNabb, D; Nikroo, A; Pino, J; Sangster, T C; Séguin, F H; Seka, W; Sio, H; Stoeckl, C; Petrasso, R D

    2016-07-15

    Light nuclei were created during big-bang nucleosynthesis (BBN). Standard BBN theory, using rates inferred from accelerator-beam data, cannot explain high levels of ^{6}Li in low-metallicity stars. Using high-energy-density plasmas we measure the T(^{3}He,γ)^{6}Li reaction rate, a candidate for anomalously high ^{6}Li production; we find that the rate is too low to explain the observations, and different than values used in common BBN models. This is the first data directly relevant to BBN, and also the first use of laboratory plasmas, at comparable conditions to astrophysical systems, to address a problem in nuclear astrophysics.

  1. Increased plasmid copy number is essential for Yersinia T3SS function and virulence.

    Science.gov (United States)

    Wang, He; Avican, Kemal; Fahlgren, Anna; Erttmann, Saskia F; Nuss, Aaron M; Dersch, Petra; Fallman, Maria; Edgren, Tomas; Wolf-Watz, Hans

    2016-07-29

    Pathogenic bacteria have evolved numerous virulence mechanisms that are essential for establishing infections. The enterobacterium Yersinia uses a type III secretion system (T3SS) encoded by a 70-kilobase, low-copy, IncFII-class virulence plasmid. We report a novel virulence strategy in Y. pseudotuberculosis in which this pathogen up-regulates the plasmid copy number during infection. We found that an increased dose of plasmid-encoded genes is indispensable for virulence and substantially elevates the expression and function of the T3SS. Remarkably, we observed direct, tight coupling between plasmid replication and T3SS function. This regulatory pathway provides a framework for further exploration of the environmental sensing mechanisms of pathogenic bacteria. Copyright © 2016, American Association for the Advancement of Science.

  2. Structural basis of eukaryotic cell targeting by type III secretion system (T3SS) effectors.

    Science.gov (United States)

    Tosi, Tommaso; Pflug, Alexander; Discola, Karen F; Neves, David; Dessen, Andréa

    2013-01-01

    Type III secretion systems (T3SS) are macromolecular complexes that translocate a wide number of effector proteins into eukaryotic host cells. Once within the cytoplasm, many T3SS effectors mimic the structure and/or function of eukaryotic proteins in order to manipulate signaling cascades, and thus play pivotal roles in colonization, invasion, survival and virulence. Structural biology techniques have played key roles in the unraveling of bacterial strategies employed for mimicry and targeting. This review provides an overall view of our current understanding of structure and function of T3SS effectors, as well as of the different classes of eukaryotic proteins that are targeted and the consequences for the infected cell. Copyright © 2013 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  3. Autophagy Protects MC3T3-E1 Cells upon Aluminum-Induced Apoptosis.

    Science.gov (United States)

    Yang, Xu; Zhang, Jian; Ji, Qiang; Wang, Fan; Song, Miao; Li, Yanfei

    2018-03-08

    Aluminum (Al) exposure has adverse effects on osteoblasts, and the effect might be through autophagy-associated apoptosis. In this study, we showed that aluminum trichloride (AlCl 3 ) could induce autophagy in MC3T3-E1 cells, as demonstrated by monodansylcadaverine (MDC) staining and the expressions of the ATG3, ATG5, and ATG9 genes. We found AlCl 3 inhibited MC3T3-E1 cell survival rate and caused apoptosis, as evidenced by CCK-8 assay, Annexin V/PI double staining, and increased expressions of Bcl-2, Bax, and Caspase-3 genes. In addition, increased autophagy induced by rapamycin further attenuated the MC3T3-E1 cell apoptosis rate after AlCl 3 exposure. These results support the hypothesis that autophagy plays a protective role in impeding apoptosis caused by AlCl 3 . Activating autophagy may be a strategy for treatment of Al-induced bone disease.

  4. Affinity of tri-iodothyronine(T3) to lymphocytes in thyroid cancer patients

    International Nuclear Information System (INIS)

    Jeevanram, R.K.; Narkar, A.A.; Ganatra, R.D.; Shah, D.H.

    1978-01-01

    Peripheral blood lymphocytes (PBL) of thyroid cancer patients and 5 normal subjects were studied for their affinity towards 125 I labelled tri-iodothyronine ( 125 I-T 3 ). The mean affinity constant K for PBL of normal subjects was 2.94 + 0.47 x 10 11 litre/mole and for thyroid cancer patients was 1.75 + 0.44 x 10 11 litre/mole. The binding affinity of the PBL of thyroid cancer patients should have been greater because of (a) lack of thyroid hormones resulting from removal of thyroid gland, and (b) higher levels of thyroid stimulating hormone (TSH) which is known to increase the binding of T 3 to the cells. The lowered T 3 binding of PBL of thyroid cancer patients is explained on the basis of the morphological changes on the lymphocytes of cancer patients. (auth.)

  5. A fundamental research on T3 radioimmunoassay by polyethylene glycol (PEG) method

    International Nuclear Information System (INIS)

    Takahashi, Tadashige; Hayakawa, Susumu; Takagi, Atsushi; Sugisawa, Yoshihiko; Kurata, Kunio

    1976-01-01

    Since Chopra, et al. produced an antibody of triiodothyronine (T 3 ), measurement of serum T 3 by radioimmunoassay has been widely used as a clinical test. However, the dextran coated charcoal (DCC) method which is most commonly used for B/F separation requires strict control of incubation temperature and time. The authors reduced this disadvantage by using polyethylene glycol (PEG) instead of DCC for B/F separation. Crossreactivities of the antibody used with thyroxine, monoiodothyrosine and diiodotyrosine are less than 0.06% which is considered to be satisfactory in comparison with those previously reported. The recovery ratios of added T 3 were between 92.4 - 105.9% and that reflected T 3 concentration well. The dilution test showed good linearity. Correlation of T 3 values measured by DCC method and PEG method in 26 sera was good (r=0.99), and linear regression function was y=0.89x + 0.30 (y: PEG method and x: DCC method). No significant differences were observed in the results obtained by 2 hr incubation at 20 - 35 0 C and 24 hr incubation at 4 0 C. As for the volume of PEG solution, 1.0 ml was most suitable. Samples should be kept at 4 0 C when centrifugation is impossible within 30 min after addition of PEG. Also in various conditions of centrifugation, the authors obtained almost same results. As described above, the authors conquered the problem involved in the DCC method, and also simplified the assay procedure by using the PEG method for B/F separation. PEG method is considered to be the methods of choice for clinical routine test for serum T 3 levels (Evans, J.)

  6. Cytotoxicity of Alpinia galanga Rhizome Crude Extract on NIH-3T3 Cells

    Directory of Open Access Journals (Sweden)

    Ferry Sandra

    2017-04-01

    Full Text Available BACKGROUND: Alpinia galanga (A. galanga was reported as a potential medicinal source due to its wide effect. A. galanga rhizome crude extract (ARCE was reported to have high cytotoxic effect in cancer cells, but low in normal cells. However half maximal inhibitory concentration (IC50 of ARCE is not clearly known yet. Hence, current study was conducted to investigate the IC50 of ARCE in normal standard fibroblast cell line, NIH-3T3 cells. METHODS: Rhizomes of A. galanga were collected, peeled, dried, milled and weighed. Extraction was performed using maceration method, then filtered and evaporated. ARCE with various concentrations were applied in NIH-3T3 cells for 24 or 48 hours. Cells were documented and counted with 3-(4,5-dimethylthiazol-2-yl-2,5-Diphenyltetrazolium bromide (MTT assay. RESULTS: Five hundreds grams of simplicia were macerated with ethanol and evaporated, 1 mg/mL crude extract with total volume of 114 mL was obtained. By addition of ARCE in NIH-3T3 cell culture, number of NIH-3T3 cells were shown less when treated with higher concentration of ARCE. Cell numbers of 0, 3.125, 6.25, 12.5, 25 and 50% ARCE treatment for 24 hours are 11,531, 11,352, 10,920, 10,365, 9,471, 8,360, respectively, meanwhile for 48 hours are 13,219, 12,686, 12,278, 11,390, 10,279, 8,390, respectively. CONCLUSION: IC50 of ARCE in 24 hours treatment was 620.5 mg/mL, while in 48 hours treatment was 666.6 mg/mL. Hence, ARCE is suggested to have low cytotoxic effect in NIH-3T3 cells. KEYWORDS: Alpinia galanga, ginger, extract, cytotoxic, MTT, NIH-3T3

  7. T3 glycoprotein is functional although structurally distinct on human T-cell receptor gamma T lymphocytes.

    OpenAIRE

    Krangel, M S; Bierer, B E; Devlin, P; Clabby, M; Strominger, J L; McLean, J; Brenner, M B

    1987-01-01

    The T-cell receptor (TCR) gamma gene product occurs in association with T3 (CD3) polypeptides on the surface of human T lymphocytes. TCR gamma lymphocytes express arrays of T3 polypeptides distinct from those typically observed on TCR alpha beta lymphocytes. This report demonstrates that identical T3 gamma, delta, and epsilon polypeptides are synthesized by TCR gamma lymphocytes and TCR alpha beta lymphocytes. However, the processing of T3 delta oligosaccharides is distinct in the two cell ty...

  8. Cytotoxic Vibrio T3SS1 Rewires Host Gene Expression to Subvert Cell Death Signaling and Activate Cell Survival Networks

    OpenAIRE

    De Nisco, Nicole J.; Kanchwala, Mohammed; Li, Peng; Fernandez, Jessie; Xing, Chao; Orth, Kim

    2017-01-01

    Bacterial effectors are potent manipulators of host signaling pathways. The marine bacterium Vibrio parahaemolyticus (V. para), delivers effectors into host cells through two type three secretion systems (T3SS). The ubiquitous T3SS1 is vital for V. para survival in the environment, whereas T3SS2 causes acute gastroenteritis in human hosts. Although the natural host is undefined, T3SS1 effectors attack highly conserved cellular processes and pathways to orchestrate non-apoptotic cell death. Mu...

  9. Escherichia coli type III secretion system 2: a new kind of T3SS?

    OpenAIRE

    Zhou, Mingxu; Guo, Zhiyan; Duan, Qiangde; Hardwidge, Philip R; Zhu, Guoqiang

    2014-01-01

    International audience; Type III secretion systems (T3SSs) are employed by Gram-negative bacteria to deliver effector proteins into the cytoplasm of infected host cells. Enteropathogenic Escherichia coli use a T3SS to deliver effector proteins that result in the creation of the attaching and effacing lesions. The genome sequence of the Escherichia coli pathotype O157:H7 revealed the existence of a gene cluster encoding components of a second type III secretion system, the E. coli type III sec...

  10. Preparation of quality control samples for thyroid hormones T3 and T4 in radioimmunoassay techniques

    International Nuclear Information System (INIS)

    Ahmed, F.O.A.

    2006-03-01

    Today, the radioimmunoassay becomes one of the best techniques for quantitative analysis of very low concentration of different substances. RIA is being widely used in medical and research laboratories. To maintain high specificity and accuracy in RIA and other related techniques the quality controls must be introduced. In this dissertation quality control samples for thyroid hormones (Triiodothyronine T3 and Thyroxin T4), using RIA techniques. Ready made chinese T4, T3 RIA kits were used. IAEA statistical package were selected.(Author)

  11. Mango fruit peel and flesh extracts affect adipogenesis in 3T3-L1 cells.

    Science.gov (United States)

    Taing, Meng-Wong; Pierson, Jean-Thomas; Hoang, Van L T; Shaw, Paul N; Dietzgen, Ralf G; Gidley, Michael J; Roberts-Thomson, Sarah J; Monteith, Gregory R

    2012-08-01

    Obesity is associated with many chronic disease states, such as diabetes mellitus, coronary disease and certain cancers, including those of the breast and colon. There is a growing body of evidence that links phytochemicals with the inhibition of adipogenesis and protection against obesity. Mangoes (Mangifera indica L.) are tropical fruits that are rich in a diverse array of bioactive phytochemicals. In this study, methanol extracts of peel and flesh from three archetypal mango cultivars; Irwin, Nam Doc Mai and Kensington Pride, were assessed for their effects on a 3T3-L1 pre-adipocyte cell line model of adipogenesis. High content imaging was used to assess: lipid droplets per cell, lipid droplet area per cell, lipid droplet integrated intensity, nuclei count and nuclear area per cell. Mango flesh extracts from the three cultivars did not inhibit adipogenesis; peel extracts from both Irwin and Nam Doc Mai, however, did so with the Nam Doc Mai extract most potent at inhibiting adipogenesis. Peel extract from Kensington Pride promoted adipogenesis. The inhibition of adipogenesis by Irwin (100 μg mL(-1)) and Nam Doc Mai peel extracts (50 and 100 μg mL(-1)) was associated with an increase in the average nuclear area per cell; similar effects were seen with resveratrol, suggesting that these extracts may act through pathways similar to resveratrol. These results suggest that differences in the phytochemical composition between mango cultivars may influence their effectiveness in inhibiting adipogenesis, and points to mango fruit peel as a potential source of nutraceuticals.

  12. Curcumin inhibits adipogenesis induced by benzyl butyl phthalate in 3T3-L1 cells.

    Science.gov (United States)

    Sakuma, Satoru; Sumida, Maki; Endoh, Yukiko; Kurita, Ayaka; Yamaguchi, Ayana; Watanabe, Tomoki; Kohda, Tetsuya; Tsukiyama, Yui; Fujimoto, Yohko

    2017-08-15

    Phthalates are a group of endocrine disrupting chemicals and may have contributed to the recent global obesity health crisis. Increased adipogenesis via the peroxisome proliferator-activated receptor γ (PPARγ)-CCAAT-enhancer binding protein α (C/EBPα) pathway could be one critical mechanism responsible for phthalate-induced weight gain. On the other hand, curcumin has been shown to inhibit adipogenesis in cells and animal models. The present study was undertaken to evaluate, for the first time, whether curcumin could reduce adipogenesis induced by benzyl butyl phthalate (BBP) via downregulation of the PPARγ-C/EBPα pathway. 3T3-L1 preadipocytes were differentiated by treating them with insulin, dexamethasone, and 3-isobutyl-1-methylxanthine in the presence of BBP, with or without curcumin. Cells that were grown in the presence of BBP alone showed a significant increase in triacylglycerol (TG) levels. In addition, the number of Oil Red O-stained cells and the mRNA expression levels of PPARγ, C/EBPα, adiponectin, and tumor necrosis factor-α (TNFα) were significantly increased. However, treatment with BBP in combination with curcumin resulted in major reductions in TG levels, the numbers of Oil Red O-stained cells, and the mRNA expression levels of the four proteins. These results suggest that curcumin might be an inhibitor of BBP-induced weight gain and inflammation via stimulation of adipocyte differentiation and TNFα generation. Curcumin may, therefore, be a potential medication for preventing the harmful effects of phthalates. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Differential expression of fatty acid uptake in 3T3-L1 cells

    International Nuclear Information System (INIS)

    Waggoner, D.; Bernlohr, D.A.

    1987-01-01

    Cultured 3T3-L1 cells have been used as a model system to investigate the mechanism of fatty acid uptake by adipose tissue. Using a 1:1 molar ratio of 14 C-oleate and defatted bovine serum albumin (BSA), fatty acid (FA) uptake was quantitated at 4 0 and 37 0 as cell associated radioactivity. The profile of FA uptake in preadipocytes and adipocytes was biphasic; an initial rapid phase (1-20s) followed by a second slower phase (60-480s). At 37 0 the initial rate of FA accumulation in preadipocytes was identical to that in adipocytes, whereas the rate of accumulation during the second phase increased 7-fold (100 μM total FA) as a consequence of adipose conversion. When uptake measurements were made at 4 0 in adipocytes, the initial rate was identical to that at 37 0 , however the rate of second phase decreased 5-fold. Incubation of 14 C-BSA and nonradiolabeled FA with adipocyte monolayers (100 μM total FA) resulted in the rapid association (t/sub 1/2/ = 20s) of the BSA-FA complex with the cell surface. Incubation of 100, 10, and 1 μM total FA with adipocytes resulted in a 50-fold change in FA accumulation during the second phase. These results suggest that (1) FA uptake is significantly increased after differentiation, suggesting the participation of specialized proteins, (2) the temperature-insensitive initial FA accumulation can be attributed to rapid association of the BSA-FA complex to the cell surface, (3) the second phase of FA accumulation represents uptake

  14. The Benefits of the 3T3 NRU Test in the Safety Assessment of Cosmetics: Long-Term Experience from Pre-Marketing Testing in the Czech Republic.

    Czech Academy of Sciences Publication Activity Database

    Jírová, D.; Kejlová, K.; Brabec, Marek; Bendová, H.; Kolářová, H.

    2003-01-01

    Roč. 174, č. 5-6 (2003), s. 791-796 ISSN 0887-2333 Source of funding: V - iné verejné zdroje Keywords : cytotoxicity * 3T3 NRU assay * irritation * nonparametric statistical model Subject RIV: BB - Applied Statistics, Operational Research Impact factor: 1.642, year: 2003

  15. Minimal SPI1-T3SS effector requirement for Salmonella enterocyte invasion and intracellular proliferation in vivo.

    Science.gov (United States)

    Zhang, Kaiyi; Riba, Ambre; Nietschke, Monika; Torow, Natalia; Repnik, Urska; Pütz, Andreas; Fulde, Marcus; Dupont, Aline; Hensel, Michael; Hornef, Mathias

    2018-03-01

    Effector molecules translocated by the Salmonella pathogenicity island (SPI)1-encoded type 3 secretion system (T3SS) critically contribute to the pathogenesis of human Salmonella infection. They facilitate internalization by non-phagocytic enterocytes rendering the intestinal epithelium an entry site for infection. Their function in vivo has remained ill-defined due to the lack of a suitable animal model that allows visualization of intraepithelial Salmonella. Here, we took advantage of our novel neonatal mouse model and analyzed various bacterial mutants and reporter strains as well as gene deficient mice. Our results demonstrate the critical but redundant role of SopE2 and SipA for enterocyte invasion, prerequisite for transcriptional stimulation and mucosal translocation in vivo. In contrast, the generation of a replicative intraepithelial endosomal compartment required the cooperative action of SipA and SopE2 or SipA and SopB but was independent of SopA or host MyD88 signaling. Intraepithelial growth had no critical influence on systemic spread. Our results define the role of SPI1-T3SS effector molecules during enterocyte invasion and intraepithelial proliferation in vivo providing novel insight in the early course of Salmonella infection.

  16. Nanostructured sol-gel coatings doped with cerium nitrate as pre-treatments for AA2024-T3

    International Nuclear Information System (INIS)

    Zheludkevich, M.L.; Serra, R.; Montemor, M.F.; Yasakau, K.A.; Salvado, I.M. Miranda; Ferreira, M.G.S.

    2005-01-01

    Nanostructured hybrid sol-gel coatings doped with cerium ions were investigated in the present work as pre-treatments for the AA2024-T3 alloy. The sol-gel films have been synthesized from tetraethylorthosilicate (TEOS) and 3-glycidoxypropyltrimethoxysilane (GPTMS) precursors. Additionally the hybrid sol was doped with zirconia nanoparticles prepared from hydrolyzed tetra-n-propoxyzirconium (TPOZ). Cerium nitrate, as corrosion inhibitor, was added into the hybrid matrix or into the oxide nanoparticles. The chemical composition and the structure of the hybrid sol-gel films were studied by XPS (X-ray photoelectron spectroscopy) and AFM (atomic force microscopy), respectively. The evolution of the corrosion protection properties of the sol-gel films was studied by EIS (electrochemical impedance spectroscopy), which can provide quantitative information on the role of the different pre-treatments. Different equivalent circuits, for different stages of the corrosion processes, were used in order to model the coating degradation. The models were supported by SEM (scanning electron microscopy) measurements. The results show that the sol-gel films containing zirconia nanoparticles present improved barrier properties. Doping the hybrid nanostructured sol-gel coatings with cerium nitrate leads to additional improvement of the corrosion protection. The zirconia particles present in the sol-gel matrix seem to act as nanoreservoirs providing a prolonged release of cerium ions. The nanostructured sol-gel films doped with cerium nitrate can be proposed as a potential candidate for substitution of the chromate pre-treatments for AA2024-T3

  17. Anti-Obesity Effects of Starter Fermented Kimchi on 3T3-L1 Adipocytes.

    Science.gov (United States)

    Lee, Kyung-Hee; Song, Jia-Le; Park, Eui-Seong; Ju, Jaehyun; Kim, Hee-Young; Park, Kun-Young

    2015-12-01

    The anti-obesity effects of starter (Leuconostoc mesenteroides+Lactobacillus plantarum) fermented kimchi on 3T3-L1 adipocyte were studied using naturally fermented kimchi (NK), a functional kimchi (FK, NK supplemented with green tea), and FK supplemented with added starters (FKS). Oil red O staining and cellular levels of triglyceride (TG) and glycerol were used to evaluate the in vitro anti-obesity effects of these kimchis in 3T3-L1 cells. The expressions of adipogenesis/lipogenesis-related genes of peroxisome proliferator-active receptor (PPAR)-γ, CCAAT/enhance-binding protein (C/EBP)-α, and fatty acid synthase (FAS) were determined by RT-PCR. Kimchis, especially FKS, markedly decreased TG levels and increased levels of intracellular glycerol and lipid lipolysis. In addition, FKS also reduced the mRNA levels of PPAR-γ, C/EBP-α, and FAS, which are related to adipogenesis/lipogenesis in 3T3-L1 cells. These results suggest the anti-obesity effects of FKS were to due to enhanced lipolysis and reduced adipogenesis/lipogenesis in 3T3-L1 adipocytes.

  18. Radioimmunoassay of serum T3, T4 and TSH during anesthesia and operation

    International Nuclear Information System (INIS)

    Gosheva-Antonova, Ts.; Zakharieva, B.; Kurtev, I.

    1987-01-01

    The serum concentrations of thyroxine (T 3 ), triiodothyronine (T 4 ) and thyroid-stimulating hormone (TSH) were determined in 31 partients before and during urologic operations on the 30th and 60th minute since the onset of the operation, performed under endotracheal halotane or neuroleptanesthesia (NLA) in assisted breathing and intravenous drip anesthesia with ketalar-diazepam in spontaneous breathing. There was statistically significant rise in T 4 level, decrease in T 3 and negligible changes in TSH level, in patients operated under halotane anesthesia. In those operated under NLA, T 4 tended initially to be elevated, with subseguent fall to starting level, with a tendency toward rise in TSH and stable unchanged T 3 level. Ketalar-diazepam anesthesia was applied only to patients subjected to transurethral resections. T 4 in them tended to be decreased, while T 3 and TSH showed negligible changes. Since the operations of patients anesthesized with halotane and NLA had similar localizations and severity, the differences in the thyroid hormone reactions could be associated with the type of anesthesia. The negligible changes in TSH are highly suggestive that this hormone is not influenced by the operation stress and anesthetics, and does hot exert regulating effect upon the thyroid status under these conditions. The milder reactions in patients operated under ketalar-diazepam anestesia may largely be associated with the milder operation stress in transurethal resection

  19. Evading plant immunity: feedback control of the T3SS inPseudomonas syringae.

    Science.gov (United States)

    Waite, Christopher; Schumacher, Jörg; Jovanovic, Milija; Bennett, Mark; Buck, Martin

    2017-03-17

    Microbes are responsible for over 10% of the global yield losses in staple crops such as wheat, rice, and maize. Understanding the decision-making strategies that enable bacterial plant pathogens to evade the host immune system and cause disease is essential for managing their ever growing threat to food security. Many utilise the needle-like type III secretion system (T3SS) to suppress plant immunity, by injecting effector proteins that inhibit eukaryotic signalling pathways into the host cell cytoplasm. Plants can in turn evolve resistance to specific pathogens via recognition and blocking of the T3SS effectors, so leading to an ongoing co-evolutionary 'arms race' between pathogen and host pairs. The extracytoplasmic function sigma factor HrpL co-ordinates the expression of the T3SS regulon in the leaf-dwelling Pseudomonas syringae and similar pathogens. Recently, we showed that association of HrpL with a target promoter directly adjacent to the hrpL gene imposes negative autogenous control on its own expression level due to overlapping regulatory elements. Our results suggest that by down-regulating T3SS function, this fine-tuning mechanism enables P. syringae to minimise effector-mediated elicitation of plant immunity.

  20. Stimulative effects of Ulmus davidiana Planch (Ulmaceae) on osteoblastic MC3T3-E1 cells.

    Science.gov (United States)

    Suh, Seok-Jong; Yun, Woo-Sik; Kim, Kap-Sung; Jin, Un-Ho; Kim, June-Ki; Kim, Myung-Sunny; Kwon, Dae Young; Kim, Cheorl-Ho

    2007-02-12

    Ulmus davidiana Planch (Ulmaceae) has long been known to have anti-inflammatory and protective effects on damaged tissue, inflammation and bone among other functions. To treat rheumatoid arthritis (RA), a herbal medicine, Ulmus davidiana Planch (Ulmaceae) extract (UD) is being used in traditional oriental medicine. The effect of UD on the proliferation and osteoblastic differentiation in non-transformed osteoblastic cells (MC3T3-E1) was studied. UD dose-dependently increased DNA synthesis (significant at 5-20 microg/ml). UD increased alkaline phosphatase (ALP) activity and prolyl hydroxylase activity of MC3T3-E1 cells (5-20 microg/ml). Antiestrogen tamoxifen eliminated the stimulation of proliferation and ALP activity of MC3T3-E1, which was induced by UD. UD at concentrations ranged from 30 to 100 microg/ml inhibited prostaglandin E2 production in MC3T3-E1. These results indicate that UD directly stimulates cell proliferation and differentiation of osteoblasts. These results also suggest and UD is effective for bone anti-resorptive action in bone cells.

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

    Science.gov (United States)

    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.

  2. Evading plant immunity: feedback control of the T3SS in Pseudomonas syringae

    Directory of Open Access Journals (Sweden)

    Christopher Waite

    2017-03-01

    Full Text Available Microbes are responsible for over 10% of the global yield losses in staple crops such as wheat, rice, and maize. Understanding the decision-making strategies that enable bacterial plant pathogens to evade the host immune system and cause disease is essential for managing their ever growing threat to food security. Many utilise the needle-like type III secretion system (T3SS to suppress plant immunity, by injecting effector proteins that inhibit eukaryotic signalling pathways into the host cell cytoplasm. Plants can in turn evolve resistance to specific pathogens via recognition and blocking of the T3SS effectors, so leading to an ongoing co-evolutionary ‘arms race’ between pathogen and host pairs. The extracytoplasmic function sigma factor HrpL co-ordinates the expression of the T3SS regulon in the leaf-dwelling Pseudomonas syringae and similar pathogens. Recently, we showed that association of HrpL with a target promoter directly adjacent to the hrpL gene imposes negative autogenous control on its own expression level due to overlapping regulatory elements. Our results suggest that by down-regulating T3SS function, this fine-tuning mechanism enables P. syringae to minimise effector-mediated elicitation of plant immunity.

  3. The Relationship between Selenium and T3 in Selenium Supplemented and Nonsupplemented Ewes and Their Lambs

    Directory of Open Access Journals (Sweden)

    Abd Elghany Hefnawy

    2014-01-01

    Full Text Available Twenty pregnant ewes were selected and classified into two groups. The first group received subcutaneous selenium supplementation (0.1 mg of sodium selenite/kg BW at the 8th and 5th weeks before birth and 1st week after birth while the other was control group without selenium injection. Maternal plasma and serum samples were collected weekly from the 8th week before birth until the 8th week after birth and milk samples were taken from ewes weekly, while plasma and serum samples were collected at 48 hours, 1st, 2nd, 3rd, 5th, and 8th weeks after birth from the newborn lambs. Results demonstrated significant positive relationship between maternal plasma selenium and serum T3 in supplemented and control ewes (r=0.69 to 0.72, P<0.05. There was significant (P<0.001 increase in T3 in supplemented ewes and their lambs until the 8th week after birth. There was positive relationship between milk, selenium concentration, and serum T3 in the newborn lambs of the supplemented group (r=0.84, P<0.01, while the relationship was negative in the control one (r=-0.89, P<0.01. Muscular and thyroid pathological changes were independent of selenium supplementation. Selenium supplementation was important for maintaining T3 in ewes and newborn lambs until the 8th week after birth.

  4. Osteogenic gene expression of murine osteoblastic (MC3T3-E1) cells under cyclic tension

    Science.gov (United States)

    Kao, C. T.; Chen, C. C.; Cheong, U.-I.; Liu, S. L.; Huang, T. H.

    2014-08-01

    Low-level laser therapy (LLLT) can promote cell proliferation. The remodeling ability of the tension side of orthodontic teeth affects post-orthodontic stability. The purpose of the present study was to investigate the osteogenic effects of LLLT on osteoblast-like cells treated with a simulated tension system that provides a mechanical tension regimen. Murine osteoblastic (MC3T3-E1) cells were cultured in a Flexcell strain unit with programmed loads of 12% elongation at a frequency of 0.5 Hz for 24 and 48 h. The cultured cells were treated with a low-level diode laser using powers of 5 J and 10 J. The proliferation of MC3T3-E1 cells was determined using the Alamar Blue assay. The expression of osteogenic genes (type I collagen (Col-1), osteopontin (OPN), osteocalcin (OC), osteoprotegerin (OPG), receptor activator of nuclear factor kappa B ligand (RANKL), bone morphologic protein (BMP-2), and bone morphologic protein (BMP-4)) in MC3T3-E1 cells was analyzed using reverse transcription polymerase chain reaction (RT-PCR). The data were analyzed using one-way analysis of variance. The proliferation rate of tension-cultured MC3T3-E1 cells under 5 J and 10 J LLLT increased compared with that of the control group (p orthodontics.

  5. Anti-Obesity Effects of Starter Fermented Kimchi on 3T3-L1 Adipocytes

    Science.gov (United States)

    Lee, Kyung-Hee; Song, Jia-Le; Park, Eui-Seong; Ju, Jaehyun; Kim, Hee-Young; Park, Kun-Young

    2015-01-01

    The anti-obesity effects of starter (Leuconostoc mesenteroides+Lactobacillus plantarum) fermented kimchi on 3T3-L1 adipocyte were studied using naturally fermented kimchi (NK), a functional kimchi (FK, NK supplemented with green tea), and FK supplemented with added starters (FKS). Oil red O staining and cellular levels of triglyceride (TG) and glycerol were used to evaluate the in vitro anti-obesity effects of these kimchis in 3T3-L1 cells. The expressions of adipogenesis/lipogenesis-related genes of peroxisome proliferator-active receptor (PPAR)-γ, CCAAT/enhance-binding protein (C/EBP)-α, and fatty acid synthase (FAS) were determined by RT-PCR. Kimchis, especially FKS, markedly decreased TG levels and increased levels of intracellular glycerol and lipid lipolysis. In addition, FKS also reduced the mRNA levels of PPAR-γ, C/EBP-α, and FAS, which are related to adipogenesis/lipogenesis in 3T3-L1 cells. These results suggest the anti-obesity effects of FKS were to due to enhanced lipolysis and reduced adipogenesis/lipogenesis in 3T3-L1 adipocytes. PMID:26770918

  6. T(3) LARYNGEAL-CANCER, PRIMARY SURGERY VS PLANNED COMBINED RADIOTHERAPY AND SURGERY

    NARCIS (Netherlands)

    TJHOHESLINGA, RE; TERHAARD, CHJ; SCHOUWENBURG, P; HILGERS, FJM; DOLSMA, WV; CROLL, GA; HOOGENHOUT, J; KNEGT, PP; LEER, JWH; HORDIJK, GJ

    1993-01-01

    The Dutch Co-operative Head and Neck Oncology Group performed a retrospective, nationwide study of laryngeal cancer between 1975 and 1984. The results for T3 laryngeal cancer treated with primary laryngectomy (n = 137) with post-operative radiotherapy when indicated or planned combined

  7. T-3, a packaging and transportation system for irradiated US breeder reactor experiments

    International Nuclear Information System (INIS)

    Hansen, L.J.; Berg, J.D.; Berger, J.D.; Weber, E.T.; Krupar, J.J.

    1980-01-01

    Irradiation testing of fast reactor fuels and materials in EBR-II, TREAT, and other US facilities required a packaging and transportation system that would ensure that changes in the specimens were irradiation induced and not the result of transport. The T-2 cask and the development of the T-3 cask are discussed

  8. The identification and investigation of synergistic combinations of corrosion inhibitors for AA2024-T3

    Science.gov (United States)

    Chambers, Brian

    2007-12-01

    The overall goal of this research was to identify and investigate possible replacements of chromate corrosion inhibitive pigments for aluminum aerospace alloys. This research project was divided into two objectives: (1) to develop high throughput screening (HTS) methods that rapidly and quantitatively assess inhibitor and inhibitor mixture efficacy on aluminum alloy 2024-T3 (AA2024-T3) for the purpose of identifying synergies, and (2) to investigate the mechanisms of inhibitor interaction for a selected combination of compounds that demonstrate synergistic or antagonistic behavior. Thirteen inorganic compounds were selected from a review of the literature as promising candidates for AA2024-T3 inhibition or synergists with other inhibitors. These thirteen compounds, a large number of combinations, and sodium chromate were evaluated at 3.4 mM in 0.6 M NaCl to simulate the harsh conditions in which inhibitive pigments must perform. Three rapid test methods were developed and used to assess corrosion inhibitor performance on AA2024-T3: (1) the current at a fixed DC potential (100 mV) between two AA2024-T3 wire electrodes (50 parallel cells), (2) the cyclic voltammetric measurement of surface Cu following 24 hour open circuit (OC) exposure (96 parallel cells), and (3) the fluorometric measurement of aluminum ion concentration resulting from 24 hour OC corrosion (96 parallel cells). These methods were utilized to assess inhibitor performance on AA2024-T3 as a function of mixture ratio, pH, and concentration. A number of binary inhibitor mixtures demonstrated both potent and broad-range synergy as identified by the HTS methods. Two binary mixtures, cerium chloride with sodium metavanadate and lanthanum chloride with sodium molybdate, were selected for more detailed investigation due to their antagonistic and synergistic interaction respectively. Experimental evidence supports the hypothesis that the antagonistic interaction between cerium and metavanadate originates from

  9. Osteogenic gene expression of murine osteoblastic (MC3T3-E1) cells under cyclic tension

    International Nuclear Information System (INIS)

    Kao, C T; Chen, C C; Cheong, U-I; Liu, S L; Huang, T H

    2014-01-01

    Low-level laser therapy (LLLT) can promote cell proliferation. The remodeling ability of the tension side of orthodontic teeth affects post-orthodontic stability. The purpose of the present study was to investigate the osteogenic effects of LLLT on osteoblast-like cells treated with a simulated tension system that provides a mechanical tension regimen. Murine osteoblastic (MC3T3-E1) cells were cultured in a Flexcell strain unit with programmed loads of 12% elongation at a frequency of 0.5 Hz for 24 and 48 h. The cultured cells were treated with a low-level diode laser using powers of 5 J and 10 J. The proliferation of MC3T3-E1 cells was determined using the Alamar Blue assay. The expression of osteogenic genes (type I collagen (Col-1), osteopontin (OPN), osteocalcin (OC), osteoprotegerin (OPG), receptor activator of nuclear factor kappa B ligand (RANKL), bone morphologic protein (BMP-2), and bone morphologic protein (BMP-4)) in MC3T3-E1 cells was analyzed using reverse transcription polymerase chain reaction (RT-PCR). The data were analyzed using one-way analysis of variance. The proliferation rate of tension-cultured MC3T3-E1 cells under 5 J and 10 J LLLT increased compared with that of the control group (p < 0.05). Prominent mineralization of the MC3T3-E1 cells was visible using a von Kossa stain in the 5 J LLLT group. Osteogenic genes (Col-1, OC, OPG and BMP-2) were significantly expressed in the MC3T3-E1 cells treated with 5 J and 10 J LLLT (p < 0.05). LLLT in tension-cultured MC3T3-E1 cells showed synergistic osteogenic effects, including increases in cell proliferation and Col-1, OPN, OC, OPG and BMP-2 gene expression. LLLT might be beneficial for bone remodeling on the tension side of orthodontics. (paper)

  10. Cardamom extract induces cell proliferation by increasing potassium currents in NIH3T3 cell line.

    Science.gov (United States)

    Siddiqui, Sonia; Hassan, Sohail; Imran, Sumaira; Khan, Faisal; Ahmed, Faheem; Dar, Asim

    2017-11-01

    Amommum subulatum (Roxb.) or Cardamom extract is known to have anti-inflammatory and neuroprotective effects towards many gastrointestinal related problems. However, uptill now different fractions of cardamom extract on fibroblasts with respect to potassium channel activity have not been investigated. Therefore, present study investigated the effects of different fractions of cardamom extract on potassium channels in non-tumor NIH3T3 cell line. Phytochemical analysis of hydroalcoholic, n-hexane, butane and ethyl acetate fractions of cardamom extracts were purified and isolated by thin layer chromatography (TLC). 3T3 cells were cultured and incubated with hydroalcohol (1-2 μ/ml), n-hexane (1 μ/ml), butane (2 μ/ml) and ethyl acetate (1-2 μ/ml) for 5 hrs at 37°C. Modulation in potassium currents were recorded by whole-cell patch clamp method. The data showed two constituents Cineol (C 10 H 18 O) and Terpinyl acetate (C10H17OOCCH3) by TLC method. The present study shows that the constituents in n-hexane, hydro alcohol (1 μ/ml) and ethyl acetate (2 μ/ml) significantly increased (p<0.01) the potassium outward rectifying currents from NIH3T3 cells when compared to untreated controls cells. Whereas, butanol fraction (2 μ/ml) significantly decreased (p<0.01) the inward rectifying currents when compared to controls. Moreover hydroalcoholic and n-hexane fractions have increased the proliferation in 3T3 cell line. On the other hand butanol and ethyl acetate did not induce proliferation in 3T3 cells. Taken together, our data suggested that cardamom extract contains constituents that increased K+ currents, cell migration and proliferation and are involved in wound healing.

  11. Isolation and characterization of NIH 3T3 cells expressing polyomavirus small T antigen

    Energy Technology Data Exchange (ETDEWEB)

    Noda, T.; Satake, M.; Robins, T.; Ito, Y.

    1986-10-01

    The polyomavirus small T-antigen gene, together with the polyomavirus promoter, was inserted into retrovirus vector pGV16 which contains the Moloney sarcoma virus long terminal repeat and neomycin resistance gene driven by the simian virus 40 promoter. This expression vector, pGVST, was packaged into retrovirus particles by transfection of PSI2 cells which harbor packaging-defective murine retrovirus genome. NIH 3T3 cells were infected by this replication-defective retrovirus containing pGVST. Of the 15 G418-resistant cell clones, 8 express small T antigen at various levels as revealed by immunoprecipitation. A cellular protein with an apparent molecular weight of about 32,000 coprecipitates with small T antigen. Immunofluorescent staining shows that small T antigen is mainly present in the nuclei. Morphologically, cells expressing small T antigen are indistinguishable from parental NIH 3T3 cells and have a microfilament pattern similar to that in parental NIH 3T3 cells. Cells expressing small T antigen form a flat monolayer but continue to grow beyond the saturation density observed for parental NIH 3T3 cells and eventually come off the culture plate as a result of overconfluency. There is some correlation between the level of expression of small T antigen and the growth rate of the cells. Small T-antigen-expressing cells form small colonies in soft agar. However, the proportion of cells which form these small colonies is rather small. A clone of these cells tested did not form tumors in nude mice within 3 months after inoculation of 10/sup 6/ cells per animal. Thus, present studies establish that the small T antigen of polyomavirus is a second nucleus-localized transforming gene product of the virus (the first one being large T antigen) and by itself has a function which is to stimulate the growth of NIH 3T3 cells beyond their saturation density in monolayer culture.

  12. Synthetic peptide TEKKRRETVEREKE derived from ezrin induces differentiation of NIH/3T3 fibroblasts.

    Science.gov (United States)

    Chulkina, Marina; Negmadjanov, Ulugbek; Lebedeva, Ekaterina; Pichugin, Aleksey; Mazurov, Dmitriy; Ataullakhanov, Ravshan; Holmuhamedov, Ekhson

    2017-09-15

    Synthetic 14 AA peptide (Gepon) derived from the hinge region of ezrin, a protein that links cell surface molecules to intracellular actin filaments, accelerates and facilitates wound and ulcer healing in clinical applications. However, the molecular mechanisms underlying this phenomenon and involved in enhanced healing of wounds with Gepon are not yet understood. The purpose of current study was to investigate intracellular signaling pathways involved in the effect of this peptide on wild type and genetically modified (CD44 KO) NIH/3T3 embryonic mouse fibroblasts. Gepon treatment of NIH/3T3 cells resulted in morphological and biochemical changes, characteristic of differentiated fibroblasts. While treatment of NIH/3T3 cells with TGF-β1 triggered the activation of both canonical and non-canonical signaling pathways, exposure of fibroblasts to Gepon activated only the ERK1/2 dependent pathway without modulating SMAD dependent signaling pathway. Knocking out hyaluronic acid CD44 receptor did not change Gepon or TGF-β1 dependent activation of intracellular signaling pathways and assembling of α-SMA-positive filaments. Gepon dependent differentiation of NIH/3T3 fibroblasts is based on activation of ERK1/2 kinase, non-canonical intracellular signaling pathway. Our data suggest that the treatment of fibroblasts with Gepon triggers activation of the non-canonical (SMAD independent) intracellular signaling pathway that involves ERK1/2kinase phosphorylation. Activation of the MAPK signaling pathway and the increase in formation of α-SMA containing stress filaments induced by Gepon were independent on presence of CD44 receptor in NIH/3T3 fibroblasts. Thus, our observation designates the significance and sufficiency of MAPK pathway mediated activation of fibroblasts with Gepon for healing of erosion, ulcers and wounds. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Clinically-staged T3N0 rectal cancer: is preoperative chemoradiotherapy the optimal treatment?

    Science.gov (United States)

    Lombardi, Raffaele; Cuicchi, Dajana; Pinto, Carmine; Di Fabio, Francesca; Iacopino, Bruno; Neri, Stefano; Tardio, Maria Lucia; Ceccarelli, Claudio; Lecce, Ferdinando; Ugolini, Giampaolo; Pini, Sara; Di Tullio, Piergiorgio; Taffurelli, Mario; Minni, Francesco; Martoni, Andrea; Cola, Bruno

    2010-03-01

    Preoperative chemoradiotherapy has been widely adopted as the standard of care for stage II-III rectal cancers. However, patients with T3N0 lesions had been shown to have a better prognosis than other categories of locally advanced tumor. Thus, neoadjuvant chemoradiation is likely to be overtreatment in this subgroup of patients. Nevertheless, the low accuracy rate of preoperative staging techniques for detection of node-negative tumors does not allow to check this hypothesis. We analyzed a group of patients with cT3N0 low rectal cancer who underwent neoadjuvant chemoradiotherapy with the purpose of evaluating the incidence of metastatic nodes in the resected specimens. Between January 2002 and February 2008, 100 patients with low rectal cancer underwent clinical staging by means of endorectal ultrasound, computed tomography, positron emission tomography, and magnetic resonance imaging. All patients received preoperative 5-fluorouracil-based chemoradiotherapy and surgical resection with curative aim. Of 100 patients with locally advanced rectal cancer, 32 were clinically staged as T3N0M0. Pathological analysis showed the presence of lymph node metastases in nine patients (28%) (node-positive group). In the remaining 23 cases, clinical N stage was confirmed at pathology (node-negative group). Node-positive and node-negative groups differ only in the number of ypT3 tumors (P cancer represents an undertreatment risk in at least 28% of cases, making necessary the use of postoperative chemoradiotherapy. Preoperative chemoradiotherapy should be the therapy of choice on the grounds of the principle that overtreatment is less hazardous than undertreatment for cT3N0 rectal cancers.

  14. The Yersinia enterocolitica type 3 secretion system (T3SS) as toolbox for studying the cell biological effects of bacterial Rho GTPase modulating T3SS effector proteins.

    Science.gov (United States)

    Wölke, Stefan; Ackermann, Nikolaus; Heesemann, Jürgen

    2011-09-01

    The bacterial effector proteins IpgB(1) and IpgB(2) of Shigella and Map of Escherichia coli activate the Rho GTPases Rac1, RhoA and Cdc42, respectively, whereas YopE and YopT of Yersinia inhibit these Rho family GTPases. We established a Yersinia toolbox which allows to study the cellular effects of these effectors in different combinations in the context of Yersinia type 3 secretion system (Ysc)-T3SS-mediated injection into HeLa cells. For this purpose hybrid proteins were constructed by fusion of YopE with the effector protein of interest. As expected, injected hybrid proteins induced membrane ruffles and Yersinia uptake for IpgB(1) , stress fibres for IpgB(2) and microspikes for Map. By co-infection experiments we could demonstrate (i) IpgB(2) -mediated and ROCK-dependent inhibition of IpgB(1) -mediated Rac1 effects, (ii) YopT-mediated suppression of IpgB(1) -induced Yersinia invasion and (iii) failure of YopE-mediated suppression of IpgB(1) -induced Yersinia invasion, presumably due to preferential inhibition of RhoG by YopE GAP function. By infecting polarized MDCK cells we could demonstrate that Map or IpgB(1) but not IpgB(2) affects cell monolayer integrity. In summary, the Yersinia toolbox is suitable to study cellular effects of effector proteins of diverse bacterial species separately or in combination in the context of bacterial T3SS-mediated injection. © 2011 Blackwell Publishing Ltd.

  15. Molecular characterisation of group IVA (cytosolic) phospholipase A2 in murine osteoblastic MC3T3-E1 cells.

    Science.gov (United States)

    Leis, Hans Jörg; Windischhofer, Werner

    2015-02-01

    Formation of the powerful osteogenic prostaglandin E2 by osteoblasts, a key modulatory event in the paracrine and autocrine regulation of bone cell activity, is preceded by release of the precursor arachidonic acid from phospholipid stores. The main routes of arachidonate liberation may involve phospholipase enzymes such as group IVA phospholipase A2 which is believed to be the main effector in many cell system due to its preference for arachidonate-containing lipids. MC3T3-E1 cells are non-transformed osteoblasts and are widely used as an in vitro model of osteoblast function. In these cells there is still no clarity about the main release pathway of arachidonic acid. Besides cytosolic phospholipase A2, phospholipase C and D pathways may play a key role in arachidonate release. Despite the crucial role of osteoblastic prostgalandin synthesis information on the occurrence of involved enzymes at the molecular level is scarse in MC3T3-E1 cells. We have characterised group IVA phospholipase A2 at the mRNA in these cells as a constitutively expressed enzyme which is cytosolic and translocates to the membrane upon endothelin-1 stimulation. Using immunopurification combined with Western blotting and high-resolution mass spectrometry, the enzyme was also identified at the protein level. Using specific gene silencing we were able to show that osteoblastic cytosolic phospholipase A2 is crucially involved in ET-1-induced prostaglandin formation.

  16. Integrability of the superstring in AdS3 × S 2 × S 2 × T 3

    Science.gov (United States)

    Wulff, Linus

    2017-06-01

    Type II supergravity admits an AdS_3× S^2× S^2× T3 solution with fluxes depending on several free parameters. We determine the constraints on these parameters imposed by the requirement of (classical) integrability of the superstring sigma model. To do this we analyze the low-energy effective action for the spinning GKP string. The absence of particle production in the tree-level S-matrix of bosonic excitations is shown to imply the vanishing of two of the four parameters in the NSNS three-form flux. This reduces the supergravity background to either the one-parameter AdS_3× S^2× S^2× T3 background preserving eight supersymmetries, or a non-supersymmetric branch, which differs only by flipping a sign in the RR flux. We show that both these branches can be obtained from AdS_3× S^3× S^3× S1 by T-dualities on the (Hopf) circle fibers of the three-spheres and therefore the integrability of the string in these backgrounds follows.

  17. Gene expression analysis and microdialysis suggest hypothalamic triiodothyronine (T3) gates daily torpor in Djungarian hamsters (Phodopus sungorus).

    Science.gov (United States)

    Bank, Jonathan H H; Cubuk, Ceyda; Wilson, Dana; Rijntjes, Eddy; Kemmling, Julia; Markovsky, Hanna; Barrett, Perry; Herwig, Annika

    2017-07-01

    Thyroid hormones play an important role in regulating seasonal adaptations of mammals. Several studies suggested that reduced availability of 3,3',5-triiodothyronine (T3) in the hypothalamus is required for the physiological adaptation to winter in Djungarian hamsters. We have previously shown that T3 is involved in the regulation of daily torpor, but it remains unclear, whether T3 affects torpor by central or peripheral mechanisms. To determine the effect of T3 concentrations within the hypothalamus in regulating daily torpor, we tested the hypothesis that low hypothalamic T3 metabolism would favour torpor and high T3 concentrations would not. In experiment 1 gene expression in torpid hamsters was assessed for transporters carrying thyroid hormones between cerebrospinal fluid and hypothalamic cells and for deiodinases enzymes, activating or inactivating T3 within hypothalamic cells. Gene expression analysis suggests reduced T3 in hypothalamic cells during torpor. In experiment 2, hypothalamic T3 concentrations were altered via microdialysis and torpor behaviour was continuously monitored by implanted body temperature transmitters. Increased T3 concentrations in the hypothalamus reduced expression of torpor as well as torpor bout duration and depth. Subsequent analysis of gene expression in the ependymal layer of the third ventricle showed clear up-regulation of T3 inactivating deiodinase 3 but no changes in several other genes related to photoperiodic adaptations in hamsters. Finally, serum analysis revealed that increased total T3 serum concentrations were not necessary to inhibit torpor expression. Taken together, our results are consistent with the hypothesis that T3 availability within the hypothalamus significantly contributes to the regulation of daily torpor via a central pathway.

  18. Escherichia coli type III secretion system 2: a new kind of T3SS?

    Science.gov (United States)

    Zhou, Mingxu; Guo, Zhiyan; Duan, Qiangde; Hardwidge, Philip R; Zhu, Guoqiang

    2014-03-19

    Type III secretion systems (T3SSs) are employed by Gram-negative bacteria to deliver effector proteins into the cytoplasm of infected host cells. Enteropathogenic Escherichia coli use a T3SS to deliver effector proteins that result in the creation of the attaching and effacing lesions. The genome sequence of the Escherichia coli pathotype O157:H7 revealed the existence of a gene cluster encoding components of a second type III secretion system, the E. coli type III secretion system 2 (ETT2). Researchers have revealed that, although ETT2 may not be a functional secretion system in most (or all) strains, it still plays an important role in bacterial virulence. This article summarizes current knowledge regarding the E. coli ETT2, including its genetic characteristics, prevalence, function, association with virulence, and prospects for future work.

  19. Volume-sensitive NADPH oxidase activity and taurine efflux in NIH3T3 mouse fibroblasts

    DEFF Research Database (Denmark)

    Friis, Martin Barfred; Vorum, Katrine Gribel; Lambert, Ian Henry

    2008-01-01

    Reactive oxygen species (ROS) are produced in NIH3T3 fibroblasts during hypotonic stress, and H(2)O(2) potentiates the concomitant release of the organic osmolyte taurine (Lambert IH. J Membr Biol 192: 19-32, 2003). The increase in ROS production [5-(and-6)-carboxy-2', 7'-dichlorodihydrofluorescein......M) but is unaffected by the nitric oxide synthase inhibitor N omega-nitro-l-arginine methyl ester, indicating that the volume-sensitive ROS production is NADPH oxidase dependent. NIH3T3 cells express the NADPH oxidase components: p22 phox, a NOX4 isotype; p47 phox; and p67 phox (real-time PCR). Exposure to the Ca2...

  20. Investigation of Droplet Deposition for Suspensions Usable for Thermoplastic 3D Printing (T3DP)

    Science.gov (United States)

    Scheithauer, Uwe; Johne, Robert; Weingarten, Steven; Schwarzer, Eric; Richter, Hans-Jürgen; Moritz, Tassilo; Michaelis, Alexander

    2018-01-01

    Thermoplastic 3D printing (T3DP) is an additive manufacturing (AM) technology, which can be used for the production of dense single- and especially multi-material components. This becomes possible because of the combination of the precise deposition of small droplets of molten thermoplastic suspensions containing ceramic or metal particles, and a curing mechanism caused on cool down increasing the viscosity. In this paper, the droplet formation behavior of zirconia suspensions for T3DP (82 and 84 wt.%) was investigated. The droplet fusion factor (dff) is introduced to calculate the necessary distance between two droplets to form filament-like structures by fusion of adjacent droplets. Filament-like structures with a smooth surface and a nearly homogeneous cross section were manufactured for both suspensions with a dff of 44% or higher.

  1. Evaluation of T3B fingerprinting for identification of clinical and environmental Sporothrix species.

    Science.gov (United States)

    Oliveira, Manoel Marques Evangelista; Franco-Duarte, Ricardo; Romeo, Orazio; Pais, Célia; Criseo, Giuseppe; Sampaio, Paula; Zancope-Oliveira, Rosely Maria

    2015-03-01

    In this study, PCR fingerprinting using the universal primer T3B was applied to distinguish among clinical and environmental species of the Sporothrix complex, Sporothrix brasiliensis, S. globosa, S. mexicana, S. pallida, S. luriei and S. schenckii sensu stricto. The T3B fingerprinting generated clearly distinct banding patterns, allowing the correct identification of all 43 clinical and environmental isolates at the species level, what was confirmed by partial calmodulin gene sequence analyses. This technique is reproducible and provides the identification of all species of the Sporothrix complex with sufficient accuracy to be applied in clinical mycology laboratories as well as in epidemiological studies in order to obtain a better understanding of the epidemiology of sporotrichosis. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  2. Development of a solid phase technic for radioimmunoassay of triiodothyronine (T3) in serum

    International Nuclear Information System (INIS)

    Hamada, M.M.; Mesquita, C.H.; Silva, C.P.G. da.

    1988-10-01

    We have developed a solid phase radioimmunoassay (RIA) system for triiodothyronine (T 3 ), by immobilizing triiodothyronine antibodies on the inner wall of reaction tubes. The antibody-coated tubes were made via reaction of antibody with glutaraldeyde residue pre coated on the inner wall of the tubes by alkaline self-polimerization. The quality of the coated tubes was tested through its performance in the RIA methodology, by analysing the following RIA parameters: minimum detectable dose (MDD), nonspecific binding (NSB), ''X 50% '', slope of the standard curve, intra and inter-assay precision, accuracy of the method and figure of merit. The serum levels of T 3 in hypothyroid and hyperthyroid patients and the normal values range were determined for the solid phase RIA system. The results are in agreement with found in the literature. (author) [pt

  3. Expression of Nanog gene promotes NIH3T3 cell proliferation

    International Nuclear Information System (INIS)

    Zhang Jingyu; Wang Xia; Chen Bing; Suo Guangli; Zhao Yanhong; Duan Ziyuan; Dai Jianwu

    2005-01-01

    Cells are the functional elements in tissue engineering and regenerative medicine. A large number of cells are usually needed for these purposes. However, there are numbers of limitations for in vitro cell proliferation. Nanog is an important self-renewal determinant in embryonic stem cells. However, it remains unknown whether Nanog will influence the cell cycle and cell proliferation of mature cells. In this study, we expressed Nanog in NIH3T3 cells and showed that expression of Nanog in NIH3T3 promoted cells to enter into S phase and enhanced cell proliferation. This suggests that Nanog gene might function in a similar fashion in mature cells as in ES cells. In addition, it may provide an approach for in vitro cell expansion

  4. Deltamethrin increases the fat accumulation in 3T3-L1 adipocytes and Caenorhabditis elegans.

    Science.gov (United States)

    Shen, Peiyi; Hsieh, Tsung-Hsiu; Yue, Yiren; Sun, Quancai; Clark, John M; Park, Yeonhwa

    2017-03-01

    Research has shown that permethrin, a Type-I pyrethroid, increases triglyceride (fat) accumulation in adipocytes. Little is known, however, about any similar effect of deltamethrin, a Type-II pyrethroid, which produces a distinct syndrome of poisoning in mammals compared with permethrin. This study was therefore aimed to explore the role of deltamethrin on fat accumulation in 3T3-L1 adipocytes and Caenorhabditis elegans. Deltamethrin (10 μM) significantly increased the fat accumulation in 3T3-L1 adipocytes and wild type C. elegans compared to respective controls. Deltamethrin decreased the ratio of phosphorylated AMP-activated kinase (pAMPKα) over AMPKα and phosphorylated acetyl-CoA carboxylase (ACC) over ACC, while it increased expression of CCAAT/enhancer-binding protein (C/EBPα) and peroxisome proliferator-activated receptor-γ (PPARγ) in 3T3-L1 adipocytes. Similarly, deltamethrin potentiated fat accumulation in C. elegans without affecting growth or pharyngeal pumping rate. Moreover, deltamethrin significantly reduced the total progeny number and locomotive activities in C. elegans in a dose-dependent manner. Deltamethrin increased fat accumulation via aak-2 (an ortholog of AMPKα) and nhr-49 (a homolog of peroxisome proliferator-activated receptor-α and also downstream target of aak-2) mediated mechanisms. The current work is the first report of the effects of deltamethrin on increased fat storage by 3T3- L1 adipocytes and C. elegans via aak-2 (AMPKα ortholog)-mediated mechanism. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. Pseudomonas aeruginosa uses T3SS to inhibit diabetic wound healing

    Science.gov (United States)

    Goldufsky, Josef; Wood, Stephen J.; Jayaraman, Vijayakumar; Majdobeh, Omar; Chen, Lin; Qin, Shanshan; Zhang, Chunxiang; DiPietro, Luisa A.; Shafikhani, Sasha H.

    2015-01-01

    Diabetic foot ulcers are responsible for more hospitalizations than any other complication of diabetes. Bacterial infection is recognized as an important factor associated with impaired healing in diabetic ulcers. Pseudomonas aeruginosa is the most frequently detected Gram-negative pathogen in diabetic ulcers. P. aeruginosa infection has been shown to impair healing in diabetic wounds in a manner that correlates with its ability to form biofilm. While the majority of infections in diabetic ulcers are biofilm associated, 33% of infections are nonbiofilm in nature. P. aeruginosa is the most prevalent Gram-negative pathogen in all diabetic wound types, which suggests that the deleterious impact of P. aeruginosa on healing in diabetic wounds goes beyond its ability to form biofilm and likely involves other factors. The Type III Secretion System (T3SS) virulence structure is required for the pathogenesis of all P. aeruginosa clinical isolates, suggesting that it may also play a role in the inhibition of wound repair in diabetic skin ulcers. We evaluated the role of T3SS in mediating P. aeruginosa–induced tissue damage in the wounds of diabetic mice. Our data demonstrate that P. aeruginosa establishes a robust and persistent infection in diabetic wounds independent of its ability to form biofilm and causes severe wound damage in a manner that primarily depends on its T3SS. PMID:25912785

  6. [Incorporation of Copper Ions into T2/T3 Centers of Two-Domain Laccases].

    Science.gov (United States)

    Gabdulkhakov, A G; Kostareva, O S; Kolyadenko, I A; Mikhaylina, A O; Trubitsina, L I; Tishchenko, S V

    2018-01-01

    Laccase belongs to the family of copper-containing oxidases. A study was made of the mechanism that sustains the incorporation of copper ions into the T2/T3 centers of recombinant two-domain laccase Streptomyces griseoflavus Ac-993. The occupancy of the T3 center by copper ions was found to increase with an increasing copper content in the culture medium and after dialysis of the protein preparation against a copper sulfate-containing buffer. The T2 center was filled only when overproducer strain cells were grown at a higher copper concentration in the medium. Two-domain laccases were assumed to possess a channel that serves to deliver copper ions to the T3 center during the formation of the three-dimensional laccase conformation and dialysis of the protein preparation. A narrower channel leads to the T2 center in two-domain laccases compared with three-domain ones, rendering the center less accessible for copper atoms. The incorporation of copper ions into the T2 center of two-domain laccases is likely to occur in the course of their biosynthesis or the formation of a functional trimer.

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

    Energy Technology Data Exchange (ETDEWEB)

    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); 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); Hiranaka, Natsumi; Suzuki, Takeshi [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); Kanazawa, Kaoru [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); 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); Ieko, Masahiro [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-γ 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-α and interleukin-1β 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-γ in 3T3-L1 adipocytes. Activation of peroxisome proliferator-activated receptor-γ 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.

  8. Characterization of V. cholerae T3SS-dependent cytotoxicity in cultured intestinal epithelial cells.

    Science.gov (United States)

    Miller, Kelly A; Chaand, Mudit; Gregoire, Stacy; Yoshida, Takeshi; Beck, Lisa A; Ivanov, Andrei I; Dziejman, Michelle

    2016-12-01

    AM-19226 is a pathogenic, non-O1/non-O139 serogroup strain of Vibrio cholerae that uses a Type 3 Secretion System (T3SS) mediated mechanism to colonize host tissues and disrupt homeostasis, causing cholera. Co-culturing the Caco2-BBE human intestinal epithelial cell line with AM-19226 in the presence of bile results in rapid mammalian cell death that requires a functional T3SS. We examined the role of bile, sought to identify the mechanism, and evaluated the contributions of T3SS translocated effectors in in vitro cell death. Our results suggest that Caco2-BBE cytotoxicity does not proceed by apoptotic or necrotic mechanisms, but rather displays characteristics consistent with osmotic lysis. Cell death was preceded by disassembly of epithelial junctions and reorganization of the cortical membrane skeleton, although neither cell death nor cell-cell disruption required VopM or VopF, two effectors known to alter actin dynamics. Using deletion strains, we identified a subset of AM-19226 Vops that are required for host cell death, which were previously assigned roles in protein translocation and colonization, suggesting that they function other than to promote cytotoxicity. The collective results therefore suggest that cooperative Vop activities are required to achieve cytotoxicity in vitro, or alternatively, that translocon pores destabilize the membrane in a bile dependent manner. © 2016 John Wiley & Sons Ltd.

  9. Pseudomonas aeruginosa uses T3SS to inhibit diabetic wound healing.

    Science.gov (United States)

    Goldufsky, Josef; Wood, Stephen J; Jayaraman, Vijayakumar; Majdobeh, Omar; Chen, Lin; Qin, Shanshan; Zhang, Chunxiang; DiPietro, Luisa A; Shafikhani, Sasha H

    2015-01-01

    Diabetic foot ulcers are responsible for more hospitalizations than any other complication of diabetes. Bacterial infection is recognized as an important factor associated with impaired healing in diabetic ulcers. Pseudomonas aeruginosa is the most frequently detected Gram-negative pathogen in diabetic ulcers. P. aeruginosa infection has been shown to impair healing in diabetic wounds in a manner that correlates with its ability to form biofilm. While the majority of infections in diabetic ulcers are biofilm associated, 33% of infections are nonbiofilm in nature. P. aeruginosa is the most prevalent Gram-negative pathogen in all diabetic wound types, which suggests that the deleterious impact of P. aeruginosa on healing in diabetic wounds goes beyond its ability to form biofilm and likely involves other factors. The Type III Secretion System (T3SS) virulence structure is required for the pathogenesis of all P. aeruginosa clinical isolates, suggesting that it may also play a role in the inhibition of wound repair in diabetic skin ulcers. We evaluated the role of T3SS in mediating P. aeruginosa-induced tissue damage in the wounds of diabetic mice. Our data demonstrate that P. aeruginosa establishes a robust and persistent infection in diabetic wounds independent of its ability to form biofilm and causes severe wound damage in a manner that primarily depends on its T3SS. © 2015 by the Wound Healing Society.

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

    International Nuclear Information System (INIS)

    Takahashi, Nobuhiko; Yoshizaki, Takayuki; Hiranaka, Natsumi; Suzuki, Takeshi; Yui, Tomoo; Akanuma, Masayoshi; Kanazawa, Kaoru; Yoshida, Mika; Naito, Sumiyoshi; Fujiya, Mikihiro; Kohgo, Yutaka; Ieko, Masahiro

    2013-01-01

    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-γ 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-α and interleukin-1β 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-γ in 3T3-L1 adipocytes. Activation of peroxisome proliferator-activated receptor-γ 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

  11. Rubi Fructus (Rubus coreanus Inhibits Differentiation to Adipocytes in 3T3-L1 Cells

    Directory of Open Access Journals (Sweden)

    Mi-Young Jeong

    2013-01-01

    Full Text Available Rubi Fructus (RF is known to exert several pharmacological effects including antitumor, antioxidant, and anti-inflammatory activities. However, its antiobesity effect has not been reported yet. This study was focused on the antidifferentiation effect of RF extract on 3T3-L1 preadipocytes. When 3T3-L1 preadipocytes were differentiating into adipocytes, 10–100 μg/mL of RF was added. Next, the lipid contents were quantified by Oil Red O staining. RF significantly reduced lipid accumulation and downregulated the expression of peroxisome proliferator-activated receptor γ (PPARγ, CCAAT0-enhancer-binding proteins α (C/EBPα, adipocyte fatty acid-binding protein 2 (aP2, resistin, and adiponectin in ways that were concentration dependent. Moreover, RF markedly upregulated liver kinase B1 and AMP-activated protein kinase (AMPK. Interestingly, pretreatment with AMPKα siRNA and RF downregulated the expression of PPARγ and C/EBPα protein as well as the adipocyte differentiation. Our study shows that RF is capable of inhibiting the differentiation of 3T3-L1 adipocytes through the modulation of PPARγ, C/EBPα, and AMPK, suggesting that it has a potential for therapeutic application in the treatment or prevention of obesity.

  12. Evaluation of cytotoxicity and gelatinases activity in 3T3 fibroblast cell by root repair materials

    Directory of Open Access Journals (Sweden)

    Varol Basak

    2016-09-01

    Full Text Available The aim of this study was to investigate the effects of calcium silicate-based products on cytotoxicity in the 3T3 fibroblast and gelatinolytic activity of matrix metalloproteinases (MMPs. 3T3 fibroblasts were incubated directly with Ortho Mineral trioxide aggregate (MTA, BioAggregate, Biodentine, MTA Plus, MTA Angelus and MTA Cerkamed for 24 hours and seven days. The cytotoxicity was determined using an MTT assay. Supernatants were collected to determine MMP-2 and MMP-9. Data were analysed using IBM SPSS 22. Seventh day extracts of Ortho MTA and Biodentine showed reduced cell viability. Specific characterization of MMPs in cell culture demonstrated that MMP-2 (62 kPa in the cell culture supernatants by gelatin zymography showed induced expression in four out of seven groups by 3T3 cells. No MMP-9 expression was observed. The cytotoxicity of materials revealed a significant difference in cell viability between the groups on the first and seventh days. The results of this study revealed minor cytotoxic effects for Ortho MTA and Biodentine. This study suggests that endodontic sealers induced production of MMP-2. MMP-9 might be expressed in small amounts when compared with MMP-2.

  13. The lipid fraction of human milk initiates adipocyte differentiation in 3T3-L1 cells.

    Science.gov (United States)

    Fujisawa, Yasuko; Yamaguchi, Rie; Nagata, Eiko; Satake, Eiichiro; Sano, Shinichiro; Matsushita, Rie; Kitsuta, Kazunobu; Nakashima, Shinichi; Nakanishi, Toshiki; Nakagawa, Yuichi; Ogata, Tsutomu

    2013-09-01

    The prevalence of childhood obesity has increased worldwide over the past decade. Despite evidence that human milk lowers the risk of childhood obesity, the mechanism is not fully understood. We investigated the direct effect of human milk on differentiation of 3T3-L1 preadipocytes. 3T3-L1 preadipocytes were treated with donated human milk only or the combination of the standard hormone mixture; insulin, dexamethasone (DEX), and 3-isobututyl-1-methylxanthine (IBMX). Furthermore, the induction of preadipocyte differentiation by extracted lipids from human milk was tested in comparison to the cells treated with lipid extracts from infant formula. Adipocyte differentiation, specific genes as well as formation of lipid droplets were examined. We clearly show that lipids present in human milk initiate 3T3-L1 preadipocyte differentiation. In contrast, this effect was not observed in response to lipids present in infant formula. The initiation of preadipocyte differentiation by human milk was enhanced by adding the adipogenic hormone, DEX or insulin. The expression of late adipocyte markers in Day 7 adipocytes that have been induced into differentiation with human milk lipid extracts was comparable to those in control cells initiated by a standard adipogenic hormone cocktail. These results demonstrate that human milk contains bioactive lipids that can initiate preadipocyte differentiation in the absence of the standard adipogenic compounds via a unique pathway. Copyright © 2013 Elsevier Ltd. All rights reserved.

  14. An experimental investigation of fatigue damage in aluminum 2024-T3 alloys

    Science.gov (United States)

    Ferguson, Milton W.

    1993-01-01

    Aluminum alloys are finding increasing use in the aerospace and automobile industries due to their attractive low density-high modulus and low density-high strength characteristics. Unfortunately, cyclic stress-strain deformation alters the microstructure of the material. These structural changes can lead to fatigue damage and ultimately service failure. Therefore, in order to assess the integrity of the alloy, a correlation between fatigue damage and a measurable microstructural property is needed. Aluminum 2024-T3, a commonly used commercial alloy, contains many grains (individual crystals) of various orientations. The sizes and orientations of these grains are known to affect the strength, hardness, and magnetic permeability of polycrystalline alloys and metals; therefore, perhaps a relationship between a grain property and the fatigue state can be established. Tension-compression cycling in aluminum alloys can also induce changes in their dislocation densities. These changes can be studied from measurements of the electrical resistivities of the materials. Consequently, the goals of this investigation were: to study the grain orientation of aluminum 2024-T3 and to seek a correlation between the grain orientation and the fatigue state of the material; and to measure the electrical resistivities of fatigued samples of aluminum 2024-T3 and to interpret the findings.

  15. Stevioside from Stevia rebaudiana Bertoni Increases Insulin Sensitivity in 3T3-L1 Adipocytes

    Directory of Open Access Journals (Sweden)

    Nabilatul Hani Mohd-Radzman

    2013-01-01

    Full Text Available Stevioside from Stevia rebaudiana has been reported to exert antihyperglycemic effects in both rat and human subjects. There have been few studies on these effects in vitro. In this paper, radioactive glucose uptake assay was implemented in order to assess improvements in insulin sensitivity in 3T3-L1 cells by elevation of glucose uptake following treatment with stevioside. Oil Red-O staining and MTT assay were utilized to confirm adipocyte differentiation and cell viability, respectively. Findings from this research showed a significant increase in absorbance values in mature adipocytes following Oil Red-O staining, confirming the differentiation process. Stevioside was noncytotoxic to 3T3-L1 cells as cell viability was reduced by a maximum of 17%, making it impossible to determine its IC50. Stevioside increased glucose uptake activities by 2.1 times (p<0.001 in normal conditions and up to 4.4 times (p<0.001 in insulin-resistant states. At times, this increase was higher than that seen in positive control group treated with rosiglitazone maleate, an antidiabetic agent. Expressions of pY20 and p-IRS1 which were measured via Western blot were improved by stevioside treatment. In conclusion, stevioside has direct effects on 3T3-L1 insulin sensitivity via increase in glucose uptake and enhanced expression of proteins involved in insulin-signalling pathway.

  16. Ginkgolide C Suppresses Adipogenesis in 3T3-L1 Adipocytes via the AMPK Signaling Pathway

    Directory of Open Access Journals (Sweden)

    Chian-Jiun Liou

    2015-01-01

    Full Text Available Ginkgolide C, isolated from Ginkgo biloba leaves, is a flavone reported to have multiple biological functions, from decreased platelet aggregation to ameliorating Alzheimer disease. The study aim was to evaluate the antiadipogenic effect of ginkgolide C in 3T3-L1 adipocytes. Ginkgolide C was used to treat differentiated 3T3-L1 cells. Cell supernatant was collected to assay glycerol release, and cells were lysed to measure protein and gene expression related to adipogenesis and lipolysis by western blot and real-time PCR, respectively. Ginkgolide C significantly suppressed lipid accumulation in differentiated adipocytes. It also decreased adipogenesis-related transcription factor expression, including peroxisome proliferator-activated receptor and CCAAT/enhancer-binding protein. Furthermore, ginkgolide C enhanced adipose triglyceride lipase and hormone-sensitive lipase production for lipolysis and increased phosphorylation of AMP-activated protein kinase (AMPK, resulting in decreased activity of acetyl-CoA carboxylase for fatty acid synthesis. In coculture with an AMPK inhibitor (compound C, ginkgolide C also improved activation of sirtuin 1 and phosphorylation of AMPK in differentiated 3T3-L1 cells. The results suggest that ginkgolide C is an effective flavone for increasing lipolysis and inhibiting adipogenesis in adipocytes through the activated AMPK pathway.

  17. Characterization of V. cholerae T3SS-dependent cytotoxicity in cultured intestinal epithelial cells

    Science.gov (United States)

    Miller, Kelly A.; Chaand, Mudit; Gregoire, Stacy; Yoshida, Takeshi; Beck, Lisa; Ivanov, Andrei I.; Dziejman, Michelle

    2016-01-01

    Summary AM-19226 is a pathogenic, non-O1/non-O139 serogroup strain of Vibrio cholerae that uses a Type 3 Secretion System (T3SS) mediated mechanism to colonize host tissues and disrupt homeostasis, causing cholera. Co-culturing the Caco2-BBE human intestinal epithelial cell line with AM-19226 in the presence of bile results in rapid mammalian cell death that requires a functional T3SS. We examined the role of bile, sought to identify the mechanism, and evaluated the contributions of T3SS translocated effectors in in vitro cell death. Our results suggest that Caco2-BBE cytotoxicity does not proceed by apoptotic or necrotic mechanisms, but rather displays characteristics consistent with osmotic lysis. Cell death was preceded by disassembly of epithelial junctions and reorganization of the cortical membrane skeleton, although neither cell death nor cell-cell disruption required VopM or VopF, two effectors known to alter actin dynamics. Using deletion strains, we identified a subset of AM-19226 Vops that are required for host cell death, which were previously assigned roles in protein translocation and colonization, suggesting that they function other than to promote cytotoxicity. The collective results therefore suggest that cooperative Vop activities are required to achieve cytotoxicity in vitro, or alternatively, that translocon pores destabilize the membrane in a bile dependent manner. PMID:27302486

  18. Conversion of the thyroxin (T4) in 3,5,3'-triiodothyronin (T3) and 3,3',5'-triiodothyronin (T3 reverse) in human leukocytes suspensions. Hyperthyroidism and hypothyroidism studies

    International Nuclear Information System (INIS)

    Bianco, A.C.; Douglas, C.R.; Marone, M.M.; Scalissi, N.M.; Correa, P.H.S.

    1984-01-01

    The peripheral metabolism of thyroid hormones was studied in suspensions of human leukocytes through the evaluation of in vitro generation of T 3 and rT 3 (RIA) from non-radioactive T 4 . Increased in vitro generation of T 3 and rT 3 was observed in suspensions from hyperthyroid patients, while a significant decrease was evidenced when leukocytes from hypothyroid patients were used. These alterations are apparently due to the excess and lack of thyroid hormones, respectively, since they could be reserved in both cases by specific clinical treatment. (author) [pt

  19. Increased thyroidal T4 to T3 conversion in autonomously functioning thyroid adenoma: from euthyroidism to thyrotoxicosis.

    LENUS (Irish Health Repository)

    Solter, M

    2012-01-31

    AIM: The aim was to investigate whether the intrathyroid conversion of T4 to T3 in autonomously functioning thyroid adenoma (AFTA) tissue could influence serum T3 levels and suppression of TSH, especially in patients with borderline thyroid function. PATIENTS AND METHODS: In ten patients with AFTA, thyroidal conversion of T4 to T3 was investigated in nodular and paranodular, TSH-suppressed tissue. All patients had normal serum T4 and suppressed TSH. Serum T3 was normal in six, and borderline or slightly increased in four. AFTA and paranodular tissues were surgically removed and frozen at -70 degrees C, then homogenized in a glass homogenizer, centrifuged at 100,000xg, and particulate fraction collected as a pellet. Analysis mixture consisted of thyroid enzyme suspension in 50 mumol\\/L TRIS buffer with 5 mumol DTT and 200 muL 1.3 mumol T4. Incubation was performed at 37 degrees C and the generation of T3 measured after 5, 10, 20 and 40 minutes respectively. RESULTS: T3 production (pmol\\/mg protein) was significantly higher in AFTA than in paranodular tissues (8.8 1.2\\/Mean +\\/- SE\\/vs. 1.8 +\\/- 0.2; p<0.01), and excessively high (9.8, 14.1, 14.2 and 15.0) in four patients with borderline or slightly supranormal serum T3. A significant correlation was found between serum T3 concentrations and T3 generation (T4 conversion) in AFTA tissues. CONCLUSION: Results suggest that increased thyroidal T4 to T3 conversion in AFTA tissue could be involved in an increased delivery of T3, increased serum T3 and suppressed serum TSH, particularly in patients with the disease evolving from euthyroid to an early hyperthyroid phase.

  20. PPARγ partial agonist GQ-16 strongly represses a subset of genes in 3T3-L1 adipocytes

    Energy Technology Data Exchange (ETDEWEB)

    Milton, Flora Aparecida [Faculdade de Ciências da Saúde, Laboratório de Farmacologia Molecular, Universidade de Brasília (Brazil); Genomic Medicine, Houston Methodist Research Institute, Houston, TX (United States); Cvoro, Aleksandra [Genomic Medicine, Houston Methodist Research Institute, Houston, TX (United States); Amato, Angelica A. [Faculdade de Ciências da Saúde, Laboratório de Farmacologia Molecular, Universidade de Brasília (Brazil); Sieglaff, Douglas H.; Filgueira, Carly S.; Arumanayagam, Anithachristy Sigamani [Genomic Medicine, Houston Methodist Research Institute, Houston, TX (United States); Caro Alves de Lima, Maria do; Rocha Pitta, Ivan [Laboratório de Planejamento e Síntese de Fármacos – LPSF, Universidade Federal de Pernambuco (Brazil); Assis Rocha Neves, Francisco de [Faculdade de Ciências da Saúde, Laboratório de Farmacologia Molecular, Universidade de Brasília (Brazil); Webb, Paul, E-mail: pwebb@HoustonMethodist.org [Genomic Medicine, Houston Methodist Research Institute, Houston, TX (United States)

    2015-08-28

    Thiazolidinediones (TZDs) are peroxisome proliferator-activated receptor gamma (PPARγ) agonists that improve insulin resistance but trigger side effects such as weight gain, edema, congestive heart failure and bone loss. GQ-16 is a PPARγ partial agonist that improves glucose tolerance and insulin sensitivity in mouse models of obesity and diabetes without inducing weight gain or edema. It is not clear whether GQ-16 acts as a partial agonist at all PPARγ target genes, or whether it displays gene-selective actions. To determine how GQ-16 influences PPARγ activity on a gene by gene basis, we compared effects of rosiglitazone (Rosi) and GQ-16 in mature 3T3-L1 adipocytes using microarray and qRT-PCR. Rosi changed expression of 1156 genes in 3T3-L1, but GQ-16 only changed 89 genes. GQ-16 generally showed weak effects upon Rosi induced genes, consistent with partial agonist actions, but a subset of modestly Rosi induced and strongly repressed genes displayed disproportionately strong GQ-16 responses. PPARγ partial agonists MLR24 and SR1664 also exhibit disproportionately strong effects on transcriptional repression. We conclude that GQ-16 displays a continuum of weak partial agonist effects but efficiently represses some negatively regulated PPARγ responsive genes. Strong repressive effects could contribute to physiologic actions of GQ-16. - Highlights: • GQ-16 is an insulin sensitizing PPARγ ligand with reduced harmful side effects. • GQ-16 displays a continuum of weak partial agonist activities at PPARγ-induced genes. • GQ-16 exerts strong repressive effects at a subset of genes. • These inhibitor actions should be evaluated in models of adipose tissue inflammation.

  1. Conventional kinesin KIF5B mediates adiponectin secretion in 3T3-L1 adipocytes

    Energy Technology Data Exchange (ETDEWEB)

    Cui, Ju, E-mail: juzi.cui@gmail.com [The Key Laboratory of Geriatrics, Beijing Hospital and Beijing Institute of Geriatrics, Beijing (China); Pang, Jing; Lin, Ya-Jun; Jiang, Ping; Gong, Huan [The Key Laboratory of Geriatrics, Beijing Hospital and Beijing Institute of Geriatrics, Beijing (China); Wang, Zai [Institute of Clinical Medical Sciences, China-Japan Friendship Hospital, Beijing (China); Li, Jian; Cai, Jian-Ping [The Key Laboratory of Geriatrics, Beijing Hospital and Beijing Institute of Geriatrics, Beijing (China); Huang, Jian-Dong, E-mail: jdhuang@hku.hk [School of Biomedical Sciences and Shenzhen Institute of Research and Innovation, The University of Hong Kong, Pokfulam (Hong Kong); The Centre for Synthetic Biology Engineering Research, Shenzhen Institutes of Advanced Technology, Shenzhen (China); Zhang, Tie-Mei, E-mail: tmzhang126@126.com [The Key Laboratory of Geriatrics, Beijing Hospital and Beijing Institute of Geriatrics, Beijing (China)

    2016-08-05

    Insulin stimulates adiponectin secretion and glucose transporter type 4 (GLUT4) translocation in adipocyte to regulate metabolism homeostasis. Similar to GLUT4 translocation, intracellular trafficking and release of adiponectin in adipocytes relies on the trans-Golgi network and endosomal system. Recent studies show that the heavy chain of conventional kinesin (KIF5B) mediates GLUT4 translocation in murine 3T3-L1 adipocytes, however, the motor machinery involved in mediating intracellular trafficking and release of adiponectin is unknown. Here, we examined the role of KIF5B in the regulation of adiponectin secretion. The KIF5B level was up-regulated during 3T3-L1 adipogenesis. This increase in cytosolic KIF5B was synchronized with the induction of adiponectin. Endogenous KIF5B and adiponectin were partially colocalized at the peri-nuclear and cytosolic regions. In addition, adiponectin-containing vesicles were co-immunoprecipitated with KIF5B. Knockdown of KIF5B resulted in a marked inhibition of adiponectin secretion and overexpression of KIF5B enhanced adiponectin release, whereas leptin secretion was not affected by changes in KIF5B expression. These data suggest that the secretion of adiponectin, but not leptin, is dependent on functional KIF5B. - Highlights: • The KIF5B level was up regulated during 3T3-L1 adipogenesis. • Endogenous KIF5B and adiponectin were partially colicalized. • Adiponectin-containing vesicles were co-immunoprecipitated with KIF5B. • The secretion of adiponectin, but not leptin, is dependent on functional KIF5B.

  2. Hydrogen sulfide promotes adipogenesis in 3T3L1 cells.

    Directory of Open Access Journals (Sweden)

    Chin-Yi Tsai

    Full Text Available The effect of hydrogen sulfide (H2S on differentiation of 3T3L1-derived adipocytes was examined. Endogenous H2S was increased after 3T3L1 differentiation. The expression of the H2S-synthesising enzymes, cystathionine γ-lyase (CSE, cystathionine β-synthase (CBS and 3-mercaptopyruvate sulfurtransferase (3-MST, was increased in a time-dependent manner during 3T3L1 differentiation. Expression of genes associated with adipogenesis related genes including fatty acid binding protein 4 (FABP4/aP2, a key regulator of this process, was increased by GYY4137 (a slow-releasing H2S donor compound and sodium hydrosulfide (NaHS, a classical H2S donor but not by ZYJ1122 or time-expired NaHS. Furthermore expression of these genes were reduced by aminooxyacetic acid (AOAA, CBS inhibitor, DL-propargylglycine (PAG, CSE inhibitor as well as by CSE small interference RNA (siCSE and siCBS. The size and number of lipid droplets in mature adipocytes was significantly increased by both GYY4137 and NaHS, which also impaired the ability of CL316,243 (β3-agonist to promote lipolysis in these cells. In contrast, AOAA and PAG had the opposite effect. Taken together, we show that the H2S-synthesising enzymes CBS, CSE and 3-MST are endogenously expressed during adipogenesis and that both endogenous and exogenous H2S modulate adipogenesis and adipocyte maturation.

  3. Nebivolol stimulates mitochondrial biogenesis in 3T3-L1 adipocytes

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Chenglin; Chen, Dongrui; Xie, Qihai [State Key Laboratory of Medical Genomics, Shanghai Key Laboratory of Vascular Biology, Department of Hypertension, Ruijin Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200025 (China); Yang, Ying, E-mail: yangying_sh@yahoo.com [Department of Endocrine and Metabolic Diseases, Shanghai Institute of Endocrine and Metabolic Diseases, Shanghai Clinical Center for Endocrine and Metabolic Diseases, Ruijin Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200025 (China); Shen, Weili, E-mail: weili_shen@hotmail.com [State Key Laboratory of Medical Genomics, Shanghai Key Laboratory of Vascular Biology, Department of Hypertension, Ruijin Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200025 (China)

    2013-08-16

    Highlights: •Nebivolol may act as a partial agonist of β3-adrenergic receptor (AR). •Nebivolol stimulates mitochondrial DNA replication and protein expression. •Nebivolol promotes mitochondrial synthesis via activation of eNOS by β3-AR. -- Abstract: Nebivolol is a third-generation β-adrenergic receptor (β-AR) blocker with additional beneficial effects, including the improvement of lipid and glucose metabolism in obese individuals. However, the underlying mechanism of nebivolol’s role in regulating the lipid profile remains largely unknown. In this study, we investigated the role of nebivolol in mitochondrial biogenesis in 3T3-L1 adipocytes. Exposure of 3T3-L1 cells to nebivolol for 24 h increased mitochondrial DNA copy number, mitochondrial protein levels and the expression of transcription factors involved in mitochondrial biogenesis, including PPAR-γ coactivator-1α (PGC-1α), Sirtuin 3 (Sirt3), mitochondrial transcription factor A (Tfam) and nuclear related factor 1 (Nrf1). These changes were accompanied by an increase in oxygen consumption and in the expression of genes involved in fatty acid oxidation and antioxidant enzymes in 3T3-L1 adipocytes, including nebivolol-induced endothelial nitric oxide synthase (eNOS), as well as an increase in the formation of cyclic guanosine monophosphate (cGMP). Pretreatment with NG-nitro-L-arginine methyl ester (l-NAME) attenuated nebivolol-induced mitochondrial biogenesis, as did the soluble guanylate cyclase inhibitor, ODQ. Treatment with nebivolol and β3-AR blocker SR59230A markedly attenuated PGC-1α, Sirt3 and manganese superoxide dismutase (MnSOD) protein levels in comparison to treatment with nebivolol alone. These data indicate that the mitochondrial synthesis and metabolism in adipocytes that is promoted by nebivolol is primarily mediated through the eNOS/cGMP-dependent pathway and is initiated by the activation of β3-AR receptors.

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

    Energy Technology Data Exchange (ETDEWEB)

    Masiello, Lisa M.; Fotos, Joseph S.; Galileo, Deni S.; Karin, Norm 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.

  5. Characterization of Catalase from Psychrotolerant Psychrobacter piscatorii T-3 Exhibiting High Catalase Activity

    OpenAIRE

    Kimoto, Hideyuki; Yoshimune, Kazuaki; Matsuyma, Hidetoshi; Yumoto, Isao

    2012-01-01

    A psychrotolerant bacterium, strain T-3 (identified as Psychrobacter piscatorii), that exhibited an extraordinarily high catalase activity was isolated from the drain pool of a plant that uses H2O2 as a bleaching agent. Its cell extract exhibited a catalase activity (19,700 U·mg protein−1) that was higher than that of Micrococcus luteus used for industrial catalase production. Catalase was approximately 10% of the total proteins in the cell extract of the strain. The catalase (PktA) was purif...

  6. Cell Volume Regulation and Signaling in 3T3-L1 Pre-adipocytes and Adipocytes

    DEFF Research Database (Denmark)

    Eduardsen, Kathrine; Larsen, Susanne; Novak, Ivana

    2011-01-01

    no detectable effect on InsR, yet increased ERK1/2 phosphorylation. In conclusion, differentiated 3T3-L1 adipocytes exhibit greatly accelerated RVD and RVI responses and increased swelling-activated taurine efflux compared to pre-adipocytes. Furthermore, in pre-adipocytes, Cav-1/caveolae integrity......) and osmotic cell shrinkage (RVI), accompanied by increased swelling-activated taurine efflux. However, caveolin-1 distribution was not detectably altered after osmotic swelling or shrinkage, and caveolae integrity, as studied by cholesterol depletion or expression of dominant negative Cav-1, was not required...

  7. Effects of dietary phytoestrogens on plasma testosterone and triiodothyronine (T3 levels in male goat kids

    Directory of Open Access Journals (Sweden)

    Ekstedt Elisabeth

    2009-12-01

    Full Text Available Abstract Background Exposure to xenoestrogens in humans and animals has gained increasing attention due to the effects of these compounds on reproduction. The present study was undertaken to investigate the influence of low-dose dietary phytoestrogen exposure, i.e. a mixture of genistein, daidzein, biochanin A and formononetin, on the establishment of testosterone production during puberty in male goat kids. Methods Goat kids at the age of 3 months received either a standard diet or a diet supplemented with phytoestrogens (3 - 4 mg/kg/day for ~3 months. Plasma testosterone and total and free triiodothyronine (T3 concentrations were determined weekly. Testicular levels of testosterone and cAMP were measured at the end of the experiment. Repeated measurement analysis of variance using the MIXED procedure on the generated averages, according to the Statistical Analysis System program package (Release 6.12, 1996, SAS Institute Inc., Cary, NC, USA was carried out. Results No significant difference in plasma testosterone concentration between the groups was detected during the first 7 weeks. However, at the age of 5 months (i.e. October 1, week 8 phytoestrogen-treated animals showed significantly higher testosterone concentrations than control animals (37.5 nmol/l vs 19.1 nmol/l. This elevation was preceded by a rise in plasma total T3 that occurred on September 17 (week 6. A slightly higher concentration of free T3 was detected in the phytoestrogen group at the same time point, but it was not until October 8 and 15 (week 9 and 10 that a significant difference was found between the groups. At the termination of the experiment, testicular cAMP levels were significantly lower in goats fed a phytoestrogen-supplemented diet. Phytoestrogen-fed animals also had lower plasma and testicular testosterone concentrations, but these differences were not statistically significant. Conclusion Our findings suggest that phytoestrogens can stimulate testosterone

  8. Nitridation Of The A A 2024 T3 Aluminium By The Glow Discharge Plasma Technique

    International Nuclear Information System (INIS)

    Mudjiman, Supardjono; Sujitno, Tjipto; Sudjatmoko

    1996-01-01

    Nitridation of A A 2024 T3 aluminium by means of plasma glow discharge technique has been carried out. For this purpose, the experiments were carried out at the temperature 30 o C, 60 o C, 100 o C, 150 o C, 200 o C, and 250 o C whereas the nitridation time were varied at 5 minutes, 15 minutes, 40 minutes, 90 minutes and 180 minutes. The results showed that the optimum temperature and time of nitridation were 60 o C and 90 minutes respectively and the hardness increased from 115 to 166 KHN

  9. Cell shrinkage as a signal to apoptosis in NIH 3T3 fibroblasts

    DEFF Research Database (Denmark)

    Friis, Martin B; Friborg, Christel R; Schneider, Linda

    2005-01-01

    Cell shrinkage is a hallmark of the apoptotic mode of programmed cell death, but it is as yet unclear whether a reduction in cell volume is a primary activation signal of apoptosis. Here we studied the effect of an acute elevation of osmolarity (NaCl or sucrose additions, final osmolarity 687...... mosmol l(-1)) on NIH 3T3 fibroblasts to identify components involved in the signal transduction from shrinkage to apoptosis. After 1.5 h the activity of caspase-3 started to increase followed after 3 h by the appearance of many apoptotic-like bodies. The caspase-3 activity increase was greatly enhanced...

  10. Preoperative chemoradiation of locally advanced T3 rectal cancer combined with an endorectal boost

    DEFF Research Database (Denmark)

    Jakobsen, Anders; Mortensen, John P; Bisgaard, Claus

    2006-01-01

    (TRG) system. TRG1 was recorded in 27% of the patients, and a further 27% were classified as TRG2. TRG3 was found in 40%, and 6% had TRG4. The toxicity was low. CONCLUSION: The results indicate that high-dose radiation with concurrent chemotherapy and endorectal brachytherapy is feasible with a high......PURPOSE: To investigate the effect and feasibility of concurrent radiation and chemotherapy combined with endorectal brachytherapy in T3 rectal cancer with complete pathologic remission as end point. METHODS AND MATERIALS: The study included 50 patients with rectal adenocarcinoma. All patients had...

  11. Familial Longevity Is Associated With Higher TSH Secretion and Strong TSH-fT3 Relationship

    DEFF Research Database (Denmark)

    Jansen, Steffy W; Roelfsema, Ferdinand; van der Spoel, Evie

    2015-01-01

    CONTEXT: Longevity is associated with changes in circulating levels of thyroid hormone (TH) and/or TSH in animals and humans, but underlying mechanisms remain elusive. OBJECTIVE: We explored in 38 offspring of nonagenarian participants from the Leiden Longevity Study, who are enriched for longevity...... properties of TSH. The temporal relationship between TSH and free T3 at zero delay was higher in offspring (0.48 ± 0.2) compared with partners (0.26 ± 0.4) (P = .05), but the feedback and forward interplay between TSH and TH did not differ. CONCLUSIONS: Familial longevity is associated with increased basal...

  12. Model remediation of contaminated sites as illustrated by the example of the TNT-remediation project Stadtallendorf, Hessen, Germany. Part 1: Final report; Part 2: Documentation and balancing of the measures; Part 3: Documentation of citizens' participation and public relations activities in Stadtallendorf; Modellhafte Sanierung von Altlasten am Beispiel des TNT-Sanierungsprojektes Stadtallendorf/Hessen. T. 1: Abschlussbericht. T. 2: Dokumentation und Bilanzierung von Auswirkungen der Sanierung. T. 3: Dokumentation der Buergerbeteiligung und Oeffentlichkeitsarbeit in Stadtallendorf

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1999-07-01

    The project comprised the following stages: 1. Development of a technology for purification of soils contaminated with explosives. The process comprised soil washing, hot steam treatment, and thermal treatment of residues; 2. Projecting, construction and two years of operation of a soil purification plant with a rated throughput of 20 t/h and a total throughput of 130,000 t; 3. Remediation of two representative sites on DAG terrain (TNT production chain and filling stations); 4. Verification of the findings in an accompanying scientific programme. The site under investigation was formerly occupied by the Dynamit AG (DAG) Stadtallendorf, where TNT, contaminated waste water and acids were produced, and contamination levels were high in several areas. Today, the area is a residential area. In addition to the investigations described here, the waste water discharge system of the DAG was modernized. [German] Das FuE-Vorhaben Stadtallendorf wurde mit folgender Zielsetzung begonnen: (1) Entwicklung einer geeigneten und moeglichst umweltvertraeglichen Technik zur Reinigung von Boeden mit sprengstoffspezifischen Kontaminationen; hierzu wurde in Anlehnung an die Ausschreibung des Bundes ein kombiniertes Bodenbehandlungsverfahren vorgesehen, das aus folgenden Komponenten bestand: - Bodenwaesche, - Heissdampfbehandlung, - Thermische Reststoffbehandlung. (2) Planung, Bau und zweijaehriger Betrieb einer entsprechenden grosstechnischen Bodenreinigungsanlage (Nenndurchsatz von 20 t/Stunde; Gesamtdurchsatz 130.000 t). (3) Bodensanierung von zwei repraesentativen Arealen im DAG-Gelaende (TNT-Produktionskette und Fuellstellenbetrieb). (4) Absicherung der Ergebnisse in einem wissenschaftlichen Begleitprogramm. Gegenstand der Phase 2 des Vorhabens war der vollstaendige Prozess der Planung und Umsetzung der Sanierung eines (Teils eines) Planungsraumes des Ruestungsaltstandortes Dynamit AG (DAG) Stadtallendorf. Es handelte sich dabei um einen Bereich, der als Standort fuer TNT

  13. Assessment of combined toxicity of heavy metals from industrial wastewaters on Photobacterium phosphoreum T3S

    Science.gov (United States)

    Zeb, BibiSaima; Ping, Zheng; Mahmood, Qaisar; Lin, Qiu; Pervez, Arshid; Irshad, Muhammad; Bilal, Muhammad; Bhatti, Zulfiqar Ahmad; Shaheen, Shahida

    2017-07-01

    This research work is focusing on the toxicities of heavy metals of industrial origin to anaerobic digestion of the industrial wastewater. Photobacterium phosphoreum T3S was used as an indicator organism. The acute toxicities of heavy metals on P. phosphoreum T3S were assessed during 15-min half inhibitory concentration (IC50) as indicator at pH 5.5-6. Toxicity assays involved the assessment of multicomponent mixtures using TU and MTI approaches. The results of individual toxicity indicated that the toxicity of Cd, Cu and Pb on P. phosphoreum increased with increasing concentrations and there was a linear correlation. The 15-min IC50 values of Cd, Cu and Pb were 0.537, 1.905 and 1.231 mg/L, respectively, and their toxic order was Cd > Pb > Cu. The combined effects of Cd, Cu and Pb were assayed by equivalent concentration mixing method. The results showed that the combined effects of Cd + Cu, Cd + Pb, Cu + Pb, Cd + Cu + Pb were antagonistic, antagonistic and partly additive. The combined effect of three heavy metals was partly additive.

  14. Hormonal evaluation of T4 and T3 through radioimmunoassay in younglings of rats with hypothyroidism

    International Nuclear Information System (INIS)

    Silveira, M.F.G.; Silva, I.M.S.; Pereira, S.S.L.; Souza, G.M.L.; Carvalho, E.F.M.B.; Cavalcante, C.V.G.; Lima Filho, G.L.; Catanho, M.T.J.A.

    2000-01-01

    The onset of fetal thyroid function occurs about 17-18 days after conception in the rat. The maternal hypothyroidism which occurs during gestation provokes alteration in the rat after birth. Due to this alteration, we decided to analyze the hormonal modification in the newborn rats. The hypothyroidism was induced in normal dams, which were being treated for 7 days with MMI (in the concentration of 0,03% in drinking water) before mating. Another dam group which was submitted to an induction of hypothyroidism maintained the treatment with MMI for 13 days during gestation. The hormones were assessed by radioimmunoassay technique. It was seen that the rats which were born from hypothyroid dams suffered alterations on its T 4 and T 3 hormone levels concerning to 10, 30 and 60 days after birth. There was also modifications on their weight and size. The growth is affected throughout post-natal life by thyroid hormones, which has a facilitator influence on growth hormone economy, as opposed to the inhibitory effects on TSH economy. The administration of MMI bars the fetal thyroid gland function, causing a decrease of both T 4 and T 3 levels, even after the birth, indicating that the maternal hypothyroidism influences on the post-natal life of the rat. (author)

  15. The transition of corrosion pitting to surface fatigue cracks in 2024-T3 aluminum alloy

    Science.gov (United States)

    Clark, Paul Nolan

    Pitting corrosion experiments were executed to characterize the dynamics. of accelerated corrosion for the combination of 2024-T3 aluminum and a 3.5% NaCl solution at room temperature. Platinum was used as a counter electrode and a silver-silver chloride electrode was used as a reference. A corrosion protocol was developed to accelerate pitting corrosion on 2024-T3 aluminum. The developed protocol allowed for the relative control of pitting depth on fatigue dog-bone specimens. A pitting damage threshold was encountered for depths beyond an average value of approximately 135mum. Corrosion fatigue experiments were performed on specimens that had been prior corroded using the developed corrosion protocol. Pitting depths from approximately 40mum-200mum were examined for fatigue characterization. Corrosion pitting to surface fatigue crack transition was captured and fatigue crack growth behavior was documented. As well as the pit to crack transition, the short crack behavior to long crack behavior transition was captured and analyzed. Fractographic analysis of corrosion fatigue specimens revealed cracks nucleating, propagating and causing subsequent failure from accelerated prior pitting corrosion damage.

  16. Characterization of dextransucrase from Leuconostoc mesenteroides T3, water kefir grains isolate

    Directory of Open Access Journals (Sweden)

    Miljković Miona G.

    2017-01-01

    Full Text Available The production of dextransucrase (DS by Leuconostoc mesenteroides T3, novel isolate from water kefir grain, was studied and optimized. Bacterial supernatant reached activity of 3.1 U/ml when the culture was grown at 23 C and under static culture condition using classical Tsuchiya medium for DS production. The increase of sucrose concentration to 7% led to an increase of DS activity by 52% compared to the control. Medium with 2% beef extract and 1% yeast extract resulted in 4.52 U/ml, which was 47% higher than in the control (with 2% yeast extract. Finally, the increase of K2HPO4 concentration from 2 to 3% resulted in the increased enzyme activity by 28%. Enzyme purified by polyethylene glycol 400 fractionation displayed maximum activity at 30°C and pH 5.4. Zymogram analysis confirmed the presence of DS of approximately 180 kDa. The addition of divalent cations Ca2+, Mg2+, Fe2+ and Co2+ led to a minor increase of DS activity, while the addition of Mn2+ was the most prominent with 73% increase. These findings classify dextransucrase from Leuconostoc mesenteroides T3 as promising candidate for production of dextran, which has numerous applications in various industries. [Project of the Serbian Ministry of Education, Science and Technological Development, Grant no. TR 31035

  17. Characterization of Catalase from Psychrotolerant Psychrobacter piscatorii T-3 Exhibiting High Catalase Activity

    Science.gov (United States)

    Kimoto, Hideyuki; Yoshimune, Kazuaki; Matsuyma, Hidetoshi; Yumoto, Isao

    2012-01-01

    A psychrotolerant bacterium, strain T-3 (identified as Psychrobacter piscatorii), that exhibited an extraordinarily high catalase activity was isolated from the drain pool of a plant that uses H2O2 as a bleaching agent. Its cell extract exhibited a catalase activity (19,700 U·mg protein−1) that was higher than that of Micrococcus luteus used for industrial catalase production. Catalase was approximately 10% of the total proteins in the cell extract of the strain. The catalase (PktA) was purified homogeneously by only two purification steps, anion exchange and hydrophobic chromatographies. The purified catalase exhibited higher catalytic efficiency and higher sensitivity of activity at high temperatures than M. luteus catalase. The deduced amino acid sequence showed the highest homology with catalase of Psycrobacter cryohalolentis, a psychrotolelant bacterium obtained from Siberian permafrost. These findings suggest that the characteristics of the PktA molecule reflected the taxonomic relationship of the isolate as well as the environmental conditions (low temperatures and high concentrations of H2O2) under which the bacterium survives. Strain T-3 efficiently produces a catalase (PktA) at a higher rate than Exiguobacterium oxidotolerans, which produces a very strong activity of catalase (EktA) at a moderate rate, in order to adapt to high concentration of H2O2. PMID:22408420

  18. CORROSION RESISTANT SOL–GEL COATING ON 2024-T3 ALUMINUM

    Directory of Open Access Journals (Sweden)

    S. Yazdani

    2016-06-01

    Full Text Available The inherent reactivity of the Al–Cu alloys is such that their use for structural, marine, and aerospace components and structures would not be possible without prior application of a corrosion resistance system. Historically these corrosion resistance coatings were based on the use of chemicals containing Cr (VI compounds. Silane coatings are of increasing interest in industry due to their potential application for the replacement of current toxic hexavalent chromate based treatments. In this study, hydrophobic coating sol was prepared with methyltriethoxysilane (MTES, methanol (MeOH, and water (as 7M NH4OH at a molar ratio of 1:25:4.31 respectively. The coatings were applied by a dip-technique to 2024-T3 Al alloy, and subsequently cured at room temperature and there after heat treated in an oven at 150°C. The anticorrosion properties of the coatings within 3.5 wt% NaCl solution were studied by Tafel polarization technique. The sol–gel coating exhibited good anticorrosion properties providing an adherent protection film on the Al 2024-T3 substrate. The surface properties were characterized by water contact angle measurement, scanning electron microscopy (SEM, and the composition was studied by Fourier transform infrared spectroscopy (FTIR.

  19. Triiodothyronine (T3) action on aquatic locomotor behavior during metamorphosis of the bullfrog Rana catesbeiana.

    Science.gov (United States)

    Fernández-Mongil, Marisabel; Venza, Celia J; Rivera, Amelia; Lasalde-Dominicci, José A; Burggren, Warren; Rojas, Legier V

    2009-01-01

    Thyroid hormones--particularly triiodothyronine, T3--play a critical role in the morphological transformations comprising metamorphosis in larval bullfrogs (Rana catesbeiana). Traditional staging criteria for anuran larvae incompletely distinguish physiological and behavioral changes during growth. We therefore first developed a new parameter to describe larval growth, the developmental index (DI), which is simply the ratio between the tail length of the larva and its head diameter. Using the DI we were able to identify two distinct populations classifying the larvae during growth along a continuous linear scale with a cutoff value of DI at 2.8. Classification based on the DI, used in this study, proved an effective complement to existing classifications based on developmental staging into pre- or pro-metamorphic stages. Exposure to T3 in the water induced a rapid (beginning within 5 min) and significant decrease (approximately 20-40%) in locomotor activity, measured as total distance traversed and velocity. The largest decrease occurred in more developed larvae (DI or =2.8). We discuss the significance of the possible role of an apoptotic volume decrease at the level of the neuromuscular junction.

  20. The depletion of nuclear glutathione impairs cell proliferation in 3t3 fibroblasts.

    Directory of Open Access Journals (Sweden)

    Jelena Markovic

    2009-07-01

    Full Text Available Glutathione is considered essential for survival in mammalian cells and yeast but not in prokaryotic cells. The presence of a nuclear pool of glutathione has been demonstrated but its role in cellular proliferation and differentiation is still a matter of debate.We have studied proliferation of 3T3 fibroblasts for a period of 5 days. Cells were treated with two well known depleting agents, diethyl maleate (DEM and buthionine sulfoximine (BSO, and the cellular and nuclear glutathione levels were assessed by analytical and confocal microscopic techniques, respectively. Both agents decreased total cellular glutathione although depletion by BSO was more sustained. However, the nuclear glutathione pool resisted depletion by BSO but not with DEM. Interestingly, cell proliferation was impaired by DEM, but not by BSO. Treating the cells simultaneously with DEM and with glutathione ethyl ester to restore intracellular GSH levels completely prevented the effects of DEM on cell proliferation.Our results demonstrate the importance of nuclear glutathione in the control of cell proliferation in 3T3 fibroblasts and suggest that a reduced nuclear environment is necessary for cells to progress in the cell cycle.

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

    Science.gov (United States)

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

    2015-03-01

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

  2. The "T3 Support Centre" (Teaching, Technology and Testing - Not just another help desk

    Directory of Open Access Journals (Sweden)

    Carol Miles

    2005-10-01

    Full Text Available Many faculty members embrace the challenge of responding to rising student demands for more technically advanced course supports by offering their courseware through a variety of media. However, it is often difficult for them to find the time required to become proficient in the use of the software packages, course management systems and web technologies at their disposal. These new realities of teaching point to the need for support systems for faculty members that go beyond the traditional computer services "help desk" with a more comprehensive support service that actually becomes involved in the development and modification of technology-based course materials and computerized test marking and analysis. Increasing demand for these types of services at Carleton University resulted in the establishment of the T3 (Teaching...Technology...Testing Support Centre. The service offers faculty members extended-hour phone-in and walk-in support as well as a variety of resources such as Scantron and Item Analysis service for multiple choice exams, the use of scanners and colour printers, as well as a variety of teaching publications and contacts. This paper details the planning, administration, and services offered of the T3 Service, including advice those attempting to establish a similar service. Usage statistics from the first year of operations will be delineated.

  3. Agarose gel electrophoresis reveals structural fluidity of a phage T3 DNA packaging intermediate.

    Science.gov (United States)

    Serwer, Philip; Wright, Elena T

    2012-01-01

    We find a new aspect of DNA packaging-associated structural fluidity for phage T3 capsids. The procedure is (i) glutaraldehyde cross-linking of in vivo DNA packaging intermediates for the stabilization of structure and then (ii) determining effective radius by two-dimensional agarose gel electrophoresis (2D-AGE). The intermediates are capsids with incompletely packaged DNA (ipDNA) and without an external DNA segment; these intermediates are called ipDNA-capsids. We initially increase the production of ipDNA-capsids by raising NaCl concentration during in vivo DNA packaging. By 2D-AGE, we find a new state of contracted shell for some particles of one previously identified ipDNA-capsid. The contracted shell-state is found when the ipDNA length/mature DNA length (F) is above 0.17, but not at lower F. Some contracted-shell ipDNA-capsids have the phage tail; others do not. The contracted-shell ipDNA-capsids are explained by premature DNA maturation cleavage that makes accessible a contracted-shell intermediate of a cycle of the T3 DNA packaging motor. The analysis of ipDNA-capsids, rather than intermediates with uncleaved DNA, provides a simplifying strategy for a complete biochemical analysis of in vivo DNA packaging. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Regulation of p53 in NIH3T3 mouse fibroblasts following hyperosmotic stress

    DEFF Research Database (Denmark)

    Lambert, Ian Henry; Enghoff, Maria Stine; Brandi, Marie-Luise

    2015-01-01

    The aim of this project was to analyze the regulation of p53 expression in NIH3T3 fibroblasts under the influence of increasing hyperosmotic stress. Expression of p53 showed a biphasic response pattern in NIH3T3 cells under increasing osmotic stress (337 mOsm to 737 mOsm) with a maximum at 587 m......Osm. Under isotonic conditions p53 expression increased after addition of the proteasome inhibitor MG132 indicating that cellular p53 levels in unperturbed cells is kept low by proteasomal degradation. However, under hypertonic conditions p53 synthesis as well as p53 degradation were significantly reduced...... and it is demonstrated that the increase in p53 expression observed when tonicity is increased from 337 to 587 mOsm reflects that degradation is more inhibited than synthesis, whereas the decrease in p53 expression at higher tonicities reflects that synthesis is more inhibited than degradation. The activity of the p53...

  5. Quantitation of T-3 (triiodothyronine) and T-4 (thyroxine) in serum and plasma. Draft report

    International Nuclear Information System (INIS)

    Ceglowski, W.; Williams, R.B.

    1981-12-01

    This report summarizes an examination of the published literature concerning the quantitation of thyroxine and triiodothyronine in the clinical laboratory. It therefore details the precision, accuracy, sensitivity, and specificity obtainable in various commercial systems and those devised in the clinical laboratory. The data produced by several of the procedures often indicate that improvements in these parameters would enhance overall assay performance and increase the reliability of the clinical interpretation derivable from assay results. For T-3 and T-4 in vitro assays a very large number of systems exist and are currently being utilized in clinical laboratories in this country. For the sake of brevity some systems, while mentioned, are not reviewed in exhaustive detail. Radioimmunoassay, as the most frequently performed assay for both T-3 and T-4 is extensively reviewed. Also discussed with particular interest are assay systems which will undoubtedly impact on the future course of thyroid hormone assessment in the clinical laboratory, namely enzyme immunoassay and fluorescent immunoassay. The state of the art in T-4 measurements in neonates, because it is such a critical area for application of in vitro thyroid testing, is given detailed review. The quantitation of free thyroxine has been discussed in detail. These assays have been gaining more frequent use in the clinical laboratory and increased commercial system development

  6. Lipopolysaccharide Modifies Glycerol Permeability and Metabolism in 3T3-L1 Adipocytes

    Directory of Open Access Journals (Sweden)

    Jeanne Durendale Chiadak

    2017-11-01

    Full Text Available Aquaglyceroporins—aquaporin membrane channels (AQP that conduct glycerol and other small neutral solutes in addition to water—play major roles in obesity. In adipocytes, aquaglyceroporins mediate glycerol uptake and release across the plasma membrane, which are two key steps for triacylglycerols (TAGs synthesis (lipogenesis and hydrolysis (lipolysis. The aim of this study was to assess both glycerol permeability and metabolism in undifferentiated 3T3-L1 cells (UDCs as well as in untreated (CTL-DCs versus lipopolysaccharide (LPS-DCs-treated differentiated 3T3-L1 adipocytes. Glycerol release, TAGs content and whole membrane glycerol permeability were significantly increased in DCs as compared to UDCs. Moreover, in DCs, LPS treatment significantly increased TAGs content and decreased glycerol permeability. In addition, a significant reduction in whole membrane glycerol permeability was observed in LPS-DCs as compared to CTL-DCs. The relative contributions of AQP3, AQP7 and AQP9 (facilitated diffusion, as well as that of the phospholipid bilayer (simple diffusion, to the whole membrane glycerol permeability, were estimated biophysically in UDCs, CTL-DCs and LPS-DCs, using selective AQP inhibitors. Further studies will be required to determine if modifications in either subcellular localization and/or activity of aquaglyceroporins could account for the data herein. Nevertheless, our findings provide novel insights in understanding the LPS-induced adipocyte hypertrophy that accompanies obesity.

  7. Low T3 syndrome in neuromyelitis optica spectrum disorder: Associations with disease activity and disability.

    Science.gov (United States)

    Cho, Eun Bin; Min, Ju-Hong; Cho, Hye-Jin; Seok, Jin Myoung; Lee, Hye Lim; Shin, Hee Young; Lee, Kwang-Ho; Kim, Byoung Joon

    2016-11-15

    Neuromyelitis optica (NMO) sometimes coexists with serological marker-positive, non-organ-specific autoimmune disorders. We evaluated the prevalence of thyroid dysfunction and anti-thyroid antibodies in patients with NMO spectrum disorder (NMOSD) and investigated the associations between thyroid dysfunction/autoimmunity and clinical features of NMOSD. Forty-nine NMOSD patients with anti-aquaporin-4 antibody and 392 age- and sex-matched healthy controls were included. We measured the levels of thyroid hormones and anti-thyroid antibodies. The prevalence of clinical hypothyroidism, subclinical hyperthyroidism, and low T3 syndrome were higher in patients with NMOSD (4.1%, 12.2%, and 20.4%, respectively) compared with healthy controls (0.3%, 2.8%, and 0.5%, respectively; p=0.034, p=0.001, and poral prednisolone use, iv methylprednisolone use, other immunosuppressive agents use, and the location of lesion (ρ=-0.416, p=0.010). Our study suggests that thyroid dysfunction is frequent in patients with NMOSD; particularly, serum T3 levels may be a useful indicator of disease activity and disability in NMOSD. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Acanthamoeba genotypes T3 and T4 as causative agents of amoebic keratitis in Mexico.

    Science.gov (United States)

    Omaña-Molina, Maritza; Vanzzini-Zago, Virginia; Hernandez-Martinez, Dolores; Gonzalez-Robles, Arturo; Salazar-Villatoro, Lizbeth; Ramirez-Flores, Elizabeth; Oregon-Miranda, Eric; Lorenzo-Morales, Jacob; Martinez-Palomo, Adolfo

    2016-02-01

    Free-living amoebae (FLA) are widely distributed worldwide. Some genera included in this group act as opportunistic pathogens causing fatal encephalitis and Acanthamoeba keratitis (AK), a sight-threatening infection of the cornea associated with the use of soft contact lenses that could even end in blindness if an early diagnosis and treatment are not achieved. Furthermore, the numbers of AK cases keep rising worldwide mainly due to an increase of contact lens wearers and lack of hygiene in the maintenance of lenses and their cases. In Mexico, no cases of AK have been described so far although the isolation of other pathogenic FLA such as Naegleria fowleri and Balamuthia mandrillaris from both clinical and environmental sources has been reported. The present study reports two cases of Acanthamoeba keratitis diagnosed in two patients admitted to the Hospital "Luis Sánchez Bulnes" for Blindness Prevention in Mexico City, Mexico. Corneal scrapes and contact lenses were checked for the presence of Acanthamoeba strains in both patients. Strains were axenized after initial isolation to classify at the genotype level. After sequencing the diagnostic fragment 3 (DF3) region located on the 18S ribosomal DNA (rDNA) gene of Acanthamoeba, genotype T3 and genotype T4 were identified in clinical case 1 and 2, respectively. To our knowledge, these are the first reported cases of AK in Mexico in the literature and the first description of Acanthamoeba genotypes T3 and T4 as causative agents of amoebic infection.

  9. MuRF1 mono-ubiquitinates TRα to inhibit T3-induced cardiac hypertrophy in vivo.

    Science.gov (United States)

    Wadosky, Kristine M; Berthiaume, Jessica M; Tang, Wei; Zungu, Makhosi; Portman, Michael A; Gerdes, A Martin; Willis, Monte S

    2016-04-01

    Thyroid hormone (TH) is recognized for its role in cellular metabolism and growth and participates in homeostasis of the heart. T3 activates pro-survival pathways including Akt and mTOR. Treatment with T3 after myocardial infarction is cardioprotective and promotes elements of physiological hypertrophic response after cardiac injury. Although T3 is known to benefit the heart, very little about its regulation at the molecular level has been described to date. The ubiquitin proteasome system (UPS) regulates nuclear hormone receptors such as estrogen, progesterone, androgen, and glucocorticoid receptors by both degradatory and non-degradatory mechanisms. However, how the UPS regulates T3-mediated activity is not well understood. In this study, we aim to determine the role of the muscle-specific ubiquitin ligase muscle ring finger-1 (MuRF1) in regulating T3-induced cardiomyocyte growth. An increase in MuRF1 expression inhibits T3-induced physiological cardiac hypertrophy, whereas a decrease in MuRF1 expression enhances T3's activity both in vitro and in cardiomyocytes in vivo MuRF1 interacts directly with TRα to inhibit its activity by posttranslational ubiquitination in a non-canonical manner. We then demonstrated that a nuclear localization apparatus that regulates/inhibits nuclear receptors by sequestering them within a subcompartment of the nucleus was necessary for MuRF1 to inhibit T3 activity. This work implicates a novel mechanism that enhances the beneficial T3 activity specifically within the heart, thereby offering a potential target to enhance cardiac T3 activity in an organ-specific manner. © 2016 Society for Endocrinology.

  10. Identification of novel substrates of Shigella T3SA through analysis of its virulence plasmid-encoded secretome.

    Directory of Open Access Journals (Sweden)

    Laurie Pinaud

    Full Text Available Many human Gram-negative bacterial pathogens express a Type Three Secretion Apparatus (T3SA, including among the most notorious Shigella spp., Salmonella enterica, Yersinia enterocolitica and enteropathogenic Escherichia coli (EPEC. These bacteria express on their surface multiple copies of the T3SA that mediate the delivery into host cells of specific protein substrates critical to pathogenesis. Shigella spp. are Gram-negative bacterial pathogens responsible for human bacillary dysentery. The effector function of several Shigella T3SA substrates has largely been studied but their potential cellular targets are far from having been comprehensively delineated. In addition, it is likely that some T3SA substrates have escaped scrutiny as yet. Indeed, sequencing of the virulence plasmid of Shigella flexneri has revealed numerous open reading frames with unknown functions that could encode additional T3SA substrates. Taking advantage of label-free mass spectrometry detection of proteins secreted by a constitutively secreting strain of S. flexneri, we identified five novel substrates of the T3SA. We further confirmed their secretion through the T3SA and translocation into host cells using β-lactamase assays. The coding sequences of two of these novel T3SA substrates (Orf13 and Orf131a have a guanine-cytosine content comparable to those of T3SA components and effectors. The three other T3SA substrates identified (Orf48, Orf86 and Orf176 have significant homology with antitoxin moieties of type II Toxin-Antitoxin systems usually implicated in the maintenance of low copy plasmids. While Orf13 and Orf131a might constitute new virulence effectors contributing to S. flexneri pathogenicity, potential roles for the translocation into host cells of antitoxins or antitoxin-like proteins during Shigella infection are discussed.

  11. Local control of T3 carcinomas after accelerated fractionation: a look at the 'gap'

    International Nuclear Information System (INIS)

    Wang, C.C.; Efird, Jimmy; Nakfoor, Bruce; Martins, Patricia

    1996-01-01

    Purpose: To study the effects of midcourse treatment break or gaps related to the local control of T3 carcinoma of the oropharynx and larynx following accelerated hyperfractionated radiation therapy. Methods and Materials: All patients were treated at the Massachusetts General Hospital from 1979 through 1994 with treatment consisting of 1.6 Gy per fraction, two fractions a day for the treatment of T3 carcinoma of the oropharynx and larynx. They were entered in the head and neck data base. Their treatment dates, treatment breaks, and doses vs. local control were analyzed and compared. A p-value of 0.05 was considered statistically significant. Results: A total of 162 patients were available for review. Due to the acute severe mucosal effects, most of the patients required a midcourse pause or 'break' after a dose of 38.4-48 Gy before treatment could be resumed and completed. The data indicate that (a) prolongation of the treatment gap for more than 14 days, (b) total treatment course longer than 45 days, (c) total dose less than 67 Gy, and (d) male gender adversely affected local control. In spite of the gaps, the female patients with advanced carcinomas enjoyed the benefits of improved local control after the accelerated hyperfractionated radiation therapy. Conclusions: Accelerated hyperfractionation radiation therapy using 1.6 Gy per fraction/twice-a-day (b.i.d.) for a total dose of 70.4 Gy in 6 weeks is effective in achieving high local control of T3 squamous cell carcinoma of the oropharynx and larynx. The midcourse treatment gap should be as short as possible with the projected total dose and time. Should the gaps be unduly prolonged due to various circumstances, further increase in the total dose, for example, 72-75 Gy, and/or increase of the fraction sizes, for example, 1.8-2.0 Gy/f b.i.d. after the gap may be necessary to compensate for the adverse effects of the tumor regeneration from the prolonged gap

  12. Ursolic acid inhibits adipogenesis in 3T3-L1 adipocytes through LKB1/AMPK pathway.

    Directory of Open Access Journals (Sweden)

    Yonghan He

    Full Text Available BACKGROUND: Ursolic acid (UA is a triterpenoid compound with multiple biological functions. This compound has recently been reported to possess an anti-obesity effect; however, the mechanisms are less understood. OBJECTIVE: As adipogenesis plays a critical role in obesity, the present study was conducted to investigate the effect of UA on adipogenesis and mechanisms of action in 3T3-L1 preadipocytes. METHODS AND RESULTS: The 3T3-L1 preadipocytes were induced to differentiate in the presence or absence of UA for 6 days. The cells were determined for proliferation, differentiation, fat accumulation as well as the protein expressions of molecular targets that regulate or are involved in fatty acid synthesis and oxidation. The results demonstrated that ursolic acid at concentrations ranging from 2.5 µM to 10 µM dose-dependently attenuated adipogenesis, accompanied by reduced protein expression of CCAAT element binding protein β (C/EBPβ, peroxisome proliferator-activated receptor γ (PPARγ, CCAAT element binding protein α (C/EBPα and sterol regulatory element binding protein 1c (SREBP-1c, respectively. Ursolic acid increased the phosphorylation of acetyl-CoA carboxylase (ACC and protein expression of carnitine palmitoyltransferase 1 (CPT1, but decreased protein expression of fatty acid synthase (FAS and fatty acid-binding protein 4 (FABP4. Ursolic acid increased the phosphorylation of AMP-activated protein kinase (AMPK and protein expression of (silent mating type information regulation 2, homolog 1 (Sirt1. Further studies demonstrated that the anti-adipogenic effect of UA was reversed by the AMPK siRNA, but not by the Sirt1 inhibitor nicotinamide. Liver kinase B1 (LKB1, the upstream kinase of AMPK, was upregulated by UA. When LKB1 was silenced with siRNA or the inhibitor radicicol, the effect of UA on AMPK activation was diminished. CONCLUSIONS: Ursolic acid inhibited 3T3-L1 preadipocyte differentiation and adipogenesis through the LKB1/AMPK

  13. Reversal of dexamethasone induced insulin resistance in 3T3L1 adipocytes by 3β-taraxerol of Mangifera indica.

    Science.gov (United States)

    Sangeetha, K N; Shilpa, K; Jyothi Kumari, P; Lakshmi, B S

    2013-02-15

    The present study investigates the efficacy of Mangifera indica ethyl acetate extract (MIEE) and its bioactive compound, 3β-taraxerol in the reversal of dexamethasone (DEX) induced insulin resistance in 3T3L1 adipocytes. MIEE and 3β-taraxerol were evaluated for their ability to restore impaired glucose uptake and, expression of molecular markers in the insulin signaling pathway induced by DEX in 3T3L1 adipocytes using 2-deoxy-D-[1-(3)H] glucose uptake assay and ELISA. An insulin resistant model has been developed using a glucocorticoid, DEX on 3T3L1 adipocytes. Insulin resistant condition was observed at 24h of DEX induction wherein a maximum degree of resistance of about 50% was measured based on inhibition of glucose uptake, which was confirmed using cytotoxicity analysis. The developed model of insulin resistance was studied in comparison to positive control rosiglitazone. DEX induced inhibition of glucose uptake and the expression of insulin signaling markers GLUT4 and PI3K were found to be restored by 3β-taraxerol and MIEE, thus delineating its mechanism of action in the reversal of insulin resistance. 3β-Taraxerol effectively restored DEX induced desensitization via restoration of PI3K and GLUT4 expression. To conclude, since 3β-taraxerol exhibits significant effect in reversing insulin resistance it can be further investigated as an insulin resistance reversal agent. Copyright © 2012 Elsevier GmbH. All rights reserved.

  14. Friction stir spot welding of 2024-T3 aluminum alloy with SiC nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Paidar, Moslem; Sarab, Mahsa Laali [Islamic Azad University, Tehran (Iran, Islamic Republic of)

    2016-01-15

    In this study, the Friction stir spot welding (FSSW) of 2024-T3 aluminum alloy with 1.6 mm thickness was investigated. The effects of the silicon carbide (SiC) nanoparticles on the metallurgical and mechanical properties were discussed. The effects of particles on tension shear and wear tests were also investigated. The process was conducted at a constant rotational speed of 1000 rpm. Results showed that adding SiC nanoparticles to the weld during FSSW had a major effect on the mechanical properties. In fact, the addition of nanoparticles as barriers prevented grain growth in the Stir zone (SZ). The data obtained in the tensile-shear and wear tests showed that tensile-shear load and wear resistance increased with the addition of SiC nanoparticles, which was attributed to the fine grain size produced in the SZ.

  15. Stimulation of protein phosphatase activity by insulin and growth factors in 3T3 cells

    International Nuclear Information System (INIS)

    Chan, C.P.; McNall, S.J.; Krebs, E.G.; Fischer, E.H.

    1988-01-01

    Incubation of Swiss mouse 3T3-D1 cells with physiological concentrations of insulin resulted in a rapid and transient activation of protein phosphatase activity as measure by using [ 32 P]phosphorylase α as substrate. Activation reached a maximum level (140% of control value) within 5 min of addition and returned to control levels within 20 min. The effect of insulin was dose-dependent with half-maximal activation occurring at ∼5 nM insulin. This activity could be completely inhibited by addition of the heat-stable protein inhibitor 2, which suggests the presence of an activated type-1 phosphatase. Similar effects on phosphatase activity were seen when epidermal growth factor and platelet-derived growth factor were tested. These results suggest that some of the intracellular effects caused by insulin and growth factors are mediated through the activation of a protein phosphatase

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

    Energy Technology Data Exchange (ETDEWEB)

    Arbeitman, C.R. [Gerencia de Investigacion y Aplicaciones, TANDAR-CNEA (Argentina); Grosso, M.F. del, E-mail: delgrosso@tandar.cnea.gov.a [Gerencia de Investigacion y Aplicaciones, TANDAR-CNEA (Argentina); Consejo Nacional de Investigaciones Cientificas y Tecnicas, CONICET (Argentina); Ibanez, I. [Consejo Nacional de Investigaciones Cientificas y Tecnicas, CONICET (Argentina); Gerencia de Aplicaciones Tecnologicas de la Energia Nuclear, Dpto. de Radiobiologia, TANDAR-CNEA (Argentina); Garcia Bermudez, G. [Gerencia de Investigacion y Aplicaciones, TANDAR-CNEA (Argentina); Consejo Nacional de Investigaciones Cientificas y Tecnicas, CONICET (Argentina); Escuela de Ciencia y Tecnologia, UNSAM (Argentina); Duran, H. [Consejo Nacional de Investigaciones Cientificas y Tecnicas, CONICET (Argentina); Gerencia de Aplicaciones Tecnologicas de la Energia Nuclear, Dpto. de Radiobiologia, TANDAR-CNEA (Argentina); Escuela de Ciencia y Tecnologia, UNSAM (Argentina); Chappa, V.C. [Gerencia de Investigacion y Aplicaciones, TANDAR-CNEA (Argentina); Consejo Nacional de Investigaciones Cientificas y Tecnicas, CONICET (Argentina); Mazzei, R. [U.A. Tecnologicas y Agropecuarias, CNEA, Dpto. Ing. Quimica, UTN FRBA, Bs. As. (Argentina); Behar, M. [Instituto de Fisica, Universidade Federal do Rio Grande do Sul, Porto Alegre (Brazil)

    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.

  17. Feasibility study of a biocompatible pneumatic dispensing system using mouse 3T3-J2 fibroblasts

    Science.gov (United States)

    Lee, Sangmin; Kim, Hojin; Kim, Joonwon

    2017-12-01

    This paper presents results for dispensing living cells using a pneumatic dispensing system to verify the feasibility of using this system to fabricate biomaterials. Living cells (i.e., mouse 3T3-J2 fibroblast) were dispensed with different dispensing pressures in order to evaluate the effect of dispensing process on cell viability and proliferation. Based on the results of a live-dead assay, more than 80% of cell viability has been confirmed which was reasonably similar to that in the control group. Furthermore, measurement of cell metabolic activity after dispensing confirmed that the dispensed cell proliferated at a rate comparable to that of the control group. These results demonstrate that the pneumatic dispensing system is a promising tool for fabrication of biomaterials.

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

    International Nuclear Information System (INIS)

    Arbeitman, C.R.; Grosso, M.F. del; Ibanez, I.; Garcia Bermudez, G.; Duran, H.; Chappa, V.C.; Mazzei, R.; Behar, M.

    2010-01-01

    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.

  19. Production of antisera and development of radioimmunoassay for serum T3, T4, and ferritin

    International Nuclear Information System (INIS)

    Elhag, Omer Mohamed Abdalla

    1998-05-01

    In this study twelve local rabbits and sixteen New-zealand rabbits were subjected to immunization against T3 and T4 immunogens. Two local sheep (ovis aris) were immunized against human liver ferritin. The T3 and T4 immunogens were prepared by conjugation of the haptens to carrier proteins (bovine serum albumin ''BSA'' and horse serum protein ''HSP''), using water soluble carboiimide as coupling agent. The local and New-zealand rabbits were immunized against these conjugates emulsified in freund's complete adjuvant (FCA) in the first and second injections, and emulsified in freund's incomplete adjuvant (FIA) in the following injections. The blood samples obtained from rabbits after each injection were tested for antibodies as well as for the effect of immunization on rabbits biochemical and haematological parameters. The blood samples obtained from sheep were tested for anti-ferritin antibodies using crude antiserum, then this antiserum was purified using ammonium sulphate. A part of it was adsorbed physically onto polystyrene beads while the other part was linked chemically to magnitisable particles inorder to develop to IRMAs. The purified antiferritin antibody was diluted 200,000 folds before being coated to polystyrene beads, and different dilutions were tried with coupling to magnetic solid phase. Optimization and validation procedures for the two IRMAs ferritin were performed. The results obtained showed poor response of rabbits to immunization against T3 and T4 immunogen conjugates, where the percent bound (B%) of tracer with the antibody ranged from (0.0-22%) for local rabbits using charcoal seperation technique, and (0.0-2.9%) using second antibody precipitation technique. The B% for the antiserum obtained from New-zealand rabbits ranged from (0.0-18.1) using second antibody precipitation technique. Serum T3, T4 and TSH of the immunized rabbits were measured and found to be not significantly different form the controls (p=0.2211, 0.098, 0.35 respectively

  20. Friction stir spot welding of 2024-T3 aluminum alloy with SiC nanoparticles

    International Nuclear Information System (INIS)

    Paidar, Moslem; Sarab, Mahsa Laali

    2016-01-01

    In this study, the Friction stir spot welding (FSSW) of 2024-T3 aluminum alloy with 1.6 mm thickness was investigated. The effects of the silicon carbide (SiC) nanoparticles on the metallurgical and mechanical properties were discussed. The effects of particles on tension shear and wear tests were also investigated. The process was conducted at a constant rotational speed of 1000 rpm. Results showed that adding SiC nanoparticles to the weld during FSSW had a major effect on the mechanical properties. In fact, the addition of nanoparticles as barriers prevented grain growth in the Stir zone (SZ). The data obtained in the tensile-shear and wear tests showed that tensile-shear load and wear resistance increased with the addition of SiC nanoparticles, which was attributed to the fine grain size produced in the SZ

  1. Corrosion of AA2024-T3 Part I: Localised corrosion of isolated IM particles

    International Nuclear Information System (INIS)

    Boag, A.; Hughes, A.E.; Glenn, A.M.; Muster, T.H.; McCulloch, D.

    2011-01-01

    Research highlights: → Corrosion of AA2024-T3 in 0.1 M NaCl was examined after various immersion times up to 120 min. → It was found that localised corrosion at isolated intermetallic (IM) particles proceeded through a hierarchy of attack. → Attack began at around 2.5 min on S-phase particles which underwent dealloying and by 15 min had developed trenches around their peripheries. The morphology of the corrosion product changed considerably during this period. → Attack on cathodic particles appeared to initiate after corrosion had finished at S-phase particles and proceeded to AlCuFeMn IM particles where the attack was complete by 30 min. Corrosion then became active around the (Al,Cu) x (Fe,Mn) y Si. IM particles and was complete by 120 min immersion. - Abstract: Polished specimens of AA2024-T3 were immersed for various times up to 120 min in 0.1 M NaCl. The development of corrosion around isolated intermetallic particles was monitored using scanning electron microscopy with energy dispersive X-ray spectroscopy (EDXS). The earliest stages of attack started with localised corrosion of the S-phase particles resulting in dealloying which was followed by trenching around these particles. Subsequently, trenching was observed around cathodic particles where trenching started with AlCuFeMn particles with Cu/Fe ratios typically around 2.5 and then progressed to AlCuFeMnSi particles. This latter category of particles had a much lower Cu/Fe ratio, typically 0.5.

  2. Overexpression of the mitochondrial T3 receptor induces skeletal muscle atrophy during aging.

    Directory of Open Access Journals (Sweden)

    François Casas

    2009-05-01

    Full Text Available In previous studies, we characterized a new hormonal pathway involving a mitochondrial T3 receptor (p43 acting as a mitochondrial transcription factor. In in vitro and in vivo studies, we have shown that p43 increases mitochondrial transcription and mitochondrial biogenesis. In addition, p43 overexpression in skeletal muscle stimulates mitochondrial respiration and induces a shift in metabolic and contractile features of muscle fibers which became more oxidative.Here we have studied the influence of p43 overexpression in skeletal muscle of mice during aging. We report that p43 overexpression initially increased mitochondrial mass. However, after the early rise in mitochondrial DNA occurring at 2 months of age in transgenic mice, we observed a progressive decrease of mitochondrial DNA content which became 2-fold lower at 23 months of age relatively to control animals. Moreover, p43 overexpression induced an oxidative stress characterized by a strong increase of lipid peroxidation and protein oxidation in quadriceps muscle, although antioxidant enzyme activities (catalase and superoxide dismutase were stimulated. In addition, muscle atrophy became detectable at 6 months of age, probably through a stimulation of the ubiquitin proteasome pathway via two muscle-specific ubiquitin ligases E3, Atrogin-1/MAFbx and MuRF1.Taken together, these results demonstrate that a prolonged stimulation of mitochondrial activity induces muscle atrophy. In addition, these data underline the importance of a tight control of p43 expression and suggest that a deregulation of the direct T3 mitochondrial pathway could be one of the parameters involved in the occurrence of sarcopenia.

  3. Microstructure Stability During Creep of Friction Stir Welded AA2024-T3 Alloy

    Science.gov (United States)

    Regev, Michael; Rashkovsky, Tal; Cabibbo, Marcello; Spigarelli, Stefano

    2018-01-01

    The poor weldability of the AA2024 aluminum alloy limits its use in industrial applications. Because friction stir welding (FSW) is a non-fusion welding process, it seems to be a promising solution for welding this alloy. In the current study, FSW was applied to butt weld AA2024-T3 aluminum alloy plates. Creep tests were conducted at 250 and at 315 °C on both the parent material and the friction stir welded specimens. The microstructures of the welded and non-welded AA2024-T3 specimens before and after the creep tests were studied and compared. A comprehensive transmission electron microscopy study together with a high-resolution scanning electron microscopy study and energy-dispersive x-ray spectroscopy analysis was conducted to investigate the microstructure stability. The parent material seems to contain two kinds of Cu-rich precipitates—coarse precipitates of a few microns each and uniformly dispersed fine nanosized precipitates. Unlike the parent material, the crept specimens were found to contain the two kinds of precipitates mentioned above together with platelet-like precipitates. In addition, extensive decoration of the grain boundaries with precipitates was clearly observed in the crept specimens. Controlled aging experiments for up to 280 h at the relevant temperatures were conducted on both the parent material and the welded specimens in order to isolate the contribution of exposure to high temperatures to the microstructure changes. TEM study showed the development of dislocation networks into a cellular dislocation structure in the case of the parent metal. Changes in the dislocation structure as a function of the creep strain and the FSW process were recorded. A detailed creep data analysis was conducted, taking into account the instability of the microstructure.

  4. [Research of differential expression of virulence-related proteins of T3SS in Pseudomonas aeruginosa].

    Science.gov (United States)

    Li, Xiao-qiang; Zhuo, Chao; Liao, Dong-jiang; Xiao, Shu-nian; Jin, Guang-yao; Zhong, Nan-shan

    2010-03-30

    To analyze the difference of virulence-related protein concerned with type III secretion system (T3SS) in Pseudomonas aeruginosa during the changes of antibiotic sensitivity and interpret the clinical patient data to explore the relationship between the changes in resistance and variance of virulence. The isolates of Pseudomonas aeruginosa was isolated from the respiratory tract of a same patient with an altered sensitivity of antibiotics. It turned out to be one clone. The homolog of isolates was determined by ERIC-PCR. The Kirby-Bauer antibiotic testing was employed to detect the sensitivity of antibiotics of isolates. PCR was used to detect the gene of T3SS and virulence of isolates and two-dimensional gel electrophoresis to compare the whole-cell proteins. The mass spectrometry was employed to analyze a variety of protein spots. The relevant information was retrieved from protein databases. Clinical record was collected to study the relationship of clinical features, bacteria resistance and virulence-associated protein. One subject was diagnosed with acute exacerbation of chronic obstructive pulmonary disease (AECOPD) complicated with pneumonia. Pseudomonas aeruginosa was isolated from his respiratory tract three times in one year. Sensitivity spectrum of isolates were as follows: sensitivity, multi-drug resistant (MDR) and pan-drug resistance (PDR). Virulence gene was exo U+/exo S-. Twenty-one differentially expressed proteins were revealed by two-dimensional gel electrophoresis in three isolates. The functions of 11 proteins were definite. Only 9 proteins were associated with basal bacterial metabolism. Disulfide oxidoreductase A (DsbA) corresponded to the variation of virulence and sensitivity spectrum. Clinical record revealed that the severe lung infection was caused by the PDR strain and the patient died within one month. The sensitivity spectrum and virulence of Pseudomonas aeruginosa may undergo changes when there is an alteration of eco

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

    Science.gov (United States)

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

    2008-01-01

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

  6. Thoracoscopic bilateral T3 sympathectomy for primary focal hyperhidrosis in children.

    Science.gov (United States)

    Laje, Pablo; Rhodes, Kali; Magee, Leanne; Klarich, Mary Kate

    2017-02-01

    Present our experience in the surgical treatment of primary focal hyperhidrosis of the hands by thoracoscopic bilateral T3 sympathectomy in pediatric patients. Retrospective chart review of all patients operated between 2013 and 2015. We operated and included in the study 28 patients, 22 females and 6 males. Mean age was 14 (6-21) years. All patients had previously tried at least one form of medical therapy with no success. All patients were extensively counseled regarding the potential side effects of the sympathectomy. The operations were done in supine position with the arms extended. All patients were intubated with a double-lumen endotracheal tube for sequential lung isolation. We used a 5-mm port for the scope and a 3-mm port for the instruments, both placed in the axilla. The third rib was identified by fluoroscopy. The sympathectomy was done with monopolar cautery. Mean operative time was 43 (25-71) minutes. No chest tubes were used. The incidence of intraoperative or postoperative complications was zero. All patients were discharged within the first 24 postoperative hours. All patients achieved immediate complete postoperative resolution of the palmar hyperhidrosis, sustained in all cases at a median follow-up of 17 (2-34) months. The mean preoperative quality of life score (based on a multifunctional self-assessment questionnaire) was 41/100, whereas after the operation, it was 92/100. Only 1 patient developed temporary compensatory sweating. All patients were satisfied with the result of the operation. Thoracoscopic bilateral T3 sympathectomy is a safe and effective treatment for children and adolescents with primary focal hyperhidrosis of the hands who failed medical management and have a very low rate of compensatory sweating. IV. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Monoterpene limonene induces brown fat-like phenotype in 3T3-L1 white adipocytes.

    Science.gov (United States)

    Lone, Jameel; Yun, Jong Won

    2016-05-15

    Several dietary compounds that are able to induce the brown fat-like phenotype in white adipocytes have been considered for treatment of obesity due to their ability to increase energy expenditure. Here, we report that limonene induces the brown fat-like phenotype in 3T3-L1 adipocytes by increasing expression of brown adipocyte-specific genes and proteins. Limonene-induced browning in white adipocytes was investigated by determining expression levels of brown fat-specific genes and proteins by real-time RT-PCR, immunoblot analysis, and immunocytochemical staining. Limonene enhanced mitochondrial biogenesis, as evidenced by increased mitochondrial content and immunofluorescent intensity. Limonene also significantly elevated protein levels of HSL, PLIN, p-AMPK, p-ACC, ACO, COX4, CPT1, and CYT C, suggesting its possible role in enhancement of lipolysis and lipid catabolism. Increased expression of PRDM16, UCP1, C/EBPβ, and other brown fat-specific markers by limonene was possibly mediated by activation of β3-adnergenic receptor (β3-AR), as inhibition of β3-AR inhibited up-regulation of brown fat-specific markers. Similarly, limonene-mediated activation of ERK and up-regulation of key brown adipocyte specific markers were eliminated by treatment with ERK antagonist. Taken together, these results suggest that limonene induces browning of 3T3-L1 adipocytes via activation of β3-AR and the ERK signaling pathway. In conclusion, our findings suggest that limonene plays a dual modulatory role in induction of the brown adipocyte-like phenotype as well as promotion of lipid metabolism and thus may have potential therapeutic implications for treatment of obesity. Copyright © 2016 Elsevier Inc. All rights reserved.

  8. Tumor budding is a strong and reproducible prognostic marker in T3N0 colorectal cancer.

    LENUS (Irish Health Repository)

    Wang, Lai Mun

    2012-02-01

    BACKGROUND: Tumor budding along the advancing front of colorectal adenocarcinoma is an early event in the metastatic process. A reproducible, prognostic budding scoring system based on outcomes in early stage colorectal cancer has not been established. DESIGN: One hundred twenty-eight T3N0M0 colorectal carcinoma patients with known outcome were identified. Tumor budding was defined as isolated tumor cells or clusters of <5 cells at the invasive tumor front. Tumor bud counts were generated in 5 regions at 200x by 2 pathologists (conventional bud count method). The median bud count per case was used to divide cases into low (median=0) and high budding (median > or =1) groups. Forty cases were reevaluated to assess reproducibility using the conventional and a novel rapid bud count method. RESULTS: Fifty-seven (45%) carcinomas had high and 71 (55%) had low budding scores. High budding was associated with an infiltrative growth pattern (P<0.0001) and lymphovascular invasion (P=0.005). Five-year cancer-specific survival was significantly poorer in high compared with low budding groups: 63% versus 91%, respectively, P<0.0001. Multivariate analysis demonstrated tumor budding to be independently prognostic (hazard ratio=4.76, P<0.001). Interobserver agreement was at least equivalent comparing the conventional to the rapid bud count methods: 87.5% agreement (kappa=0.75) versus 92.5% agreement (kappa=0.85), respectively. CONCLUSIONS: Tumor budding is a strong, reproducible, and independent prognostic marker of outcome that is easily assessed on hematoxylin and eosin slides. This may be useful for identifying the subset of T3N0M0 patients at high risk of recurrence who may benefit from adjuvant therapy.

  9. ATP-Driven Contraction of Phage T3 Capsids with DNA Incompletely Packaged In Vivo

    Directory of Open Access Journals (Sweden)

    Philip Serwer

    2017-05-01

    Full Text Available Adenosine triphosphate (ATP cleavage powers packaging of a double-stranded DNA (dsDNA molecule in a pre-assembled capsid of phages that include T3. Several observations constitute a challenge to the conventional view that the shell of the capsid is energetically inert during packaging. Here, we test this challenge by analyzing the in vitro effects of ATP on the shells of capsids generated by DNA packaging in vivo. These capsids retain incompletely packaged DNA (ipDNA and are called ipDNA-capsids; the ipDNA-capsids are assumed to be products of premature genome maturation-cleavage. They were isolated via preparative Nycodenz buoyant density centrifugation. For some ipDNA-capsids, Nycodenz impermeability increases hydration and generates density so low that shell hyper-expansion must exist to accommodate associated water. Electron microscopy (EM confirmed hyper-expansion and low permeability and revealed that 3.0 mM magnesium ATP (physiological concentration causes contraction of hyper-expanded, lowpermeability ipDNA-capsids to less than mature size; 5.0 mM magnesium ATP (border of supraphysiological concentration or more disrupts them. Additionally, excess sodium ADP reverses 3.0 mM magnesium ATP-induced contraction and re-generates hyper-expansion. The Nycodenz impermeability implies assembly perfection that suggests selection for function in DNA packaging. These findings support the above challenge and can be explained via the assumption that T3 DNA packaging includes a back-up cycle of ATP-driven capsid contraction and hyper-expansion.

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

    International Nuclear Information System (INIS)

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

    2012-01-01

    Highlights: ► Overexpression of ATF3 inhibits adipocyte differentiation in 3T3-L1 cells. ► Overexpression of ATF3 represses C/EBPα expression. ► ATF3 directly binds to mouse C/EBPα promoter spanning from −1928 to −1907. ► 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/EBPα transcript and repressed the activity of the 3.6-kb mouse C/EBPα promoter, demonstrating that ATF3 downregulates C/EBPα expression. Transfection studies using mutant constructs containing 5′-deletions in the C/EBPα 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/EBPα promoter spanning from −1928 to −1907. Both chemical hypoxia-mimetics or physical hypoxia led to reduce the C/EBPα mRNA and repress the promoter activity of the C/EBPα gene, whereas increase ATF3 mRNA, suggesting that ATF3 may contribute to the inhibition of adipocyte differentiation in hypoxia through downregulation of C/EBPα expression. Collectively, these results demonstrate that ATF3 represses the C/EBPα gene, resulting in inhibition of adipocyte differentiation, and thus plays a role in hypoxia-mediated inhibition of adipocyte differentiation.

  11. Analysis of the Rana catesbeiana tadpole tail fin proteome and phosphoproteome during T3-induced apoptosis: identification of a novel type I keratin.

    Science.gov (United States)

    Domanski, Dominik; Helbing, Caren C

    2007-08-06

    Thyroid hormones (THs) are vital in the maintenance of homeostasis and in the control of development. One postembryonic developmental process that is principally regulated by THs is amphibian metamorphosis. This process has been intensively studied at the genomic level yet very little information at the proteomic level exists. In addition, there is increasing evidence that changes in the phosphoproteome influence TH action. Here we identify components of the proteome and phosphoproteome in the tail fin that changed within 48 h of exposure of premetamorphic Rana catesbeiana tadpoles to 10 nM 3,5,3'-triiodothyronine (T3). To this end, we developed a cell and protein fractionation method combined with two-dimensional gel electrophoresis and phosphoprotein-specific staining. Altered proteins were identified using mass spectrometry (MS). We identified and cloned a novel Rana larval type I keratin, RLK I, which may be a target for caspase-mediated proteolysis upon exposure to T3. In addition, the RLK I transcript is reduced during T3-induced and natural metamorphosis which is consistent with a larval keratin. Furthermore, GILT, a protein involved in the immune system, is changed in phosphorylation state which is linked to its activation. Using a complementary MS technique for the analysis of differentially-expressed proteins, isobaric tags for relative and absolute quantitation (iTRAQ) revealed 15 additional proteins whose levels were altered upon T3 treatment. The success of identifying proteins whose levels changed upon T3 treatment with iTRAQ was enhanced through de novo sequencing of MS data and homology database searching. These proteins are involved in apoptosis, extracellular matrix structure, immune system, metabolism, mechanical function, and oxygen transport. We have demonstrated the ability to derive proteomics-based information from a model species for postembryonic development for which no genome information is currently available. The present study

  12. Analysis of the Rana catesbeiana tadpole tail fin proteome and phosphoproteome during T3-induced apoptosis: identification of a novel type I keratin

    Directory of Open Access Journals (Sweden)

    Helbing Caren C

    2007-08-01

    Full Text Available Abstract Background Thyroid hormones (THs are vital in the maintenance of homeostasis and in the control of development. One postembryonic developmental process that is principally regulated by THs is amphibian metamorphosis. This process has been intensively studied at the genomic level yet very little information at the proteomic level exists. In addition, there is increasing evidence that changes in the phosphoproteome influence TH action. Results Here we identify components of the proteome and phosphoproteome in the tail fin that changed within 48 h of exposure of premetamorphic Rana catesbeiana tadpoles to 10 nM 3,5,3'-triiodothyronine (T3. To this end, we developed a cell and protein fractionation method combined with two-dimensional gel electrophoresis and phosphoprotein-specific staining. Altered proteins were identified using mass spectrometry (MS. We identified and cloned a novel Rana larval type I keratin, RLK I, which may be a target for caspase-mediated proteolysis upon exposure to T3. In addition, the RLK I transcript is reduced during T3-induced and natural metamorphosis which is consistent with a larval keratin. Furthermore, GILT, a protein involved in the immune system, is changed in phosphorylation state which is linked to its activation. Using a complementary MS technique for the analysis of differentially-expressed proteins, isobaric tags for relative and absolute quantitation (iTRAQ revealed 15 additional proteins whose levels were altered upon T3 treatment. The success of identifying proteins whose levels changed upon T3 treatment with iTRAQ was enhanced through de novo sequencing of MS data and homology database searching. These proteins are involved in apoptosis, extracellular matrix structure, immune system, metabolism, mechanical function, and oxygen transport. Conclusion We have demonstrated the ability to derive proteomics-based information from a model species for postembryonic development for which no genome

  13. Wakefield Computations for the CLIC PETS using the Parallel Finite Element Time-Domain Code T3P

    Energy Technology Data Exchange (ETDEWEB)

    Candel, A; Kabel, A.; Lee, L.; Li, Z.; Ng, C.; Schussman, G.; Ko, K.; /SLAC; Syratchev, I.; /CERN

    2009-06-19

    In recent years, SLAC's Advanced Computations Department (ACD) has developed the high-performance parallel 3D electromagnetic time-domain code, T3P, for simulations of wakefields and transients in complex accelerator structures. T3P is based on advanced higher-order Finite Element methods on unstructured grids with quadratic surface approximation. Optimized for large-scale parallel processing on leadership supercomputing facilities, T3P allows simulations of realistic 3D structures with unprecedented accuracy, aiding the design of the next generation of accelerator facilities. Applications to the Compact Linear Collider (CLIC) Power Extraction and Transfer Structure (PETS) are presented.

  14. Development of an 'In vitro' system for the caption essay of T3; Desarrollo de un sistema 'In vitro' para el ensayo de captacion de T3

    Energy Technology Data Exchange (ETDEWEB)

    Lavalley, C.; Ferro, G.; Zambrano, F.; Lezama, J

    1990-02-15

    Triiodothyronine uptake (T3U) is a qualitative technique for evaluation of the unsaturation capacity of thyroid binding globulin (TBG). This paper presents results related to a T3 standardized serum and the integration of T3-I-125, and adsorbent for labelled hormone. Labelled hormone were prepared by the chloramine T method and then purified by high performance liquid chromatography. The specific activity was 500 {mu} Ci/ {mu} g. Various adsorbents such as: Norit A Charcoal, calcium silicate, talc, bovine serum albumin macroaggregated (BSAM) were used in different buffers as: Tris-HCl, barbital and Michaelis. Standardized serum was prepared by mixing different euthyroid sera. Best conditions for T3U assays were achieved with 15 mg/ml. BSAM at pH 8.6 in presence of Tris-HCl buffer for hypothyroid and hyperthyroid sera, for which we obtained < 0.9 {+-} 0.04 and > 1.1 {+-} 0.05 respectively as a T3U index with a 3.0 % of coefficient variation. The reagents so prepared can be conveniently used for T3U assays. (Author)

  15. Archive of Geosample Data and Information from the University of Wisconsin Ice Island T3 Core Collection

    Data.gov (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — From 1963 to 1972, 349 piston cores were collected from the Arctic Ocean using Ice Island T3 as a sampling platform and sent to the University of Wisconsin-Madison...

  16. [Effects of triterpenoid from Psidium guajava leaves ursolic acid on proliferation, differentiation of 3T3-L1 preadipocyte and insulin resistance].

    Science.gov (United States)

    Lin, Juan-Na; Kuang, Qiao-Ting; Ye, Kai-He; Ye, Chun-Ling; Huang, Yi; Zhang, Xiao-Qi; Ye, Wen-Cai

    2013-08-01

    To investigate the influences of triterpenoid from Psidium guajava Leaves (ursolic acid) on the proliferation, differentiation of 3T3-L1 preadipocyte, and its possible mechanism treat for insulin resistance. 3T3-L1 preadipocyte was cultured in vitro. After adding ursolic acid to the culture medium for 48h, the cell viability was tested by MTT assay. Induced for 6 days, the lipid accumulation of adipocyte was measured by Oil Red O staining. The insulin resistant cell model was established with Dexamethasone. Cellular glucose uptake was determined with GOD-POD assays and FFA concentration was determined at the time of 48h. Secreted adiponectin were measured by ELISA. The protein levels of PPARgamma and PTP1B in insulin resistant adipocyte were measured by Western Blotting. Compared with medium control group, 30, 100 micromol/L ursolic acid could increase its proliferation and differentiation significantly (P 0.05). Ursolic acid can improve the proliferation and differentiation of 3T3-L1 preadipocyte, enhance cellular glucose uptake, inhibit the production of FFA, promote the secretion of adiponectin insulin resistant adipocyte, its mechanism may be related to upregulating the expression of PPARgamma protein.

  17. Troponin T3 regulates nuclear localization of the calcium channel Cavβ1a subunit in skeletal muscle

    International Nuclear Information System (INIS)

    Zhang, Tan; Taylor, Jackson; Jiang, Yang; Pereyra, Andrea S.; Messi, Maria Laura; Wang, Zhong-Min; Hereñú, Claudia; Delbono, Osvaldo

    2015-01-01

    The voltage-gated calcium channel (Ca v ) β 1a subunit (Ca v β 1a ) plays an important role in excitation–contraction coupling (ECC), a process in the myoplasm that leads to muscle-force generation. Recently, we discovered that the Ca v β 1a subunit travels to the nucleus of skeletal muscle cells where it helps to regulate gene transcription. To determine how it travels to the nucleus, we performed a yeast two-hybrid screening of the mouse fast skeletal muscle cDNA library and identified an interaction with troponin T3 (TnT3), which we subsequently confirmed by co-immunoprecipitation and co-localization assays in mouse skeletal muscle in vivo and in cultured C2C12 muscle cells. Interacting domains were mapped to the leucine zipper domain in TnT3 COOH-terminus (160–244 aa) and Ca v β 1a NH 2 -terminus (1–99 aa), respectively. The double fluorescence assay in C2C12 cells co-expressing TnT3/DsRed and Ca v β 1a /YFP shows that TnT3 facilitates Ca v β 1a nuclear recruitment, suggesting that the two proteins play a heretofore unknown role during early muscle differentiation in addition to their classical role in ECC regulation. - Highlights: • Previously, we demonstrated that Ca v β 1a is a gene transcription regulator. • Here, we show that TnT3 interacts with Ca v β 1a . • We mapped TnT3 and Ca v β 1a interaction domain. • TnT3 facilitates Ca v β 1a nuclear enrichment. • The two proteins play a heretofore unknown role during early muscle differentiation

  18. Supplementation of T3 Recovers Hypothyroid Rat Liver Cells from Oxidatively Damaged Inner Mitochondrial Membrane Leading to Apoptosis

    Science.gov (United States)

    Mukherjee, Sutapa; Samanta, Luna; Roy, Anita; Bhanja, Shravani; Chainy, Gagan B. N.

    2014-01-01

    Hypothyroidism is a growing medical concern. There are conflicting reports regarding the mechanism of oxidative stress in hypothyroidism. Mitochondrial oxidative stress is pivotal to thyroid dysfunction. The present study aimed to delineate the effects of hepatic inner mitochondrial membrane dysfunction as a consequence of 6-n-propyl-2-thiouracil-induced hypothyroidism in rats. Increased oxidative stress predominance in the submitochondrial particles (SMP) and altered antioxidant defenses in the mitochondrial matrix fraction correlated with hepatocyte apoptosis. In order to check whether the effects caused by hypothyroidism are reversed by T3, the above parameters were evaluated in a subset of T3-treated hypothyroid rats. Complex I activity was inhibited in hypothyroid SMP, whereas T3 supplementation upregulated electron transport chain complexes. Higher mitochondrial H2O2 levels in hypothyroidism due to reduced matrix GPx activity culminated in severe oxidative damage to membrane lipids. SMP and matrix proteins were stabilised in hypothyroidism but exhibited increased carbonylation after T3 administration. Glutathione content was higher in both. Hepatocyte apoptosis was evident in hypothyroid liver sections; T3 administration, on the other hand, exerted antiapoptotic and proproliferative effects. Hence, thyroid hormone level critically regulates functional integrity of hepatic mitochondria; hypothyroidism injures mitochondrial membrane lipids leading to hepatocyte apoptosis, which is substantially recovered upon T3 supplementation. PMID:24987693

  19. Supplementation of T3 Recovers Hypothyroid Rat Liver Cells from Oxidatively Damaged Inner Mitochondrial Membrane Leading to Apoptosis

    Directory of Open Access Journals (Sweden)

    Sutapa Mukherjee

    2014-01-01

    Full Text Available Hypothyroidism is a growing medical concern. There are conflicting reports regarding the mechanism of oxidative stress in hypothyroidism. Mitochondrial oxidative stress is pivotal to thyroid dysfunction. The present study aimed to delineate the effects of hepatic inner mitochondrial membrane dysfunction as a consequence of 6-n-propyl-2-thiouracil-induced hypothyroidism in rats. Increased oxidative stress predominance in the submitochondrial particles (SMP and altered antioxidant defenses in the mitochondrial matrix fraction correlated with hepatocyte apoptosis. In order to check whether the effects caused by hypothyroidism are reversed by T3, the above parameters were evaluated in a subset of T3-treated hypothyroid rats. Complex I activity was inhibited in hypothyroid SMP, whereas T3 supplementation upregulated electron transport chain complexes. Higher mitochondrial H2O2 levels in hypothyroidism due to reduced matrix GPx activity culminated in severe oxidative damage to membrane lipids. SMP and matrix proteins were stabilised in hypothyroidism but exhibited increased carbonylation after T3 administration. Glutathione content was higher in both. Hepatocyte apoptosis was evident in hypothyroid liver sections; T3 administration, on the other hand, exerted antiapoptotic and proproliferative effects. Hence, thyroid hormone level critically regulates functional integrity of hepatic mitochondria; hypothyroidism injures mitochondrial membrane lipids leading to hepatocyte apoptosis, which is substantially recovered upon T3 supplementation.

  20. Coculture with BJ fibroblast cells inhibits the adipogenesis and lipogenesis in 3T3-L1 cells

    International Nuclear Information System (INIS)

    Jeong, Hyun Jeong; Park, Sahng Wook; Kim, Hojeong; Park, Sang-Kyu; Yoon, Dojun

    2010-01-01

    Mouse or human fibroblasts are commonly used as feeder cells to prevent differentiation in stem or primary cell culture. In the present study, we addressed whether fibroblasts can affect the differentiation of adipocytes. We found that the differentiation of 3T3-L1 preadipocytes was strongly suppressed when the cells were cocultured with human fibroblast (BJ) cells. BrdU incorporation analysis indicated that mitotic clonal expansion, an early event required for 3T3-L1 cell adipogenesis, was not affected by BJ cells. The 3T3-L1 cell expression levels of peroxisome proliferator-activated receptor γ2, CCAAT/enhancer-binding protein alpha (C/EBPα), sterol regulatory element binding protein-1c, and Krueppel-like factor 15, but not those of C/EBPβ or C/EBPδ, were decreased by coculture with BJ cells. When mature 3T3-L1 adipocytes were cocultured with BJ cells, their lipid contents were significantly reduced, with decreased fatty acid synthase expression and increased phosphorylated form of acetyl-CoA carboxylase 1. Our data indicate that coculture with BJ fibroblast cells inhibits the adipogenesis of 3T3-L1 preadipocytes and decreases the lipogenesis of mature 3T3-L1 adipocytes.

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

    Energy Technology Data Exchange (ETDEWEB)

    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); Seong, Je Kyung [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

  2. Plasticizers used in food-contact materials affect adipogenesis in 3T3-L1 cells.

    Science.gov (United States)

    Pomatto, Valentina; Cottone, Erika; Cocci, Paolo; Mozzicafreddo, Matteo; Mosconi, Gilberto; Nelson, Erik Russel; Palermo, Francesco Alessandro; Bovolin, Patrizia

    2018-04-01

    Recent studies suggest that exposure to some plasticizers, such as Bisphenol A (BPA), play a role in endocrine/metabolic dispruption and can affect lipid accumulation in adipocytes. Here, we investigated the adipogenic activity and nuclear receptor interactions of four plasticizers approved for the manufacturing of food-contact materials (FCMs) and currently considered safer alternatives. Differentiating 3T3-L1 mouse preadipocytes were exposed to scalar concentrations (0.01-25 μM) of DiNP (Di-iso-nonyl-phthalate), DiDP (Di-iso-decyl-phthalate), DEGDB (Diethylene glycol dibenzoate), or TMCP (Tri-m-cresyl phosphate). Rosiglitazone, a well-known pro-adipogenic peroxisome proliferator activated receptor gamma (PPARγ) agonist, and the plasticizer BPA were included as reference compounds. All concentrations of plasticizers were able to enhance lipid accumulation, with TMCP being the most effective one. Accordingly, when comparing in silico the ligand binding efficiencies to the nuclear receptors PPARγ and retinoid-X-receptor-alpha (RXRα), TMPC displayed the highest affinity to both receptors. Differently from BPA, the four plasticizers were most effective in enhancing lipid accumulation when added in the mid-late phase of differentiation, thus suggesting the involvement of different intracellular signalling pathways. In line with this, TMCP, DiDP, DiNP and DEGDB were able to activate PPARγ in transient transfection assays, while previous studies demonstrated that BPA acts mainly through other nuclear receptors. qRT-PCR studies showed that all plasticizers were able to increase the expression of CCAAT/enhancer binding protein β (Cebpβ) in the early steps of adipogenesis, and the adipogenesis master gene Pparγ2 in the middle phase, with very similar efficacy to that of Rosiglitazone. In addition, TMCP was able to modulate the expression of both Fatty Acid Binding Protein 4/Adipocyte Protein 2 (Fabp4/Ap2) and Lipoprotein Lipase (Lpl) transcripts in the late phase

  3. Enhancement of BALB/c 3T3 cells transformation by 1,2-dibromoethane promoting effect.

    Science.gov (United States)

    Colacci, A; Vaccari, M; Perocco, P; Da Vià, C; Silingardi, P; Manzini, E; Horn, W; Grilli, S

    1996-02-01

    Two of the most representative halogenated aliphatic hydrocarbons, 1,2-dibromoethane and 1,1,2,2-tetrachloroethane, were tested in the two-stage cell transformation model for analysing the promoting ability. Both of these compounds had previously been found to exert genotoxic effects, probably acting as moderate initiators. BALB/c 3T3 cells were initiated with subtransforming doses of N-methyl-N-nitro-N-nitrosoguanidine or 3-methylcholanthrene and then exposed to a chronic treatment with different non-transforming dosages of the two haloalkanes. 1,1,2,2-Tetrachloroethane did not exert any promoting activity in that system. By contrast, significant promoting effects by 1,2-dibromoethane were observed both in cells treated with N-methyl-N-nitro-N-nitrosoguanidine and in cells treated with 3-methylcholanthrene. Promotion of the transformation process initiated with 3-methylcholanthrene was detectable when confluent cells in the chemical-treated plates were replated in the level-II amplification test. This experimental procedure allowed cells to perform further rounds of replications and transformed foci to became detectable. Results gave evidence for a promoting role of 1,2-dibromoethane in multistep carcinogenesis, probably responsible for the higher oncogenic ability of this compound with respect to 1,1,2,2-tetrachloroethane.

  4. Retroviral-mediated gene transfer of human phenylalanine hydroxylase into NIH 3T3 and hepatoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Ledley, F.D.; Grenett, H.E.; McGinnis-Shelnutt, M.; Woo, S.L.C.

    1986-01-01

    Phenylketonuria (PKU) is caused by deficiency of the hepatic enzyme phenylalanine hydroxylase (PAH). A full-length human PAH cDNA sequence has been inserted into pzip-neoSV(X), which is a retroviral vector containing the bacterial neo gene. The recombinant has been transfected into Psi2 cells, which provide synthesis of the retroviral capsid. Recombinant virus was detected in the culture medium of the transfected Psi2 cells, which is capable of transmitting the human PAH gene into mouse NIH 3T3 cells by infection leading to stable incorporation of the recombinant provirus. Infected cells express PAH mRNA, immunoreactive PAH protein, and exhibit pterin-dependent phenylaline hydroxylase activity. The recombinant virus is also capable of infecting a mouse hepatoma cell line that does not normal synthesize PAH. PAH activity is present in the cellular extracts and the entire hydroxylation system is reconstituted in the hepatoma cells infected with the recombinant viruses. Thus, recombinant viruses containing human PAH cDNA provide a means for introducing functional PAH into mammalian cells of hepatic origin and can potentially be introduced into whole animals as a model for somatic gene therapy for PKU.

  5. Corrosion of AA2024-T3 Part II: Co-operative corrosion

    International Nuclear Information System (INIS)

    Hughes, A.E.; Boag, A.; Glenn, A.M.; McCulloch, D.; Muster, T.H.; Ryan, C.; Luo, C.; Zhou, X.; Thompson, G.E.

    2011-01-01

    Research highlights: → Corrosion of AA2024 in 0.1 M NaCl was examined for immersion times up to 120 min. → Rings of corrosion products with H 2 evolution developed as early as 5 min after immersion. → The rings were distinct from corrosion around isolated intermetallics elsewhere on the surface. → Analyses of chloride containing sites (red) showed a significant level of IM particle clustering. → The number of neighbors was much higher at these sites than the average particle number density. - Abstract: Polished specimens of AA2024-T3 alloy were immersed for up to 120 min in 0.1 M NaCl. The development of corrosion was monitored using scanning electron microscopy with energy dispersive X-ray spectroscopy (EDXS) and particle induced X-ray emission spectroscopy (PIXE). Both techniques revealed the intermetallic (IM) particle distributions and attack sites as distinguished by detection of chloride species. The earliest stages of attack involved localized attack around isolated IM particles as reported in Part I. Additionally attack occurred on a larger scale developing rapidly with rings of corrosion product surrounding clusters of IM particles. There were significantly higher numbers of IM particles within the corrosion rings, indicating that local clustering played an important role in co-operative corrosion.

  6. Dietary xenoestrogens differentially impair 3T3-L1 preadipocyte differentiation and persistently affect leptin synthesis.

    Science.gov (United States)

    Phrakonkham, Pascal; Viengchareun, Say; Belloir, Christine; Lombès, Marc; Artur, Yves; Canivenc-Lavier, Marie-Chantal

    2008-05-01

    Recent observations have highlighted adipogenesis alterations under exposure to several xenoestrogens at critical stages, and pointed at their possible involvement in the pathogenesis of obesity. However, it remains unclear whether these effects are mediated by classical estrogen receptor (ER) binding and subsequent transcriptional modulation. The aim of this study was to determine the (anti-)adipogenic impact of apigenin, bisphenol A, genistein and 17beta-estradiol at the onset of adipose cell maturation, and to correlate it to their estrogenic potential. In steroid-free conditions, 3T3-L1 preadipocytes were induced to differentiate in the presence of xenoestrogens for 2 days. DNA and triglyceride levels, leptin secretion and expression of Pref-1, C/EBPbeta, PPARgamma2, FAS, leptin and ERs were measured on days 0, 3 and 8 of differentiation. Genistein potently blocked mitotic clonal expansion and all markers of maturation. Bisphenol A and estradiol did not modify triglyceride accumulation but increased the expression of differentiation genes. Apigenin caused a weak but reversible delay in adipogenesis although it unexpectedly enhanced leptin synthesis. However, the expression of steroid hormone receptors was not associated with these differential effects. In conclusion, we could not put a clear estrogen-dependent mechanism forward, but early exposure to xenoestrogens persistently disrupted adipocyte gene expression and leptin synthesis.

  7. Influence of friction stir welding parameters on properties of 2024 T3 aluminium alloy joints

    Directory of Open Access Journals (Sweden)

    Eramah Abdsalam M.

    2014-01-01

    Full Text Available The aim of this work is to analyse the process of friction stir welding (FSW of 3mm thick aluminium plates made of high strength aluminium alloy - 2024 T3, as well as to assess the mechanical properties of the produced joints. FSW is a modern procedure which enables joining of similar and dissimilar materials in the solid state, by the combined action of heat and mechanical work. This paper presents an analysis of the experimental results obtained by testing the butt welded joints. Tensile strength of the produced joints is assessed, as well as the distribution of hardness, micro-and macrostructure through the joints (in the base material, nugget, heat affected zone and thermo-mechanically affected zone. Different combinations of the tool rotation speed and the welding speed are used, and the dependence of the properties of the joints on these parameters of welding technology is determined. [Projekat Ministarstva nauke Republike Srbije, br. TR 34018 i br. TR 35006

  8. Differentiation of the insulin-sensitive glucose transporter in 3T3-L1 adipocytes

    International Nuclear Information System (INIS)

    Frost, S.C.; Baly, D.L.; Cushman, S.W.; Lane, M.D.; Simpson, I.A.

    1986-01-01

    3T3-L1 fibroblasts differentiate in culture to resemble adipocytes both morphologically and biochemically. Insulin-sensitive glucose transport, as measured by 2-deoxy-[1- 14 C]- glucose uptake in the undifferentiated cell is small (2X). In contrast, the rate of glucose transport in fully differentiated cells is elevated 15-fold over basal in the presence of insulin. To determine if this is due to an increase in the number of transporters/cell or accessibility to the transporters, the number of transporters was measured in subcellular fractions over differentiation using a 3 H-cytochalasin B binding assay. The increase in the rate of insulin-sensitive glucose transport directly parallels an increase in the number of transporters which reside in an insulin-responsive intracellular compartment. This observation was confirmed by identifying the transporters by immunoblotting using an antibody generated against the human erythrocyte transporter. The molecular weight of this transporter increases over differentiation from a single band of 40kDa to a heterogeneous triplet of 40, 44 and 48kDa. These data suggest that the transporter undergoes differential processing and that the functional, insulin-responsive transporter may be different from the insulin-insensitive (basal) transporter

  9. Microstructural effects on the initiation of zinc phosphate coatings on 2024-T3 aluminum alloy

    Energy Technology Data Exchange (ETDEWEB)

    Susac, D.; Sun, X.; Li, R.Y.; Wong, K.C.; Wong, P.C.; Mitchell, K.A.R.; Champaneria, R

    2004-12-15

    The initiation of coatings deposited on to 2024-T3 aluminum alloy from supersaturated zinc phosphating solutions has been studied using scanning Auger microscopy (SAM), scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). The alloy microstructure, especially associated with the second-phase particles, strongly affects the formation stages of the coating process, where etching of the substrate has a significant role. At the start, zinc phosphate (ZPO) crystals form on the Al-Cu-Mg second-phase particles, rather than on the matrix or on the Al-Cu-Fe-Mn particles, with the initial nucleation appearing at interfaces between Al-Cu-Mg particles and the matrix. In contrast, the formation of the ZPO coating is delayed on the cathodic Al-Cu-Fe-Mn particles, compared to those of the Al-Cu-Mg composition. When the coating process is completed, the whole sample surface is covered with ZPO although its thickness varies at the different micro-regions.

  10. Quantification of Electroporation-Mediated Propidium Iodide Delivery into 3T3 Cells

    Science.gov (United States)

    Sadik, Mohamed M.; Li, Jianbo; Shan, Jerry W.; Shreiber, David I.; Lin, Hao

    2010-11-01

    Electroporation is an effective means to deliver exogenous molecules into the cellular cytoplasm, while simultaneously maintaining cell viability and functionality. In this technique, an applied electric field transiently permeabilizes the cellular membrane to enable molecular exchange. The main objective of the current work is to identify the transport mechanisms involved during electroporation, and to quantify the amount of molecules delivered into the cellular cytoplasm. An optical diagnostic system is developed to examine the transport of Propidium Iodide (PI) into 3T3 mouse fibroblast cells. Upon entering the permeabilized cell, PI binds to DNA/RNA within the cytoplasm to emit fluorescence, which is measured to track the dynamic accumulation of the dye within the cell. The results show that the total fluorescence intensity increases with a decreasing buffer electrical conductivity. The data are compared with numerical simulations, which reveals good agreement. The experimental observations and numerical analysis demonstrate that: 1) Electrophoresis plays a dominant role in mediating the transport. 2) An electrokinetic mechanism, field-amplified sample stacking, controls the achievable delivery efficiency. The study in this work is an important step toward the quantification as well as the eventual improvement of this useful technique.

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

    International Nuclear Information System (INIS)

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

    2001-01-01

    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 μm 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 deg. 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

  12. Large-Scale Automated Analysis of Location Patterns in Randomly-Tagged 3T3 Cells

    Science.gov (United States)

    Osuna, Elvira García; Hua, Juchang; Bateman, Nicholas W.; Zhao, Ting; Berget, Peter B.; Murphy, Robert F.

    2010-01-01

    Location proteomics is concerned with the systematic analysis of the subcellular location of proteins. In order to perform high-resolution, high-throughput analysis of all protein location patterns, automated methods are needed. Here we describe the use of such methods on a large collection of images obtained by automated microscopy to perform high-throughput analysis of endogenous proteins randomly-tagged with a fluorescent protein in NIH 3T3 cells. Cluster analysis was performed to identify the statistically significant location patterns in these images. This allowed us to assign a location pattern to each tagged protein without specifying what patterns are possible. To choose the best feature set for this clustering, we have used a novel method that determines which features do not artificially discriminate between control wells on different plates and uses Stepwise Discriminant Analysis (SDA) to determine which features do discriminate as much as possible among the randomly-tagged wells. Combining this feature set with consensus clustering methods resulted in 35 clusters among the first 188 clones we obtained. This approach represents a powerful automated solution to the problem of identifying subcellular locations on a proteome-wide basis for many different cell types. PMID:17285363

  13. Exogenous lactate interferes with cell-cycle control in BALB/3T3 mouse fibroblasts

    International Nuclear Information System (INIS)

    Rutz, H. Peter; Little, John B.

    1995-01-01

    Purpose: Previous studies have shown that exogenous lactate may influence proliferation rates, radiation sensitivity, and postirradiation repair capacity of mammalian cells. In the present study, we addressed the question of potential underlying mechanisms and, therefore, examined effects of exogenous lactate on proliferation rates and cell-cycle distribution in immortal but nontumorigenic mammalian cells. Methods and Materials: Cells were grown at 37 deg. C in an incubator with 5% CO 2 and 95% air, in a culture medium supplemented or not with lactate at a 10 mM concentration. Daily, we changed the culture medium and counted cells per dish. On selected days, cell-cycle distribution was determined by flow cytometry. Balb/3T3 mouse fibroblasts were used. Results: During the exponential phase of cell proliferation, mean population doubling time was significantly increased from 17.7 to 19.9 h, due to selective prolongation of G 2 /M. However, in density-inhibited cultures, exogenous lactate stimulated entry into S and proliferation to a significantly higher saturation density. Conclusions: These findings indicate that exogenous lactate interferes with mechanisms of cell-cycle control at two different points in the cell-cycle, depending on cell density and the resulting absence or presence of inhibition of cell proliferation. Interference with cell-cycle control may underlay the modification by exogenous lactate of radiosensitivity and postirradiation repair capacity in mammalian cells

  14. Adhesion, Proliferation and Migration of NIH/3T3 Cells on Modified Polyaniline Surfaces.

    Science.gov (United States)

    Rejmontová, Petra; Capáková, Zdenka; Mikušová, Nikola; Maráková, Nela; Kašpárková, Věra; Lehocký, Marián; Humpolíček, Petr

    2016-09-15

    Polyaniline shows great potential and promises wide application in the biomedical field thanks to its intrinsic conductivity and material properties, which closely resemble natural tissues. Surface properties are crucial, as these predetermine any interaction with biological fluids, proteins and cells. An advantage of polyaniline is the simple modification of its surface, e.g., by using various dopant acids. An investigation was made into the adhesion, proliferation and migration of mouse embryonic fibroblasts on pristine polyaniline films and films doped with sulfamic and phosphotungstic acids. In addition, polyaniline films supplemented with poly (2-acrylamido-2-methyl-1-propanesulfonic) acid at various ratios were tested. Results showed that the NIH/3T3 cell line was able to adhere, proliferate and migrate on the pristine polyaniline films as well as those films doped with sulfamic and phosphotungstic acids; thus, utilization of said forms in biomedicine appears promising. Nevertheless, incorporating poly (2-acrylamido-2-methyl-1-propanesulfonic) acid altered the surface properties of the polyaniline films and significantly affected cell behavior. In order to reveal the crucial factor influencing the surface/cell interaction, cell behavior is discussed in the context of the surface energy of individual samples. It was clearly demonstrated that the lesser the difference between the surface energy of the sample and cell, the more cyto-compatible the surface is.

  15. Polyaniline-lignosulfonate/epoxy coating for corrosion protection of AA2024-T3

    International Nuclear Information System (INIS)

    Gupta, G.; Birbilis, N.; Cook, A.B.; Khanna, A.S.

    2013-01-01

    Highlights: ► Synthesis of corrosion resistant, adherent Pani-LGS coating. ► 5% Pani-LGS/epoxy shows highest corrosion protection values after 30 days immersion. ► Higher corrosion protection was due to barrier effect and anodic protection. ► A mechanism for the corrosion protection is presented. - Abstract: Corrosion protection arising from epoxy coatings incorporating lignosulfonate-doped polyaniline (Pani-LGS) upon AA2024-T3 was studied in 0.6 M NaCl. Synthesized Pani-LGS particles were investigated using TEM, FTIR, TGA and conductivity, whilst coatings were also physically examined using SEM. The coating performance was studied using a combination of potentiodynamic polarisation, EIS, FTIR spectroscopy and X-ray photoelectron spectroscopy. The performance of Pani-LGS/epoxy blends is discussed more generally, with tests revealing that on exposure to 0.6 M NaCl solution for 30 days, a 5 wt% Pani-LGS/epoxy coating resulted in low levels of corrosion. A mechanism for the postulated mode of corrosion protection is presented.

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

    International Nuclear Information System (INIS)

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

    1987-01-01

    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-β-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

  17. Importância do co-cultivo com fibroblastos de camundongo 3T3 para estabelecer cultura de suspensão de células epiteliais do limbo humano Importance of 3T3 feeder layer to establish epithelial cultures from cell suspension obtained from corneo-scleral rims

    Directory of Open Access Journals (Sweden)

    Priscila Cardoso Cristovam

    2008-10-01

    segments was placed with the epithelial side up on the bottom of a 6-well culture plate (Group A. The other two fragments were trypsinized and the obtained cell suspension was cultured with (Group B or without (Group C irradiaded 3T3 cells. The cells were cultured in supplemental hormonal epithelial medium (SHEM, the epithelial migration and clone formation in groups A, B and C were evaluated with phase contrast microscopy and rodamine B staining. RESULTS: Epithelial cell growth was observed in 4/6 rims (Group A. All epithelial cell suspensions that were cultured with 3T3 cells (Group B formed clones. No adhesion or true clone formation (holo- or meroclones was observed in the cell suspensions that were cultivated without 3T3 (Group C (p=0.009. CONCLUSIONS: Epithelial cell suspension obtained from corneo-scleral rims in this model needs to be cultivated with 3T3 cells in order to form clones and establish limbal epithelial cell colonies with the potential to be used for ocular surface reconstruction.

  18. Evaluation of the prognostic significance of perirenal fat invasion and tumor size in patients with pT1-pT3a localized renal cell carcinoma in a comprehensive multicenter study of the CORONA project. Can we improve prognostic discrimination for patients with stage pT3a tumors?

    Science.gov (United States)

    Brookman-May, Sabine D; May, Matthias; Wolff, Ingmar; Zigeuner, Richard; Hutterer, Georg C; Cindolo, Luca; Schips, Luigi; De Cobelli, Ottavio; Rocco, Bernardo; De Nunzio, Cosimo; Tubaro, Andrea; Coman, Ioman; Truss, Michael; Dalpiaz, Orietta; Feciche, Bogdan; Figenshau, Robert S; Madison, Kerry; Sánchez-Chapado, Manuel; Santiago Martin, Maria del Carmen; Salzano, Luigi; Lotrecchiano, Giuseppe; Zastrow, Stefan; Wirth, Manfred; Sountoulides, Petros; Shariat, Shahrokh; Waidelich, Raphaela; Stief, Christian; Gunia, Sven

    2015-05-01

    The current TNM system for renal cell carcinoma (RCC) merges perirenal fat invasion (PFI) and renal vein invasion (RVI) as stage pT3a despite limited evidence concerning their prognostic equivalence. In addition, the prognostic value of PFI compared to pT1-pT2 tumors remains controversial. To analyze the prognostic significance of PFI, RVI, and tumor size in pT1-pT3a RCC. Data for 7384 pT1a-pT3a RCC patients were pooled from 12 centers. Patients were grouped according to stages and PFI/RVI presence as follows: pT1-2N0M0 (n=6137; 83.1%), pT3aN0M0 + PFI (n=1036; 14%), and pT3aN0M0 (RVI ± PFI; n=211; 2.9%). Radical nephrectomy or nephron-sparing surgery (NSS) (1992-2010). Cancer-specific survival was estimated using the Kaplan-Meier method. Univariate and multivariate Cox proportional-hazards regression models, as well as sensitivity and discrimination analyses, were used to evaluate the impact of clinicopathologic parameters on cancer-specific mortality (CSM). Compared to stage pT1-2, patients with stage pT3a RCC were significantly more often male (59.4% vs 53.1%) and older (64.9 vs 62.1 yr), more often had clear cell RCC (85.2% vs 77.7%), Fuhrman grade 3-4 (29.4% vs 13.4%), and tumor size >7 cm (39.1% vs 13%), and underwent NSS less often (7.5% vs 36.6%; all p<0.001). According to multivariate analysis, CSM was significantly higher for the PFI and RVI ± PFI groups compared to pT1-2 patients (hazard ratio [HR] 1.94 and 2.12, respectively; p<0.001), whereas patients with PFI only and RVI ± PFI did not differ (HR 1.17; p=0.316). Tumor size instead enhanced CSM by 7% per cm in stage pT3a (HR 1.07; p<0.001) with a 7 cm cutoff yielding the highest prediction accuracy. Since the prognostic impact of PFI and RVI on CSM seems to be comparable, merging both as stage pT3a RCC might be justified. Enhanced prognostic discrimination of stage pT3a RCC appears to be possible by applying a tumor size cutoff of 7 cm within an alternative staging system. Prognosis prediction for

  19. Blueberry peel extracts inhibit adipogenesis in 3T3-L1 cells and reduce high-fat diet-induced obesity.

    Directory of Open Access Journals (Sweden)

    Yuno Song

    Full Text Available 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/EBPβ, as well as the C/EBPα and PPARγ 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-GSK3β, was downregulated by BPE treatment in 3T3-L1 cells. Together, these data indicated that BP exerted anti-adipogenic activity by inhibiting the expression of PPARγ and C/EBPβ 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/EBPβ, C/EBPα, and PPARγ and the reduction of the phospho-Akt adipogenic factor in 3T3-L1 cells. Moreover, BPE reduced body weight gain

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

    International Nuclear Information System (INIS)

    Kohler, C.; Petersen, R.

    1986-01-01

    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 3 H inositol for 18-20 hours, washed and suspended in Herpes + Li + 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 μg/ml) increased total IP to a greater degree in Balb/c cells. Epidermal growth factor (10 ng/ml - 10 μg/ml) had no effect on PI turnover and fibroblast growth factor (10 ng/ml - 10 μg/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

  1. Response of MC3T3-E1cells on microroughen bioactive glass coated zirconia

    Directory of Open Access Journals (Sweden)

    Sapna Laxmi Tuladhar

    2017-10-01

    Full Text Available Background & Objectives:The objective of this study was to determine the cellular response of micro-roughened bioactive glass coated zirconia substrate (ZBR and non roughen bioactive glass coated zirconia substrate (ZB, and compare them with uncoated zirconia substrate (Z.  Materials & Methods:Surface micro-roughening was obtained using an Al2O3 sandblasting method. Abrasive blasting of zirconia coated bioactive glass produced an irregular finish with surface roughness average Ra 0.85 µm as determined by profilometer and scan electron microscope. Surface roughness of the samples in ascending order was ZBR>ZB>Z. Murine derived preosteoblast (MC3T3-E1 cells were seeded on the samples, and the cell morphology, growth, differentiation, were observed. Cell morphology was evaluated by means of scanning electron microscope (SEM, while cell proliferation and differentiation using MTT (3-(4,5-Dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide test and alkaline phosphates activity respectively. Results:The cell growth on all the samples continual increase with culturing up to 5days, showing good cell viability. However, there was no significant difference (p>0.05 with respect to the Z, ZB, and ZBR at day 5 at MTT assay. In particular, the alkaline phosphatase (ALP activity of the cells was significantly higher on the ZB and ZBR than Z samples at both 7 and 14 days.   Conclusion:Our findings demonstrate that bioactive glass coated surface was found to have better surface conditions to regulate bone cell differentiation 

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

    Directory of Open Access Journals (Sweden)

    Sinclair Andrew J

    2010-06-01

    Full Text Available Abstract 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 μM FA during 7-day differentiation. Results EPA markedly reduced LD size and total lipid accumulation, suppressing PPARγ, 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.

  3. Effect of acetaminophen on osteoblastic differentiation and migration of MC3T3-E1 cells.

    Science.gov (United States)

    Nakatsu, Yoshihiro; Nakagawa, Fumio; Higashi, Sen; Ohsumi, Tomoko; Shiiba, Shunji; Watanabe, Seiji; Takeuchi, Hiroshi

    2018-02-01

    N-acetyl-p-aminophenol (APAP, acetaminophen, paracetamol) is a widely used analgesic/antipyretic with weak inhibitory effects on cyclooxygenase (COX) compared to non-steroidal anti-inflammatory drugs (NSAIDs). The mechanism of action of APAP is mediated by its metabolite that activates transient receptor potential channels, including transient receptor potential vanilloid 1 (TRPV1) and TRP ankyrin 1 (TRPA1) or the cannabinoid receptor type 1 (CB1). However, the exact molecular mechanism and target underlying the cellular actions of APAP remain unclear. Therefore, we investigated the effect of APAP on osteoblastic differentiation and cell migration, with a particular focus on TRP channels and CB1. Effects of APAP on osteoblastic differentiation and cell migration of MC3T3-E1, a mouse pre-osteoblast cell line, were assessed by the increase in alkaline phosphatase (ALP) activity, and both wound-healing and transwell-migration assays, respectively. APAP dose-dependently inhibited osteoblastic differentiation, which was well correlated with the effects on COX activity compared with other NSAIDs. In contrast, cell migration was promoted by APAP, and this effect was not correlated with COX inhibition. None of the agonists or antagonists of TRP channels and the CB receptor affected the APAP-induced cell migration, while the effect of APAP on cell migration was abolished by down-regulating TRPV4 gene expression. APAP inhibited osteoblastic differentiation via COX inactivation while it promoted cell migration independently of previously known targets such as COX, TRPV1, TRPA1 channels, and CB receptors, but through the mechanism involving TRPV4. APAP may have still unidentified molecular targets that modify cellular functions. Copyright © 2017 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

    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.

  5. Troponin T3 regulates nuclear localization of the calcium channel Cavβ1a subunit in skeletal muscle.

    Science.gov (United States)

    Zhang, Tan; Taylor, Jackson; Jiang, Yang; Pereyra, Andrea S; Messi, Maria Laura; Wang, Zhong-Min; Hereñú, Claudia; Delbono, Osvaldo

    2015-08-15

    The voltage-gated calcium channel (Cav) β1a subunit (Cavβ1a) plays an important role in excitation-contraction coupling (ECC), a process in the myoplasm that leads to muscle-force generation. Recently, we discovered that the Cavβ1a subunit travels to the nucleus of skeletal muscle cells where it helps to regulate gene transcription. To determine how it travels to the nucleus, we performed a yeast two-hybrid screening of the mouse fast skeletal muscle cDNA library and identified an interaction with troponin T3 (TnT3), which we subsequently confirmed by co-immunoprecipitation and co-localization assays in mouse skeletal muscle in vivo and in cultured C2C12 muscle cells. Interacting domains were mapped to the leucine zipper domain in TnT3 COOH-terminus (160-244 aa) and Cavβ1a NH2-terminus (1-99 aa), respectively. The double fluorescence assay in C2C12 cells co-expressing TnT3/DsRed and Cavβ1a/YFP shows that TnT3 facilitates Cavβ1a nuclear recruitment, suggesting that the two proteins play a heretofore unknown role during early muscle differentiation in addition to their classical role in ECC regulation. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. The human NAIP-NLRC4-inflammasome senses the Pseudomonas aeruginosa T3SS inner-rod protein.

    Science.gov (United States)

    Grandjean, Teddy; Boucher, Anne; Thepaut, Marion; Monlezun, Laura; Guery, Benoit; Faudry, Eric; Kipnis, Eric; Dessein, Rodrigue

    2017-08-01

    While NLRC4-dependent sensing of intracellular Gram-negative pathogens such as Salmonella enterica serovar typhimurium is a beneficial host response, NLRC4-dependent sensing of the Pseudomonas aeruginosa type 3 secretion system (T3SS) has been shown to be involved in pathogenicity. In mice, different pathogen-associated microbial patterns are sensed by the combination of the NLRC4-inflammasome with different neuronal apoptosis inhibitory proteins (NAIPs). NAIP2 is involved in sensing PscI, an inner-rod protein of the P. aeruginosa T3SS. Surprisingly, only a single human NAIP (hNAIP) has been found. Moreover, there is no description of hNAIP-NLRC4 inflammasome recognition of T3SS inner-rod proteins in humans. Here, we show that the P. aeruginosa T3SS inner-rod protein PscI and needle protein PscF are both sensed by the hNAIP-NLRC4 inflammasome in human macrophages and PBMCs from healthy donors, allowing caspase-1 and IL-1β maturation and resulting in a robust inflammatory response. TLR4 and TLR2 are involved in redundantly sensing these two T3SS components. © The Japanese Society for Immunology. 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  7. Sustained Benefit Lasting One Year from T4 Instead of T3-T4 Sympathectomy for Isolated Axillary Hyperhidrosis

    Science.gov (United States)

    Munia, Marco Antonio S.; Wolosker, Nelson; Kaufmann, Paulo; de Campos, José Ribas Milanes; Puech-Leão, Pedro

    2008-01-01

    INTRODUCTION Level T4 video-assisted thoracoscopic sympathectomy proved superior to T3-T4 treatment for controlling axillary hyperhidrosis at the initial and six-month follow-ups of these patients. OBJECTIVE To compare the results of two levels of sympathectomy (T3-T4 vs. T4) for treating axillary sudoresis over one year of follow-up. METHODS Sixty-four patients with axillary hyperhidrosis were randomized to denervation of T3-T4 or T4 alone and followed prospectively. All patients were examined preoperatively and were followed postoperatively for one year. Axillary hyperhidrosis treatment was evaluated, along with the presence, location, and severity of compensatory hyperhidrosis and self-reported quality of life. RESULTS According to patient reports after one year, all cases of axillary hyperhidrosis were successfully treated by surgery. There were no instances of treatment failure. After six months, compensatory hyperhidrosis was present in 27 patients of the T3-T4 group (87.1%) and in 16 patients of the T4 group (48.5%). After one year, all T3-T4 patients experienced some degree of compensatory hyperhidrosis, compared to only 14 patients in the T4 group (42.4%). In addition, compensatory hyperhidrosis was less severe in the T4 patients (p hyperhidrosis, but the T4 group showed milder compensatory hyperhidrosis and greater patient satisfaction at the one-year follow-up. PMID:19060999

  8. Action of Thyroid Hormones, T3 and T2, on Hepatic Fatty Acids: Differences in Metabolic Effects and Molecular Mechanisms

    Directory of Open Access Journals (Sweden)

    Fabrizio Damiano

    2017-03-01

    Full Text Available The thyroid hormones (THs 3,3′,5,5′-tetraiodo-l-thyronine (T4 and 3,5,3′-triiodo-l-thyronine (T3 influence many metabolic pathways. The major physiological function of THs is to sustain basal energy expenditure, by acting primarily on carbohydrate and lipid catabolism. Beyond the mobilization and degradation of lipids, at the hepatic level THs stimulate the de novo fatty acid synthesis (de novo lipogenesis, DNL, through both the modulation of gene expression and the rapid activation of cell signalling pathways. 3,5-Diiodo-l-thyronine (T2, previously considered only a T3 catabolite, has been shown to mimic some of T3 effects on lipid catabolism. However, T2 action is more rapid than that of T3, and seems to be independent of protein synthesis. An inhibitory effect on DNL has been documented for T2. Here, we give an overview of the mechanisms of THs action on liver fatty acid metabolism, focusing on the different effects exerted by T2 and T3 on the regulation of the DNL. The inhibitory action on DNL exerted by T2 makes this compound a potential and attractive drug for the treatment of some metabolic diseases and cancer.

  9. Growth hormone induced stimulation of the T4 to T3 conversion in fed and fasting dwarf goats.

    Science.gov (United States)

    Iqbal, A; Cheema, A M; Kühn, E R

    1990-11-01

    Fed and food deprived (7 days) adult dwarf goats were injected intravenously with 75 micrograms/kg b. w. of ovine growth hormone (o-GH). Blood samples were collected from the jugular vein -2, -1 and 0 hr prior to and 30, 60, 90 and 120 min after injection and assayed for thyroxine (T4) and triiodothyronine (T3) concentrations. The 5'-monodeiodination (5'-D) activity was consequently determined in liver and kidney samples following slaughtering. Fasting alone increased plasma concentrations of T3 and T4, whereas injection of o-GH raised T3 additionally and more profoundly in food deprived animals compared to fed ones. A small increase in plasma T4 was also observed following o-GH injection, but only in starved goats. At the same time the hepatic, but not the kidney, 5'-D activity was stimulated in food deprived and GH injected animals. It is concluded that during prolonged fasting an increased peripheral T4 to T3 conversion is occurring contrary to the known decrease in T3 production during short periods of food deprivation. This increased conversion may be under the control of GH.

  10. Modulation of adipogenesis and glucose uptake by Curcuma longa extract in 3T3L1 and L6 cell lines - An in vitro study

    Directory of Open Access Journals (Sweden)

    A. Prathapan

    2012-05-01

    Full Text Available Objective: To evaluate the effects of ethyl acetate extract of Curcuma longa against modulation of glucose uptake and adipogenesis in cell line models. Methods: We used 3T3L1 and L6 cells to investigate cytotoxicity, glucose uptake with 2-NBDG as probe and adipogenesis. All the analysis was done with flowcytometry. Results: The results showed that the extract did not possess any significant glucose uptake activity but it exhibited significant adipocyte differentiation potential. Conclusions: Ethyl acetate extract of Curcuma longa exhibits significant antiadipogenesis and can be used as an effective drug for the treatment of obesity and other associated complications.

  11. Severe TSH Elevation and Pituitary Enlargement After Changing Thyroid Replacement to Compounded T4/T3 Therapy

    Directory of Open Access Journals (Sweden)

    Adlai L. Pappy BA

    2016-07-01

    Full Text Available We present the first case of iatrogenic hypothyroidism as a result of compounded thyroid hormone (T4/T3 therapy. The thyroid replacement was changed from 175 µg levothyroxine (LT4 to 57/13.5 µg compounded T4/T3 daily in order to improve the T3 level, despite normal thyroid-stimulating hormone (TSH. This resulted in clinical manifestations of hypothyroidism and high TSH level (150 µIU/mL. Six months later, the patient was referred to our clinic for abnormal pituitary magnetic resonance imaging. On reinitiating a physiologic dose of LT4, clinical and biochemical abnormalities resolved and the pituitary gland size decreased. Our case emphasizes the importance of using TSH level to gauge dose adjustments in primary hypothyroidism. Also, it underscores the current American Thyroid Association recommendation against routine use of compounded thyroid hormone therapy.

  12. 2012 ETA Guidelines: The Use of L-T4 + L-T3 in the Treatment of Hypothyroidism

    DEFF Research Database (Denmark)

    Nygaard, Birte

    2012-01-01

    Background: Data suggest symptoms of hypothyroidism persist in 5–10% of levothyroxine (L-T4)-treated hypothyroid patients with normal serum thyrotrophin (TSH). The use of L-T4 + liothyronine (L-T3) combination therapy in such patients is controversial. The ETA nominated a task force to review....... There is insufficient evidence that L-T4 + L-T3 combination therapy is better than L-T4 monotherapy, and it is recommended that L-T4 monotherapy remains the standard treatment of hypothyroidism. L-T4 + L-T3 combination therapy might be considered as an experimental approach in compliant L-T4-treated hypothyroid...

  13. Serum T3 level can predict cardiovascular events and all-cause mortality rates in CKD patients with proteinuria.

    Science.gov (United States)

    Yang, Jae Won; Han, Seung Tae; Song, Shin Han; Kim, Min Keun; Kim, Jae Seok; Choi, Seung Ok; Han, Byoung-Geun

    2012-01-01

    Patients with proteinuria frequently show changes in thyroid hormone levels. Serum T3 depression predicts a negative outcome in chronic kidney disease (CKD) patients and may be associated with cardiovascular complications or chronic inflammation. Few studies have explored the relationship between thyroid hormone dysregulation and clinical outcome in patients with proteinuria. We reviewed thyroid function test results obtained from 211 patients with 24 h urinary protein excretion greater than 150 mg/day and found a correlation of thyroid hormone level with cardiovascular events and mortality. T3 decreased with age (p = 0.001) and 24 h urine albumin (p = 0.028). Free T4 decreased in accordance with 24 h urine protein and serum creatinine (p = 0.034 and p = 0.033, respectively). In the Kaplan-Meier survival analysis, lower cumulative survival, higher cardiovascular events, and mortality were found in the low T3 group compared with the normal T3 group (p = 0.000, p = 0.013, and p = 0.001, respectively). In Cox regression analysis, we observed that, with low T3, decreased sodium, and old age, the incidence of cardiovascular complications (p = 0.000, p = 0.016, and p = 0.000, respectively), cardiovascular mortality (p = 0.000, p = 0.048, and p = 0.001, respectively), and all-cause mortality (p = 0.000, p = 0.017, and p = 0.000, respectively) increased. In CKD patients with proteinuria, low T3 concentration predicted all-cause mortality and cardiovascular event independently of the severity of proteinuria.

  14. Identification of candidates for postmastectomy radiotherapy in patients with pT3N0M0 breast cancer

    International Nuclear Information System (INIS)

    Hamamoto, Yasushi; Ohsumi, Shozo; Aogi, Kenjiro; Takashima, Shigemitsu; Shinohara, Shuichi; Nakajima, Naomi; Kataoka, Masaaki

    2013-01-01

    There is still controversy concerning the indication of postmastectomy radiotherapy (PMRT) for pT3N0M0 breast cancer. To identify the candidates for PMRT in this subset, we investigated failure patterns, and searched for risk factors for isolated locoregional failure in pT3N0M0 breast cancer after mastectomy without PMRT. Among 1,176 patients who received mastectomy without PMRT for untreated unilateral breast cancer between 1990 and 2002, 64 patients (5%) had pT3N0M0 breast cancer (age 30-81 years; median 52.5 years). Isolated locoregional failure as the initial failure occurred in three patients. For all 64 patients, the 8-year failure-free survival rate, the isolated locoregional failure-free rate, and the distant failure-free rate were 76, 93, and 82%, respectively. Incidence of isolated locoregional failure as the initial failure was 18% (2/11) for patients 40 years or younger and 2% (1/53) for patients older than 40 years. The 8-year isolated locoregional failure-free rates were 73% for patients 40 years or younger and 98% for patients older than 40 years (p=0.0135). Concerning pT3N0M0 breast cancer, incidence of isolated locoregional failure was comparatively low after mastectomy without PMRT. Routine use of PMRT for all pT3N0M0 patients seemed to be unacceptable. PMRT may be useful for younger patients because of the comparatively high incidence of isolated locoregional failure. Because of the small number of cases in our series, further studies are necessary to determine the usefulness of PMRT for younger patients with pT3N0M0 breast cancer. (author)

  15. Intervalos de referencia para concentraciones séricas de T3 y T4. Estudio preliminar

    Directory of Open Access Journals (Sweden)

    Cecilia Miranda Pantoja

    2013-12-01

    Full Text Available Fundamento: la determinación cuantitativa de las hormonas tiroideas T3 y T4 reviste gran importancia en el diagnóstico y la evaluación del hipertiroidismo, en especial del hipertiroidismo aislado causado por T3.Objetivo: establecer los intervalos de referencia de T3 y T4 en el laboratorio de medicina nuclear del Hospital General Universitario Dr. Gustavo Aldereguía Lima de Cienfuegos. Métodos: estudio descriptivo y prospectivo realizado mediante el método de radioinmunoanálisis, competencia que se establece entre la T3 y T4 sin marcar, y la marcada por un número limitado de los sitios de unión del anticuerpo específico. Al hacer reaccionar una cantidad fija de trazador y anticuerpo con diferentes cantidades del ligando sin marcar, la cantidad de trazador unido por el anticuerpo será inversamente proporcional a la concentración del ligando sin marcar. Resultados: los valores obtenidos se describen según una distribución gaussiana (media aritmética = 117, desviación estándar =31 para T4; media aritmética = 2,64, desviación estándar = 0,64 para T3, comprobado mediante un test de Chi cuadrado. Los rangos de valores normales obtenidos fueron de de 55 – 178 nmol/L y 1,4 – 3,9 nmol/L para T4 y T3 respectivamente. Conclusiones: los intervalos de referencia obtenidos resultaron más amplios que los propuestos por el productor, sobre todo en el caso de T4.

  16. Nitric Oxide-Mediated Regulation of GLUT by T3 and Follicle-Stimulating Hormone in Rat Granulosa Cells.

    Science.gov (United States)

    Tian, Ye; Ding, Yu; Liu, Juan; Heng, Dai; Xu, Kaili; Liu, Wenbo; Zhang, Cheng

    2017-06-01

    Thyroid hormones are important for normal reproductive function. Although 3,5,3'-triiodothyronine (T3) enhances follicle-stimulating hormone (FSH)-induced preantral follicle growth and granulosa cells development in vitro, little is known about the molecular mechanisms regulating ovarian development via glucose. In this study, we investigated whether and how T3 combines with FSH to regulate glucose transporter protein (GLUT) expression and glucose uptake in granulosa cells. In this study, we present evidence that T3 and FSH cotreatment significantly increased GLUT-1/GLUT-4 expression, and translocation in cells, as well as glucose uptake. These changes were accompanied by upregulation of nitric oxide (NO) synthase (NOS)3 expression, total NOS and NOS3 activity, and NO content in granulosa cells. Furthermore, we found that activation of the mammalian target of rapamycin (mTOR) and phosphoinositide 3-kinase (PI3K)/Akt pathway is required for the regulation of GLUT expression, translocation, and glucose uptake by hormones. We also found that l-arginine upregulated GLUT-1/GLUT-4 expression and translocation, which were related to increased glucose uptake; however, these responses were significantly blocked by N(G)-nitro-l-arginine methylester. In addition, inhibiting NO production attenuated T3- and FSH-induced GLUT expression, translocation, and glucose uptake in granulosa cells. Our data demonstrate that T3 and FSH cotreatment potentiates cellular glucose uptake via GLUT upregulation and translocation, which are mediated through the activation of the mTOR/PI3K/Akt pathway. Meanwhile, NOS3/NO are also involved in this regulatory system. These findings suggest that GLUT is a mediator of T3- and FSH-induced follicular development. Copyright © 2017 Endocrine Society.

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

    Science.gov (United States)

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

    2013-04-01

    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 demonstrate in osteoblast cultures producing an abundant collagenous matrix that normally show 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 β-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 10μM. 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

  18. Possibilities and limitations of combined radio-surgical treatment of large orofacial T3/T4-tumors. Moeglichkeiten und Grenzen der kombiniert radiotherapeutisch-chirurgischen Behandlung grosser orofazialer T3-/T4-Tumoren

    Energy Technology Data Exchange (ETDEWEB)

    Kaercher, K.H. (Universitaetsklinik fuer Strahlentherapie und Strahlenbiologie, Vienna (Austria)); Kaercher, H. (Abteilung fuer Mund-, Kiefer- und Gesichtschirurgie, Univ. Graz (Austria))

    1993-05-01

    The possibilities, limits and good cosmetic and functional results of combined radiotherapy-surgical treatment of large orofacial tumors (T3) are prescribed. The pre- and postoperative splitted radiotherapy with operation in between is favoured. The radical en-bloc-resection with radical dissection is the surgical therapy of choice. The defects have been reconstructed with osteomusculocutaneous flaps. (orig.)

  19. The pathogenicity factor HrpF interacts with HrpA and HrpG to modulate type III secretion system (T3SS) function and t3ss expression in Pseudomonas syringae pv. averrhoi.

    Science.gov (United States)

    Huang, Yi-Chiao; Lin, Yuan-Chuen; Wei, Chia-Fong; Deng, Wen-Ling; Huang, Hsiou-Chen

    2016-09-01

    To ensure the optimal infectivity on contact with host cells, pathogenic Pseudomonas syringae has evolved a complex mechanism to control the expression and construction of the functional type III secretion system (T3SS) that serves as a dominant pathogenicity factor. In this study, we showed that the hrpF gene of P. syringae pv. averrhoi, which is located upstream of hrpG, encodes a T3SS-dependent secreted/translocated protein. Mutation of hrpF leads to the loss of bacterial ability on elicitation of disease symptoms in the host and a hypersensitive response in non-host plants, and the secretion or translocation of the tested T3SS substrates into the bacterial milieu or plant cells. Moreover, overexpression of hrpF in the wild-type results in delayed HR and reduced t3ss expression. The results of protein-protein interactions demonstrate that HrpF interacts directly with HrpG and HrpA in vitro and in vivo, and protein stability assays reveal that HrpF assists HrpA stability in the bacterial cytoplasm, which is reduced by a single amino acid substitution at the 67th lysine residue of HrpF with alanine. Taken together, the data presented here suggest that HrpF has two roles in the assembly of a functional T3SS: one by acting as a negative regulator, possibly involved in the HrpSVG regulation circuit via binding to HrpG, and the other by stabilizing HrpA in the bacterial cytoplasm via HrpF-HrpA interaction prior to the secretion and formation of Hrp pilus on the bacterial surface. © 2015 BSPP and John Wiley & Sons Ltd.

  20. Cell competition in mouse NIH3T3 embryonic fibroblasts is controlled by the activity of Tead family proteins and Myc.

    Science.gov (United States)

    Mamada, Hiroshi; Sato, Takashi; Ota, Mitsunori; Sasaki, Hiroshi

    2015-02-15

    Cell competition is a short-range communication originally observed in Drosophila. Relatively little is known about cell competition in mammals or in non-epithelial cells. Hippo signaling and its downstream transcription factors of the Tead family, control cell proliferation and apoptosis. Here, we established an in vitro model system that shows cell competition in mouse NIH3T3 embryo fibroblast cells. Co-culture of Tead-activity-manipulated cells with normal (wild-type) cells caused cell competition. Cells with reduced Tead activity became losers, whereas cells with increased Tead activity became super-competitors. Tead directly regulated Myc RNA expression, and cells with increased Myc expression also became super-competitors. At low cell density, cell proliferation required both Tead activity and Myc. At high cell density, however, reduction of either Tead activity or Myc was compensated for by an increase in the other, and this increase was sufficient to confer 'winner' activity. Collectively, NIH3T3 cells have cell competition mechanisms similar to those regulated by Yki and Myc in Drosophila. Establishment of this in vitro model system should be useful for analyses of the mechanisms of cell competition in mammals and in fibroblasts. © 2015. Published by The Company of Biologists Ltd.

  1. Binding mode analysis of a major T3SS translocator protein PopB with its chaperone PcrH from Pseudomonas aeruginosa.

    Science.gov (United States)

    Banerjee, Anindyajit; Dey, Supratim; Chakraborty, Abhijit; Datta, Aohona; Basu, Abhishek; Chakrabarti, Saikat; Datta, Saumen

    2014-12-01

    Pseudomonas aeruginosa, a Gram-negative pathogen uses a specialized set of Type III secretion system (T3SS) translocator proteins to establish virulence in the host cell. An understanding of the factors that govern translocation by the translocator protein-chaperone complex is thus of immense importance. In this work, experimental and computational techniques were used to probe into the structure of the major translocator protein PopB from P. aeruginosa and to identify the important regions involved in functioning of the translocator protein. This study reveals that the binding sites of the common chaperone PcrH, needed for maintenance of the translocator PopB within the bacterial cytoplasm, which are primarily localized within the N-terminal domain. However, disordered and flexible residues located both at the N- and C-terminal domains are also observed to be involved in association with the chaperone. This intrinsic disorderliness of the terminal domains is conserved for all the major T3SS translocator proteins and is functionally important to maintain the intrinsically disordered state of the translocators. Our experimental and computational analyses suggest that a "disorder-to-order" transition of PopB protein might take place upon PcrH binding. The long helical coiled-coil part of PopB protein perhaps helps in pore formation while the flexible apical region is involved in chaperone interaction. Thus, our computational model of translocator protein PopB and its binding analyses provide crucial functional insights into the T3SS translocation mechanism. © 2014 Wiley Periodicals, Inc.

  2. Spa47 is an oligomerization-activated type three secretion system (T3SS) ATPase from Shigella flexneri.

    Science.gov (United States)

    Burgess, Jamie L; Jones, Heather B; Kumar, Prashant; Toth, Ronald T; Middaugh, C Russell; Antony, Edwin; Dickenson, Nicholas E

    2016-05-01

    Gram-negative pathogens often use conserved type three secretion systems (T3SS) for virulence. The Shigella type three secretion apparatus (T3SA) penetrates the host cell membrane and provides a unidirectional conduit for injection of effectors into host cells. The protein Spa47 localizes to the base of the apparatus and is speculated to be an ATPase that provides the energy for T3SA formation and secretion. Here, we developed an expression and purification protocol, producing active Spa47 and providing the first direct evidence that Spa47 is a bona fide ATPase. Additionally, size exclusion chromatography and analytical ultracentrifugation identified multiple oligomeric species of Spa47 with the largest greater than 8 fold more active for ATP hydrolysis than the monomer. An ATPase inactive Spa47 point mutant was then engineered by targeting a conserved Lysine within the predicted Walker A motif of Spa47. Interestingly, the mutant maintained a similar oligomerization pattern as active Spa47, but was unable to restore invasion phenotype when used to complement a spa47 null S. flexneri strain. Together, these results identify Spa47 as a Shigella T3SS ATPase and suggest that its activity is linked to oligomerization, perhaps as a regulatory mechanism as seen in some related pathogens. Additionally, Spa47 catalyzed ATP hydrolysis appears to be essential for host cell invasion, providing a strong platform for additional studies dissecting its role in virulence and providing an attractive target for anti-infective agents. © 2016 The Protein Society.

  3. Thyroxine (T 4-RIA) and triiodothyronine (T 3-RIA) serum levels in sheep fed on Leucaena Leucocephala

    International Nuclear Information System (INIS)

    Pessoa, J.M.; Rodriguez, N.M.; Cardoso, W.M.; Velez, C.E.S.

    1988-01-01

    The aim of this work is to study the toxicity of Leucaena leucocephala on thyroxine and triiodothyronine serum concentration. Results indicate that ovine T 4 and T 3 serum levels do not decrease with Leucaena leucocephala feeding, sohen proreided for 41 days. (author). 10 refs, 1 tab

  4. [The effect of microencapsulated NGF-expressing NIH-3T3 cells on bioengineered dermis function in vitro].

    Science.gov (United States)

    Hu, Zhaohua; Chen, Shaozong; Jin, Yan; Xiong, Ying; Wang, Wei; Ma, Xiaojun; Zhao, Yu; Li, Wangzhou; Lei, Zhanjun

    2007-02-01

    Nerve growth factor (NGF) can promote the regeneration of peripheral nerve as well as contraction and reepithelization of wound. We constructed a bioengineered dermis containing microencapsulated NGF-expressing NIH-3T3 cells and study the effect of the microencapsule to the bioengineered dermis and seed cells. NGF gene was transfected into NIH-3T3 cells and enclosed into alginate-poly-L-lysine-alginate (APA) microencapsulation and cultivated in vitro. Content of NGF in microencapsules culturing supernatant was measured by enzyme linked immunosorbent assay (ELISA) method. These microencapsules were co-cultured with epidermic cells and fibroblast cells. Bioengineered dermis was constructed with NGF-expressing micorencapsules as seed cells using tissue engineering method. NIH-3T3 microencapsules, empty microencapsules, normal culture media were control groups. After one week culture, the characteristics of the dermis were described by MTT test, the content of hydroxyproline (HP), HE staining and ultrastructure photograph. We found the NGF-expressing microencapsulates can secret NGF steadly after cultured 8w in vitro, promot the proliferation of epidermic cells and secret collagen of fibroblast cells. These functions can maintaine in bioengineered dermis. So NGF-expressing NIH-3T3 microencapsulates can promote the quality of bioengineered dermis.

  5. Thoracoscopic sympathectomy of T2 and T3 ganglions for palmar hyperhydrosis

    Directory of Open Access Journals (Sweden)

    Toolabi K

    2008-05-01

    Full Text Available Background: Hyperhydrosis, excessive sweating, can profoundly affect the quality of life of the patient, with severe impairment of daily activities, social relationships and occupational activities. The purpose of this study was to evaluate the outcome of thoracoscopic sympathectomy in patients with palmar hyperhydrosis.Methods: In a clinical trial at Imam Khomeini, Milad and Velenjak Hospitals from 2003 to 2006, 33 patients older than five years of age with palmar hyperhydrosis underwent thoracoscopic sympathectomy of T2 and T3 ganglions. The variables regarding operation duration, length of hospital stay and early post-op complications were recorded. In addition, the quality of life of the patients was evaluated using the DLQI questionnaire, while the satisfaction of the patients was evaluated using the visual analogue scale before, immediately after and six months after the operation.Results: The mean age of the patients was 23 years and 63.6% of our patients were female. The sites of hyperhydrosis were hand, foot and armpit in 72.7% of the patients. In 90.91% of the cases, disease onset occurred during childhood and 78.79% of the patients had a family history of hyperhydrosis. Hyperhydrosis impaired function and had social, interpersonal and emotional effects in 72.7% of the cases. The mean duration of the operation was 40.3 minutes and the mean hospital stay was 1.45 days. The most common early post-op complications were atelectasis (7.57%, short-term pulmonary complications (3.03% and pleural effusion (3.03%. Compensatory hyperhydrosis occurred in 60% of the cases, while 18.75% experienced gustatory sweating. Palms were reported to be totally dry immediately after and six months after the operation in 81.82% of the cases, while 18.18% reported acceptable levels of perspiration. The mean level of satisfaction immediately after and six months after the procedure was 0.81. All patients felt improvement in all aspects of their lives

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

    International Nuclear Information System (INIS)

    Liang, Qiu-Hua; Liu, Yuan; Wu, Shan-Shan; Cui, Rong-Rong; Yuan, Ling-Qing; Liao, Er-Yuan

    2013-01-01

    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

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

    Energy Technology Data Exchange (ETDEWEB)

    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

  8. Pengaruh Suplementasi Ganda Iodium dan Zat Besi (Fe terhadap Kadar Tsh, Ft4, T3 dan Ferritin Anak Sekolah Dasar

    Directory of Open Access Journals (Sweden)

    Donny Kristanto Mulyantoro

    2016-01-01

    Full Text Available PENGARUH SUPLEMENTASI GANDA IODIUM DAN ZAT BESI (Fe TERHADAP KADAR TSH, fT4, T3 DAN FERRITIN ANAK SEKOLAH DASAR   Effect of Iodine and Iron (Fe Dual Supplementation on The Levels of TSH, fT4, T3 and Ferritin in Primary School Children ABSTRACT Background. Intercorrelations between iodine and iron (Fe on the function of the thyroid gland and the status of iron (Fe affects the effectiveness of Iodine deficiency disorders (IDD prevention and anemia programs. Communities group prone to nutritional problems are school age children who are in growth period. Objective. This study aims to compare the effect of iodine iron (I+Fe double supplementation with single supplementation of iodine (I and single supplementation of iron (Fe on the function of the thyroid gland and the status of iron (Fe. Method. The study was a randomized double-blind controlled trial. Provision of interventions done by randomization with block permutations dividing study subjects into four groups: Iodine+iron (I+Fe dual supplement group, iodine (I only supplement group, iron (Fe only suplement group and placebo group. Subjects were primary school children aged 9-12 years. Interventions conducted for 13 weeks and thyroid function measured by serum levels of TSH, fT4 and T3, while the iron status based on ferritin levels. Results. Supplementation of Iodine and Iron can increase ferritin and T3 levels, while supplementation of I+FeI, I or Fe can increase levels of fT4 although it is not statistically significant. Ancova showed there is a mechanism of mutual influence between ferritin, fT4 and T3. Conclusion. There is no differences between dual supplementation of iodine and iron with single supplementation of iodine or iron in alteration of ferritin, TSH, fT4, and T3 serum. Keywords: iodine, iron, supplementation.   ABSTRAK Latar belakang. Interkorelasi antara iodium dan zat besi (Fe terhadap fungsi kelenjar tiroid dan status zat besi (Fe mempengaruhi efektifitas program

  9. Effect of Ganoderma applanatum mycelium extract on the inhibition of adipogenesis in 3T3-L1 adipocytes.

    Science.gov (United States)

    Kim, Ji-Eun; Park, Sung-Jin; Yu, Mi-Hee; Lee, Sam-Pin

    2014-10-01

    Ganoderma applanatum (GA) and related fungal species have been used for over 2000 years in China to prevent and treat various human diseases. However, there is no critical research evaluating the functionality of GA grown using submerged culture technology. This study aimed to evaluate the effects of submerged culture GA mycelium (GAM) and its active components (protocatechualdehyde [PCA]) on preadipocyte differentiation of 3T3-L1 cells. Mouse-derived preadipocyte 3T3-L1 cells were treated with differentiation inducers in the presence or absence of GAM extracts. We determined triglyceride accumulations, glycerol-3-phosphate dehydrogenase (GPDH) activities, and differentiation makers. PCA, the active component of GAM extract, was also used to treat 3T3-L1 cells. The MTT assay showed that the GAM extract (0.01-1 mg/mL) was not toxic to 3T3-L1 preadipocyte. Treatment of cells with GAM extracts and its active components significantly decreased the GPDH activity and lipid accumulation, a marker of adipogenesis, in a dose-dependent manner. Western blot analysis results showed that the protein expression levels of peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer-binding protein α (C/EBPα), and sterol regulatory element-binding protein 1 (SREBP1) were inhibited by the GAM extract. In addition, adipogenic-specific genes such as perilipin, fatty acid synthase (FAS), fatty acid transport protein 1 (FATP1), and fatty acid-binding protein 4 (FABP4) decreased in a dose-dependent manner. Quantitative high-performance liquid chromatography analysis showed that the GAM extract contained 1.14 mg/g PCA. GAM extracts suppressed differentiation of 3T3-L1 preadipocytes, in part, through altered regulation of PPARγ, C/EBPα, and SREBP1. These results suggest that GAM extracts and PCA may suppress adipogenesis by inhibiting differentiation of preadipocytes.

  10. Hh/Gli antagonist in acute myeloid leukemia with CBFA2T3-GLIS2 fusion gene.

    Science.gov (United States)

    Masetti, Riccardo; Bertuccio, Salvatore Nicola; Astolfi, Annalisa; Chiarini, Francesca; Lonetti, Annalisa; Indio, Valentina; De Luca, Matilde; Bandini, Jessica; Serravalle, Salvatore; Franzoni, Monica; Pigazzi, Martina; Martelli, Alberto Maria; Basso, Giuseppe; Locatelli, Franco; Pession, Andrea

    2017-01-21

    CBFA2T3-GLIS2 is a fusion gene found in 17% of non-Down syndrome acute megakaryoblastic leukemia (non-DS AMKL, FAB M7) and in 8% of pediatric cytogenetically normal acute myeloid leukemia (CN-AML, in association with several French-American-British (FAB) subtypes). Children with AML harboring this aberration have a poor outcome, regardless of the FAB subtype. This fusion gene drives a peculiar expression pattern and leads to overexpression of some of Hedgehog-related genes. GLI-similar protein 2 (GLIS2) is closely related to the GLI family, the final effectors of classic Hedgehog pathway. These observations lend compelling support to the application of GLI inhibitors in the treatment of AML with the aberration CBFA2T3-GLIS2. GANT61 is, nowadays, the most potent inhibitor of GLI family proteins. We exposed to GANT61 AML cell lines and primary cells positive and negative for CBFA2T3-GLIS2 and analyzed the effect on cellular viability, induction of apoptosis, cell cycle, and expression profile. As compared to AML cells without GLIS2 fusion, GANT61 exposure resulted in higher sensitivity of both cell lines and primary AML cells carrying CBFA2T3-GLIS2 to undergo apoptosis and G1 cell cycle arrest. Remarkably, gene expression studies demonstrated downregulation of GLIS2-specific signature genes in both treated cell lines and primary cells, in comparison with untreated cells. Moreover, chromatin immunoprecipitation analysis revealed direct regulation by GLIS2 chimeric protein of DNMT1 and DNMT3B, two genes implicated in important epigenetic functions. Our findings indicate that the GLI inhibitor GANT61 may be used to specifically target the CBFA2T3-GLIS2 fusion gene in pediatric AML.

  11. Rhizobium-legume symbiosis in the absence of Nod factors: two possible scenarios with or without the T3SS.

    Science.gov (United States)

    Okazaki, Shin; Tittabutr, Panlada; Teulet, Albin; Thouin, Julien; Fardoux, Joël; Chaintreuil, Clémence; Gully, Djamel; Arrighi, Jean-François; Furuta, Noriyuki; Miwa, Hiroki; Yasuda, Michiko; Nouwen, Nico; Teaumroong, Neung; Giraud, Eric

    2016-01-01

    The occurrence of alternative Nod factor (NF)-independent symbiosis between legumes and rhizobia was first demonstrated in some Aeschynomene species that are nodulated by photosynthetic bradyrhizobia lacking the canonical nodABC genes. In this study, we revealed that a large diversity of non-photosynthetic bradyrhizobia, including B. elkanii, was also able to induce nodules on the NF-independent Aeschynomene species, A. indica. Using cytological analysis of the nodules and the nitrogenase enzyme activity as markers, a gradient in the symbiotic interaction between bradyrhizobial strains and A. indica could be distinguished. This ranged from strains that induced nodules that were only infected intercellularly to rhizobial strains that formed nodules in which the host cells were invaded intracellularly and that displayed a weak nitrogenase activity. In all non-photosynthetic bradyrhizobia, the type III secretion system (T3SS) appears required to trigger nodule organogenesis. In contrast, genome sequence analysis revealed that apart from a few exceptions, like the Bradyrhizobium ORS285 strain, photosynthetic bradyrhizobia strains lack a T3SS. Furthermore, analysis of the symbiotic properties of an ORS285 T3SS mutant revealed that the T3SS could have a positive or negative role for the interaction with NF-dependent Aeschynomene species, but that it is dispensable for the interaction with all NF-independent Aeschynomene species tested. Taken together, these data indicate that two NF-independent symbiotic processes are possible between legumes and rhizobia: one dependent on a T3SS and one using a so far unknown mechanism.

  12. Stage T3a renal cell carcinoma: staging accuracy of CT for sinus fat, perinephric fat or renal vein invasion.

    Science.gov (United States)

    Sokhi, H K; Mok, W Y; Patel, U

    2015-01-01

    To study the accuracy of CT for staging T3a (TNM 2009) renal cell carcinoma (RCC). Unenhanced and nephrographic phase CT studies of 117 patients (male:female = 82:35; age range, 21-86 years) with T1-T3a RCC were independently reviewed by 2 readers. The presence of sinus or perinephric fat, or renal vein invasion and tumour characteristics were noted. Median (range) tumour size was 5.5 (0.9-19.0) cm; and 46 (39%), 16 (14%) and 55 (47%) tumours were pT1, pT2 and pT3a RCC, respectively. The sensitivity/specificity for sinus fat, perinephric fat and renal vein invasion were 71/79%, 83/76% and 59/93% (Reader 1) and 88/71%, 68/72% and 69/91% (Reader 2) with κ = 0.41, 0.43 and 0.61, respectively. Sinus fat invasion was seen in 47/55 (85%) cases with T3a RCC vs 16/55 (29%) and 33/55 (60%) for perinephric fat and renal vein invasion. Tumour necrosis, irregularity of tumour edge and direct tumour contact with perirenal fascia or sinus fat increased the odds of local invasion [odds ratio (OR), 2.5-3.7; p fat invasion was the most common invasive feature. Centrally situated renal tumours with an irregular tumour edge, inseparable from sinus structures or the perirenal fascia and CT features of tumour necrosis should alert the reader to the possibility of Stage T3a RCC (OR, 2.5-3.9).

  13. Long-term results of adjuvant hormonal therapy plus radiotherapy following radical prostatectomy for patients with pT3N0 or pT3N1 prostate cancer

    International Nuclear Information System (INIS)

    Miyake, Hideaki; Sakai, Iori; Harada Ken-ichi; Eto, Hiroshi; Hara, Isao

    2004-01-01

    The objective of the present study was to evaluate the efficacy of adjuvant androgen suppression in conjunction with external beam irradiation after radical prostatectomy in patients with pathologically confirmed extraprostatic disease. Between July 1988 and October 1999, 38 patients with pT3N0 or pT3N1 prostate cancer received adjuvant hormonal therapy and external beam irradiation following radical retropubic prostatectomy and pelvic lymphadenectomy. Administration of luteinizing hormone-releasing hormone analog or castration were initiated as an adjuvant androgen suppression within 4 weeks after surgery, whereas pelvic irradiation was performed at a median dose of 50 G within 3 months after surgery. The prognostic advantage of this combined adjuvant therapy was analyzed. During the median observation period of 92 months, biochemical recurrence occurred in four of the 38 patients and five patients died. Of these five patients, only one died of prostate cancer progression. The 10-year biochemical recurrence-free, cancer-specific and overall survival rates of the 38 patients were 86.7%, 90.9% and 78.7%, respectively. Among several factors examined, only tumor grade was significantly associated with biochemical recurrence-free survival in these patients; however, there were no factors that were independent predictors for biochemical recurrence, based on multivariate analysis. Furthermore, biochemical recurrence-free survival in the 38 patients was significantly superior to that in 54 patients with locally advanced disease who did not receive any postoperative therapies until biochemical recurrence; however, there was no significant difference in cancer-specific and overall survival between these two groups. Despite retrospective analysis with a relatively small number of patients, results of the present study suggest favorable effects of the combined adjuvant treatments with androgen ablation and pelvic irradiation on cancer control for patients with pT3N0 or pT3N1

  14. Amelioration of Mitochondrial Dysfunction-Induced Insulin Resistance in Differentiated 3T3-L1 Adipocytes via Inhibition of NF-κB Pathways

    Directory of Open Access Journals (Sweden)

    Mohamad Hafizi Abu Bakar

    2014-12-01

    Full Text Available A growing body of evidence suggests that activation of nuclear factor kappa B (NF-κB signaling pathways is among the inflammatory mechanism involved in the development of insulin resistance and chronic low-grade inflammation in adipose tissues derived from obese animal and human subjects. Nevertheless, little is known about the roles of NF-κB pathways in regulating mitochondrial function of the adipose tissues. In the present study, we sought to investigate the direct effects of celastrol (potent NF-κB inhibitor upon mitochondrial dysfunction-induced insulin resistance in 3T3-L1 adipocytes. Celastrol ameliorates mitochondrial dysfunction by altering mitochondrial fusion and fission in adipocytes. The levels of oxidative DNA damage, protein carbonylation and lipid peroxidation were down-regulated. Further, the morphology and quantification of intracellular lipid droplets revealed the decrease of intracellular lipid accumulation with reduced lipolysis. Moreover, massive production of the pro-inflammatory mediators tumor necrosis factor-α (TNF-α and interleukin-1β (IL-1β were markedly depleted. Insulin-stimulated glucose uptake activity was restored with the enhancement of insulin signaling pathways. This study signified that the treatments modulated towards knockdown of NF-κB transcription factor may counteract these metabolic insults exacerbated in our model of synergy between mitochondrial dysfunction and inflammation. These results demonstrate for the first time that NF-κB inhibition modulates mitochondrial dysfunction induced insulin resistance in 3T3-L1 adipocytes.

  15. MicroRNA-24 promotes 3T3-L1 adipocyte differentiation by directly targeting the MAPK7 signaling

    Energy Technology Data Exchange (ETDEWEB)

    Jin, Min, E-mail: min_jin@zju.edu.cn [Division of Reproductive Medicine & Infertility, The Second Affiliated Hospital, School of Medicine, Zhejiang University, 88#, Jiefang Rd., Hangzhou, Zhejiang, 310009 (China); Wu, Yutao; Wang, Jing [School of Medicine, Zhejiang University, 288# Yuhangtang Rd, Hangzhou, Zhejiang, 310003 (China); Chen, Jian; Huang, Yiting; Rao, Jinpeng; Feng, Chun [Division of Reproductive Medicine & Infertility, The Second Affiliated Hospital, School of Medicine, Zhejiang University, 88#, Jiefang Rd., Hangzhou, Zhejiang, 310009 (China)

    2016-05-20

    Over the past years, MicroRNAs (miRNAs) act as a vital role in harmony with gene regulation and maintaining cellular homeostasis. It is well testified that miRNAshave been involved in numerous physiological and pathological processes, including embryogenesis, cell fate decision, and cellular differentiation. Adipogenesis is an organized process of cellular differentiation by which pre-adipocytes differentiate towards mature adipocytes, and it is tightly modulated by a series of transcription factors such as peroxisome proliferator-activated receptor γ (PPAR-γ) and sterol regulatory-element binding proteins 1 (SREBP1). However, the molecular mechanisms underlying the connection between miRNAs and adipogenesis-related transcription factors remain obscure. In this study, we unveiled that miR- 24 was remarkably upregulated during 3T3-L1 adipogenesis. Overexpression of miR-24 significantly promoted 3T3-L1 adipogenesis, as evidenced by its ability to increase the expression of PPAR-γ and SREBP1, lipid droplet formation and triglyceride (TG) accumulation. Furthermore, we found that neither ectopic expression of miR-24nor miR-24 inhibitor affect cell proliferation and cell cycle progression. Finally, we demonstrated that miR-24 plays the modulational role by directly repressing MAPK7, a key number in the MAPK signaling pathway. These data indicate that miR-24 is a novel positive regulator of adipocyte differentiation by targeting MAPK7, which provides new insights into the molecular mechanism of miRNA-mediated cellular differentiation. -- Highlights: •We firstly found miR-24 was upregulated in 3T3-L1 pre-adipocytes differentiation. •miR-24 promoted 3T3-L1 pre-adipocytes differentiation while silencing the expression of miR-24 had an opposite function. •miR-24 regulated 3T3-L1 differentiation by directly targeting MAPK7 signaling pathway. •miR-24did not affect 3T3-L1 pre-adipocytes cellular proliferation.

  16. A dynamic and adaptive network of cytosolic interactions governs protein export by the T3SS injectisome.

    Science.gov (United States)

    Diepold, Andreas; Sezgin, Erdinc; Huseyin, Miles; Mortimer, Thomas; Eggeling, Christian; Armitage, Judith P

    2017-06-27

    Many bacteria use a type III secretion system (T3SS) to inject effector proteins into host cells. Selection and export of the effectors is controlled by a set of soluble proteins at the cytosolic interface of the membrane spanning type III secretion 'injectisome'. Combining fluorescence microscopy, biochemical interaction studies and fluorescence correlation spectroscopy, we show that in live Yersinia enterocolitica bacteria these soluble proteins form complexes both at the injectisome and in the cytosol. Binding to the injectisome stabilizes these cytosolic complexes, whereas the free cytosolic complexes, which include the type III secretion ATPase, constitute a highly dynamic and adaptive network. The extracellular calcium concentration, which triggers activation of the T3SS, directly influences the cytosolic complexes, possibly through the essential component SctK/YscK, revealing a potential mechanism involved in the regulation of type III secretion.

  17. T3SS effector VopL inhibits the host ROS response, promoting the intracellular survival of Vibrio parahaemolyticus.

    Science.gov (United States)

    de Souza Santos, Marcela; Salomon, Dor; Orth, Kim

    2017-06-01

    The production of antimicrobial reactive oxygen species by the nicotinamide dinucleotide phosphate (NADPH) oxidase complex is an important mechanism for control of invading pathogens. Herein, we show that the gastrointestinal pathogen Vibrio parahaemolyticus counteracts reactive oxygen species (ROS) production using the Type III Secretion System 2 (T3SS2) effector VopL. In the absence of VopL, intracellular V. parahaemolyticus undergoes ROS-dependent filamentation, with concurrent limited growth. During infection, VopL assembles actin into non-functional filaments resulting in a dysfunctional actin cytoskeleton that can no longer mediate the assembly of the NADPH oxidase at the cell membrane, thereby limiting ROS production. This is the first example of how a T3SS2 effector contributes to the intracellular survival of V. parahaemolyticus, supporting the establishment of a protective intracellular replicative niche.

  18. T3SS effector VopL inhibits the host ROS response, promoting the intracellular survival of Vibrio parahaemolyticus.

    Directory of Open Access Journals (Sweden)

    Marcela de Souza Santos

    2017-06-01

    Full Text Available The production of antimicrobial reactive oxygen species by the nicotinamide dinucleotide phosphate (NADPH oxidase complex is an important mechanism for control of invading pathogens. Herein, we show that the gastrointestinal pathogen Vibrio parahaemolyticus counteracts reactive oxygen species (ROS production using the Type III Secretion System 2 (T3SS2 effector VopL. In the absence of VopL, intracellular V. parahaemolyticus undergoes ROS-dependent filamentation, with concurrent limited growth. During infection, VopL assembles actin into non-functional filaments resulting in a dysfunctional actin cytoskeleton that can no longer mediate the assembly of the NADPH oxidase at the cell membrane, thereby limiting ROS production. This is the first example of how a T3SS2 effector contributes to the intracellular survival of V. parahaemolyticus, supporting the establishment of a protective intracellular replicative niche.

  19. Casein kinase 2 regulates the active uptake of the organic osmolyte taurine in NIH3T3 mouse fibroblasts

    DEFF Research Database (Denmark)

    Jacobsen, Jack H; Clement, Christian A; Friis, Martin B

    2008-01-01

    Inhibition of the constitutively active casein kinase 2 (CK2) with 2-dimethyl-amino-4,5,6,7-tetrabromo-1H-benzimidasole stimulates the Na(+)-dependent taurine influx via the taurine transporter TauT in NIH3T3 cells. CK2 inhibition reduces the TauT mRNA level and increases the localization of Tau......T to ER but has no detectable effect on TauT protein expression. On the other hand, CK2 inhibition increases the affinity of TauT towards Na(+ )and reduces the Na(+)/taurine stoichiometry for active taurine uptake. It is suggested that CK2 controls the cellular taurine uptake in unperturbated NIH3T3 cells......, i.e., inhibition of CK2 increases the affinity of TauT towards Na(+) and hence Na(+)-dependent taurine uptake....

  20. Downregulation of the taurine transporter TauT during hypo-osmotic stress in NIH3T3 mouse fibroblasts

    DEFF Research Database (Denmark)

    Hansen, Daniel Bloch; Friis, Martin Barfred; Hoffmann, Else Kay

    2012-01-01

    The present work was initiated to investigate regulation of the taurine transporter TauT by reactive oxygen species (ROS) and the tonicity-responsive enhancer binding protein (TonEBP) in NIH3T3 mouse fibroblasts during acute and long-term (4 h) exposure to low-sodium/hypo-osmotic stress. Taurine...... are significantly increased following hyperosmotic exposure. Swelling-induced ROS production in NIH3T3 fibroblasts is generated by NOX4 and by increasing total ROS, by either exogenous application of H(2)O(2) or overexpressing NOX4, we demonstrate that TonEBP activity and taurine influx are regulated negatively...... by ROS under hypo-osmotic, low-sodium conditions, whereas the TauT mRNA level is unaffected. Acute exposure to ROS reduces taurine uptake as a result of modulated TauT transport kinetics. Thus, swelling-induced ROS production could account for the reduced taurine uptake under low...

  1. Nuclear phosphoproteome analysis of 3T3-L1 preadipocyte differentiation reveals system-wide phosphorylation of transcriptional regulators

    DEFF Research Database (Denmark)

    Rabiee, Atefeh; Schwämmle, Veit; Sidoli, Simone

    2017-01-01

    of which were assigned as regulators of gene expression. Among 288 identified transcriptional regulators, 49 were regulated within four hours of adipogenic stimulation including several known and many novel potential adipogenic regulators. A kinase-substrate database for 3T3-L1 preadipocytes established...... stages (4 hours) of preadipocyte differentiation. We identified a total of 4072 proteins including 2434 phosphorylated proteins, a majority of which were assigned as regulators of gene expression. Our results demonstrate that adipogenic stimuli increase the nuclear abundance and/or the phosphorylation...... for 3T3-L1 preadipocytes by investigating the relationship between protein kinases and protein phosphorylation sites identified in our dataset. A majority of the putative protein kinases belong to the cyclin-dependent kinase (CDK) family and the mitogen activated protein kinase (MAPK) family including P...

  2. Inhibition of T3 production in levothyroxine-treated female mice by the root extract of Convolvulus pluricaulis.

    Science.gov (United States)

    Panda, S; Kar, A

    2001-01-01

    An investigation was made to evaluate the role of Convolvulus pluricaulis root extract in the regulation of hyperthyroidism in female mice. Its possible site of action was also studied. L-Thyroxine treatment for 30 days increased serum concentrations of thyroxine (T4) and triodothyronine (T3). The activity of hepatic 5'-monodeiodinase (5'-DI) and glucose-6-phosphatase (G-6-Pase) was also enhanced. On the other hand, administration of the plant extract either alone or with L-T4, decreased serum T3 concentration and the activity of hepatic 5'-DI and G-6-phase, without marked alteration in hepatic lipid peroxidation, indicating the possible regulation of hyperthyroidism by the plant extract. It appears that the action of the plant extract on thyroid function is primarily mediated through the inhibition of 5'-DI enzyme activity.

  3. Activation of liver X receptors prevents statin-induced death of 3T3-L1 preadipocytes

    DEFF Research Database (Denmark)

    Madsen, Lise; Petersen, Rasmus K; Steffensen, Knut R

    2008-01-01

    The biological functions of liver X receptors (LXRs) alpha and beta have primarily been linked to pathways involved in fatty acid and cholesterol homeostasis. Here we report a novel role of LXR activation in protecting cells from statin-induced death. When 3T3-L1 preadipocytes were induced...... of LXRalpha, we demonstrate that the response to LXR agonists is LXR-dependent. Interestingly, LXR-mediated rescue of statin-induced apoptosis was not related to up-regulation of genes previously shown to be involved in the antiapoptotic action of LXR. Furthermore, forced expression of Bcl-2 did not prevent...... statin-induced apoptosis; nor did LXR action depend on protein kinase B, whose activation by insulin was impaired in statin-treated cells. Rather, LXR-dependent rescue of statin-induced apoptosis in 3T3-L1 preadipocytes required NF-kappaB activity, since expression of a dominant negative version...

  4. HUBUNGAN KANDUNGAN KLOR SERUM DENGAN HORMON T3/T4 PADA ANAK SEKOLAH DI DAERAH GONDOK ENDEMIK

    Directory of Open Access Journals (Sweden)

    Sukati Saidin

    2012-11-01

    Full Text Available ASSOCIATION OF SERUM CHLOR CONTENT WITH T3/T4 HORMONEIN SCHOOL CHILDREN IN IODINE DEFICIENCY REGION.Background: The National Mapping Survey of IDD (1998 found that 7% of sub districts In Indonesia was regarded as severe endemic goitre area (TGR>30%. The high TGR prevalence, beside as a result of low iodized salt consumption (< 30 ppm, It was assumed as the effect of exposure of goitrogenic agent such as chlorine. Based on observation in Karawang sub district showed people had food habit to consume fish contaminated by insecticide used for killing milk fish predator or salted fish which had also contaminated by insecticide used during process of fish drying. Insecticide raw material consists of chlorine which can not be broken by heat or oxidation. Previous study by Gaitan E. (1986 found that chlorine component could inhibit iodine metabolism to form mono and di-iodotyrosine as precursor of T3 and T4 hormones.Objectives: The aim of this study was to find an association of serum chlorine as a reflection of chlorine consumption from daily food with T3 and T4 hormone.Methods: Research design was case control. Study was conducted in Karawang district, West Java. The subject were elementary school children in the fourth, fifth and sixth grades with positive goitre at grade I and II by palpation. Sample size was 140 children divided into two groups, case group (70 children and control group (70 children. Main data collected was chlorine consumption from daily food, serum chlorine, serum T3 and T4 hormones as well as anthropometries.Results: The result showed that chlorine consumption from food was relatively greater in case group (135.9 ugr/day than in control group (129.9 ug/day but statistically it was not significant. Serum chlorine content in case group (1 14.8 mmol/L was significantly higher than in control group (102.1 mmol/L. Serum T4 hormone in case group (7.3 ug/dl was significantly lower than in control group (9.5 ug/dl. Serum T3 hormone in

  5. Crack Initiation and Growth Behavior at Corrosion Pit in 2024-T3 Aluminum Alloy

    Science.gov (United States)

    2014-09-01

    Air Force Institute of Technology Air University Air Education and Training Command...Mesh created by Abaqus for the uni-axial specimen with a crack on a hole with refined mesh near the crack tip... Abaqus . This approach is adapted as there are neither closed form solutions nor accepted models of the previously mentioned shape of specimens

  6. The location of locoregional recurrence in pathologic T3N0, non-irradiated lower rectal cancer

    International Nuclear Information System (INIS)

    Kim, Mi Sun; Keum, Ki Chang; Rhee, Woo Joong; Kim, Hyun Ju; Kim, Min Ji; Choi, Seo Hee; Nam, Ki Chang; Koom, Woong Sub

    2013-01-01

    To investigate the patterns of locoregional recurrence of pathologic T3N0 (pT3N0) lower rectal cancer omitting postoperative radiotherapy (RT) and explore the potential of modification of a RT field. From Jan 2003 to Nov 2011, 35 patients omitting preoperative or postoperative RT for pT3N0 lower rectal cancer were included. We defined the lower rectal cancer as the tumor with the inferior margin located below the virtual line-a convergent level between rectal wall and levator ani muscle. All patients had radiologic examinations for recurrence evaluation during the follow-up duration. The median follow-up duration was 66.4 months (range, 1.4 to 126.1 months). Eight (22.9%) of the 35 patients had recurrence. Three (8.6%) was local recurrence (LR) only, 3 (8.6%) was distant metastasis (DM) only, and 2 (5.7%) was LR with DM. All LR were located at primary tumor sites. The overall survival rate, LR-free survival rate, and DM-free survival rate at 5 years was 79.8%, 83%, and 87%, respectively. All LR developed from tumors over 5 cm. However, there was no statistical significance (p = 0.065). There was no other risk factor for LR. Even though the patients included in this study had pathologically favorable pT3N0 rectal cancer, LR developed in 14.3% of patients. Most of the LR was located at primary tumor sites prior to surgery. Based on these findings, it might seem reasonable to consider postoperative RT with a smaller radiation field to the primary tumor site rather than the conventional whole pelvic irradiation.

  7. Inhibition of adipogenesis and leptin production in 3T3-L1 adipocytes by a derivative of meridianin C

    International Nuclear Information System (INIS)

    Park, Yu-Kyoung; Lee, Tae-Yoon; Choi, Jong-Soon; Hong, Victor Sukbong; Lee, Jinho; Park, Jong-Wook; Jang, Byeong-Churl

    2014-01-01

    Highlights: • Compound 7b, a meridianin C derivative, inhibits adipogenesis. • Compound 7b inhibits C/EBP-α, PPAR-γ, FAS, STAT-3, and STAT-5 in 3T3-L1 adipocytes. • Compound 7b inhibits leptin, but not adiponectin, expression in 3T3-L1 adipocytes. • Compound 7b thus may have therapeutic potential against obesity. - Abstract: Meridianin C, a marine alkaloid, is a potent protein kinase inhibitor and has anti-cancer activity. We have recently developed a series of meridianin C derivatives (compound 7a–7j) and reported their proviral integration Moloney Murine Leukemia Virus (pim) kinases’ inhibitory and anti-proliferative effects on human leukemia cells. Here we investigated the effect of these meridianin C derivatives on adipogenesis. Strikingly, among the derivatives tested, compound 7b most strongly inhibited lipid accumulation during the differentiation of 3T3-L1 preadipocytes into adipocytes. However, meridianin C treatment was largely cytotoxic to 3T3-L1 adipocytes. On mechanistic levels, compound 7b reduced not only the expressions of CCAAT/enhancer-binding protein-α (C/EBP-α), peroxisome proliferator-activated receptor-γ (PPAR-γ), and fatty acid synthase (FAS) but also the phosphorylation levels of signal transducer and activator of transcription-3 (STAT-3) and STAT-5 during adipocyte differentiation. Moreover, compound 7b repressed leptin, but not adiponectin, expression during adipocyte differentiation. Collectively, these findings demonstrate that a meridianin C derivative inhibits adipogenesis by down-regulating expressions and/or phosphorylations of C/EBP-α, PPAR-γ, FAS, STAT-3 and STAT-5

  8. Inhibition of adipogenesis and leptin production in 3T3-L1 adipocytes by a derivative of meridianin C

    Energy Technology Data Exchange (ETDEWEB)

    Park, Yu-Kyoung [Department of Molecular Medicine, College of Medicine, Keimyung University, 1095 Dalgubeoldaero, Dalseo-gu, Daegu 704-701 (Korea, Republic of); Lee, Tae-Yoon [Department of Microbiology, College of Medicine, Yeungnam University, 170 Hyunchung-Ro, Nam-gu, Daegu 705-717 (Korea, Republic of); Choi, Jong-Soon [Division of Life Science, Korea Basic Science Institute, 169-148 Gwahakro, Yuseong-gu, Daejeon 305-333 (Korea, Republic of); Hong, Victor Sukbong [Department of Chemistry, College of Natural Sciences, Keimyung University, 1095 Dalgubeoldaero, Dalseo-gu, Daegu 704-701 (Korea, Republic of); Lee, Jinho, E-mail: jinho@gw.kmu.ac.kr [Department of Chemistry, College of Natural Sciences, Keimyung University, 1095 Dalgubeoldaero, Dalseo-gu, Daegu 704-701 (Korea, Republic of); Park, Jong-Wook, E-mail: j303nih@dsmc.or.kr [Department of Immunology, College of Medicine, Keimyung University, 1095 Dalgubeoldaero, Dalseo-gu, Daegu 704-701 (Korea, Republic of); Jang, Byeong-Churl, E-mail: jangbc123@gw.kmu.ac.kr [Department of Molecular Medicine, College of Medicine, Keimyung University, 1095 Dalgubeoldaero, Dalseo-gu, Daegu 704-701 (Korea, Republic of)

    2014-10-03

    Highlights: • Compound 7b, a meridianin C derivative, inhibits adipogenesis. • Compound 7b inhibits C/EBP-α, PPAR-γ, FAS, STAT-3, and STAT-5 in 3T3-L1 adipocytes. • Compound 7b inhibits leptin, but not adiponectin, expression in 3T3-L1 adipocytes. • Compound 7b thus may have therapeutic potential against obesity. - Abstract: Meridianin C, a marine alkaloid, is a potent protein kinase inhibitor and has anti-cancer activity. We have recently developed a series of meridianin C derivatives (compound 7a–7j) and reported their proviral integration Moloney Murine Leukemia Virus (pim) kinases’ inhibitory and anti-proliferative effects on human leukemia cells. Here we investigated the effect of these meridianin C derivatives on adipogenesis. Strikingly, among the derivatives tested, compound 7b most strongly inhibited lipid accumulation during the differentiation of 3T3-L1 preadipocytes into adipocytes. However, meridianin C treatment was largely cytotoxic to 3T3-L1 adipocytes. On mechanistic levels, compound 7b reduced not only the expressions of CCAAT/enhancer-binding protein-α (C/EBP-α), peroxisome proliferator-activated receptor-γ (PPAR-γ), and fatty acid synthase (FAS) but also the phosphorylation levels of signal transducer and activator of transcription-3 (STAT-3) and STAT-5 during adipocyte differentiation. Moreover, compound 7b repressed leptin, but not adiponectin, expression during adipocyte differentiation. Collectively, these findings demonstrate that a meridianin C derivative inhibits adipogenesis by down-regulating expressions and/or phosphorylations of C/EBP-α, PPAR-γ, FAS, STAT-3 and STAT-5.

  9. Enhancing osteogenic differentiation of MC3T3-E1 cells by immobilizing RGD onto liquid crystal substrate

    International Nuclear Information System (INIS)

    Wu, Shaopeng; Yang, Xiaohui; Li, Wenqiang; Du, Lin; Zeng, Rong; Tu, Mei

    2017-01-01

    To understand the effects of GRGDF modification on MC3T3-E1 cell behavior, we cultured these cells onto a biomimetic liquid crystalline matrix modified with GRGDF peptide (OPC-GA-RGD). Successful immobilization of GRGDF on the liquid crystalline surface was verified by fluorescent labeling, attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy and X-ray photoelectron spectroscopy (XPS). OPC-GA-RGDs retained its liquid crystalline feature after surface modification. The RGD-immobilized OPC substrate was hardly beneficial to initial cell adhesion but could support long-term cell survival. The enhancement in cell proliferation did not correlate with RGD density. The lower GRGDF density immobilized on the liquid crystalline OPC matrix (OPC-GA-RGD3) promoted cell adhesion, proliferation, ALP expression level and mineralization, suggesting that both the viscoelasticity-based mechanical stimuli and receptor/ligand-based biochemical cue synergistically modulate MC3T3-E1 cell behavior. - Highlight: • A novel type of GRGDF-immobilized liquid crystalline matrices was fabricated and served as a substrate for the in vitro culture of MC3T3-E1 cells. • The lower RGD density might provide a better condition for initial cell adhesion and proliferation, up-regulation of ALP expression levels, and mineralization. • The intrinsic liquid crystalline feature of OPC matrix, instead of RGD efficiency, promoted initial cell adhesion. • Properties of the liquid crystalline OPC matrix together with the stable receptor-ligand binging synergistically modulated MC3T3-E1 cell behavior.

  10. Citrus aurantium flavonoids inhibit adipogenesis through the Akt signaling pathway in 3T3-L1 cells

    Directory of Open Access Journals (Sweden)

    Kim Gon-Sup

    2012-04-01

    Full Text Available Abstract Background Obesity is a health hazard that is associated with a number of diseases and metabolic abnormalities, such as type-2 diabetes, hypertension, dyslipidemia, and coronary heart disease. In the current study, we investigated the effects of Citrus aurantium flavonoids (CAF on the inhibition of adipogenesis and adipocyte differentiation in 3T3-L1 cells. Methods During adipocyte differentiation, 3T3-L1 cells were treated with 0, 10, and 50 μg/ml CAF, and then the mRNA and protein expression of adipogenesis-related genes was assayed. We examined the effect of CAF on level of phosphorylated Akt in 3T3-L1 cells treated with CAF at various concentrations during adipocyte differentiation. Results The insulin-induced expression of C/EBPβ and PPARγ mRNA and protein were significantly down-regulated in a dose-dependent manner following CAF treatment. CAF also dramatically decreased the expression of C/EBPα, which is essential for the acquisition of insulin sensitivity by adipocytes. Moreover, the expression of the aP2 and FAS genes, which are involved in lipid metabolism, decreased dramatically upon treatment with CAF. Interestingly, CAF diminished the insulin-stimulated serine phosphorylation of Akt (Ser473 and GSK3β (Ser9, which may reduce glucose uptake in response to insulin and lipid accumulation. Furthermore, CAF not only inhibited triglyceride accumulation during adipogenesis but also contributed to the lipolysis of adipocytes. Conclusions In the present study, we demonstrate that CAF suppressed adipogenesis in 3T3-L1 adipocytes. Our results indicated that CAF down-regulates the expression of C/EBPβ and subsequently inhibits the activation of PPARγ and C/EBPα. The anti-adipogenic activity of CAF was mediated by the inhibition of Akt activation and GSK3β phosphorylation, which induced the down-regulation of lipid accumulation and lipid metabolizing genes, ultimately inhibiting adipocyte differentiation.

  11. Studies on the MxiH protein in T3SS needles using DNP-enhanced ssNMR spectroscopy.

    Science.gov (United States)

    Fricke, Pascal; Demers, Jean-Philippe; Becker, Stefan; Lange, Adam

    2014-01-13

    Bacterial T3SS needles formed by the protein MxiH are studied using DNP-enhanced ssNMR spectroscopy at 14.1 T (600 MHz). This technique provides spectra of good resolution, allowing us to draw conclusions about the protein dynamics. With the obtained signal enhancement, samples of limited quantity now get within reach of ssNMR studies. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Production of d, t, 3He, anti d, anti t and anti 3He by 200 GeV protons

    International Nuclear Information System (INIS)

    Bozzoli, W.; Giacomelli, G.; Rimondi, F.; Zylberajch, S.; Lesquoy, E.; Meunier, R.; Moscoso, L.; Muller, A.; Bussiere, A.

    1978-01-01

    Data are presented on the yields of d, t, 3 He, anti d, anti t, and anti 3 He with laboratory momenta between 12 and 37 GeV/c produced by 200 GeV protons on beryllium and aluminium. The production yield of nuclei depends significantly on the target nucleus, while the anti d production is independent of target material. The mass dependence of the production cross section is exponential for both nuclei and antinuclei

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

    International Nuclear Information System (INIS)

    Yuan, Guoyue; Jia, Jue; Di, Liangliang; Zhou, Libin; Dong, Sijing; Ye, Jingjing; Wang, Dong; Yang, Ling; Wang, Jifang; Li, Lianxi; Yang, Ying; Mao, Chaoming; Chen, Mingdao

    2012-01-01

    Highlights: ► CRP increases TNF-α and IL-6 genes expression in matured 3T3-L1 adipocytes. ► CRP suppresses adiponectin, leptin and PPAR-γ mRNA levels in matured 3T3-L1 cells. ► Wortmannin reverses effects of CRP on adiponectin, TNF-α and leptin mRNA levels. ► 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-γ) genes expression and raised tumor necrosis factor-α (TNF-α) 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-α and leptin genes expression. These results collectively suggest that CRP regulates adiponectin, TNF-α, leptin, IL-6 and PPAR-γ genes expression, and that might represent a mechanism by which CRP regulates insulin resistance, obesity and metabolic syndrome.

  14. Inhibitory potential of rambutan seeds extract and fractions on adipogenesis in 3T3-L1 cell line

    OpenAIRE

    Sylvia Soeng; Endang Evacuasiany; Wahyu Widowati; Nurul Fauziah; Visi Tinta Manik; Maesaroh Maesaroh

    2015-01-01

    Objective: Type 2 diabetes is a global health problem with increasing prevalence related to several conditions; one of these is due to obesity. Rambutan (Nephelium lappaceum L) seeds contain various phenolic compounds. The present study was designed to evaluate the phytochemical content and the inhibitory potential of rambutan seeds extract and fractions on glucose-6-phosphate dehydrogenase (G6PDH), and #945;-glucosidase, and triglyceride activities ex vivo in 3T3-L1 cell line (pre-adipocyte...

  15. Functional expression of 5-HT{sub 2A} receptor in osteoblastic MC3T3-E1 cells

    Energy Technology Data Exchange (ETDEWEB)

    Hirai, Takao; Kaneshige, Kota; Kurosaki, Teruko [Department of Molecular Pharmacology, Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University, 1 Gakuen-cho, Fukuyama, Hiroshima 729-0292 (Japan); Nishio, Hiroaki, E-mail: nishio@fupharm.fukuyama-u.ac.jp [Department of Molecular Pharmacology, Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University, 1 Gakuen-cho, Fukuyama, Hiroshima 729-0292 (Japan)

    2010-05-28

    In the previous study, we reported the gene expression for proteins related to the function of 5-hydroxytryptamine (5-HT, serotonin) and elucidated the expression patterns of 5-HT{sub 2} receptor subtypes in mouse osteoblasts. In the present study, we evaluated the possible involvement of 5-HT receptor subtypes and its inactivation system in MC3T3-E1 cells, an osteoblast cell line. DOI, a 5-HT{sub 2A} and 5-HT{sub 2C} receptor selective agonist, as well as 5-HT concentration-dependently increased proliferative activities of MC3T3-E1 cells in their premature period. This effect of 5-HT on cell proliferation were inhibited by ketanserin, a 5-HT{sub 2A} receptor specific antagonist. Moreover, both DOI-induced cell proliferation and phosphorylation of ERK1 and 2 proteins were inhibited by PD98059 and U0126, selective inhibitors of MEK in a concentration-dependent manner. Furthermore, treatment with fluoxetine, a 5-HT specific re-uptake inhibitor which inactivate the function of extracellular 5-HT, significantly increased the proliferative activities of MC3T3-E1 cells in a concentration-dependent manner. Our data indicate that 5-HT fill the role for proliferation of osteoblast cells in their premature period. Notably, 5-HT{sub 2A} receptor may be functionally expressed to regulate mechanisms underlying osteoblast cell proliferation, at least in part, through activation of ERK/MAPK pathways in MC3T3-E1 cells.

  16. Citrus aurantium flavonoids inhibit adipogenesis through the Akt signaling pathway in 3T3-L1 cells.

    Science.gov (United States)

    Kim, Gon-Sup; Park, Hyoung Joon; Woo, Jong-Hwa; Kim, Mi-Kyeong; Koh, Phil-Ok; Min, Wongi; Ko, Yeoung-Gyu; Kim, Chung-Hei; Won, Chung-Kil; Cho, Jae-Hyeon

    2012-04-03

    Obesity is a health hazard that is associated with a number of diseases and metabolic abnormalities, such as type-2 diabetes, hypertension, dyslipidemia, and coronary heart disease. In the current study, we investigated the effects of Citrus aurantium flavonoids (CAF) on the inhibition of adipogenesis and adipocyte differentiation in 3T3-L1 cells. During adipocyte differentiation, 3T3-L1 cells were treated with 0, 10, and 50 μg/ml CAF, and then the mRNA and protein expression of adipogenesis-related genes was assayed. We examined the effect of CAF on level of phosphorylated Akt in 3T3-L1 cells treated with CAF at various concentrations during adipocyte differentiation. The insulin-induced expression of C/EBPβ and PPARγ mRNA and protein were significantly down-regulated in a dose-dependent manner following CAF treatment. CAF also dramatically decreased the expression of C/EBPα, which is essential for the acquisition of insulin sensitivity by adipocytes. Moreover, the expression of the aP2 and FAS genes, which are involved in lipid metabolism, decreased dramatically upon treatment with CAF. Interestingly, CAF diminished the insulin-stimulated serine phosphorylation of Akt (Ser473) and GSK3β (Ser9), which may reduce glucose uptake in response to insulin and lipid accumulation. Furthermore, CAF not only inhibited triglyceride accumulation during adipogenesis but also contributed to the lipolysis of adipocytes. In the present study, we demonstrate that CAF suppressed adipogenesis in 3T3-L1 adipocytes. Our results indicated that CAF down-regulates the expression of C/EBPβ and subsequently inhibits the activation of PPARγ and C/EBPα. The anti-adipogenic activity of CAF was mediated by the inhibition of Akt activation and GSK3β phosphorylation, which induced the down-regulation of lipid accumulation and lipid metabolizing genes, ultimately inhibiting adipocyte differentiation.

  17. Influence of Process Parameters in the Friction Surfacing of AA 6082-T6 over AA 2024-T3

    OpenAIRE

    Gandra, J.; Pereira, D.; Miranda, R.M.; Vilaça, P.

    2013-01-01

    VK: T20309 Friction Surfacing is a solid state coating technique with applications in hardfacing, corrosion protection and repair. Since it doesn’t require the fusion of the materials involved, it is suitable to join aluminium alloys while avoiding several of their processing difficulties. The present study addresses the deposition of AA 6082-T6 coatings on AA 2024-T3 substrates, while focusing on the effect of process parameters, such as, axial force, rotation and travel speed. Sound alum...

  18. Abnormalities of Thyroid Hormone Metabolism during Systemic Illness: The Low T3 Syndrome in Different Clinical Settings

    OpenAIRE

    Arnaldo Moura Neto; Denise Engelbrecht Zantut-Wittmann

    2016-01-01

    Thyroid hormone abnormalities are common in critically ill patients. For over three decades, a mild form of these abnormalities has been described in patients with several diseases under outpatient care. These alterations in thyroid hormone economy are a part of the nonthyroidal illness and keep an important relationship with prognosis in most cases. The main feature of this syndrome is a fall in free triiodothyronine (T3) levels with normal thyrotropin (TSH). Free thyroxin (T4) and reverse T...

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

    Energy Technology Data Exchange (ETDEWEB)

    Yuan, Guoyue, E-mail: yuanguoyue@hotmail.com [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); 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); Dong, Sijing; Ye, Jingjing; Wang, Dong; Yang, Ling; Wang, Jifang [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); 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); Mao, Chaoming [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)

    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.

  20. Performance of Software for the Full Symmetric Eigenproblem on CRAY T3E and T90 Systems

    OpenAIRE

    Gutheil, Inge; Zimmermann, Ruth

    2000-01-01

    MPP systems with a message-passing programming paradigm such as CRAY T3E still play an important role in high performance scientific computing and so there is need for basic numerical software for those systems. There are several libraries for numerical linear algebra computations with message-passing available most of which are public domain libraries. The eigensystem solvers of some of these libraries are compared taking into account not only performance but also user-friendliness. Furtherm...

  1. Uniform Corrosion and General Dissolution of Aluminum Alloys 2024-T3, 6061-T6, and 7075-T6

    Science.gov (United States)

    Huang, I.-Wen

    Uniform corrosion and general dissolution of aluminum alloys was not as well-studied in the past, although it was known for causing significant amount of weight loss. This work comprises four chapters to understand uniform corrosion of aluminum alloys 2024-T3, 6061-T6, and 7075-T6. A preliminary weight loss experiment was performed for distinguishing corrosion induced weight loss attributed to uniform corrosion and pitting corrosion. The result suggested that uniform corrosion generated a greater mass loss than pitting corrosion. First, to understand uniform corrosion mechanism and kinetics in different environments, a series of static immersion tests in NaCl solutions were performed to provide quantitative measurement of uniform corrosion. Thereafter, uniform corrosion development as a function of temperature, pH, Cl-, and time was investigated to understand the influence of environmental factors. Faster uniform corrosion rate has been found at lower temperature (20 and 40°C) than at higher temperature (60 and 80°C) due to accelerated corrosion product formation at high temperatures inhibiting corrosion reactions. Electrochemical tests including along with scanning electron microscopy (SEM) were utilized to study the temperature effect. Second, in order to further understand the uniform corrosion influence on pit growth kinetics, a long term exposures for 180 days in both immersion and ASTM-B117 test were performed. Uniform corrosion induced surface recession was found to have limited impact on pit geometry regardless of exposure methods. It was also found that the competition for limited cathodic current from uniform corrosion the primary rate limiting factor for pit growth. Very large pits were found after uniform corrosion growth reached a plateau due to corrosion product coverage. Also, optical microscopy and focused ion beam (FIB) imaging has provided more insights of distinctive pitting geometry and subsurface damages found from immersion samples and B117

  2. Lipolytic efficacy of alginate double-layer nanoemulsion containing oleoresin capsicum in differentiated 3T3-L1 adipocytes.

    Science.gov (United States)

    Lee, Mak-Soon; Jung, Sunyoon; Shin, Yoonjin; Lee, Seohyun; Kim, Chong-Tai; Kim, In-Hwan; Kim, Yangha

    2017-01-01

    Background : Oleoresin capsicum (OC) is an organic extract from fruits of the genus Capsicum , and has been reported to have an anti-obesity effect. Objective : This study comparatively investigated lipolytic effects of single-layer nanoemulsion (SN) and alginate double-layer nanoemulsion (AN) containing OC in 3T3-L1 adipocytes. Methods : SN and AN were compared by analyzing the intracellular lipid accumulation, triglyceride (TG) content, release of free fatty acids (FFAs) and glycerol, and mRNA expression of genes related to adipogenesis and lipolysis were analyzed in fully differentiated 3T3-L1 adipocytes. Results : Compared with SN, AN exhibited higher efficiency in inhibiting the intracellular lipid accumulation and TG content, and enhanced the release of FFAs and glycerol into the medium. In AN-treated cells, mRNA levels of peroxisome proliferator-activated receptor-γ and the fatty acid-binding protein adipocyte protein-2, which are involved in adipogenesis, were down-regulated, whereas those of genes related to lipolysis, including hormone-sensitive lipase and carnitine palmitoyl transferase-1α, were up-regulated compared with SN-treated cells. Conclusion : The lipolytic effect of AN was greater than that of SN; this was partly associated with the increased TG hydrolysis via induction of lipolytic gene expression and suppression of adipogenic gene expression in 3T3-L1 adipocytes.​​​​.

  3. Injection of T3SS effectors not resulting in invasion is the main targeting mechanism ofShigellatoward human lymphocytes.

    Science.gov (United States)

    Pinaud, Laurie; Samassa, Fatoumata; Porat, Ziv; Ferrari, Mariana L; Belotserkovsky, Ilia; Parsot, Claude; Sansonetti, Philippe J; Campbell-Valois, François-Xavier; Phalipon, Armelle

    2017-09-12

    The enteroinvasive bacterium Shigella is a facultative intracellular bacterium known, in vitro, to invade a large diversity of cells through the delivery of virulence effectors into the cell cytoplasm via a type III secretion system (T3SS). Here, we provide evidence that the injection of T3SS effectors does not necessarily result in cell invasion. Indeed, we demonstrate through optimization of a T3SS injection reporter that effector injection without subsequent cell invasion, termed the injection-only mechanism, is the main strategy used by Shigella to target human immune cells. We show that in vitro-activated human peripheral blood B, CD4 + T, and CD8 + T lymphocytes as well as switched memory B cells are mostly targeted by the injection-only mechanism. B and T lymphocytes residing in the human colonic lamina propria, encountered by Shigella upon its crossing of the mucosal barrier, are also mainly targeted by injection-only. These findings reveal that cells refractory to invasion can still be injected, thus extending the panel of host cells manipulated to the benefit of the pathogen. Future analysis of the functional consequences of the injection-only mechanism toward immune cells will contribute to the understanding of the priming of adaptive immunity, which is known to be altered during the course of natural Shigella infection.

  4. Allelic Tests and Sequence Analysis of Three Genes for Resistance to Xanthomonas perforans Race T3 in Tomato

    Directory of Open Access Journals (Sweden)

    Zhao Baimei

    2015-07-01

    Full Text Available Three crosses, Hawaii7981 × PI128216, Hawaii7981 × LA1589, and PI128216 × LA1589, were made to develop F2 populations for testing allelism among three genes Xv3, Rx4, and RxLA1589 conferring resistance to bacterial spot caused by Xanthomonas perforans race T3 in tomato. Each population consisted of 535–1 655 individuals. An infiltration method was used to inoculate the leaves of the parental and F2 plants as well as the susceptible control OH88119 for detecting hypersensitive resistance (HR. The results showed that all the tomato plants except OH88119 had HR to race T3, indicating that Xv3, Rx4, and RxLA1589 were allelic genes. Genomic DNA fragments of the Rx4 alleles from Hawaii7981, PI128216, and LA1589 were amplified using gene-specific primers and sequenced. No sequence variation was observed in the coding region of Rx4 in the three resistant lines. Based on the published map positions of these loci as well as the allelic tests and sequence data obtained in this study, we speculated that Xv3, Rx4, and RxLA1589 were the same gene. The results will provide useful information for understanding the mechanism of resistance to race T3 and developing resistant tomato varieties.

  5. Characterization and expression of the human T cell receptor-T3 complex by monoclonal antibody F101.01

    DEFF Research Database (Denmark)

    Geisler, C; Plesner, T; Pallesen, G

    1988-01-01

    A murine monoclonal antibody (MoAb) F101.01 reacting with the T cell receptor (TCR)-T3 complex is presented. Immunohistological studies showed that F101.01 specifically stains T-zone lymphocytes in lymph nodes, tonsils, and splenic tissue. Two-colour immunofluorescence and flow cytometry...... demonstrated co-expression of the antigen defined by F101.01 and the pan-T cell antigens defined by CD2, CD3, CD5, and CD7 antibodies. Cells stained with CD4 and CD8 antibodies were both included in the F101.01-positive population, whereas CD16-positive natural killer cells (NK), B cells (CD19 and CD20......), and myeloid cells (CD13 and CD33) were excluded. The target antigen of F101.01 co-modulated with the CD3-defined antigen (T3) and the TCR recognized by the MoAb WT-31. CD3 antibody and WT-31 both blocked binding of F101.01. F101.01 precipitated the TCR-T3 complex from lysates of 125I-labelled peripheral blood...

  6. Effects of PEMF exposure at different pulses on osteogenesis of MC3T3-E1 cells.

    Science.gov (United States)

    Li, Kangchu; Ma, Shirong; Li, Yurong; Ding, Guirong; Teng, Zenghui; Liu, Junye; Ren, Dongqing; Guo, Yao; Ma, Lei; Guo, Guozhen

    2014-09-01

    Pulsed electromagnetic fields (PEMFs) were considered to be a factor which may affect osteogenesis of osteoblasts, but the effects were diverse with different PEMF parameters. The aim of the current study is to explore the effects of exposure to PEMFs at different pulse number on osteogenesis of osteoblasts. The mouse osteoblast-like MC3T3-E1 cells were exposed to 0, 400 or 2800 pulses 400kV/m PEMF and the proliferation, differentiation and mineralization of cells were observed after PEMF exposure by the methods of MTT, biochemical measurement, real-time PCR and Alizarin Red assay. Compared with 0 pulses groups, the growth curve, alkaline phosphatase (ALP) activity, mRNA level of osteocalcin (OCN) and mineralized nodule formation of MC3T3-E1 cells did not change after 400 pulses PEMF exposure, but decreased after 2800 pulses PEMF exposure. It suggested that under our experimental conditions, only 2800 pulses 400kV/m PEMF exposure can suppress the proliferation, differentiation and mineralization of MC3T3-E1 cells, but 400 pulses 400kV/m PEMF exposure cannot. Pulse number is another involved parameter which may influence the effects of PEMF on osteogenesis of osteoblasts. Copyright © 2014 Elsevier Ltd. All rights reserved.

  7. Current algebra results for Ksub(t3) beyond the Callan Treiman relation

    International Nuclear Information System (INIS)

    Kimel, I.

    1976-01-01

    In order to obtain improved theoretical information on the decay form factors for Ksub(l3), current algebra is used taking into account terms of first order in the pion momentum. It is shown, in a fairly model independent manner, that the form factor has, to a good extent a linear behaviour. It is pointed out that explicit expansion in chiral symmetry-breaking parameters (or the commitment to a specific model of that breaking) are not really necessary for obtaining most of the features of the Ksub(l3) decays that can be compared with experiment. Current algebra with pions that can have s-amm four-momentum is sufficient

  8. Computational approach to predict species-specific type III secretion system (T3SS) effectors using single and multiple genomes.

    Science.gov (United States)

    Hobbs, Christopher K; Porter, Vanessa L; Stow, Maxwell L S; Siame, Bupe A; Tsang, Herbert H; Leung, Ka Yin

    2016-12-19

    Many gram-negative bacteria use type III secretion systems (T3SSs) to translocate effector proteins into host cells. T3SS effectors can give some bacteria a competitive edge over others within the same environment and can help bacteria to invade the host cells and allow them to multiply rapidly within the host. Therefore, developing efficient methods to identify effectors scattered in bacterial genomes can lead to a better understanding of host-pathogen interactions and ultimately to important medical and biotechnological applications. We used 21 genomic and proteomic attributes to create a precise and reliable T3SS effector prediction method called Genome Search for Effectors Tool (GenSET). Five machine learning algorithms were trained on effectors selected from different organisms and a trained (voting) algorithm was then applied to identify other effectors present in the genome testing sets from the same (GenSET Phase 1) or different (GenSET Phase 2) organism. Although a select group of attributes that included the codon adaptation index, probability of expression in inclusion bodies, N-terminal disorder, and G + C content (filtered) were better at discriminating between positive and negative sets, algorithm performance was better when all 21 attributes (unfiltered) were used. Performance scores (sensitivity, specificity and area under the curve) from GenSET Phase 1 were better than those reported for six published methods. More importantly, GenSET Phase 1 ranked more known effectors (70.3%) in the top 40 ranked proteins and predicted 10-80% more effectors than three available programs in three of the four organisms tested. GenSET Phase 2 predicted 43.8% effectors in the top 40 ranked proteins when tested on four related or unrelated organisms. The lower prediction rates from GenSET Phase 2 may be due to the presence of different translocation signals in effectors from different T3SS families. The species-specific GenSET Phase 1 method offers an alternative

  9. Modest hypoxia significantly reduces triglyceride content and lipid droplet size in 3T3-L1 adipocytes

    International Nuclear Information System (INIS)

    Hashimoto, Takeshi; Yokokawa, Takumi; Endo, Yuriko; Iwanaka, Nobumasa; Higashida, Kazuhiko; Taguchi, Sadayoshi

    2013-01-01

    Highlights: •Long-term hypoxia decreased the size of LDs and lipid storage in 3T3-L1 adipocytes. •Long-term hypoxia increased basal lipolysis in 3T3-L1 adipocytes. •Hypoxia decreased lipid-associated proteins in 3T3-L1 adipocytes. •Hypoxia decreased basal glucose uptake and lipogenic proteins in 3T3-L1 adipocytes. •Hypoxia-mediated lipogenesis may be an attractive therapeutic target against obesity. -- Abstract: Background: A previous study has demonstrated that endurance training under hypoxia results in a greater reduction in body fat mass compared to exercise under normoxia. However, the cellular and molecular mechanisms that underlie this hypoxia-mediated reduction in fat mass remain uncertain. Here, we examine the effects of modest hypoxia on adipocyte function. Methods: Differentiated 3T3-L1 adipocytes were incubated at 5% O 2 for 1 week (long-term hypoxia, HL) or one day (short-term hypoxia, HS) and compared with a normoxia control (NC). Results: HL, but not HS, resulted in a significant reduction in lipid droplet size and triglyceride content (by 50%) compared to NC (p < 0.01). As estimated by glycerol release, isoproterenol-induced lipolysis was significantly lowered by hypoxia, whereas the release of free fatty acids under the basal condition was prominently enhanced with HL compared to NC or HS (p < 0.01). Lipolysis-associated proteins, such as perilipin 1 and hormone-sensitive lipase, were unchanged, whereas adipose triglyceride lipase and its activator protein CGI-58 were decreased with HL in comparison to NC. Interestingly, such lipogenic proteins as fatty acid synthase, lipin-1, and peroxisome proliferator-activated receptor gamma were decreased. Furthermore, the uptake of glucose, the major precursor of 3-glycerol phosphate for triglyceride synthesis, was significantly reduced in HL compared to NC or HS (p < 0.01). Conclusion: We conclude that hypoxia has a direct impact on reducing the triglyceride content and lipid droplet size via

  10. Modest hypoxia significantly reduces triglyceride content and lipid droplet size in 3T3-L1 adipocytes

    Energy Technology Data Exchange (ETDEWEB)

    Hashimoto, Takeshi, E-mail: thashimo@fc.ritsumei.ac.jp [Faculty of Sport and Health Science, Ritsumeikan University, 1-1-1 Nojihigashi, Kusatsu, Shiga 525-8577 (Japan); Yokokawa, Takumi; Endo, Yuriko [Faculty of Sport and Health Science, Ritsumeikan University, 1-1-1 Nojihigashi, Kusatsu, Shiga 525-8577 (Japan); Iwanaka, Nobumasa [Ritsumeikan Global Innovation Research Organization, Ritsumeikan University, 1-1-1 Nojihigashi, Kusatsu, Shiga 525-8577 (Japan); Higashida, Kazuhiko [Faculty of Sport and Health Science, Ritsumeikan University, 1-1-1 Nojihigashi, Kusatsu, Shiga 525-8577 (Japan); Faculty of Sport Science, Waseda University, 2-579-15 Mikajima, Tokorozawa, Saitama 359-1192 (Japan); Taguchi, Sadayoshi [Faculty of Sport and Health Science, Ritsumeikan University, 1-1-1 Nojihigashi, Kusatsu, Shiga 525-8577 (Japan)

    2013-10-11

    Highlights: •Long-term hypoxia decreased the size of LDs and lipid storage in 3T3-L1 adipocytes. •Long-term hypoxia increased basal lipolysis in 3T3-L1 adipocytes. •Hypoxia decreased lipid-associated proteins in 3T3-L1 adipocytes. •Hypoxia decreased basal glucose uptake and lipogenic proteins in 3T3-L1 adipocytes. •Hypoxia-mediated lipogenesis may be an attractive therapeutic target against obesity. -- Abstract: Background: A previous study has demonstrated that endurance training under hypoxia results in a greater reduction in body fat mass compared to exercise under normoxia. However, the cellular and molecular mechanisms that underlie this hypoxia-mediated reduction in fat mass remain uncertain. Here, we examine the effects of modest hypoxia on adipocyte function. Methods: Differentiated 3T3-L1 adipocytes were incubated at 5% O{sub 2} for 1 week (long-term hypoxia, HL) or one day (short-term hypoxia, HS) and compared with a normoxia control (NC). Results: HL, but not HS, resulted in a significant reduction in lipid droplet size and triglyceride content (by 50%) compared to NC (p < 0.01). As estimated by glycerol release, isoproterenol-induced lipolysis was significantly lowered by hypoxia, whereas the release of free fatty acids under the basal condition was prominently enhanced with HL compared to NC or HS (p < 0.01). Lipolysis-associated proteins, such as perilipin 1 and hormone-sensitive lipase, were unchanged, whereas adipose triglyceride lipase and its activator protein CGI-58 were decreased with HL in comparison to NC. Interestingly, such lipogenic proteins as fatty acid synthase, lipin-1, and peroxisome proliferator-activated receptor gamma were decreased. Furthermore, the uptake of glucose, the major precursor of 3-glycerol phosphate for triglyceride synthesis, was significantly reduced in HL compared to NC or HS (p < 0.01). Conclusion: We conclude that hypoxia has a direct impact on reducing the triglyceride content and lipid droplet size via

  11. Reassessment of MxiH subunit orientation and fold within native Shigella T3SS needles using surface labelling and solid-state NMR.

    Science.gov (United States)

    Verasdonck, Joeri; Shen, Da-Kang; Treadgold, Alexander; Arthur, Christopher; Böckmann, Anja; Meier, Beat H; Blocker, Ariel J

    2015-12-01

    T3SSs are essential virulence determinants of many Gram-negative bacteria, used to inject bacterial effectors of virulence into eukaryotic host cells. Their major extracellular portion, a ∼50 nm hollow, needle-like structure, is essential to host cell sensing and the conduit for effector secretion. It is formed of a small, conserved subunit arranged as a helical polymer. The structure of the subunit has been studied by electron cryomicroscopy within native polymers and by solid-state NMR in recombinant polymers, yielding two incompatible atomic models. To resolve this controversy, we re-examined the native polymer used for electron cryomicroscopy via surface labelling and solid-state NMR. Our data show the orientation and overall fold of the subunit within this polymer is as established by solid-state NMR for recombinant polymers. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  12. Femtoscopy measurements in a p(T = 3.5 GeV) + Nb system

    Energy Technology Data Exchange (ETDEWEB)

    Arnold, Oliver [Physik Department E12 and Excellence Cluster ' ' Universe' ' , Technische Universitaet Muenchen, Garching (Germany); Collaboration: HADES-Collaboration

    2015-07-01

    In the 1950s, Hanburry-Brown and Twiss realized that there is a chance that photons are correlated after their emission from a light emitting astrophysical object like a star. They constructed a formalism, which allowed them to determine the angular size of the object by measuring the correlation signal between the emitted photons. Independently of this discovery, Goldhaber et al. measured later on an angular correlation for like-sign pion pairs in nuclear physics. We use the technique of two-particle correlations in a femtoscopy measurement of proton and Lambda pairs, which were produced in proton-niobium collisions and detected with HADES, where the proton had a kinetic beam energy of 3.5 GeV. This allows us to extract the region of homogeneity for proton-lambda pairs and additionally study their interaction. For comparisons of the source size we constructed also the correlation functions of proton and pion pairs and measured the k{sub T} dependence of the source. Our measurements allow us also to confront the experimental data with predictions of theoretical transport model calculations (UrQMD) to gain information about the emission dynamics of the particles.

  13. Maf-dependent bacterial flagellin glycosylation occurs before chaperone binding and flagellar T3SS export.

    Science.gov (United States)

    Parker, Jennifer L; Lowry, Rebecca C; Couto, Narciso A S; Wright, Phillip C; Stafford, Graham P; Shaw, Jonathan G

    2014-04-01

    Bacterial swimming is mediated by rotation of a filament that is assembled via polymerization of flagellin monomers after secretion via a dedicated flagellar Type III secretion system. Several bacteria decorate their flagellin with sialic acid related sugars that is essential for motility. Aeromonas caviae is a model organism for this process as it contains a genetically simple glycosylation system and decorates its flagellin with pseudaminic acid (Pse). The link between flagellin glycosylation and export has yet to be fully determined. We examined the role of glycosylation in the export and assembly process in a strain lacking Maf1, a protein involved in the transfer of Pse onto flagellin at the later stages of the glycosylation pathway. Immunoblotting, established that glycosylation is not required for flagellin export but is essential for filament assembly since non-glycosylated flagellin is still secreted. Maf1 interacts directly with its flagellin substrate in vivo, even in the absence of pseudaminic acid. Flagellin glycosylation in a flagellin chaperone mutant (flaJ) indicated that glycosylation occurs in the cytoplasm before chaperone binding and protein secretion. Preferential chaperone binding to glycosylated flagellin revealed its crucial role, indicating that this system has evolved to favour secretion of the polymerization competent glycosylated form. © 2014 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.

  14. Delivery of the autofluorescent protein R-phycoerythrin by calcium phosphate nanoparticles into four different eukaryotic cell lines (HeLa, HEK293T, MG-63, MC3T3): Highly efficient, but leading to endolysosomal proteolysis in HeLa and MC3T3 cells.

    Science.gov (United States)

    Kopp, Mathis; Rotan, Olga; Papadopoulos, Chrisovalantis; Schulze, Nina; Meyer, Hemmo; Epple, Matthias

    2017-01-01

    Nanoparticles can be used as carriers to transport biomolecules like proteins and synthetic molecules across the cell membrane because many molecules are not able to cross the cell membrane on their own. The uptake of nanoparticles together with their cargo typically occurs via endocytosis, raising concerns about the possible degradation of the cargo in the endolysosomal system. As the tracking of a dye-labelled protein during cellular uptake and processing is not indicative of the presence of the protein itself but only for the fluorescent label, a label-free tracking was performed with the red-fluorescing model protein R-phycoerythrin (R-PE). Four different eukaryotic cell lines were investigated: HeLa, HEK293T, MG-63, and MC3T3. Alone, the protein was not taken up by any cell line; only with the help of calcium phosphate nanoparticles, an efficient uptake occurred. After the uptake into HeLa cells, the protein was found in early endosomes (shown by the marker EEA1) and lysosomes (shown by the marker Lamp1). There, it was still intact and functional (i.e. properly folded) as its red fluorescence was detected. However, a few hours after the uptake, proteolysis started as indicated by the decreasing red fluorescence intensity in the case of HeLa and MC3T3 cells. 12 h after the uptake, the protein was almost completely degraded in HeLa cells and MC3T3 cells. In HEK293T cells and MG-63 cells, no degradation of the protein was observed. In the presence of Bafilomycin A1, an inhibitor of acidification and protein degradation in lysosomes, the fluorescence of R-PE remained intact over the whole observation period in the four cell lines. These results indicate that despite an efficient nanoparticle-mediated uptake of proteins by cells, a rapid endolysosomal degradation may prevent the desired (e.g. therapeutic) effect of a protein inside a cell.

  15. Catabolism of Branched Chain Amino Acids Contributes Significantly to Synthesis of Odd-Chain and Even-Chain Fatty Acids in 3T3-L1 Adipocytes.

    Directory of Open Access Journals (Sweden)

    Scott B Crown

    Full Text Available The branched chain amino acids (BCAA valine, leucine and isoleucine have been implicated in a number of diseases including obesity, insulin resistance, and type 2 diabetes mellitus, although the mechanisms are still poorly understood. Adipose tissue plays an important role in BCAA homeostasis by actively metabolizing circulating BCAA. In this work, we have investigated the link between BCAA catabolism and fatty acid synthesis in 3T3-L1 adipocytes using parallel 13C-labeling experiments, mass spectrometry and model-based isotopomer data analysis. Specifically, we performed parallel labeling experiments with four fully 13C-labeled tracers, [U-13C]valine, [U-13C]leucine, [U-13C]isoleucine and [U-13C]glutamine. We measured mass isotopomer distributions of fatty acids and intracellular metabolites by GC-MS and analyzed the data using the isotopomer spectral analysis (ISA framework. We demonstrate that 3T3-L1 adipocytes accumulate significant amounts of even chain length (C14:0, C16:0 and C18:0 and odd chain length (C15:0 and C17:0 fatty acids under standard cell culture conditions. Using a novel GC-MS method, we demonstrate that propionyl-CoA acts as the primer on fatty acid synthase for the production of odd chain fatty acids. BCAA contributed significantly to the production of all fatty acids. Leucine and isoleucine contributed at least 25% to lipogenic acetyl-CoA pool, and valine and isoleucine contributed 100% to lipogenic propionyl-CoA pool. Our results further suggest that low activity of methylmalonyl-CoA mutase and mass action kinetics of propionyl-CoA on fatty acid synthase result in high rates of odd chain fatty acid synthesis in 3T3-L1 cells. Overall, this work provides important new insights into the connection between BCAA catabolism and fatty acid synthesis in adipocytes and underscores the high capacity of adipocytes for metabolizing BCAA.

  16. ClC-3 Promotes Osteogenic Differentiation in MC3T3-E1 Cell After Dynamic Compression.

    Science.gov (United States)

    Wang, Dawei; Wang, Hao; Gao, Feng; Wang, Kun; Dong, Fusheng

    2017-06-01

    ClC-3 chloride channel has been proved to have a relationship with the expression of osteogenic markers during osteogenesis, persistent static compression can upregulate the expression of ClC-3 and regulate osteodifferentiation in osteoblasts. However, there was no study about the relationship between the expression of ClC-3 and osteodifferentiation after dynamic compression. In this study, we applied dynamic compression on MC3T3-E1 cells to detect the expression of ClC-3, runt-related transcription factor 2 (Runx2), bone morphogenic protein-2 (BMP-2), osteopontin (OPN), nuclear-associated antigen Ki67 (Ki67), and proliferating cell nuclear antigen (PCNA) in biopress system, then we investigated the expression of these genes after dynamic compression with Chlorotoxin (specific ClC-3 chloride channel inhibitor) added. Under transmission electron microscopy, there were more cell surface protrusions, rough surfaced endoplasmic reticulum, mitochondria, Golgi apparatus, abundant glycogen, and lysosomes scattered in the cytoplasm in MC3T3-E1 cells after dynamic compression. The nucleolus was more obvious. We found that ClC-3 was significantly up-regulated after dynamic compression. The compressive force also up-regulated Runx2, BMP-2, and OPN after dynamic compression for 2, 4 and 8 h. The proliferation gene Ki67 and PCNA did not show significantly change after dynamic compression for 8 h. Chlorotoxin did not change the expression of ClC-3 but reduced the expression of Runx2, BMP-2, and OPN after dynamic compression compared with the group without Cltx added. The data from the current study suggested that ClC-3 may promotes osteogenic differentiation in MC3T3-E1 cell after dynamic compression. J. Cell. Biochem. 118: 1606-1613, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  17. Key steps in type III secretion system (T3SS) towards translocon assembly with potential sensor at plant plasma membrane.

    Science.gov (United States)

    Ji, Hongtao; Dong, Hansong

    2015-09-01

    Many plant- and animal-pathogenic Gram-negative bacteria employ the type III secretion system (T3SS) to translocate effector proteins from bacterial cells into the cytosol of eukaryotic host cells. The effector translocation occurs through an integral component of T3SS, the channel-like translocon, assembled by hydrophilic and hydrophobic proteinaceous translocators in a two-step process. In the first, hydrophilic translocators localize to the tip of a proteinaceous needle in animal pathogens, or a proteinaceous pilus in plant pathogens, and associate with hydrophobic translocators, which insert into host plasma membranes in the second step. However, the pilus needs to penetrate plant cell walls in advance. All hydrophilic translocators so far identified in plant pathogens are characteristic of harpins: T3SS accessory proteins containing a unitary hydrophilic domain or an additional enzymatic domain. Two-domain harpins carrying a pectate lyase domain potentially target plant cell walls and facilitate the penetration of the pectin-rich middle lamella by the bacterial pilus. One-domain harpins target plant plasma membranes and may play a crucial role in translocon assembly, which may also involve contrapuntal associations of hydrophobic translocators. In all cases, sensory components in the target plasma membrane are indispensable for the membrane recognition of translocators and the functionality of the translocon. The conjectural sensors point to membrane lipids and proteins, and a phosphatidic acid and an aquaporin are able to interact with selected harpin-type translocators. Interactions between translocators and their sensors at the target plasma membrane are assumed to be critical for translocon assembly. © 2014 BSPP AND JOHN WILEY & SONS LTD.

  18. Long-term outcome of hand-assisted laparoscopic nephroureterectomy for pathologic T3 upper urinary tract urothelial carcinoma.

    Science.gov (United States)

    Chung, Shiu-Dong; Chen, Shyh-Chyan; Wang, Shuo-Meng; Chueh, Shih-Chieh; Lai, Ming-Kuen; Huang, Chao-Yuan; Pu, Yeong-Shiau; Huang, Kuo-How; Yu, Hong-Jeng

    2009-01-01

    To determine the feasibility and long-term outcomes of hand-assisted laparoscopic nephroureterectomy (HALNU) compared with open nephroureterectomy (ONU) in the management of pT(3)N0 upper urinary tract urothelial carcinoma (UUT-UC). Between January 1994 and December 2005, 21 patients who underwent HALNU for stage pT(3)N0 UTT-UC were matched and compared with 31 patients who underwent ONU. The oncologic out-comes, including bladder recurrence, recurrence-free survival, cancer-specific survival, and overall survival, were statistically analyzed. The median follow-up period in the HALNU group was 72 months (range 33-111 months) and 115 months in the ONU group (range 24-161 months). Patient age, sex, body mass index, tumor size, specimen weight, and American Society of Anesthesiologists classification showed no significant difference between the two groups. The HALNU group had statistically less blood loss than the ONU group (113 mL versus 487 mL; P = 0.02). The average hospital stay and doses of narcotic analgesics were significantly less in the HALNU group than the ONU group. The complication and bladder recurrence rates were similar between the two groups. The 5-year recurrence-free survival, cancer-specific survival, and overall survival were also comparable in both groups. HALNU is a safe and efficacious procedure with comparable long-term oncologic outcomes in comparison with ONU in treating patients with locally advanced pT(3)N0UUT-UC.

  19. Potential role of marine algae extract on 3T3-L1 cell proliferation and differentiation: an in vitro approach

    Directory of Open Access Journals (Sweden)

    Soundharrajan Ilavenil

    Full Text Available BACKGROUND: From ancient times, marine algae have emerged as alternative medicine and foods, contains the rich source of natural products like proteins, vitamins, and secondary metabolites, especially Chlorella vulgaris (C. vulgaris contains numerous anti-inflammatory, antioxidants and wound healing substances. Type 2 diabetes mellitus is closely associated with adipogenesis and their factors. Hence, we aimed to investigate the chemical constituents and adipo-genic modulatory properties of C. vulgaris in 3T3-L1 pre-adipocytes. RESULTS: We analysed chemical constituents in ethanolic extract of C. vulgaris (EECV by LC-MS. Results revealed that the EECV contains few triterpenoids and saponin compounds. Further, the effect of EECV on lipid accumulation along with genes and proteins expressions which are associated with adipogenesis and lipogenesis were evaluated using oil red O staining, qPCR and western blot techniques. The data indicated that that EECV treatment increased differentiation and lipid accumulation in 3T3-L1 cells, which indicates positive regulation of adipogenic and lipogenic activity. These increases were associated with up-regulation of PPAR-γ2, C/EBP-α, adiponectin, FAS, and leptin mRNA and protein expressions. Also, EECV treatments increased the concentration of glycerol releases as compared with control cells. Troglitazone is a PPAR-γ agonist that stimulates the PPAR-y2, adiponectin, and GLUT-4 expressions. Similarly, EECV treatments significantly upregulated PPAR-γ2, adiponectin, GLUT-4 expressions and glucose utilization. Further, EECV treatment decreased AMPK-α expression as compared with control and metformin treated cells. CONCLUSION: The present research findings confirmed that the EECV effectively modulates the lipid accumulation and differentiation in 3T3-L1 cells through AMPK-α mediated signalling pathway.

  20. Could a wait and see policy be justified in T3/4 rectal cancers after chemo-radiotherapy?

    International Nuclear Information System (INIS)

    Hughes, Robert; Harrison, Mark; Glynne-Jones, Robert

    2010-01-01

    Chemoradiotherapy (CRT) followed by total mesorectal excision is the standard when MRI staging demonstrates threatened surgical margins in locally advanced rectal cancer (LARC). Interest in non-surgical management of LARC as an alternative to a resection has been provoked by published excellent long-term outcomes of patients who achieve clinical complete responses (cCR) after CRT. The present retrospective study aimed to determine whether similar rates of local disease control are seen in a UK cancer centre in patients with T3-4 tumours, who obtained a cCR after preoperative CRT, but did not undergo surgery. Method. The outcome and treatment details of 266 patients who underwent CRT for clinically staged T3-4 rectal adenocarcinomas between 1993 and 2005 were reviewed. Results. Fifty-eight patients did not proceed to surgery, 10 of whom were identified as having a cCR. Six of these 10 patients subsequently developed intrapelvic recurrent disease with a median time to local progression of 20 months. Local relapse preceded the development of metastatic disease or occurred simultaneously. No patients underwent salvage resection. Conclusion. CRT alone in cT3/T4 rectal cancers has a high rate of local relapse even after cCR. Delaying or avoiding surgery might be appropriate for cT1 or cT2 tumours, or elderly and frail patients with co-morbidity, but these results do not support the current uncritical move to extrapolate this approach to all surgically fit patients with rectal cancer

  1. Optimal response of key enzymes and uncoupling protein to cold in BAT depends on local T3 generation

    International Nuclear Information System (INIS)

    Bianco, A.C.; Silva, J.E.

    1987-01-01

    The authors have examined the activity of three lipogenic enzymes [malic enzyme (ME), glucose-6-phosphate dehydrogenase (G-6-PD), and acetyl coenzyme A (CoA) carboxylase], the activity of the mitochondrial FAD-dependent α-glycerolphosphate dehydrogenase (α-GPD), and the mitochondrial concentration of uncoupling protein (UCP) in brown adipose tissue (BAT) of euthyroid and hypothyroid rats, both at room temperature and in response to acute cold stress. These enzymes and UCP are important for the thermogenic response of BAT in adaptation to cold. The basal level of the lipogenic enzymes was normal or slightly elevated in hypothyroid rats maintained at 23 0 C, but the levels of α-GPD and UCP were markedly reduced. Forty-eight hours at 4 0 C resulted in an increase in the activity of G-6-PD, acetyl-CoA carboxylase, and α-GPD and in the concentration of UCP both in euthyroid and hypothyroid animals, but the levels reached were invariably less in hypothyroid animals, indicating that thyroid hormone is necessary for a full metabolic response of BAT under maximal demands. Of all variables measured, the most affected was UCP followed by α-GDP. Dose-response relationship analysis of the UCP response to T 3 indicated that the normalization of the response to cold requires saturation of the nuclear T 3 receptors. They concluded, therefore, that the activation of the BAT 5'-deiodinase induced by cold exposure is essential to provide the high levels of nuclear T 3 required for the full expression of BAT thermogenic potential

  2. Overexpression of the mitochondrial T3 receptor p43 induces a shift in skeletal muscle fiber types.

    Directory of Open Access Journals (Sweden)

    François Casas

    Full Text Available In previous studies, we have characterized a new hormonal pathway involving a mitochondrial T3 receptor (p43 acting as a mitochondrial transcription factor and consequently stimulating mitochondrial activity and mitochondrial biogenesis. We have established the involvement of this T3 pathway in the regulation of in vitro myoblast differentiation. We have generated mice overexpressing p43 under control of the human alpha-skeletal actin promoter. In agreement with the previous characterization of this promoter, northern-blot and western-blot experiments confirmed that after birth p43 was specifically overexpressed in skeletal muscle. As expected from in vitro studies, in 2-month old mice, p43 overexpression increased mitochondrial genes expression and mitochondrial biogenesis as attested by the increase of mitochondrial mass and mt-DNA copy number. In addition, transgenic mice had a body temperature 0.8 degrees C higher than control ones and displayed lower plasma triiodothyronine levels. Skeletal muscles of transgenic mice were redder than wild-type animals suggesting an increased oxidative metabolism. In line with this observation, in gastrocnemius, we recorded a strong increase in cytochrome oxidase activity and in mitochondrial respiration. Moreover, we observed that p43 drives the formation of oxidative fibers: in soleus muscle, where MyHC IIa fibers were partly replaced by type I fibers; in gastrocnemius muscle, we found an increase in MyHC IIa and IIx expression associated with a reduction in the number of glycolytic fibers type IIb. In addition, we found that PGC-1alpha and PPARdelta, two major regulators of muscle phenotype were up regulated in p43 transgenic mice suggesting that these proteins could be downstream targets of mitochondrial activity. These data indicate that the direct mitochondrial T3 pathway is deeply involved in the acquisition of contractile and metabolic features of muscle fibers in particular by regulating PGC-1alpha

  3. Compressive force induces osteoclast differentiation via prostaglandin E(2) production in MC3T3-E1 cells.

    Science.gov (United States)

    Sanuki, Rina; Shionome, Chieko; Kuwabara, Akiko; Mitsui, Narihiro; Koyama, Yuki; Suzuki, Naoto; Zhang, Fan; Shimizu, Noriyoshi; Maeno, Masao

    2010-04-01

    In orthodontic tooth movement, prostaglandin E(2) (PGE(2)) released from osteoblasts can alter the normal process of bone remodeling. We previously showed that compressive force (CF) controls bone formation by stimulating the production of PGE(2) and Ep2 and/or Ep4 receptors in osteoblasts. The present study was undertaken to examine the effect of CF on the production of PGE(2), cyclooxygenase-2 (COX-2), macrophage colony-stimulating factor (M-CSF), receptor activator of NF-kappaB ligand (RANKL), and osteoprotegerin (OPG) using osteoblastic MC3T3-E1 cells and to examine the indirect effect of CF on osteoclast differentiation using RAW264.7 cells as osteoclast precursors. MC3T3-E1 cells were cultured with or without continuous CF (1.0 or 3.0 g/cm(2)) for 24 hr, and PGE(2) production was determined using ELISA. The expression of COX-2, M-CSF, RANKL, and OPG genes and proteins was determined using real-time PCR and ELISA, respectively. Osteoclast differentiation was estimated using tartrate-resistant acid phosphatase (TRAP) staining of RAW 264.7 cells cultured for 10 days with conditioned medium from CF-treated MC3T3-E1 cells and soluble RANKL. As CF increased, PGE(2) production and the expression of COX-2, M-CSF, and RANKL increased, whereas OPG expression decreased. The number of TRAP-positive cells increased as CF increased. Celecoxib, a specific inhibitor of COX-2, blocked the stimulatory effect of CF on TRAP staining and the production of PGE(2), M-CSF, RANKL, and OPG. These results suggest that CF induces osteoclast differentiation by increasing M-CSF production and decreasing OPG production via PGE(2) in osteoblasts.

  4. Systematic review on T3 laryngeal squamous cell carcinoma; still far from a consensus on the optimal organ preserving treatment.

    Science.gov (United States)

    Riga, M; Chelis, L; Danielides, V; Vogiatzaki, T; Pantazis, T-L; Pantazis, D

    2017-01-01

    The optimal treatment of patients with T3 laryngeal carcinoma is controversially challenged by open partial laryngectomies (OPL), transoral laser microsurgery (TLM) and radiation therapy alone (RT) or combined with chemotherapy (ChRT). Treatment guidelines, experts' opinions and clinical studies are highly contradictory. The aim of this study is to compare the primary outcomes of the available treatment methods and identify the sources of variance among studies. A review of the literature published in the time period 2003-2015 was conducted via the PubMed database (www.pubmed.org) and Scopus database (www.scopus.com) with the search terms "T3 laryngeal squamous cell cancer treatment". Data from clinical studies involving patients with T3 laryngeal cancer (n > 10) subjected to TLM, OPL, ChRT or RT, were pooled. In the absence of controlled studies, prospective and retrospective clinical trials with minimum 5-year follow-up were acceptable, provided that they included a description of patient eligibility criteria, so as to exclude studies with serious selection bias. Literature lacks studies with homogenous populations regarding TNM staging, preoperative/postoperative treatment or anatomical subsite. This raises substantial controversies and prohibits the conduction of a meta-analysis. Data for qualitative analysis were pooled from 8 studies (n = 1226). OPL and TLM both offer patients high survival and organ preservation rates. Preoperative induction chemotherapy seems to significantly compromise overall survival. Multicenter studies referring to homogenous populations, at least regarding staging and anatomical subsite, are needed. No safe conclusions can be drawn given the heterogeneity in patient cohorts, study design and evaluation of results in the existing literature. Copyright © 2016 Elsevier Ltd and British Association of Surgical Oncology/European Society of Surgical Oncology. All rights reserved.

  5. Role of imaging in the follow-up of T2-T3 glottic cancer treated by transoral laser microsurgery.

    Science.gov (United States)

    Marchi, Filippo; Piazza, Cesare; Ravanelli, Marco; Gaggero, Giovanna; Parrinello, Giampiero; Paderno, Alberto; Perotti, Pietro; Filauro, Marta; Maroldi, Roberto; Peretti, Giorgio

    2017-10-01

    An unblinded retrospective analysis of prospectively collected data was carried out on 138 patients affected by glottic pT2 and selected pT3 squamous cell carcinomas (SCC) treated by transoral laser microsurgery (TLM). The entire cohort was divided into two groups: Group A included 78 "high-risk" patients (pT2 with impaired vocal cord mobility, pT3 for anterior paraglottic and/or pre-epiglottic space invasion, presence of angioembolization, perineural spread, and positive lymph nodes in the neck) who underwent postoperative surveillance by endoscopy and imaging (CT or MR), while Group B included 60 "low-risk" patients (pT2 with absence of the above-mentioned features) who underwent endoscopic follow-up alone. Aim of the present study was to assess the diagnostic gain in performing combined endoscopic and radiologic surveillance in the "high-risk" group compared to endoscopy alone in the "low-risk" one. There was no significant difference in terms of overall and disease-specific survivals between the two follow-up policies in spite of their different risk profiles. The same was true for organ preservation rate, which was 81% in the "high-risk" and 89% in the "low-risk" group. In contrast, the cumulative probability of disease-free survival was 54% for Group A and 65% for Group B (p = 0.0023). Therefore, our combined endoscopy and imaging surveillance protocol allowed increasing the submucosal recurrence detection rate in "high-risk" pT2-pT3 glottic SCC to 43%. An earlier detection of submucosal recurrences made salvage surgery by TLM feasible in at least half of cases, thus closing the gap between oncologic outcomes obtained in "high-"- vs. "low-risk" patients and leading to organ preservation rates that are favorably comparable to those obtained with open-neck partial laryngectomies and non-surgical organ preservation protocols.

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

    Energy Technology Data Exchange (ETDEWEB)

    He, Yonghan [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); Zhang, Shuocheng [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); 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); 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); 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)

    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 γ (PPAR{sub γ}) and CCAAT element binding protein α (C/EBP{sub α}), 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 β-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.

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

    International Nuclear Information System (INIS)

    He, Yonghan; Li, Ying; Zhang, Shuocheng; Perry, Ben; Zhao, Tiantian; Wang, Yanwen; Sun, Changhao

    2013-01-01

    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 γ (PPAR γ ) and CCAAT element binding protein α (C/EBP α ), 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 β-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

  8. High glucose impaired estrogen receptor alpha signaling via β-catenin in osteoblastic MC3T3-E1.

    Science.gov (United States)

    Wang, Rui; Gao, Dong; Zhou, Yin; Chen, Lu; Luo, Bin; Yu, Yanrong; Li, Hao; Hu, Jiawei; Huang, Qiren; He, Ming; Peng, Weijie; Luo, Dan

    2017-11-01

    Diabetic Mellitus is a risk factor for osteoporosis. It has been suggested that altered estrogen or estrogen receptor α/β (ERα/β) signaling may be involved in diabetic osteoporosis. The present study is to investigate the effects of high glucose on ERα/β signaling in osteoblastic MC3T3-E1 and how the altered signaling of ERα/β affect osteoblastic bone formation. ERα/β signaling was demonstrated as ERα/β protein expression (Western Blotting) and ER transcription activity (Luciferase Reporter assays). Proliferation (WSK-1 assaying), differentiation (ALP staining) and mineralization (Alizalard Red staining) of MC3T3-E1 were examined to evaluate bone formation function. It has been found that high glucose increased ERα/β expression dose-dependently and time-dependently, but high glucose (33mM) decreased ERα transcription activity. 17β-estradiol increased the ERα/β expression dose-dependently in normal medium, but decreased the ERα/β expression dose-dependently in medium with high glucose (33mM). High glucose decreased bone formation and also decreased the osteogenic effects of 17β-estradiol (10 -8 M). High glucose decreased β-catenin expression dose-dependently and time-dependently. LiCl, an inhibitor of β-catenin degradation, decreased ERα expression but increased ERα transcription activity. When compared with high glucose treatment, LiCl (5mM) increased ALP activity and calcified nodes. Besides, high glucose also decreased the protein expression PI-3K, pAKT/AKT, GSK-3β. In conclusion, the present study suggested that high glucose may impair ERα transcription activity by inhibiting β-catenin signaling in osteoblastic MC3T3-E1, leading decreased bone formation ligand-dependently or ligand-independently. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. High-level expression of human insulin receptor cDNA in mouse NIH 3T3 cells

    Energy Technology Data Exchange (ETDEWEB)

    Whittaker, J.; Okamoto, A.K.; Thys, R.; Bell, G.I.; Steiner, D.F.; Hofmann, C.A.

    1987-08-01

    In order to develop a simple, efficient system for the high-level expression of human insulin receptors in eukaryotic cells, a full-length human kidney insulin receptor cDNA was inserted into a bovine papilloma virus vector under the control of the mouse metallothionein promoter. After transfection of mouse NIH 3T3 cells with this construct, seven cell lines expressing insulin receptors were isolated; two cell lines had more than 10/sup 6/ receptors per cell. The cell line with the highest /sup 125/I-insulin binding (NIH 3T3 HIR3.5) had 6 x 10/sup 6/ receptors with a K/sub d/ of 10/sup -9/ M. This level was not dependent on exposure to metals but could be increased further to 2 x 10/sup 7/ receptors per cell by addition of sodium butyrate to the culture medium. The ..cap alpha.. and ..beta.. subunits had apparent molecular weights of 147,000 and 105,000, respectively (compared to 135,000 and 95,000 in IM-9 human lymphocytes), values identical to those of the ..cap alpha.. and ..beta.. subunits of the insulin receptors of nontransformed NIH 3T3 cells. This size difference was due to altered carbohydrate composition, as N-glycanase digestion reduced the apparent receptor subunit size of the transfected cells and IM-9 lymphocytes to identical values. The alteration in N-linked oligosaccharide composition could not be ascribed to differences in the kinetics of posttranslational processing of the insulin receptors, which was comparable to that of other cells studied. The basal rate of glycogen synthesis in the cells overexpressing insulin receptors was increased 4- to 5-fold compared with controls. Low levels of added insulin (0.1 nM) caused a 50% increase in the rate of glycogen synthesis

  10. High-level expression of human insulin receptor cDNA in mouse NIH 3T3 cells

    International Nuclear Information System (INIS)

    Whittaker, J.; Okamoto, A.K.; Thys, R.; Bell, G.I.; Steiner, D.F.; Hofmann, C.A.

    1987-01-01

    In order to develop a simple, efficient system for the high-level expression of human insulin receptors in eukaryotic cells, a full-length human kidney insulin receptor cDNA was inserted into a bovine papilloma virus vector under the control of the mouse metallothionein promoter. After transfection of mouse NIH 3T3 cells with this construct, seven cell lines expressing insulin receptors were isolated; two cell lines had more than 10 6 receptors per cell. The cell line with the highest 125 I-insulin binding (NIH 3T3 HIR3.5) had 6 x 10 6 receptors with a K/sub d/ of 10 -9 M. This level was not dependent on exposure to metals but could be increased further to 2 x 10 7 receptors per cell by addition of sodium butyrate to the culture medium. The α and β subunits had apparent molecular weights of 147,000 and 105,000, respectively (compared to 135,000 and 95,000 in IM-9 human lymphocytes), values identical to those of the α and β subunits of the insulin receptors of nontransformed NIH 3T3 cells. This size difference was due to altered carbohydrate composition, as N-glycanase digestion reduced the apparent receptor subunit size of the transfected cells and IM-9 lymphocytes to identical values. The alteration in N-linked oligosaccharide composition could not be ascribed to differences in the kinetics of posttranslational processing of the insulin receptors, which was comparable to that of other cells studied. The basal rate of glycogen synthesis in the cells overexpressing insulin receptors was increased 4- to 5-fold compared with controls. Low levels of added insulin (0.1 nM) caused a 50% increase in the rate of glycogen synthesis

  11. α-Tocopherol protected against cobalt nanoparticles and cocl2induced cytotoxicity and inflammation in Balb/3T3 cells.

    Science.gov (United States)

    Yan, Xin; Liu, Yake; Xie, Tian; Liu, Fan

    2018-04-01

    Currently, tissue damage induced by cobalt nanoparticles (CoNPs) and cobalt ions (Co 2+ ) are the most serious adverse effect in the patients with metal-on-metal hip prostheses. Therefore, an urgent need exists for the identification of the mechanisms and the development of therapeutic strategies to limit it. We aimed to explore the mechanisms of cytotoxicity of CoNPs and Co 2+ and developed strategies to reduce this cytotoxicity with α-tocopherol treatment. To evaluate the protective effect of α-tocopherol, Balb/3T3 cells were pretreated with 10 μM α-tocopherol for 24 h. The cells were then exposed to different concentrations of CoNPs and Co 2+ for 12 h, 24 h and 48 h. The cell viabilities, reactive oxygen species (ROS), inflammatory cytokines and MAP kinase (MAPK) levels were measured. CoNPs and Co 2+ can induce the increase of ROS and inflammatory cytokines in Balb/3T3 cells, such as tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6). However, α-tocopherol pretreatment can significantly prevent cytotoxicity induced by CoNPs and Co 2+ , decrease ROS production and decrease levels of inflammatory cytokines in Balb/3T3 cells. Additionally, MAPK pathway may be involved in the protection of α-tocopherol against cytotoxicity induced by CoNPs and Co 2+ in vitro. Our results provide new insights into the potential therapeutic use of α-tocopherol in the prevention and treatment of various oxidative- or inflammatory stress-related inflammation and injuries.

  12. T3 Regulates a Human Macrophage-Derived TSH-β Splice Variant: Implications for Human Bone Biology.

    Science.gov (United States)

    Baliram, R; Latif, R; Morshed, S A; Zaidi, M; Davies, T F

    2016-09-01

    TSH and thyroid hormones (T3 and T4) are intimately involved in bone biology. We have previously reported the presence of a murine TSH-β splice variant (TSH-βv) expressed specifically in bone marrow-derived macrophages and that exerted an osteoprotective effect by inducing osteoblastogenesis. To extend this observation and its relevance to human bone biology, we set out to identify and characterize a TSH-β variant in human macrophages. Real-time PCR analyses using human TSH-β-specific primers identified a 364-bp product in macrophages, bone marrow, and peripheral blood mononuclear cells that was sequence verified and was homologous to a human TSH-βv previously reported. We then examined TSH-βv regulation using the THP-1 human monocyte cell line matured into macrophages. After 4 days, 46.1% of the THP-1 cells expressed the macrophage markers CD-14 and macrophage colony-stimulating factor and exhibited typical morphological characteristics of macrophages. Real-time PCR analyses of these cells treated in a dose-dependent manner with T3 showed a 14-fold induction of human TSH-βv mRNA and variant protein. Furthermore, these human TSH-βv-positive cells, induced by T3 exposure, had categorized into both M1 and M2 macrophage phenotypes as evidenced by the expression of macrophage colony-stimulating factor for M1 and CCL-22 for M2. These data indicate that in hyperthyroidism, bone marrow resident macrophages have the potential to exert enhanced osteoprotective effects by oversecreting human TSH-βv, which may exert its local osteoprotective role via osteoblast and osteoclast TSH receptors.

  13. Isolation of reovirus T3D mutants capable of infecting human tumor cells independent of junction adhesion molecule-A.

    Directory of Open Access Journals (Sweden)

    Diana J M van den Wollenberg

    Full Text Available Mammalian Reovirus is a double-stranded RNA virus with a distinctive preference to replicate in and lyse transformed cells. On that account, Reovirus type 3 Dearing (T3D is clinically evaluated as oncolytic agent. The therapeutic efficacy of this approach depends in part on the accessibility of the reovirus receptor Junction Adhesion Molecule-A (JAM-A on the target cells. Here, we describe the isolation and characterization of reovirus T3D mutants that can infect human tumor cells independent of JAM-A. The JAM-A-independent (jin mutants were isolated on human U118MG glioblastoma cells, which do not express JAM-A. All jin mutants harbour mutations in the S1 segments close to the region that encodes the sialic acid-binding pocket in the shaft of the spike protein. In addition, two of the jin mutants encode spike proteins with a Q336R substitution in their head domain. The jin mutants can productively infect a wide range of cell lines that resist wt reovirus T3D infection, including chicken LMH cells, hamster CHO cells, murine endothelioma cells, human U2OS and STA-ET2.1 cells, but not primary human fibroblasts. The jin-mutants rely on the presence of sialic-acid residues on the cell surface for productive infection, as is evident from wheat germ agglutinin (WGA inhibition experiments, and from the jin-reovirus resistance of CHO-Lec2 cells, which have a deficiency of sialic-acids on their glycoproteins. The jin mutants may be useful as oncolytic agents for use in tumors in which JAM-A is absent or inaccessible.

  14. Systemic translocation of Salmonella enterica serovar Dublin in cattle occurs predominantly via efferent lymphatics in a cell-free niche and requires type III secretion system 1 (T3SS-1) but not T3SS-2.

    Science.gov (United States)

    Pullinger, Gillian D; Paulin, Susan M; Charleston, Bryan; Watson, Patricia R; Bowen, Alison J; Dziva, Francis; Morgan, Eirwen; Villarreal-Ramos, Bernardo; Wallis, Timothy S; Stevens, Mark P

    2007-11-01

    Salmonella enterica is an important diarrheal pathogen, and infections may involve severe systemic sequelae depending on serovar- and host-specific factors. The molecular mechanisms underlying translocation of host-restricted and -specific serovars of S. enterica from the intestines to distal organs are ill defined. By surgical cannulation of lymph and blood vessels draining the distal ileum in cattle, S. enterica serovar Dublin was observed to translocate predominantly via mesenteric lymph nodes to efferent lymphatics in a manner that correlates with systemic virulence, since the fowl typhoid-associated serovar Gallinarum translocated at a significantly lower level. While both S. enterica serovars Dublin and Gallinarum were intracellular while in the intestinal mucosa and associated with major histocompatibility complex class II-positive cells, the bacteria were predominantly extracellular within efferent lymph. Screening of a library of signature-tagged serovar Dublin mutants following oral inoculation of calves defined the role of 36 virulence-associated loci in enteric and systemic phases of infection. The number and proportion of tagged clones reaching the liver and spleen early after oral infection were identical to the values in efferent lymph, implying that this may be a relevant mode of dissemination. Coinfection studies confirmed that lymphatic translocation requires the function of type III secretion system 1 (T3SS-1) but, remarkably, not T3SS-2. This is the first description of the mode and genetics of systemic translocation of serovar Dublin in its natural host.

  15. Trip report on IAEA Training Workshop on Implementation of Integrated Management Systems for Research Reactors (T3-TR-45496).

    Energy Technology Data Exchange (ETDEWEB)

    Pratt, Richard J.

    2013-11-01

    From 17-21 June 2013, Sandia National Laboratories, Technical Area-V (SNL TA-V) represented the United States Department of Energy/National Nuclear Security Administration (DOE/NNSA) at the International Atomic Energy Agency (IAEA) Training Workshop (T3-TR-45486). This report gives a breakdown of the IAEA regulatory structure for those unfamiliar, and the lessons learned and observations that apply to SNL TA-V that were obtained from the workshop. The Safety Report Series, IAEA workshop final report, and SNL TA-V presentation are included as attachments.

  16. Andrographolide inhibits adipogenesis of 3T3-L1 cells by suppressing C/EBPβ expression and activation

    International Nuclear Information System (INIS)

    Chen, Ching-Chu; Chuang, Wei-Ting; Lin, Ai-Hsuan; Tsai, Chia-Wen; Huang, Chin-Shiu; Chen, Yun-Ting; Chen, Haw-Wen; Lii, Chong-Kuei

    2016-01-01

    Andrographolide, a diterpenoid, is the most abundant terpenoid in Andrographis paniculata, a popular Chinese herbal medicine. Andrographolide displays diverse biological activities including hypoglycemia, hypolipidemia, anti-inflammation, and anti-tumorigenesis. Recent evidence indicates that andrographolide displays anti-obesity property by inhibiting lipogenic gene expression, however, the underlying mechanisms remain to be elucidated. In this study, the effects of andrographolide on transcription factor cascade and mitotic clonal expansion in 3T3-L1 preadipocyte differentiation into adipocyte were determined. Andrographolide dose-dependently (0–15 μM) inhibited CCAAT/enhancer-binding protein α (C/EBPα) and C/EBPβ mRNA and protein expression as well as peroxisome proliferator-activated receptor γ (PPARγ) protein level during the adipogenesis of 3T3-L1 cells. Concomitantly, fatty acid synthase and stearoyl-CoA desaturase expression and lipid accumulation were attenuated by andrographolide. Oil-red O staining further showed that the first 48 h after the initiation of differentiation was critical for andrographolide inhibition of adipocyte formation. Andrographolide inhibited the phosphorylation of PKA and the activation of cAMP response element-binding protein (CREB) in response to a differentiation cocktail, which led to attenuated C/EBPβ expression. In addition, ERK and GSK3β-dependent C/EBPβ phosphorylation was attenuated by andrographolide. Moreover, andrographolide suppressed cyclin A, cyclin E, and CDK2 expression and impaired the progression of mitotic clonal expansion (MCE) by arresting the cell cycle at the Go/G1 phase. Taken together, these results indicate that andrographolide has a potent anti-obesity action by inhibiting PKA-CREB-mediated C/EBPβ expression as well as C/EBPβ transcriptional activity, which halts MCE progression and attenuates C/EBPα and PPARγ expression. - Highlights: • Andrographolide is a diterpenoid phytochemical.

  17. t(3;11)(q12;p15)/NUP98-LOC348801 fusion transcript in acute myeloid leukemia.

    Science.gov (United States)

    Gorello, Paolo; Brandimarte, Lucia; La Starza, Roberta; Pierini, Valentina; Bury, Loredana; Rosati, Roberto; Martelli, Massimo F; Vandenberghe, Peter; Wlodarska, Iwona; Mecucci, Cristina

    2008-09-01

    In a case of acute myeloid leukemia we report molecular cytogenetic findings of a t(3;11)(q12;p15), characterized as a new NUP98 translocation rearranging with LOC348801 at chromosome 3. NUP98 involvement was detected by fluorescence in situ hybridization. 3'-RACE-PCR showed nucleotide 1718 (exon 13) of NUP98 was fused in-frame with nucleotide 1248 (exon 2) of LOC348801. RT-PCR and cloning experiments detected two in-frame spliced NUP98-LOC348801 transcripts and the reciprocal LOC348801-NUP98. A highly specific double-color double-fusion FISH assay reliably detects NUP98-LOC348801.

  18. Adjuvant radiotherapy for pathologic stage T3/4 adenocarcinoma of the prostate: Ten-year update

    International Nuclear Information System (INIS)

    Anscher, Mitchell S.; Robertson, Cary N.; Prosnitz, Leonard R.

    1995-01-01

    Purpose: To determine the role of adjuvant postoperative radiotherapy (RT) following radical prostatectomy (RP) in a group of patients with pathologic Stage T3/4 adenocarcinoma of the prostate followed for a median of 10 years after treatment. Methods and Materials: Between 1970 and 1983, 159 patients underwent RP for newly diagnosed adenocarcinoma of the prostate and were found to have pathologic Stage T3/4 tumors. Forty-six received adjuvant RT and 113 did not. Radiotherapy usually consisted of 45-50 Gy to the whole pelvis followed by a boost to the prostate bed of 10-15 Gy, to a total dose of 55-65 Gy. Patients were analyzed with respect to survival, disease-free survival, local control, and freedom from distant metastases. A rising prostate-specific antigen in the absence of other evidence of relapse was scored as a separate category of recurrence. Results: Both groups of patients have been followed for a median of 10 years. The actuarial survival at 10 and 15 years was 62% and 62% for the RT group compared to 52% and 37%, respectively, for the RP group (p = 0.18). The disease-free survival for the RT group was 55% and 48% at 10 and 15 years, respectively, compared to 37% and 33% for the RP group (p = 0.16). Similarly, there was no difference in the rate of distant metastases between the two groups. In contrast, the local relapse rate was significantly reduced by the addition of postoperative radiotherapy. The actuarial local control rate at 10 and 15 years was 92% and 82%, respectively, for the RT group vs. 60% and 53% for the RP group (p 0.002). Conclusions: While postoperative pelvic RT significantly improves local control compared to RP alone for pathologic Stage T3/4 prostate cancer, it has no impact on distant metastases and consequently does not improve survival. These data are consistent with the conclusion that many patients with pathologic Stage T3/4 prostate cancer have occult metastases at presentation and will not be cured by local therapies alone

  19. Preparation of quality control samples for its use in the radioimmunoassay de T3, T4 and TSH

    International Nuclear Information System (INIS)

    Lavalley E, C.; Delgado S, B.; Ruiz J, A.; Zambrano A, F.

    1991-09-01

    The use of quality control samples is necessary to evaluate, in a very simple way, the quality of the assays in the radioimmunoanalysis, since allows to settle down a quality control intra and inter analysis. In this work the methodology used for the preparation of these samples with low, media and high concentration for hormones related with the thyroid is shown, being obtained the following concentrations: 50, 200 and 500 ng/dl for T 3 ; 5.6, 7.8 and 14.4 μ g/dl for T 4 and 5.4, 13.4 and >50 μ U I/ml for TSH. (Author)

  20. Quantifying the roles of random motility and directed motility using advection-diffusion theory for a 3T3 fibroblast cell migration assay stimulated with an electric field.

    Science.gov (United States)

    Simpson, Matthew J; Lo, Kai-Yin; Sun, Yung-Shin

    2017-03-17

    Directed cell migration can be driven by a range of external stimuli, such as spatial gradients of: chemical signals (chemotaxis); adhesion sites (haptotaxis); or temperature (thermotaxis). Continuum models of cell migration typically include a diffusion term to capture the undirected component of cell motility and an advection term to capture the directed component of cell motility. However, there is no consensus in the literature about the form that the advection term takes. Some theoretical studies suggest that the advection term ought to include receptor saturation effects. However, others adopt a much simpler constant coefficient. One of the limitations of including receptor saturation effects is that it introduces several additional unknown parameters into the model. Therefore, a relevant research question is to investigate whether directed cell migration is best described by a simple constant tactic coefficient or a more complicated model incorporating saturation effects. We study directed cell migration using an experimental device in which the directed component of the cell motility is driven by a spatial gradient of electric potential, which is known as electrotaxis. The electric field (EF) is proportional to the spatial gradient of the electric potential. The spatial variation of electric potential across the experimental device varies in such a way that there are several subregions on the device in which the EF takes on different values that are approximately constant within those subregions. We use cell trajectory data to quantify the motion of 3T3 fibroblast cells at different locations on the device to examine how different values of the EF influences cell motility. The undirected (random) motility of the cells is quantified in terms of the cell diffusivity, D, and the directed motility is quantified in terms of a cell drift velocity, v. Estimates D and v are obtained under a range of four different EF conditions, which correspond to normal

  1. Study of the continuous spectrum of 6Li and the reaction t(3He,d)α by the algebraic version of the RGM

    International Nuclear Information System (INIS)

    Filippov, G.F.; Lopez Trujillo, A.; Rybkin, I.Yu.

    1992-01-01

    Using the algebraic version of the resonating group method, the continuous spectrum of 6 Li states with zero isospin is studied. The decay channels t+ 3 He and α+α are taken into account. The astrophysical S-factor of the t( 3 He,d)α reaction is calculated. 20 refs.; 6 figs.; 2 tab. (author)

  2. Effects of LPS and dietary free fatty acids on MCP-1 in 3T3-L1 adipocytes and macrophages in vitro

    DEFF Research Database (Denmark)

    Cullberg, K B; Larsen, Jens Ølholm; Pedersen, S B

    2014-01-01

    Effects of LPS and dietary free fatty acids on MCP-1 in 3T3-L1 adipocytes and macrophages in vitro......Effects of LPS and dietary free fatty acids on MCP-1 in 3T3-L1 adipocytes and macrophages in vitro...

  3. 18O-Tracer Metabolomics Reveals Protein Turnover and CDP-Choline Cycle Activity in Differentiating 3T3-L1 Pre-Adipocytes.

    Directory of Open Access Journals (Sweden)

    Jay S Kirkwood

    Full Text Available The differentiation of precursor cells into mature adipocytes (adipogenesis has been an area of increased focus, spurred by a rise in obesity rates. Though our understanding of adipogenesis and its regulation at the cellular level is growing, many questions remain, especially regarding the regulation of the metabolome. The 3T3-L1 cell line is the most well characterized cellular model of adipogenesis. Using a time course metabolomics approach, we show that the 3T3-L1 preadipocyte metabolome is greatly altered during the first 48 hours of differentiation, where cells go through about two rounds of cell division, a process known as mitotic clonal expansion. Short-chain peptides were among several small molecules that were increased during mitotic clonal expansion. Additional indicators of protein turnover were also increased, including bilirubin, a degradation product of heme-containing proteins, and 3-methylhistidine, a post-translationally modified amino acid that is not reutilized for protein synthesis. To study the origin of the peptides, we treated differentiating preadipocytes with 18O labeled water and found that 18O was incorporated into the short chain peptides, confirming them, at least in part, as products of hydrolysis. Inhibitors of the proteasome or matrix metalloproteinases affected the peptide levels during differentiation, but inhibitors of autophagy or peptidases did not. 18O was also incorporated into several choline metabolites including cytidine 5'-diphosphocholine (CDP-choline, glycerophosphocholine, and several phosphatidylcholine species, indicative of phosphatidylcholine synthesis/degradation and of flux through the CDP-choline cycle, a hallmark of proliferating cells. 18O-Tracer metabolomics further showed metabolic labeling of glutamate, suggestive of glutaminolysis, also characteristic of proliferating cells. Together, these results highlight the utility of 18O isotope labeling in combination with metabolomics to

  4. Dehydrodiconiferyl Alcohol Isolated from Cucurbita moschata Shows Anti-adipogenic and Anti-lipogenic Effects in 3T3-L1 Cells and Primary Mouse Embryonic Fibroblasts*

    Science.gov (United States)

    Lee, Junghun; Kim, Donghyun; Choi, Jonghyun; Choi, Hyounjeong; Ryu, Jae-Ha; Jeong, Jinhyun; Park, Eun-Jin; Kim, Seon-Hee; Kim, Sunyoung

    2012-01-01

    A water-soluble extract from the stems of Cucurbita moschata, code named PG105, was previously found to contain strong anti-obesity activities in a high fat diet-induced obesity mouse model. One of its biological characteristics is that it inhibits 3T3-L1 adipocyte differentiation. To isolate the biologically active compound(s), conventional solvent fractionation was performed, and the various fractions were tested for anti-adipogenic activity using Oil Red O staining method. A single spot on thin layer chromatography of the chloroform fraction showed a potent anti-adipogenic activity. When purified, the structure of its major component was resolved as dehydrodiconiferyl alcohol (DHCA), a lignan, by NMR and mass spectrometry analysis. In 3T3-L1 cells, synthesized DHCA significantly reduced the expression of several adipocyte marker genes, including peroxisome proliferator-activated receptor γ (Pparg), CCAAT/enhancer-binding protein α (Cebpa), fatty acid-binding protein 4 (Fabp4), sterol response element-binding protein-1c (Srebp1c), and stearoyl-coenzyme A desaturase-1 (Scd), and decreased lipid accumulation without affecting cell viability. DHCA also suppressed the mitotic clonal expansion of preadipocytes (an early event of adipogenesis), probably by suppressing the DNA binding activity of C/EBPβ, and lowered the production level of cyclinA and cyclin-dependent kinase 2 (Cdk2), coinciding with the decrease in DNA synthesis and cell division. In addition, DHCA directly inhibited the expression of SREBP-1c and SCD-1. Similar observations were made, using primary mouse embryonic fibroblasts. Taken together, our data indicate that DHCA may contain dual activities, affecting both adipogenesis and lipogenesis. PMID:22262865

  5. No role for tri-iodothyronine (T3) testing in the assessment of levothyroxine (T4) over-replacement in hypothyroid patients.

    Science.gov (United States)

    Livingston, M; Birch, K; Guy, M; Kane, J; Heald, A H

    2015-01-01

    Tri-iodothyronine (T3) is a sensitive marker of endogenous hyperthyroidism. In levothyroxine (T4)-induced hyperthyroidism, there is no reason for T3 to be elevated, but this test is often requested in over-treated hypothyroid patients. This study investigated how informative T3 levels are in these patients. Our hypothesis is that T3 measurement would not add anything to the assessment of T4 over-replacement in primary hypothyroidism. Over a 15-week period, consecutive thyroid function test requests in patients on levothyroxine had T3 levels measured if thyroid-stimulating hormone (TSH) was below the reference range (RR; 27 pmol/L) were defined as being over-replaced, while those with low, but measurable TSH and a normal free T4 were defined as unlikely to be over- replaced (control group). Receiver operating characteristic (ROC) curve analysis was used to assess the discriminant power of T3 to detect over-replacement. Of the 542 patients examined, 33 were included in the over-replaced group and 236 patients in the control group. A total of 273 patients were excluded for not fulfilling the criteria for either of these groups. In the over-replaced group, none had a raised T3. The most discriminant T3 level, using ROC curve analysis, was 1.6 nmol/L (RR=1.3-2.6 nmol/L), with a corresponding sensitivity and specificity of 58% and 71%, respectively (P=0.16). T3 levels bear little relation to thyroid status in patients on levothyroxine replacement, and normal levels can be seen in over-replaced patients. Measurement of T3 in this situation is of doubtful clinical value. WHAT'S ALREADY KNOWN ABOUT THIS TOPIC?: Thyroid function tests are the way that adequacy of levothyroxine replacement is determined. Where the test is available, T3 is often requested together with T4 and TSH by clinicians. The question is whether T3 measurement adds any further information. WHAT DOES THIS ARTICLE ADD?: The presented data supports the position that T3 measurement does not add anything to

  6. Alliin, a Garlic (Allium sativum Compound, Prevents LPS-Induced Inflammation in 3T3-L1 Adipocytes

    Directory of Open Access Journals (Sweden)

    Saray Quintero-Fabián

    2013-01-01

    Full Text Available Garlic (Allium sativum L. has been used to alleviate a variety of health problems due to its high content of organosulfur compounds and antioxidant activity. The main active component is alliin (S-allyl cysteine sulfoxide, a potent antioxidant with cardioprotective and neuroprotective actions. In addition, it helps to decrease serum levels of glucose, insulin, triglycerides, and uric acid, as well as insulin resistance, and reduces cytokine levels. However its potential anti-inflammatory effect is unknown. We examined the effects of alliin in lipopolysaccharide- (LPS- stimulated 3T3-L1 adipocytes by RT-PCR, Western blot, and microarrays analysis of 22,000 genes. Incubation of cells for 24 h with 100 μmol/L alliin prevented the increase in the expression of proinflammatory genes, IL-6, MCP-1, and Egr-1 in 3T3-L1 adipocytes exposed to 100 ng/mL LPS for 1 h. Interestingly, the phosphorylation of ERK1/2, which is involved in LPS-induced inflammation in adipocytes, was decreased following alliin treatment. Furthermore, the gene expression profile by microarrays evidentiate an upregulation of genes involved in immune response and downregulation of genes related with cancer. The present results have shown that alliin is able to suppress the LPS inflammatory signals by generating an anti-inflammatory gene expression profile and by modifying adipocyte metabolic profile.

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

    Directory of Open Access Journals (Sweden)

    Hung Yao-Ching

    2009-01-01

    Full Text Available Abstract 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.

  8. Effects of phytoestrogens and environmental estrogens on osteoblastic differentiation in MC3T3-E1 cells

    International Nuclear Information System (INIS)

    Kanno, Sanae; Hirano, Seishiro; Kayama, Fujio

    2004-01-01

    Phytoestrogens and environmental estrogens, which have in part some structural similarity to 17β-estradiol, are reported to act as agonists/antagonists of estrogen in animals and humans. Estrogen is known to play an important role in maintaining bone mass, since the concentration of serum estrogen decreases after menopause and the estrogen deficiency results in bone loss. In this study, we report the effects of phytoestrogens (genistein, daidzein, and coumestrol) and environmental estrogens (bisphenol A (BPA), p-n-nonylphenol (NP) and bis(2-ethylhexyl)phthalate (DEHP)) on osteoblast differentiation using MC3T3-E1 cells, a mouse calvaria osteoblast-like cell line. Coumestrol (10 -10 to 10 -6 M) slightly enhanced cell proliferation, while neither the other phytoestrogens (daidzein, genistein) nor environmental estrogens increased cell proliferation. Alkaline phosphatase (ALP) activity and cellular calcium (Ca) and phosphorus (P) contents were increased by phytoestrogens and BPA; however, neither NP nor DEHP affected those osteoblastic indicators. The effects of estrogenic potency, using the cell proliferation of MCF-7 cells, an estrogen receptor (ER)-positive human breast cancer cell line, indicate that coumestrol has the highest estrogenic potency among those phytoestrogens and environmental estrogens. The estrogenic potency of NP and DEHP were lower than the others. In conclusion, phytoestrogens, such as coumestrol, genistein and daidzein, and BPA increased ALP activity and enhanced bone mineralization in MC3T3-E1 cells, suggesting that not only phytoestrogen but also BPA, an environmental estrogen, is implicated in bone metabolism

  9. Generation and Characterization of a scFv Antibody Against T3SS Needle of Vibrio parahaemolyticus.

    Science.gov (United States)

    Wang, Rongzhi; Fang, Sui; Xiang, Shuangshuang; Ling, Sumei; Yuan, Jun; Wang, Shihua

    2014-06-01

    Vibrio parahaemolyticus, a halophilic gram-negative bacterium, is a food-borne pathogen that largely inhabits marine and estuarine environments, and poses a serious threat to human and animal health all over the world. The hollow "needle" channel, a specific assemble of T3SS which exists in most of gram-negative bacteria, plays a key role in the transition of virulence effectors to host cells. In this study, needle protein VP1694 was successfully expressed and purified, and the fusion protein Trx-VP1694 was used to immunize Balb/c mice. Subsequently, a phage single-chain fragment variable antibody (scFv) library was constructed, and a specific scFv against VP1694 named scFv-FA7 was screened by phage display panning. To further identify the characters of scFv, the soluble expression vector pACYC-scFv-skp was constructed and the soluble scFv was purified by Ni(2+) affinity chromatography. ELISA analysis showed that the scFv-FA7 was specific to VP1694 antigen, and its affinity constant was 1.07 × 10(8 )L/mol. These results offer a molecular basis to prevent and cure diseases by scFv, and also provide a new strategy for further research on virulence mechanism of T3SS in V. parahaemolyticus by scFv.

  10. A Vibrio parahaemolyticus T3SS effector mediates pathogenesis by independently enabling intestinal colonization and inhibiting TAK1 activation.

    Science.gov (United States)

    Zhou, Xiaohui; Gewurz, Benjamin E; Ritchie, Jennifer M; Takasaki, Kaoru; Greenfeld, Hannah; Kieff, Elliott; Davis, Brigid M; Waldor, Matthew K

    2013-05-30

    Vibrio parahaemolyticus type III secretion system 2 (T3SS2) is essential for the organism's virulence, but the effectors required for intestinal colonization and induction of diarrhea by this pathogen have not been identified. Here, we identify a type III secretion system (T3SS2)-secreted effector, VopZ, that is essential for V. parahaemolyticus pathogenicity. VopZ plays distinct, genetically separable roles in enabling intestinal colonization and diarrheagenesis. Truncation of VopZ prevents V. parahaemolyticus colonization, whereas deletion of VopZ amino acids 38-62 abrogates V. parahaemolyticus-induced diarrhea and intestinal pathology but does not impair colonization. VopZ inhibits activation of the kinase TAK1 and thereby prevents the activation of MAPK and NF-κB signaling pathways, which lie downstream. In contrast, the VopZ internal deletion mutant cannot counter the activation of pathways regulated by TAK1. Collectively, our findings suggest that VopZ's inhibition of TAK1 is critical for V. parahaemolyticus to induce diarrhea and intestinal pathology. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

  11. Three-dimensional reconstruction of the Shigella T3SS transmembrane regions reveals 12-fold symmetry and novel features throughout.

    Science.gov (United States)

    Hodgkinson, Julie L; Horsley, Ashley; Stabat, David; Simon, Martha; Johnson, Steven; da Fonseca, Paula C A; Morris, Edward P; Wall, Joseph S; Lea, Susan M; Blocker, Ariel J

    2009-05-01

    Type III secretion systems (T3SSs) mediate bacterial protein translocation into eukaryotic cells, a process essential for virulence of many Gram-negative pathogens. They are composed of a cytoplasmic secretion machinery and a base that bridges both bacterial membranes, into which a hollow, external needle is embedded. When isolated, the latter two parts are termed the 'needle complex'. An incomplete understanding of the structure of the needle complex has hampered studies of T3SS function. To estimate the stoichiometry of its components, we measured the mass of its subdomains by scanning transmission electron microscopy (STEM). We determined subunit symmetries by analysis of top and side views within negatively stained samples in low-dose transmission electron microscopy (TEM). Application of 12-fold symmetry allowed generation of a 21-25-A resolution, three-dimensional reconstruction of the needle complex base, revealing many new features and permitting tentative docking of the crystal structure of EscJ, an inner membrane component.

  12. A Vibrio parahaemolyticus T3SS Effector Mediates Pathogenesis by Independently Enabling Intestinal Colonization and Inhibiting TAK1 Activation

    Directory of Open Access Journals (Sweden)

    Xiaohui Zhou

    2013-05-01

    Full Text Available Vibrio parahaemolyticus type III secretion system 2 (T3SS2 is essential for the organism’s virulence, but the effectors required for intestinal colonization and induction of diarrhea by this pathogen have not been identified. Here, we identify a type III secretion system (T3SS2-secreted effector, VopZ, that is essential for V. parahaemolyticus pathogenicity. VopZ plays distinct, genetically separable roles in enabling intestinal colonization and diarrheagenesis. Truncation of VopZ prevents V. parahaemolyticus colonization, whereas deletion of VopZ amino acids 38–62 abrogates V. parahaemolyticus-induced diarrhea and intestinal pathology but does not impair colonization. VopZ inhibits activation of the kinase TAK1 and thereby prevents the activation of MAPK and NF-κB signaling pathways, which lie downstream. In contrast, the VopZ internal deletion mutant cannot counter the activation of pathways regulated by TAK1. Collectively, our findings suggest that VopZ’s inhibition of TAK1 is critical for V. parahaemolyticus to induce diarrhea and intestinal pathology.

  13. Three-dimensional reconstruction of the Shigella T3SS transmembrane regions reveals 12-fold symmetry and novel features throughout

    Energy Technology Data Exchange (ETDEWEB)

    Hodgkinson, J.L.; Wall, J.; Horsley, A.; Stabat, S.; Simon, M.; Johnson, S.; da Fonseca, P. C. A.; Morris, E. P.; Lea, S. M.; Blocker, A. J.

    2009-05-18

    Type III secretion systems (T3SSs) mediate bacterial protein translocation into eukaryotic cells, a process essential for virulence of many Gram-negative pathogens. They are composed of a cytoplasmic secretion machinery and a base that bridges both bacterial membranes, into which a hollow, external needle is embedded. When isolated, the latter two parts are termed the 'needle complex'. An incomplete understanding of the structure of the needle complex has hampered studies of T3SS function. To estimate the stoichiometry of its components, we measured the mass of its subdomains by scanning transmission electron microscopy (STEM). We determined subunit symmetries by analysis of top and side views within negatively stained samples in low-dose transmission electron microscopy (TEM). Application of 12-fold symmetry allowed generation of a 21-25-{angstrom} resolution, three-dimensional reconstruction of the needle complex base, revealing many new features and permitting tentative docking of the crystal structure of EscJ, an inner membrane component.

  14. Environmental Endocrine Disruptors Promote Adipogenesis in the 3T3-L1 Cell Line through Glucocorticoid Receptor Activation

    Science.gov (United States)

    Sargis, Robert M.; Johnson, Daniel N.; Choudhury, Rashikh A.; Brady, Matthew J.

    2014-01-01

    The burgeoning obesity and diabetes epidemics threaten health worldwide, yet the molecular mechanisms underlying these phenomena are incompletely understood. Recently, attention has focused on the potential contributions of environmental pollutants that act as endocrine disrupting chemicals (EDCs) in the pathogenesis of metabolic diseases. Because glucocorticoid signaling is central to adipocyte differentiation, the ability of EDCs to stimulate the glucocorticoid receptor (GR) and drive adipogenesis was assessed in the 3T3-L1 cell line. Various EDCs were screened for glucocorticoid-like activity using a luciferase reporter construct, and four (bisphenol A (BPA), dicyclohexyl phthalate (DCHP), endrin, and tolylfluanid (TF)) were shown to significantly stimulate GR without significant activation of the peroxisome proliferator-activated receptor-γ. 3T3-L1 preadipocytes were then treated with EDCs and a weak differentiation cocktail containing dehydrocorticosterone (DHC) in place of the synthetic dexamethasone. The capacity of these compounds to promote adipogenesis was assessed by quantitative oil red O staining and immunoblotting for adipocyte-specific proteins. The four EDCs increased lipid accumulation in the differentiating adipocytes and also upregulated the expression of adipocytic proteins. Interestingly, proadipogenic effects were observed at picomolar concentrations for several of the EDCs. Because there was no detectable adipogenesis when the preadipocytes were treated with compounds alone, the EDCs are likely promoting adipocyte differentiation by synergizing with agents present in the differentiation cocktail. Thus, EDCs are able to promote adipogenesis through the activation of the GR, further implicating these compounds in the rising rates of obesity and diabetes. PMID:19927138

  15. Estradiol determines the effects of PTH on ERα-dependent transcription in MC3T3-E1 cells.

    Science.gov (United States)

    Christensen, Monika H E; Fenne, Ingvild S; Flågeng, Marianne H; Almås, Bjørg; Lien, Ernst A; Mellgren, Gunnar

    2014-07-18

    Bone remodeling is a continuous process regulated by several hormones such as estrogens and parathyroid hormone (PTH). Here we investigated the influence of PTH on estrogen receptor alpha (ERα)-dependent transcriptional activity in MC3T3-E1 osteoblasts. Cells that were transfected with an ER-responsive reporter plasmid and treated with PTH showed increased luciferase activity. However, in the presence of 17β-estradiol, we observed that PTH inhibited ERα-mediated transcription. cAMP mimicked the effects by PTH, and the findings were confirmed in COS-1 cells transfected with expression vector encoding the catalytic subunit of cAMP-dependent protein kinase (PKA). Furthermore, PTH exhibited specific effects on the mRNA expression of the decoy receptor osteoprotegerin (OPG) and the receptor activator of NF kappa-B ligand (RANKL) in MC3T3-E1 osteoblasts. In the absence of 17β-estradiol, PTH and cAMP enhanced the OPG/RANKL ratio, whereas, OPG/RANKL was suppressed when estradiol was present. In conclusion, our results indicate that the presence of estradiol determines whether PTH and cAMP stimulates or inhibits ERα-dependent activity and the OPG/RANKL mRNA expression in an osteoblastic cell line. Copyright © 2014 Elsevier Inc. All rights reserved.

  16. T=3/2 Isobaric Analog States in 9Be populated via 8Li+p resonance scattering

    Science.gov (United States)

    Hunt, Curtis; Rogachev, G. V.; Almaraz-Calderon, S.; Aprahamian, A.; Bucher, B.; Tan, W.; Johnson, E. D.; Mitchell, J. P.; Avila, M.; Kuchera, A.; Baby, L. T.

    2017-09-01

    Proton resonance scattering with rare isotope beams, studied using the Thick Target and Inverse Kinematics (TTIK) approach, has been a very productive tool to study the structure of exotic, mostly proton rich nuclei and has key advantages ex. high efficiency and excellent energy resolution. Direct implementation of TTIK for the neutron rich case would require neutron resonance scattering with a radioactive beam which is not possible at present. It has been suggested by V. Goldberg 20 years ago to study neutron rich nuclei using TTIK through corresponding isobaric analog states populated in proton resonance scattering, which has been done for a few cases. The main goal of this experiment is to benchmark the application of the TTIK technique for studies of neutron rich nuclei. The A=9 T=3/2 isobaric multiplet provides an ideal opportunity. Experimental data on proton resonances in 9C and on the structure of low lying states in 9Li are available. We have populated T=3/2 states in 9Be using 8Li+p resonance scattering, performed modified R-matrix analysis and compared the results to the available data for 9C and 9Li. Applicability and challenges of TTIK for neutron rich nuclei studies will be discussed.

  17. The molecular mechanism regulating the autonomous circadian expression of Topoisomerase I in NIH3T3 cells.

    Science.gov (United States)

    Yang, Fang; Nakajima, Yoshihiro; Kumagai, Megumi; Ohmiya, Yoshihiro; Ikeda, Masaaki

    2009-02-27

    To identify whether Topoisomerase I (TopoI) has autonomous circadian rhythms regulated by clock genes, we tested mouse TopoI (mTopoI) promoter oscillation in NIH3T3 cells using a real-time monitoring assay and TopoI mRNA oscillations using real-time RT-PCR. Analysis of the mTopoI promoter region with Matlnspector software revealed two putative E-box (E1 and E2) and one DBP/E4BP4-binding element (D-box). Luciferase assays indicated that mTopoI gene expression was directly regulated by clock genes. The real-time monitoring assay showed that E-box and D-box response elements participate in the regulation of the circadian expression of mTopoI. Furthermore, a gel-shift assay showed that E2 is a direct target of the BMAL1/CLOCK heterodimer and DBP binds to the putative D-site. These results indicate that TopoI is expressed in an autonomous circadian rhythm in NIH3T3 cells.

  18. Spindlin1, a novel nuclear protein with a role in the transformation of NIH3T3 cells

    International Nuclear Information System (INIS)

    Gao Yanhong; Yue Wen; Zhang Peng; Li Li; Xie Xiaoyan; Yuan Hongfeng; Chen Lin; Liu Daqing; Yan Fang; Pei Xuetao

    2005-01-01

    spindlin1, a novel human gene recently isolated by our laboratory, is highly homologous to mouse spindlin gene. In this study, we cloned cDNA full-length of this novel gene and send it to GenBank database as spindlin1 (Homo sapiens spindlin1) with Accession No. AF317228. In order to investigate the function of spindlin1, we studied further the subcellular localization of Spindlin1 protein and the effects of spindlin1 overexpression in NIH3T3 cells. The results showed that the fusion protein pEGFP-N1-spindlin1 was located in the nucleus and the C-terminal is correlated with nuclear localization of Spindlin1 protein. NIH3T3 cells which could stably express spindlin1 as a result of RT-PCR analysis compared with the control cells displayed a complete morphological change; made cell growth faster; and increased the percentage of cells in G 2 /M and S phase. Furthermore, overexpressed spindlin1 cells formed colonies in soft agar in vitro and formed tumors in nude mice. Our findings provide direct evidence that spindlin1 gene may contribute to tumorigenesis

  19. Abnormalities of Thyroid Hormone Metabolism during Systemic Illness: The Low T3 Syndrome in Different Clinical Settings

    Directory of Open Access Journals (Sweden)

    Arnaldo Moura Neto

    2016-01-01

    Full Text Available Thyroid hormone abnormalities are common in critically ill patients. For over three decades, a mild form of these abnormalities has been described in patients with several diseases under outpatient care. These alterations in thyroid hormone economy are a part of the nonthyroidal illness and keep an important relationship with prognosis in most cases. The main feature of this syndrome is a fall in free triiodothyronine (T3 levels with normal thyrotropin (TSH. Free thyroxin (T4 and reverse T3 levels vary according to the underlying disease. The importance of recognizing this condition in such patients is evident to physicians practicing in a variety of specialties, especially general medicine, to avoid misdiagnosing the much more common primary thyroid dysfunctions and indicating treatments that are often not beneficial. This review focuses on the most common chronic diseases already known to present with alterations in serum thyroid hormone levels. A short review of the common pathophysiology of the nonthyroidal illness is followed by the clinical and laboratorial presentation in each condition. Finally, a clinical case vignette and a brief summary on the evidence about treatment of the nonthyroidal illness and on the future research topics to be addressed are presented.

  20. Linarin isolated from Buddleja officinalis prevents hydrogen peroxide-induced dysfunction in osteoblastic MC3T3-E1 cells.

    Science.gov (United States)

    Kim, Young Ho; Lee, Young Soon; Choi, Eun Mi

    2011-01-01

    The flowers and leaves buds of Buddleja officinalis MAXIM (Buddlejaceae) are used to treat eye troubles, hernia, gonorrhea and liver troubles in Asia. To elucidate the protective effects of linarin isolated from B. officinalis on the response of osteoblast to oxidative stress, osteoblastic MC3T3-E1 cells were pre-incubated with linarin for 1h before treatment with 0.3mM H(2)O(2) for 48h, and markers of osteoblast function and oxidative damage were examined. Linarin significantly (P<0.05) increased cell survival, alkaline phosphatase (ALP) activity, collagen content, calcium deposition, and osteocalcin secretion and decreased the production of receptor activator of nuclear factor-kB ligand (RANKL), protein carbonyl (PCO), and malondialdehyde (MDA) of osteoblastic MC3T3-E1 cells in the presence of hydrogen peroxide. These results demonstrate that linarin can protect osteoblasts against hydrogen peroxide-induced osteoblastic dysfunction and may exert anti-resorptive actions, at least in part, via the reduction of RANKL and oxidative damage. 2011 Elsevier Inc. All rights reserved.

  1. Radical prostatectomy for clinical T3 disease: expanding indications while optimizing cancer control and quality of life.

    Science.gov (United States)

    Hill, Jennifer R; Fine, Samson W; Zhang, Jingbo; Eastham, James A

    2007-08-01

    A 61-year-old male presented with a history of gradually rising PSA over several years. His PSA had increased from 3.3 to 7 ng/ml over 3 years. Digital rectal examination, prostate biopsy, bone scan, and endorectal MRI were carried out to confirm the diagnosis and evaluate the extent of the cancer, with subsequent histopathologic examination of the radical prostatectomy specimen. Preoperative clinical stage (based on the digital rectal examination alone) was cT3a. Biopsy demonstrated extraprostatic extension in one core (Gleason 9 [4 + 5]). Endorectal MRI suggested a large volume cancer with probable extraprostatic extension in the posterior midline at the apex. Examination of the radical prostatectomy specimen demonstrated a pT3aN0 prostate cancer (Gleason 4 + 3 = 7 with a minor component of Gleason pattern 5). Extraprostatic extension was seen in the right posterior apex in the area of the dominant tumor mass. Surgical margins and seminal vesicles were negative for cancer. Radical retropubic prostatectomy with preservation of both neurovascular bundles plus bilateral pelvic lymph node dissection was performed. Postoperative PSA has been nondetectable. The patient is continent and, with the aid of vardenafil HCl, has erections sufficient for intercourse.

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

    International Nuclear Information System (INIS)

    Takahashi, Nobuhiko; Yoshizaki, Takayuki; Hiranaka, Natsumi; Suzuki, Takeshi; Yui, Tomoo; Akanuma, Masayasu; Oka, Kazuya; Kanazawa, Kaoru; Yoshida, Mika; Naito, Sumiyoshi; Fujiya, Mikihiro; Kohgo, Yutaka; Ieko, Masahiro

    2011-01-01

    Highlights: ► Lipin-1 affects lipid metabolism, adipocyte differentiation, and transcription. ► Adipose lipin-1 expression is reduced in obesity. ► Lipin-1 depletion using siRNA in 3T3-L1 adipocytes increased MCP-1 expression. ► 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-κB 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.

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

    Energy Technology Data Exchange (ETDEWEB)

    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); 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); Hiranaka, Natsumi; Suzuki, Takeshi [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); Kanazawa, Kaoru [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); 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); Ieko, Masahiro [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.

  4. Reactive oxygen species regulatory mechanisms associated with rapid response of MC3T3-E1 cells for vibration stress

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Ling; Gan, Xueqi; Zhu, Zhuoli; Yang, Yang; He, Yuting; Yu, Haiyang, E-mail: yhyang6812@scu.edu.cn

    2016-02-12

    Although many previous studies have shown that refractory period-dependent memory effect of vibration stress is anabolic for skeletal homeostasis, little is known about the rapid response of osteoblasts simply derived from vibration itself. In view of the potential role of reactive oxygen species (ROS) in mediating differentiated activity of osteoblasts, whether and how ROS regulates the rapid effect of vibration deserve to be demonstrated. Our findings indicated that MC3T3-E1 cells underwent decreased gene expression of Runx2, Col-I and ALP and impaired ALP activity accompanied by increased mitochondrial fission immediately after vibration loading. Moreover, we also revealed the involvement of ERK-Drp1 signal transduction in ROS regulatory mechanisms responsible for the rapid effect of vibration stress. - Highlights: • ROS contributed to the rapid response of MC3T3-E1 cells for vibration stress. • Imbalance of mitochondrial dynamics were linked to the LMHFV-derived rapid response. • The role of ERK-Drp1 signal pathway in the LMHFV-derived osteoblast rapid response.

  5. Serum levels of triiodothyronine (T3) and thyroxine (T4) in buffalo (Bubalus bubalis Lin.) raised in Amazon region

    International Nuclear Information System (INIS)

    Silva, A.O.A. da.

    1991-08-01

    Through the use of radioimmunoassay (RIA) it was determined blood serum concentration of triiodothyronine (T 3 ) and thyroxine (T 4 ) (n=78) for two different water buffalo racial groups. Blood serum was collected from young and adult animals belonging to two farms in Castanhal country, state of Para, Brazil, through the year of 1988. The serum levels of T 3 and T 4 were statistically correlated with climatic parameters, e.g., pluviometric precipitation, environmental temperature, humidity, light intensity variation and physiological factors such as age, breed and sex. It was identified two seasons during experiment, one season the rainfall period with high precipitation rates and the other one was considered as dry season, with low precipitation rates. The average rate of temperature and humidity have shown no significant statistic difference between the two seasons. On the other hand, it was found a significant relationship between luminosity and seasons, since when the luminosity decreases the pluviometric rates increases. (author). 51 refs, 15 figs, 15 tabs

  6. Deoxyactein Isolated from Cimicifuga racemosa protects osteoblastic MC3T3-E1 cells against antimycin A-induced cytotoxicity.

    Science.gov (United States)

    Choi, Eun Mi

    2013-06-01

    Deoxyactein is one of the major constituents isolated from Cimicifuga racemosa. In the present study, we investigated the protective effects of deoxyactein on antimycin A (mitochondrial electron transport inhibitor)-induced toxicity in osteoblastic MC3T3-E1 cells. Exposure of MC3T3-E1 cells to antimycin A caused significant cell viability loss, as well as mitochondrial membrane potential dissipation, complex IV inactivation, ATP loss, intracellular calcium ([Ca(2+) ]i ) elevation and oxidative stress. Pretreatment with deoxyactein prior to antimycin A exposure significantly reduced antimycin A-induced cell damage by preventing mitochondrial membrane potential dissipation, complex IV inactivation, ATP loss, [Ca(2+) ]i elevation and oxidative stress. Moreover, deoxyactein increased the activation of PI3K (phosphoinositide 3-kinase), Akt (protein kinase B) and CREB (cAMP-response element-binding protein) inhibited by antimycin A. All these data indicate that deoxyactein may reduce or prevent osteoblasts degeneration in osteoporosis or other degenerative disorders. Copyright © 2011 John Wiley & Sons, Ltd.

  7. DNA from radiation resistant human tumor cells transfers resistance to NIH/3T3 cells with varying degrees of penetrance

    International Nuclear Information System (INIS)

    Kasid, U.; Dritschilo, A.; Weichselbaum, R.

    1987-01-01

    Experimental evidence suggests that clinical radiation resistance may correlate with in vitro radiation survival parameters. Specifically, they isolated several cell lines from radioresistant head and neck carcinomas with D/sub 0/ values greater than 2 Gy. The authors co-transfected DNA from cell line SQ2OB (D/sub 0/ = 2.4 Gy) with the rhoSVNeO plasmid into NIH/3T3 cells (D/sub 0/ = 1.7 Gy). Antibiotic G418 resistant, transformed clones were isolated and confirmed by Southern blotting to contain human alu, as well as rhoSVNeO sequences. Screening for radiation resistance with 8Gy (Cs-137) revealed that 3 of 4 tested hybrid clones show a radiation survival intermediate between NIH/3T3 and SQ2OB. This suggests that radiation resistance is a dominant, transfectable phenotype of mammalian cells and can be expressed in more sensitive cells. Karyotyping of resistant hybrid clones shows the presence of double minute chromosomes. Secondary transfection results and experiments to clone the genetic factors responsible for radiation resistance are in progress and results will be reported

  8. Survival of T4aN0 and T3N+ laryngeal cancer patients: a retrospective institutional study and systematic review.

    Science.gov (United States)

    Khoueir, Nadim; Matar, Nayla; Farah, Chadi; Francis, Evana; Tabchy, Bassam; Haddad, Amine

    2015-01-01

    We aim to assess the correlation of tumor and nodal staging to survival in pT3N+ and T4aN0 laryngeal cancer with subgroup analysis within stage IVa (pT4N0 and pT3N2). Retrospective cohort study with systematic review of the literature. Hotel Dieu de France University Hospital (tertiary referral center). Laryngeal cancer patients' registries were reviewed from 1998 to 2012 selecting pT3N+ and pT4aN0 patients treated by primary total layngectomy. Overall survivals were compared using Log rank and Kaplan-Meier analysis. A systematic review was performed by 2 reviewers including all the articles reporting the outcome of these categories of patients. Online databases, including PubMed and EMBASE, were used. Reference sections of identified studies were examined for additional articles. Thirteen T3N+ patients and 19 T4aN0 patients treated by primary total laryngectomy were included. Five-year overall survival for T3N+, T3N2 and T4aN0 was respectively 33%, 32.1% and 73.7%. Due to the small sample, the difference was not significant. The systematic review revealed three articles reporting overall survival outcome for the T4N0 group and 6 articles for the T3N+. At 5years, the survival ranged from 62.5% to 73% in T4N0 and from 32.2% to 77% in T3N+. In advanced stage laryngeal cancer, T4aN0 tends toward a better survival than T3N+ especially when compared to T3N2 although they are grouped in the same TNM stage IVa. Copyright © 2015 Elsevier Inc. All rights reserved.

  9. Involvement of NpdA, a Putative 2-Nitropropane Dioxygenase, in the T3SS Expression and Full Virulence inRalstonia solanacearumOE1-1.

    Science.gov (United States)

    Zhang, Weiqi; Li, Jing; Tang, Yu; Chen, Kai; Shi, Xiaojun; Ohnishi, Kouhei; Zhang, Yong

    2017-01-01

    Previously, we isolated several genes that potentially affected the expression of type III secretion system (T3SS) in Ralstonia solanacearum OE1-1. Here, we focused on the rsp0316 , which encodes a putative 2-nitropropane dioxygenase (hereafter designated NpdA). The deletion of npdA substantially reduced the T3SS expression and virulence in OE1-1, and the complementation with functional NpdA could completely restore its reduced T3SS expression and virulence to that of wild type. The NpdA was highly conserved among diverse R. solanacearum species and the NpdA-dependent expression of T3SS was not specific to OE1-1 strain, but not the virulence. The NpdA was important for the T3SS expression in planta , while it was not required for the bacterial growth in planta . Moreover, the NpdA was not required for the elicitation of hypersensitive response (HR) of R. solanacearum strains in tobacco leaves. The T3SS in R. solanacearum is directly controlled by the AraC-type transcriptional regulator HrpB and regulated by a complex regulation network. The NpdA affected the T3SS expression mediated with HrpB but through some novel pathway. All these results from genetic studies demonstrate that NpdA is a novel factor for the T3SS expression in diverse R. solanacearum species in medium, but specifically for the T3SS expression in strain OE1-1 in planta . And the NpdA-dependent expression of T3SS in planta plays an important role in pathogenicity of R. solanacearum OE1-1 in host plants.

  10. T3 test

    Science.gov (United States)

    ... this test if you have signs of a thyroid disorder, including: The pituitary gland does not produce normal ... Thyroid physiology and diagnostic evaluation of patients with thyroid disorders. In: Melmed S, Polonsky KS, Larsen PR, Kronenberg HM, ...

  11. T3 (Triiodothyronine) Test

    Science.gov (United States)

    ... and Iron-binding Capacity (TIBC, UIBC) Trichomonas Testing Triglycerides Troponin Tryptase Tumor Markers Uric Acid Urinalysis Urine ... and symptoms associated with hypothyroidism and a slowed metabolism , such as weight gain, dry skin, fatigue, and ...

  12. Fatigue crack growth rate behaviour of friction-stir aluminium alloy AA2024-T3 welds under transient thermal tensioning

    International Nuclear Information System (INIS)

    Ilman, M.N.; Kusmono,; Iswanto, P.T.

    2013-01-01

    Highlights: • FSW enables unweldable aircraft material AA2024-T3 to be welded without cracking. • FSW applied to aircraft structure is required to have superior fatigue resistance. • Transient thermal tensioning (TTT) is being developed for stress relieving in FSW. • The fatigue crack growth rates of FSW joints under TTT are studied. - Abstract: Friction stir welding (FSW) has become a serious candidate technology to join metallic fuselage panels for the next generation of civil aircrafts. However, residual stress introduced during welding which subsequently affects fatigue performance is still a major problem that needs to be paid attention. The present investigation aims to improve fatigue crack growth resistance of friction stir aluminium alloy AA2024-T3 welds using transient thermal tensioning (TTT) treatment. In this investigation, aluminium alloy AA2024-T3 plates were joined using FSW process with and without TTT. The welding parameters used including tool rotation speed (Rt) and the plate travelling speed (v) were 1450 rpm and 30 mm/min respectively. The TTT treatments were carried out by heating both sides of friction stir weld line using moving electric heaters ahead of, beside and behind the tool at a heating temperature of 200 °C. Subsequently, a sequence of tests was carried out including microstructural examination, hardness measurement, tensile test and fatigue crack growth rate (FCGR) test in combination with fractography using scanning electron microscopy (SEM). The FCGR test was carried out using a constant amplitude fatigue experiment with stress ratio (R) of 0.1 and frequency (f) of 11 Hz whereas specimens used were centre-crack tension (CCT) type with the initial crack located at the weld nugget. Results of this investigation showed that at low ΔK, typically below 9 MPa m 0.5 , the friction stir welds under TTT treatments lowered fatigue crack growth rate (da/dN) and the lowest (da/dN) was achieved as the heaters were located ahead of

  13. Evaluation of 99mTc-ior t3 MoAb as a radiotracer in renal graft acute rejection

    International Nuclear Information System (INIS)

    Zayas, F.; Fraxedas, R.; Reyes, L.; Manalich, R.; Aragon, L.; Perera, A.

    1998-01-01

    Diagnosis of renal graft acute rejection is made mainly by clinical signs, humoral and functional changes, and is confirmed by histopathological studies. Many efforts have been made to find a radiopharmaceutical for early diagnosis of renal graft rejection. 99mTc-ior t3 monoclonal antibody was evaluated as a radiotracer in renal graft acute rejection. Schwarz's method was used with different molar relations 2ME:IgG and different tin-chelates were explored. The selected formulation was studied by chromatography and challenge studies. Once proved the quality of the radiopharmaceutical a freeze-dried Kit was performed. Its radiochemical purity and stability was studied for several months. FPLC and immunoreactivity studies were included. The sterility and apirogenicity was certified by the Center of Research and Development of Drugs. A clinical trial was started with patients who have received a renal graft eleven days before. An amount of 1.1 GBq of 99mTc-ior t3 MoAb was administered and planar scintigraphic images were recorded. A molar relation of 2000:1 (2ME:IgG) with 30 min produced the best reduction of the MoAb. From the different tin-chelates, the tin-pyrophosphate produced the highest radiochemical purities. The MoAb freeze dried Kit contains 1.0 mg of protein and 14 ug of Sn+2, this quantities guaranteed a shelf life of 6 months. The quality controls and challenge studies showed purity higher than 95.0% and a per cent of dissociation close to 20% was seen with DTPA, 80% with HSA and 48% with Cys. Our preliminary immunoscintigraphic results showed a good correlation among scintigraphy, a high rate of counts Renal Graft/Opposite side and the cause of graft loss. The small number of cases does not permit to make a definite conclusion. However, in some clinical situations the use of 99mTc-ior t3 MoAb could help as an early and specific non-invasive diagnosis of renal graft rejection. (author)

  14. Radical prostatectomy and postoperative irradiation in patients with pathological stage C (T3) carcinoma of the prostate

    International Nuclear Information System (INIS)

    Petrovich, Zbigniew; Lieskovsky, Gary; Langholz, Bryan; Formenti, Silvia; Baert, Luc; Streeter, Oscar; Skinner, Donald G.

    1998-01-01

    Purpose: Adenocarcinoma of the prostate is the most common human cancer of internal organs. Radical surgery is regarded by many to be the treatment of choice for capsule confined disease. Since accurate preoperative assessment of tumor stage is difficult to define, many patients are subsequently found to have pathological stage C (T3) disease. These patients should be considered for adjuvant radiotherapy. Methods and Materials: A group of 201 PS C (T3) unselected patients, treated with radical prostatectomy and limited pelvic lymphadenectomy, received postoperative irradiation to the prostate bed. This radiotherapy was given between 42-90 days after surgery and consisted of a median dose of 48 Gy. Patient survival, disease free survival, time to clinical and chemical relapse and the incidence of local and systemic relapse were analyzed. The influence of multiple parameters on the treatment outcome including patient age, treatment period, clinical stage, pathological stage, Gleason's score, prostate specific antigen (PSA), radiotherapy techniques and radiation dose were examined using univariate and multivariate analysis. Follow-up ranged from 3 to 15 years, with a median of 5 years. Results: The overall 5- and 10-year actuarial survival was 92% and 83% (median > 10 years), respectively and the 5- and 10-year disease-free survival (clinical and PSA) was 67% and 53% (median > 10 years), respectively. A total of 61 (30%) patients had a recurrence, including 23 (11%) patients who had clinical and 38 (19%) who had PSA recurrence. Of the 23 patients with clinical recurrence, 10 (5%) had local recurrence, including two patients who had local and systemic recurrence. Pathological stage and Gleason's score were independently predictive of recurrence (each with p 25 ng/ml) was also an important independent factor predicting tumor recurrence, p = 0.05. All other investigated parameters were not significant in predicting tumor recurrence. This treatment program was very well

  15. Local control related to Kerma rate in T2/T3 breast cancers treated by external irradiation and brachytherapy

    International Nuclear Information System (INIS)

    Campana, F.; Danhier, S.; Kouz, J.; Asselain, B.; Vilcoq, J.R.; Gautier, C; Fourquet, A.

    1996-01-01

    Introduction: This study was conducted to determine the impact of the brachytherapy parameters on local control in patients treated with irradiation alone for breast cancer. Material and Methods: From 1985 to 1991, 241 patients with T2 (187pts) T3 (54 pts) N0 (196 pts) N1 (45 pts) M0 breast cancer have been treated with the combination of external irradiation and brachytherapy boost without surgery. Doses delivered to the whole breast and the regional nodes were 54 Gy and 50 Gy respectively (1.8 or 2 Gy/fraction, 5 fr/wk). Twenty Gy were then boosted to the primary tumor, according to the tumor shrinkage, by Ir 192 implants. Plastic tubes and rigid needles were used in 85 % and 15 % of the cases respectively. The brachytherapy boost was delivered at 85% of the basal dose rate (Reference dose rate) according to the Paris system. In some instances, dose distribution heterogeneities were observed leading to the choice of another reference isodose. Results: Median follow-up for the entire population was 75 months (9-122). Local control at 6 years follow-up for T2 and T3 was 76% (+/- 6%). Age, SBR grading and steroid receptors were found to be significantly correlated to local control. The role of the brachytherapy boost was analysed. Reference dose rate was not found to have any impact on local control, (≤ 0.6 vs > 0.6 Gy/h). This result was perhaps linked to the particular choice of the reference isodose. To avoid this bias we have evaluated the impact of the Kerma rate (Activity x Time x total length) on local control. Median kerma rate was 8600 μGy h -1 cm -1 m 2 (2200-16600). Local control rate for T2 at 6 years was 77% +/- 8% (Kerma >8600) and 73 %+/- 10% (Kerma ≤ 8600). For T3 local control rate was 82% +/-12% (Kerma >8600) and 37 % +/- 34 % (p<0.0002). Conclusion: This study demonstrated that the impact of brachytherapy on local control was better evaluated by the Kerma rate than by the reference dose rate. Whereas the basal dose rate cannot be changed

  16. Regulation of myosin light chain kinase during insulin-stimulated glucose uptake in 3T3-L1 adipocytes.

    Directory of Open Access Journals (Sweden)

    Shelly Woody

    Full Text Available Myosin II (MyoII is required for insulin-responsive glucose transporter 4 (GLUT4-mediated glucose uptake in 3T3-L1 adipocytes. Our previous studies have shown that insulin signaling stimulates phosphorylation of the regulatory light chain (RLC of MyoIIA via myosin light chain kinase (MLCK. The experiments described here delineate upstream regulators of MLCK during insulin-stimulated glucose uptake. Since 3T3-L1 adipocytes express two MyoII isoforms, we wanted to determine which isoform was required for insulin-stimulated glucose uptake. Using a siRNA approach, we demonstrate that a 60% decrease in MyoIIA protein expression resulted in a 40% inhibition of insulin-stimulated glucose uptake. We also show that insulin signaling stimulates the phosphorylation of MLCK. We further show that MLCK can be activated by calcium as well as signaling pathways. We demonstrate that adipocytes treated with the calcium chelating agent, 1,2-b (iso-aminophenoxy ethane-N,N,N',N'-tetra acetic acid, (BAPTA (in the presence of insulin impaired the insulin-induced phosphorylation of MLCK by 52% and the RLC of MyoIIA by 45% as well as impairing the recruitment of MyoIIA to the plasma membrane when compared to cells treated with insulin alone. We further show that the calcium ionophore, A23187 alone stimulated the phosphorylation of MLCK and the RLC associated with MyoIIA to the same extent as insulin. To identify signaling pathways that might regulate MLCK, we examined ERK and CaMKII. Inhibition of ERK2 impaired phosphorylation of MLCK and insulin-stimulated glucose uptake. In contrast, while inhibition of CaMKII did inhibit phosphorylation of the RLC associated with MyoIIA, inhibition of CAMKIIδ did not impair MLCK phosphorylation or translocation to the plasma membrane or glucose uptake. Collectively, our results are the first to delineate a role for calcium and ERK in the activation of MLCK and thus MyoIIA during insulin-stimulated glucose uptake in 3T3-L1 adipocytes.

  17. Heterogeneity of type III secretion system (T3SS)-1-independent entry mechanisms used by Salmonella Enteritidis to invade different cell types.

    Science.gov (United States)

    Rosselin, Manon; Abed, Nadia; Virlogeux-Payant, Isabelle; Bottreau, Elisabeth; Sizaret, Pierre-Yves; Velge, Philippe; Wiedemann, Agnès

    2011-03-01

    Salmonella causes a wide range of diseases from acute gastroenteritis to systemic typhoid fever, depending on the host. To invade non-phagocytic cells, Salmonella has developed different mechanisms. The main invasion system requires a type III secretion system (T3SS) known as T3SS-1, which promotes a Trigger entry mechanism. However, other invasion factors have recently been described in Salmonella, including Rck and PagN, which were not expressed under our bacterial culture conditions. Based on these observations, we used adhesion and invasion assays to analyse the respective roles of Salmonella Enteritidis T3SS-1-dependent and -independent invasion processes at different times of infection. Diverse cell lines and cell types were tested, including endothelial, epithelial and fibroblast cells. We demonstrated that cell susceptibility to the T3SS-1-independent entry differs by a factor of nine between the most and the least permissive cell lines tested. In addition, using scanning electron and confocal microscopy, we showed that T3SS-1-independent entry into cells was characterized by a Trigger-like alteration, as for the T3SS-1-dependent entry, and also by Zipper-like cellular alteration. Our results demonstrate for what is believed to be the first time that Salmonella can induce Trigger-like entry independently of T3SS-1 and can induce Zipper-like entry independently of Rck. Overall, these data open new avenues for discovering new invasion mechanisms in Salmonella.

  18. Ferulic Acid, But Not All Hydroxycinnamic Acids, Is a Novel T3SS Inducer ofRalstonia solanacearumand Promotes Its Infection Process in Host Plants under Hydroponic Condition.

    Science.gov (United States)

    Zhang, Yong; Li, Jing; Zhang, Weiqi; Wang, Rongsheng; Qiu, Qiaoqing; Luo, Feng; Hikichi, Yasufumi; Ohnishi, Kouhei; Ding, Wei

    2017-01-01

    Hydroxycinnamic acids (HCAs) are typical monocyclic phenylpropanoids, including cinnamic acid (Cin), coumaric acid (Cou), caffeic acid (Caf), ferulic acid (FA) and their isomers, and involved in the interactions between pathogens and host plants. Here, we focused on the impact of HCAs on expression of type III secretion system (T3SS) in Ralstonia solanacearum . FA significantly induced the expression of the T3SS and some type III effectors (T3Es) genes in hrp -inducing medium, while did not the other HCAs. However, exogenously supplemented FA did not affect the T3SS expression in planta and the elicitation of the hypersensitive response (HR) in tobacco leaves. Consistent with its central roles in pathogenicity, the FA-induced expression of the T3SS led to significant promotion on infection process of R. solanacearum in tomato plants under hydroponics cultivation. Moreover, the FA-induced expression of the T3SS was specifically mediated by the well-characterized signaling cascade PrhA-prhI/R-PrhJ-HrpG-HrpB, independent of the other known regulatory pathways. In summary, our results demonstrated that FA, a novel inducer of the T3SS in R. solanacearum , was able to promote its infection process in host plants under hydroponics condition.

  19. Ferulic Acid, But Not All Hydroxycinnamic Acids, Is a Novel T3SS Inducer of Ralstonia solanacearum and Promotes Its Infection Process in Host Plants under Hydroponic Condition

    Directory of Open Access Journals (Sweden)

    Yong Zhang

    2017-09-01

    Full Text Available Hydroxycinnamic acids (HCAs are typical monocyclic phenylpropanoids, including cinnamic acid (Cin, coumaric acid (Cou, caffeic acid (Caf, ferulic acid (FA and their isomers, and involved in the interactions between pathogens and host plants. Here, we focused on the impact of HCAs on expression of type III secretion system (T3SS in Ralstonia solanacearum. FA significantly induced the expression of the T3SS and some type III effectors (T3Es genes in hrp-inducing medium, while did not the other HCAs. However, exogenously supplemented FA did not affect the T3SS expression in planta and the elicitation of the hypersensitive response (HR in tobacco leaves. Consistent with its central roles in pathogenicity, the FA-induced expression of the T3SS led to significant promotion on infection process of R. solanacearum in tomato plants under hydroponics cultivation. Moreover, the FA-induced expression of the T3SS was specifically mediated by the well-characterized signaling cascade PrhA-prhI/R-PrhJ-HrpG-HrpB, independent of the other known regulatory pathways. In summary, our results demonstrated that FA, a novel inducer of the T3SS in R. solanacearum, was able to promote its infection process in host plants under hydroponics condition.

  20. Novel T3SS effector EseK in Edwardsiella piscicida is chaperoned by EscH and EscS to express virulence.

    Science.gov (United States)

    Cao, Huifang; Yang, Cuiting; Quan, Shu; Hu, Tianjian; Zhang, Lingzhi; Zhang, Yuanxing; Yang, Dahai; Liu, Qin

    2018-01-01

    Bacterium usually utilises type III secretion systems (T3SS) to deliver effectors directly into host cells with the aids of chaperones. Hence, it is very important to identify bacterial T3SS effectors and chaperones for better understanding of host-pathogen interactions. Edwardsiella piscicida is an invasive enteric bacterium, which infects a wide range of hosts from fish to human. Given E. piscicida encodes a functional T3SS to promote infection, very few T3SS effectors and chaperones have been identified in this bacterium so far. Here, we reported that EseK is a new T3SS effector protein translocated by E. piscicida. Bioinformatic analysis indicated that escH and escS encode two putative class I T3SS chaperones. Further investigation indicated that EscH and EscS can enhance the secretion and translocation of EseK. EscH directly binds EseK through undetermined binding domains, whereas EscS binds EseK via its N-terminal α-helix. We also found that EseK has an N-terminal chaperone-binding domain, which binds EscH and EscS to form a ternary complex. Zebrafish infection experiments showed that EseK and its chaperones EscH and EscS are necessary for bacterial colonisation in zebrafish. This work identified a new T3SS effector, EseK, and its two T3SS chaperones, EscH and EscS, in E. piscicida, which enriches our knowledge of bacterial T3SS effector-chaperone interaction and contributes to our understanding of bacterial pathogenesis. © 2017 John Wiley & Sons Ltd.

  1. Ubiquitin and ubiquitin-modified proteins activate the Pseudomonas aeruginosa T3SS cytotoxin, ExoU.

    Science.gov (United States)

    Anderson, David M; Schmalzer, Katherine M; Sato, Hiromi; Casey, Monika; Terhune, Scott S; Haas, Arthur L; Feix, Jimmy B; Frank, Dara W

    2011-12-01

    Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen that possesses a type III secretion system (T3SS) critical for evading innate immunity and establishing acute infections in compromised patients. Our research has focused on the structure-activity relationships of ExoU, the most toxic and destructive type III effector produced by P. aeruginosa. ExoU possesses phospholipase activity, which is detectable in vitro only when a eukaryotic cofactor is provided with membrane substrates. We report here that a subpopulation of ubiquitylated yeast SOD1 and other ubiquitylated mammalian proteins activate ExoU. Phospholipase activity was detected using purified ubiquitin of various chain lengths and linkage types; however, free monoubiquitin is sufficient in a genetically engineered dual expression system. The use of ubiquitin by a bacterial enzyme as an activator is unprecedented and represents a new aspect in the manipulation of the eukaryotic ubiquitin system to facilitate bacterial replication and dissemination. © 2011 Blackwell Publishing Ltd.

  2. STUDY THE EFFECTS OF PRESTRAINS IN UNIAXIAL TENSION ON THE FORMING LIMIT DIAGRAM OF ALUMINUM ALLOY SHEETS(2024 T3

    Directory of Open Access Journals (Sweden)

    Waleed J. Ali

    2015-02-01

    Full Text Available           The strain path for sheet metal may be changed during forming , this may be affect the forming limit curve (FLC . In this work the FLC before and after prestraining was determined for aluminum alloy (2024 T3 to study the effect of this type of prestraining and in different values on the FLC. This alloy was chosen because it is used widely , specially in aircraft structures .It was shown that the using of uniaxial tension prestrain affects the FLC . The major strain in right side is increased with the increasing in the prestrain , while in the left side the effect is small .

  3. Effect of injection molded micro-structured polystyrene surfaces on proliferation of MC3T3-E1 cells

    Directory of Open Access Journals (Sweden)

    G. Lucchetta

    2015-04-01

    Full Text Available In this work, osteoinductive micro-pillared polystyrene surfaces were mass-produced for bone replacement applications, by means of the micro injection molding process. Firstly, the molding process parameters were optimized with a two-level, three-factor central composite face-centered plan to increase the quality of polystyrene micro pillars replication and to maximize the pillars height uniformity over the molded part. Secondly, osteoblastic MC3T3-E1 cells adhesion and proliferation on the replicated substrates were assessed as a function of micro topography parameters, such as pillars diameter, aspect ratio and spacing. Cell morphology and proliferation were evaluated through MTS test after 1, 3 and 7 days from seeding. The experimental results showed that cells adhesion and proliferation is more positively promoted on micro-pillared surfaces compared to flat surfaces, but no correlations were observed between cell proliferation and pillar diameter and spacing.

  4. Graphene Oxide Hybridized nHAC/PLGA Scaffolds Facilitate the Proliferation of MC3T3-E1 Cells

    Science.gov (United States)

    Liang, Chunyong; Luo, Yongchao; Yang, Guodong; Xia, Dan; Liu, Lei; Zhang, Xiaomin; Wang, Hongshui

    2018-01-01

    Biodegradable porous biomaterial scaffolds play a critical role in bone regeneration. In this study, the porous nano-hydroxyapatite/collagen/poly(lactic-co-glycolic acid)/graphene oxide (nHAC/PLGA/GO) composite scaffolds containing different amount of GO were fabricated by freeze-drying method. The results show that the synthesized scaffolds possess a three-dimensional porous structure. GO slightly improves the hydrophilicity of the scaffolds and reinforces their mechanical strength. Young's modulus of the 1.5 wt% GO incorporated scaffold is greatly increased compared to the control sample. The in vitro experiments show that the nHAC/PLGA/GO (1.5 wt%) scaffolds significantly cell adhesion and proliferation of osteoblast cells (MC3T3-E1). This present study indicates that the nHAC/PLGA/GO scaffolds have excellent cytocompatibility and bone regeneration ability, thus it has high potential to be used as scaffolds in the field of bone tissue engineering.

  5. Proteomic analysis of cAMP-mediated signaling during differentiation of 3 T3-L1 preadipocytes

    DEFF Research Database (Denmark)

    Borkowski, Kamil; Wrzesinski, Krzysztow; Rogowska-Wrzesinska, Adelina

    2014-01-01

    Initiation of adipocyte differentiation is promoted by the synergistic action of insulin/insulin-like growth factor, glucocorticoids, and agents activating cAMP-dependent signaling. The action of cAMP is mediated via PKA and Epac, where at least part of the PKA function relates to strong repression...... of Rho kinase activity, whereas Epac counteracts the reduction in insulin/insulin-like growth factor signaling associated with complete repression of Rho kinase activity. However, detailed knowledge of the Epac-dependent branch and the interplay with PKA is still limited. In the present study, we present...... a comprehensive evaluation of Epac-mediated processes and their interplay with PKA during the initiation of 3 T3-L1 preadipocyte differentiation using a combination of proteomics, molecular approaches, and bioinformatics. Proteomic analyses revealed 7 proteins specifically regulated in response to Epac activation...

  6. A 7-month-old male with Allan-Herndon-Dudley syndrome and the power of T3.

    Science.gov (United States)

    Langley, Katherine G; Trau, Steven; Bean, Lora J H; Narravula, Alekhya; Schrier Vergano, Samantha A

    2015-05-01

    Allan-Herndon-Dudley syndrome (AHDS, MIM 300523) is an X-linked neurodegenerative disorder characterized by intellectual disability, severe hypotonia, diminished muscle mass, and progressive spastic paraplegia. All affected males have pathognomonic thyroid profiles with an elevated T3 , low-normal free T4 , and normal TSH. Mutations in the monocarboxylate transporter 8 (MCT8) gene, SLC16A2, have been found to be causative. Here, we describe a proband whose extensive evaluation and ultimate diagnosis of AHDS unmasked three previously undiagnosed generations of affected individuals in one family. This case illustrates the need for clinicians to consider obtaining full thyroid studies on individuals with the non-specific findings of severe hypotonia, failure to thrive, and gross motor delay. © 2015 Wiley Periodicals, Inc.

  7. A proteomic approach for identification of secreted proteins during the differentiation of 3T3-L1 preadipocytes to adipocytes

    DEFF Research Database (Denmark)

    Kratchmarova, Irina; Kalume, Dario E; Blagoev, Blagoy

    2002-01-01

    cholinergic neurostimulating peptide, neutrophil gelatinase-associated lipocalin, and haptoglobin to be expressed highly by mature adipocytes. We also used liquid chromatography-based separation followed by automated tandem mass spectrometry to identify proteins secreted by mature adipocytes. Several......We have undertaken a systematic proteomic approach to purify and identify secreted factors that are differentially expressed in preadipocytes versus adipocytes. Using one-dimensional gel electrophoresis combined with nanoelectrospray tandem mass spectrometry, proteins that were specifically...... secreted by 3T3-L1 preadipocytes or adipocytes were identified. In addition to a number of previously reported molecules that are up- or down-regulated during this differentiation process (adipsin, adipocyte complement-related protein 30 kDa, complement C3, and fibronectin), we identified four secreted...

  8. Impregnation of cotton fabric with silver nanoparticles synthesized by dextran isolated from bacterial species Leuconostoc mesenteroides T3.

    Science.gov (United States)

    Davidović, Slađana; Miljković, Miona; Lazić, Vesna; Jović, Danica; Jokić, Bojan; Dimitrijević, Suzana; Radetić, Maja

    2015-10-20

    This study was aimed to highlight the possibility of cotton fabric impregnation with silver nanoparticles synthesized by dextran isolated from Leuconostoc mesenteroides T3 in order to obtain antimicrobial properties. The fabrication of dextran was proved by FTIR spectroscopy. Particle sizes of synthesized dextran and silver nanoparticles were measured by dynamic light scattering method. The presence of silver nanoparticles on the surface of cotton fabric was confirmed by scanning electron microscopy, X-ray diffraction measurements and reflectance spectrophotometry. Antimicrobial activity of cotton fabric impregnated with silver nanoparticles was tested against bacteria Escherichia coli and Staphylococcus aureus, and fungus Candida albicans. The results indicated that synthesized silver nanoparticles can provide satisfactory antimicrobial activity. However, maximum reduction (99.9%) of all tested microorganisms can be obtained only when 1.0mmolL(-1) colloid consisting of silver nanoparticles is applied. Copyright © 2015 Elsevier Ltd. All rights reserved.

  9. Cytotoxicity of MEIC chemicals Nos. 11-30 in 3T3 mouse fibroblasts with and without microsomal activation

    DEFF Research Database (Denmark)

    Rasmussen, Eva

    1999-01-01

    The cytotoxicity of MEIC chemicals Nos, 11-30 was evaluated by determination of neutral red uptake in Balb/c 3T3 mouse fibroblasts with and without the addition of a microsomal activation mixture. The use of microsomes significantly decreased the cytotoxicity of malathion, 2,4-dichlorophenoxyacetic...... acid, propranolol, thioridazine, lithium sulfate, copper sulfate and thallium sulfate, whereas the cytotoxicity of 1,1,1-trichloroethylene, phenol, nicotine, and paraquat was significantly increased by use of the microsomal activation mixture. These cytotoxicity data are in line with observations...... in other studies on microsomal modulation of the cytotoxicity of the test substances. Moderate to good correlations were found between the cytotoxicity data and rodent lethality data, and the addition of microsomes slightly improved the in vitro/in vivo concordance. The evidence to support the relevance...

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

    Directory of Open Access Journals (Sweden)

    Jeong-Eun Park

    2013-01-01

    Full Text Available 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 γ and CCAAT/enhancer-binding protein (C/EBP α 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.

  11. Modification of heterotrimeric G-proteins in Swiss 3T3 cells stimulated with Pasteurella multocida toxin.

    Directory of Open Access Journals (Sweden)

    Rebecca C Babb

    Full Text Available Many bacterial toxins covalently modify components of eukaryotic signalling pathways in a highly specific manner, and can be used as powerful tools to decipher the function of their molecular target(s. The Pasteurella multocida toxin (PMT mediates its cellular effects through the activation of members of three of the four heterotrimeric G-protein families, G(q, G(12 and G(i. PMT has been shown by others to lead to the deamidation of recombinant Gα(i at Gln-205 to inhibit its intrinsic GTPase activity. We have investigated modification of native Gα subunits mediated by PMT in Swiss 3T3 cells using 2-D gel electrophoresis and antibody detection. An acidic change in the isoelectric point was observed for the Gα subunit of the G(q and G(i families following PMT treatment of Swiss 3T3 cells, which is consistent with the deamidation of these Gα subunits. Surprisingly, PMT also induced a similar modification of Gα(11, a member of the G(q family of G-proteins that is not activated by PMT. Furthermore, an alkaline change in the isoelectric point of Gα(13 was observed following PMT treatment of cells, suggesting differential modification of this Gα subunit by PMT. G(s was not affected by PMT treatment. Prolonged treatment with PMT led to a reduction in membrane-associated Gα(i, but not Gα(q. We also show that PMT inhibits the GTPase activity of G(q.

  12. The Outcome of Postoperative Radiation Therapy for Patients with Stage II Pancreatic Cancer (T3 or N1 Disease)

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Sang Won; Chun, Misun; Kim, Myung Wook; Kim, Wook Hwan; Kang, Seok Yun; Kang, Seung Hee; Oh, Young Taek; Lee, Sunyoung; Yang, Juno [Ajou University School of Medicine, Suwon (Korea, Republic of)

    2007-12-15

    Purpose: To analyze retrospectively the outcome of postoperative radiation therapy with or without concurrent chemotherapy for curatively resected stage II pancreatic cancer with T3 or N1 disease. Materials and Methods: Between January 1996 and December 2005, twenty-eight patients completed adjuvant radiation therapy at Ajou University Hospital. The patients had either pathologic T3 stage or N1 stage. The radiation target volume encompassed the initial tumor bed identified preoperatively, resection margin area and celiac nodal area. In the case of N1 patients, the radiation field extended to the lower margin of the L3 vertebra for covering both para-aortic lymph nodes bearing area. The median total radiation dose was 50 Gy. Ten patients received concurrent chemotherapy. Results: Thirteen patients (46%) showed loco-regional recurrences. The celiac axis nodal area was the most frequent site (4 patients). Five patients showed both loco-regional recurrence and a distant metastasis. Patients with positive lymph nodes had a relatively high probability of a distant metastasis (57.1%). Patients that had a positive resection margin showed a relatively high local failure rate (57.1%). The median disease-free survival period of all patients was 6 months and the 1- and 2-year disease free survival rates were 27.4% and 8.2%, respectively. The median overall survival period was 9 months. The 2- and 3-year overall survival rates were 31.6% and 15.8%, respectively. Conclusion: The pancreatic cancer patients with stage II had a high risk of local failure and a high risk of a distant metastasis. We suggest the concurrent use of an effective radiation-sensitizing chemotherapeutic drug and adjuvant chemotherapy after postoperative radiation therapy for the treatment of patients with stage II pancreatic cancer.

  13. Andrographolide inhibits adipogenesis of 3T3-L1 cells by suppressing C/EBPβ expression and activation.

    Science.gov (United States)

    Chen, Ching-Chu; Chuang, Wei-Ting; Lin, Ai-Hsuan; Tsai, Chia-Wen; Huang, Chin-Shiu; Chen, Yun-Ting; Chen, Haw-Wen; Lii, Chong-Kuei

    2016-09-15

    Andrographolide, a diterpenoid, is the most abundant terpenoid in Andrographis paniculata, a popular Chinese herbal medicine. Andrographolide displays diverse biological activities including hypoglycemia, hypolipidemia, anti-inflammation, and anti-tumorigenesis. Recent evidence indicates that andrographolide displays anti-obesity property by inhibiting lipogenic gene expression, however, the underlying mechanisms remain to be elucidated. In this study, the effects of andrographolide on transcription factor cascade and mitotic clonal expansion in 3T3-L1 preadipocyte differentiation into adipocyte were determined. Andrographolide dose-dependently (0-15μM) inhibited CCAAT/enhancer-binding protein α (C/EBPα) and C/EBPβ mRNA and protein expression as well as peroxisome proliferator-activated receptor γ (PPARγ) protein level during the adipogenesis of 3T3-L1 cells. Concomitantly, fatty acid synthase and stearoyl-CoA desaturase expression and lipid accumulation were attenuated by andrographolide. Oil-red O staining further showed that the first 48h after the initiation of differentiation was critical for andrographolide inhibition of adipocyte formation. Andrographolide inhibited the phosphorylation of PKA and the activation of cAMP response element-binding protein (CREB) in response to a differentiation cocktail, which led to attenuated C/EBPβ expression. In addition, ERK and GSK3β-dependent C/EBPβ phosphorylation was attenuated by andrographolide. Moreover, andrographolide suppressed cyclin A, cyclin E, and CDK2 expression and impaired the progression of mitotic clonal expansion (MCE) by arresting the cell cycle at the Go/G1 phase. Taken together, these results indicate that andrographolide has a potent anti-obesity action by inhibiting PKA-CREB-mediated C/EBPβ expression as well as C/EBPβ transcriptional activity, which halts MCE progression and attenuates C/EBPα and PPARγ expression. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. Co-ordinate regulation of the cytoskeleton in 3T3 cells overexpressing thymosin-beta4.

    Science.gov (United States)

    Golla, R; Philp, N; Safer, D; Chintapalli, J; Hoffman, R; Collins, L; Nachmias, V T

    1997-01-01

    In several cell types, short-term increases in the concentration of the G-actin-sequestering peptide thymosin-beta4 (Tbeta4) cause the disassembly of F-actin bundles. To determine the extent of cell adaptability to these reductions in F-actin, we overexpressed Tbeta4 in NIH 3T3 cells. In cell lines with Tbeta4 levels twice those of vector controls, G-actin increased approximately twofold as expected. However, F-actin did not decrease as in short-term experiments but rather also increased approximately twofold so that the G-F ratio remained constant. Surprisingly, the cytoskeletal proteins myosin IIA, alpha-actinin, and tropomyosin also increased nearly twofold. These increases were specific; DNA, total protein, lactic dehydrogenase, profilin, and actin depolymerizing factor levels were unchanged in the overexpressing cells. The Tbeta4 lines spread more fully and adhered to the dish more strongly than vector controls; this altered phenotype correlated with a twofold increase in talin and alpha5-integrin and a nearly threefold increase in vinculin. Focal adhesions, detected by indirect immunofluorescence with antivinculin, were increased in size over the controls. Northern blotting showed that mRNAs for both beta-actin and vinculin were increased twofold in the overexpressing lines. We conclude that 1) NIH 3T3 cells adapt to increased levels of G-actin sequestered by increased Tbeta4 by increasing their total actin so that the F-actin/G-actin ratio remains constant; 2) these cells coordinately increase several cytoskeletal and adhesion plaque proteins; and 3) at least for actin and vinculin, this regulation is at the transcriptional level. We therefore propose that the proteins of this multimember interacting complex making up the actin-based cytoskeleton, are coordinately regulated by factors that control the expression of several proteins. The mechanism may bear similarities to the control of synthesis of another multimember interacting complex, the myofibril of

  15. Characterization of a bombesin receptor on Swiss mouse 3T3 cells by affinity cross-linking

    International Nuclear Information System (INIS)

    Sinnett-Smith, J.; Zachary, I.; Rozengurt, E.

    1988-01-01

    We have previously identified by chemical cross-linking a cell surface protein in Swiss 3T3 cells of apparent Mr 75,000-85,000, which may represent a major component of the receptor for peptides of the bombesin family in these cells. Because bombesin-like peptides may interact with other cell surface molecules, it was important to establish the correlation between receptor binding and functions of this complex and further characterize the Mr 75,000-85,000 cross-linked protein. Detailed time courses carried out at different temperatures demonstrated that the Mr 75,000-85,000 affinity-labelled band was the earliest cross-linked complex detected in Swiss 3T3 cells incubated with 125I-labelled gastrin-releasing peptide (125I-GRP). Furthermore, the ability of various nonradioactive bombesin agonists and antagonists to block the formation of the Mr 75,000-85,000 cross-linked complex correlated extremely well (r = 0.994) with the relative capacity of these peptides to inhibit 125I-GRP specific binding. Pretreatment with unlabelled GRP for up to 6 h caused only a slight decrease in both specific 125I-GRP binding and the affinity labelling of the Mr 75,000-85,000 protein. We also show that the cross-linked complex is a glycoprotein. First, solubilized affinity labelled Mr 75,000-85,000 complex applied to wheat germ lectin-sepharose columns was eluted by addition of 0.3 M N-acetyl-D-glucosamine. Second, treatment with endo-beta-N-acetylglucosaminidase F reduced the apparent molecular weight of the affinity-labelled band from 75,000-85,000 to 43,000, indicating the presence of N-linked oligosaccharide groups

  16. Curcumin attenuates lipolysis stimulated by tumor necrosis factor-α or isoproterenol in 3T3-L1 adipocytes.

    Science.gov (United States)

    Xie, Xiao-yun; Kong, Po-Ren; Wu, Jin-feng; Li, Ying; Li, Yan-xiang

    2012-12-15

    Curcumin, an active component derived from dietary spice turmeric (Curcuma longa), has been demonstrated antihyperglycemic, antiinflammatory and hypocholesterolemic activities in obesity and diabetes. These effects are associated with decreased level of circulating free fatty acids (FFA), however the mechanism has not yet been elucidated. The flux of FFA and glycerol from adipose tissue to the blood stream primarily depends on the lipolysis of triacylglycerols in the adipocytes. Adipocyte lipolysis is physiologically stimulated by catecholamine hormones. Tumor necrosis factor-α (TNFα) stimulates chronic lipolysis in obesity and type 2 diabetes. In this study, we examined the role of curcumin in inhibiting lipolytic action upon various stimulations in 3T3-L1 adipocytes. Glycerol release from TNFα or isoproterenol-stimulated 3T3-L1 adipocytes in the absence or presence of curcumin was determined using a colorimetric assay (GPO-Trinder). Western blotting was used to investigate the TNFα-induced phosphorylation of MAPK and perilipin expression. Fatcake and cytosolic fractions were prepared to examine the isoproterenol-stimulated hormone-sensitive lipase translocation. Treatment with curcumin attenuated TNFα-mediated lipolysis by suppressing phosphorylation of extracellular signal-related kinase 1/2 (ERK1/2) and reversing the downregulation of perilipin protein in TNFα-stimulated adipocytes (p<0.05). The acute lipolytic response to adrenergic stimulation of isoproterenol was also restricted by curcumin (10-20 μM, p<0.05), which was compatible with reduced perilipin phosphorylation(29%, p<0.05) and hormone-sensitive lipase translocation(20%, p<0.05). This study provides evidence that curcumin acts on adipocytes to suppress the lipolysis response to TNFα and catecholamines. The antilipolytic effect could be a cellular basis for curcumin decreasing plasma FFA levels and improving insulin sensitivity. Copyright © 2012 Elsevier GmbH. All rights reserved.

  17. Effect of germinated brown rice extracts on pancreatic lipase, adipogenesis and lipolysis in 3T3-L1 adipocytes.

    Science.gov (United States)

    Lim, See Meng; Goh, Yong Meng; Kuan, Wen Bin; Loh, Su Peng

    2014-11-03

    This study investigated anti-obesity effects of seven different solvent (n-hexane, toluene, dicholoromethane, ethyl acetate, absolute methanol, 80% methanol and deionized water) extracts of germinated brown rice (GBR) on pancreatic lipase activity, adipogenesis and lipolysis in 3T3-L1 adipocytes. GBR were extracted separately by employing different solvents with ultrasound-assisted. Pancreatic lipase activity was determined spectrophotometrically by measuring the hydrolysis of p-nitrophenyl butyrate (p-NPB) to p-nitrophenol at 405 nm. Adipogenesis and lipolysis were assayed in fully differentiated 3T3-L1 adipocytes by using Oil Red O staining and glycerol release measurement. GBR extract using hexane showed the highest inhibitory effect (13.58 ± 0.860%) at concentration of 200 μg/ml followed by hexane extract at 100 μg/ml (9.98 ± 1.048%) while ethyl acetate extract showed the lowest (2.62 ± 0.677%) at concentration of 200 μg/ml on pancreatic lipase activity. Water extract at 300 μg/ml showed 61.55 ± 3.824% of Oil Red O staining material (OROSM), a marker of adipogenesis. It significantly decrease (p < 0.05) lipid accumulation than control (OROSM = 100%), follow by ethyl acetate extract at 300 μg/ml (OROSM = 65.17 ± 3.131%). All the GBR extracts induced lipolysis with 1.22-1.83 fold of greater glycerol release than control. GBR extracts especially the least polar and intermediate polar solvent extracts exhibited inhibitory effect on pancreatic lipase, decrease fat accumulation by adipocyte differentiation inhibition, and stimulate lipolysis on adipocytes. Therefore, GBR could be furthered study and developed as a functional food in helping the treatment and/or prevention of obesity.

  18. Comparison of levels of T3, T4 and TSH in maternal and cord blood taken during delivery between a minority and the Han tribe

    International Nuclear Information System (INIS)

    Maimaitijiang Abudurexiti; Rezhiwanguli Wushiman

    2001-01-01

    Objective: To compare the serum level of T 3 , T 4 and TSH in blood of mother and umbilical cord between the pregnant Uigur women and pregnant Han women. Methods: The serum levels of T 3 , T 4 and TSH in between the two tribes were analyzed by RIA method. Results: There was remarkable difference between the levels of mother blood and of umbilical cord blood (P 0.05). Conclusion: In certain similar habitation, the nationality and living habit have no distinct effect on the serum level of T 3 , T 4 and TSH

  19. Obese gene expression at in vivo levels by fat pads derived from s.c. implanted 3T3-F442A preadipocytes

    DEFF Research Database (Denmark)

    Mandrup, S; Loftus, T M; MacDougald, O A

    1997-01-01

    3T3-F442A preadipocytes implanted s.c. into athymic mice develop into fat pads that are indistinguishable from normal adipose tissue. Implanted preadipocytes harboring a beta-galactosidase transgene gave rise to fat pads in which almost all adipocytes expressed beta-galactosidase. This finding...... proved that the implanted 3T3-F442A preadipocytes, rather than endogenous preadipose cells, gave rise to the newly developed "adipose tissue." 3T3-F442A preadipocytes, when differentiated into adipocytes in cell culture, express the obese gene at an unexpectedly low level, i.e.,...

  20. Obese gene expression at in vivo levels by fat pads derived from s.c. implanted 3T3-F442A preadipocytes

    DEFF Research Database (Denmark)

    Mandrup, S; Loftus, T M; MacDougald, O A

    1997-01-01

    proved that the implanted 3T3-F442A preadipocytes, rather than endogenous preadipose cells, gave rise to the newly developed "adipose tissue." 3T3-F442A preadipocytes, when differentiated into adipocytes in cell culture, express the obese gene at an unexpectedly low level, i.e.,......3T3-F442A preadipocytes implanted s.c. into athymic mice develop into fat pads that are indistinguishable from normal adipose tissue. Implanted preadipocytes harboring a beta-galactosidase transgene gave rise to fat pads in which almost all adipocytes expressed beta-galactosidase. This finding...

  1. Panax ginseng total protein promotes proliferation and secretion of collagen in NIH/3T3 cells by activating extracellular signal-related kinase pathway

    Directory of Open Access Journals (Sweden)

    Xuenan Chen

    2017-07-01

    Conclusion: Our studies suggest that GTP promoted proliferation and secretion of collagen in NIH/3T3 cells by activating the ERK signal pathway, which shed light on a potential function of GTP in promoting wound healing.

  2. Effect of Turmeric and its Active Principle Curcumin on T3-Induced Oxidative Stress and Hyperplasia in Rat Kidney: A Comparison

    OpenAIRE

    Samanta, Luna; Panigrahi, Jogamaya; Bhanja, Shravani; Chainy, Gagan B. N.

    2010-01-01

    The present study was designed to compare the potential of turmeric and its active principle curcumin on T3-induced oxidative stress and hyperplasia. Adult male Wistar strain rats were rendered hyperthyroid by T3 treatment (10 μg · 100 g−1 · day−1 intraperitoneal for 15 days in 0.1 mM NaOH) to induce renal hyperplasia. Another two groups were treated similarly with T3 along with either turmeric or curcumin (30 mg kg−1 body weight day−1 orally for 15 days). The results indicate that T3 induces...

  3. Type 3 Secretion System (T3SS) ofBradyrhizobiumsp. DOA9 and Its Roles in Legume Symbiosis and Rice Endophytic Association.

    Science.gov (United States)

    Songwattana, Pongpan; Noisangiam, Rujirek; Teamtisong, Kamonluck; Prakamhang, Janpen; Teulet, Albin; Tittabutr, Panlada; Piromyou, Pongdet; Boonkerd, Nantakorn; Giraud, Eric; Teaumroong, Neung

    2017-01-01

    The Bradyrhizobium sp. DOA9 strain isolated from a paddy field has the ability to nodulate a wide spectrum of legumes. Unlike other bradyrhizobia, this strain has a symbiotic plasmid harboring nod , nif , and type 3 secretion system (T3SS) genes. This T3SS cluster contains all the genes necessary for the formation of the secretory apparatus and the transcriptional activator (TtsI), which is preceded by a nod -box motif. An in silico search predicted 14 effectors putatively translocated by this T3SS machinery. In this study, we explored the role of the T3SS in the symbiotic performance of DOA9 by evaluating the ability of a T3SS mutant (Ω rhcN ) to nodulate legumes belonging to Dalbergioid, Millettioid, and Genistoid tribes. Among the nine species tested, four ( Arachis hypogea , Vigna radiata , Crotalaria juncea , and Macroptilium atropurpureum ) responded positively to the rhcN mutation (ranging from suppression of plant defense reactions, an increase in the number of nodules and a dramatic improvement in nodule development and infection), one ( Stylosanthes hamata ) responded negatively (fewer nodules and less nitrogen fixation) and four species ( Aeschynomene americana , Aeschynomene afraspera , Indigofera tinctoria , and Desmodium tortuosum ) displayed no phenotype. We also tested the role of the T3SS in the ability of the DOA9 strain to endophytically colonize rice roots, but detected no effect of the T3SS mutation, in contrast to what was previously reported in the Bradyrhizobium SUTN9-2 strain. Taken together, these data indicate that DOA9 contains a functional T3SS that interferes with the ability of the strain to interact symbiotically with legumes but not with rice.

  4. Studi Eksperimental Pengendalian Korosi pada Aluminium 2024-T3 di Lingkungan Air Laut Melalui Penambahan Inhibitor Kalium Kromat (K2CrO4)

    OpenAIRE

    Wibowo, Waris; Ilman, Mochammad Noer

    2013-01-01

    Aluminium paduan seri 2024-T3 banyak digunakan di industri manufaktur seperti pesawat terbang, otomotif, dan kapal laut karena aluminium merupakan logam ringan dan memiliki sifat mekanik yang baik. Namun demikian aluminium paduan 2024-T3 rentan terhadap korosi bila dioperasikan di lingkungan yang korosif, seperti air laut. Salah satu upaya untuk mengendalikan laju korosi adalah dengan penambahan inhibitor. Penelitian ini bertujuan untuk mempelajari pengaruh inhibitor kalium kromat (K2CrO4) da...

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

    International Nuclear Information System (INIS)

    Jozaki, K.; Kuriu, A.; Hirota, S.; Onoue, H.; Ebi, Y.; Adachi, S.; Ma, J.Y.; Tarui, S.; Kitamura, Y.

    1991-01-01

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

  6. Co-localization of zinc transporter 3 (ZnT3) with sensory neuromediators and/or neuromodulators in the enteric nervous system of the porcine esophagus.

    Science.gov (United States)

    Wojtkiewicz, Joanna; Rytel, Liliana; Makowska, Krystyna; Gonkowski, Sławomir

    2017-06-01

    Zinc transporter 3 (ZnT3) is one of the zinc transporters family. It is closely connected to the nervous system, where enables the transport of zinc ions from the cytoplasm to synaptic vesicles. This substance has been described within the central and peripheral nervous system, especially in the enteric nervous system (ENS). The aim of the present study was to describe the co-localization of ZnT3 with selected neuromediators and/or neuromodulators participating in sensory stimuli conduction in neurons of the ENS within the porcine esophagus. Co-localization of ZnT3 with substance P (SP), leucine enkephalin (LENK) and calcitonin gene-related peptide (CGRP) was studied using standard double-immunofluorescence technique. The obtained results show that ZnT3, SP and/or LENK may occur in the same enteric neurons, and the degree of co-localization of these substances clearly depends on the fragment of esophagus studied and the type of enteric ganglia. In contrast, the co-localization of ZnT3 with CGRP was not observed during the present investigation. The obtained results suggest that ZnT3 in the ENS may be involved in the conduction of sensory and/or pain stimuli.

  7. Effects of obesity, total fasting and re-alimentation on L-thyroxine (T4), 3,5,3'-L-triiodothyronine (T3), 3,3',5'-L-triiodothyronine (rT3), thyroxine binding globulin (TBG), cortisol, thyrotrophin, cortisol binding globulin (CBG), transferrin, alpha 2-haptoglobin and complement C'3 in serum.

    Science.gov (United States)

    Scriba, P C; Bauer, M; Emmert, D; Fateh-Moghadam, A; Hofmann, G G; Horn, K; Pickardt, C R

    1979-08-01

    The effects of total fasting for 31 +/- 10 days followed by re-alimentation with an 800 calorie diet on thyroid function, i.e. T4,T3,rT3,RT3U (resin T3 uptake), and TSH, and on TBG levels in serum were studied sequentially in obese hospitalized patients (N=18). Additionally, cortisol, growth hormone, prolactin, parathyrin and free fatty acids were followed as hormonal and metabolic parameters, respectively. Further, CBG, transferrin, alpha 2-haptoglobin and complement C'3 were measured as representatives of other serum proteins. Results before fasting: T4, T3, TBG, cortisol, CBG, alpha 2-haptoglobin and complement C'3 of the obese patients were elevated when compared with healthy normal weight controls, whereas rT3, T4/TBG ratio, T3/TBG ratio, TSH, coritsol/cbg ratio, growth hormone, prolactin, parathyrin and transferrin of the obese group were normal. RT3U and fT4 index were decreased in the obese patients. Results during fasting: Significant decreases were observed during fasting for the following parameters -- T3, TBG, T3/TBG ratio, transferrin, alpha 2-haptoglobin complement C'3. rT3, T4/TBG ratio, RT3U, fT4 index and FFA increased. T4, tsh response to TRH stimulation, cortisol, CBG, cortisol/cbg ratio, parathyrin, growth hormone and prolactin did not change. Results during re-alimentation: T3, TBG, T3/TBG ratio, TSH response to TRH, transferrin, alpha 2-haptoglobin and complement C'3 increased. Conversely, fT3, RT3U, FFA, cortisol and cortisol/cbg ratio decreased whereas the other parameters did not change. 1) There is no evidence for primary hypothyroidism in obese patients during prolonged fasting and re-alimentation. 2) The rapid decrease of T3 and increase of RT3U after initiation of fasting are not fully explained by the observed slower decreases in TBG. 3) The alterations of T3, rT3 and RT3U resemble in their kinetics the changes in FFA levels. 4) Fasting reduced the levels of only certain serum proteins, interestingly TBG, transferrin, alpha 2

  8. Impact of therapeutic factors on local control in T2-T3 anal carcinoma treated by radiation or radiochemotherapy

    International Nuclear Information System (INIS)

    Allal, Abdelkarim S.; Mermillod, Bernadette; Kurtz, John M.; Marti, Marc-Claude

    1996-01-01

    Purpose: To investigate the influence of therapeutic parameters on local control in T2-T3 anal carcinoma treated by chemo-radiotherapy or radiation therapy alone. Materials and Methods: From 1976 to 1993, 137 patients with anal carcinoma staged T2 (85) or T3 (52) completed curative sphincter-conservating treatment, 54 with radiotherapy alone and 83 with concomitant chemo-radiotherapy. Radiation therapy was delivered in two sequences with a median gap of 46 days. The two main techniques used for the first sequence were a direct perineal cobalt field ± a sacral arc field with a median dose of 30 Gy/10 fractions/19 days (27.7%) and two antero-posterior opposed pelvic fields (≥ 6 MV photons) with a median dose of 40 Gy/20 fractions/31 days (62%). Iridium-192 implant boost was used in 116 patients (median dose 20 Gy, Paris system) and external radiation boost in 21 patients (median dose 20 Gy/10 fractions/13 days). Generally the chemoradiotherapy patients received starting on day 1 an IV bolus of Mitomycin-C (0.4 mg/kg, maximum 20 mg) and a 5-day continuous infusion of 5-fluorourcil 600-800 mg/m 2 /day. For surviving patients median follow-up was 65 months. Univariate and multivariate analyses were carried out to determine therapeutic parameters affecting local control after adjustment for clinical factors. Results: The 5-year actuarial local control was 77%. Factors associated with a decrease of local control in univariate analysis included: age less than 66 years (67% vs 85%), male gender (65% vs 81%), tumor extension more than (1(3)) circumference of the anal canal (68% vs 90%), lymph node involvement (64% vs 81%), use of external irradiation for the boost (62% vs 79%), and overall treatment time more than 74 days (69% vs 85%). In a multivariate analysis none of the therapeutic parameters remained significant when adjusted for the four significant clinical factors. The only therapeutic factor which might have had an impact was overall treatment time (p = .09

  9. Preoperative chemoradiation with cisplatin and 5-fluorouracil for extraperitoneal T3 rectal cancer: acute toxicity, tumor response, sphincter preservation

    International Nuclear Information System (INIS)

    Valentini, Vincenzo; Coco, Claudio; Cellini, Numa; Picciocchi, Aurelio; Rosetto, Maria Elena; Mantini, Giovanna; Marmiroli, Luca; Barbaro, Brunella; Cogliandolo, Santa; Nuzzo, Gennaro; Tedesco, Manfredo; Ambesi-Impiombato, Fabrizio; Cosimelli, Maurizio; Rotman, Marvin

    1999-01-01

    Purpose: To evaluate the impact of preoperative external radiation therapy intensified by systemic chemotherapy including bolus cisplatin (c-DDP) and 4-day infusional 5-fluorouracil (PLAFUR-4) on tumor response and sphincter preservation in patients with extraperitoneal T3 rectal cancer with acceptable toxicity, and to compare the results to our previous experience with bolus mitomycin c (MMC) and 4-day infusion 5-FU (FUMIR). Methods and Materials: Between October 1995 and March 1998, 40 consecutive patients with resectable extraperitoneal adenocarcinoma of the rectum were treated with preoperative chemoradiation: slow infusion iv c-DDP, 60 mg/m 2 , day 1 and 29 plus 24-h continuous infusion iv 5-fluorouracil (5-FU) 1000 mg/m 2 , days 1-4 and 29-32, and concurrent external beam radiotherapy (45 Gy whole pelvis followed by 5.4 Gy boost). All but 3 patients had T3 disease. Surgery was performed 6-8 weeks after the end of chemoradiation. Results: No patient had Grade 4 acute toxicity. Grade 3 hematological toxicity was observed only in 2 (5%) patients. No patient had major gastrointestinal, skin, or urological acute toxicity. All patients had radical surgery. There was no perioperative mortality; perioperative morbidity rate was 12%. Overall, 23% (9 of 40) of patients had a complete pathological response and 10% (4 of 40) of patients had rare isolated residual cancer cells (Tmic). Comparing the stage at the diagnostic workup with the pathological stage, tumor downstaging was observed in 27 (68%) patients; nodal status downstaging was detected in 24 (60%) patients. Thirty-four (85%) patients had a sphincter-saving surgical procedure. In 4 of 10 (40%) patients who were definitive candidates for an abdominoperineal resection (APR), the sphincter was preserved, as it was in 13 of 13 (100%) probable candidates. Lengthening of the distance between the anorectal ring and the lower pole of the tumor ≥ 20 mm was observed in 9 (23%) patients. None of the patients had soilage

  10. Delivery of the autofluorescent protein R-phycoerythrin by calcium phosphate nanoparticles into four different eukaryotic cell lines (HeLa, HEK293T, MG-63, MC3T3: Highly efficient, but leading to endolysosomal proteolysis in HeLa and MC3T3 cells.

    Directory of Open Access Journals (Sweden)

    Mathis Kopp

    Full Text Available Nanoparticles can be used as carriers to transport biomolecules like proteins and synthetic molecules across the cell membrane because many molecules are not able to cross the cell membrane on their own. The uptake of nanoparticles together with their cargo typically occurs via endocytosis, raising concerns about the possible degradation of the cargo in the endolysosomal system. As the tracking of a dye-labelled protein during cellular uptake and processing is not indicative of the presence of the protein itself but only for the fluorescent label, a label-free tracking was performed with the red-fluorescing model protein R-phycoerythrin (R-PE. Four different eukaryotic cell lines were investigated: HeLa, HEK293T, MG-63, and MC3T3. Alone, the protein was not taken up by any cell line; only with the help of calcium phosphate nanoparticles, an efficient uptake occurred. After the uptake into HeLa cells, the protein was found in early endosomes (shown by the marker EEA1 and lysosomes (shown by the marker Lamp1. There, it was still intact and functional (i.e. properly folded as its red fluorescence was detected. However, a few hours after the uptake, proteolysis started as indicated by the decreasing red fluorescence intensity in the case of HeLa and MC3T3 cells. 12 h after the uptake, the protein was almost completely degraded in HeLa cells and MC3T3 cells. In HEK293T cells and MG-63 cells, no degradation of the protein was observed. In the presence of Bafilomycin A1, an inhibitor of acidification and protein degradation in lysosomes, the fluorescence of R-PE remained intact over the whole observation period in the four cell lines. These results indicate that despite an efficient nanoparticle-mediated uptake of proteins by cells, a rapid endolysosomal degradation may prevent the desired (e.g. therapeutic effect of a protein inside a cell.

  11. First multi-epitope subunit vaccine against extraintestinal pathogenic Escherichia coli delivered by a bacterial type-3 secretion system (T3SS).

    Science.gov (United States)

    Wieser, Andreas; Magistro, Giuseppe; Nörenberg, Dominik; Hoffmann, Christiane; Schubert, Sören

    2012-01-01

    Infections due to extraintestinal pathogenic E. coli (ExPEC) are very common in humans as well as in animals. In humans ExPEC infections include urinary tract infections (UTI), septicemia, and wound infections, which result in significant morbidity, mortality, and substantial healthcare costs. In view of the increasing number of ExPEC infections caused by more and more resistant strains, effective prevention would be desirable. Given the rising treatment costs, a vaccine may be cost-effective in selected patient groups, such as women with recurrent UTI, patients with neurologic disorders impairing bladder function and men with prostate hyperplasia. Previous vaccine studies used single target proteins or whole inactivated ExPEC cells. Here, we describe a vaccine system for oral application based on artificial multiple subunit vaccine proteins. Those multi-epitope proteins are composed of predicted epitopes derived from ExPEC virulence-associated proteins. As ExPEC are known to form intracellular biofilms in the urothelium and can also resist killing by non-activated macrophages, T-cell responses are supposed to be an important measure to counteract these stages of ExPEC during infection. Therefore, a live bacterial antigen delivery system based upon the Salmonella type-III secretion system (T3SS) was used in this study to directly deliver the vaccine proteins into the cytoplasm of the host cells. Epitope-rich domains of the proteins FyuA, IroN, ChuA, IreA, Iha, and Usp were expressed in an attenuated Salmonella enterica serovar Typhimurium strain and translocated into target cells for extended periods of time inducing a strong T-cell response. No significant antibody titre increase against the secreted vaccine proteins could be detected in vaginal wash or serum. Despite that, one of the vaccine proteins was able to significantly reduce bacterial load in the challenge model of intraperitoneal sepsis. This study shows that a vaccine encompassing distinct epitopes of

  12. Cinnamon extract enhances glucose uptake in 3T3-L1 adipocytes and C2C12 myocytes by inducing LKB1-AMP-activated protein kinase signaling.

    Directory of Open Access Journals (Sweden)

    Yan Shen

    Full Text Available 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.

  13. Molecular characterization of the t(3;9) associated with immortalization in the MCF10A cell line.

    Science.gov (United States)

    Cowell, John K; LaDuca, Jeffrey; Rossi, Michael R; Burkhardt, Tania; Nowak, Norma J; Matsui, Sei-ichi

    2005-11-01

    The t(3;9)(p14;p21) in the MCF10A human mammary gland epithelial cell line was the single cytogenetic event that accompanied the transition from primary culture to immortalized cell line, suggesting that it is related to the development of the immortalization phenotype. To study the molecular consequences of the breakpoints in this rearrangement, we used a combination of fluorescence in situ hybridization (FISH) and array comparative genomic hybridization (CGH). The 3p14 translocation breakpoint occurs within BAC RP11-795e22, which accommodates only the TAFA1 gene, a novel cysteine-rich secreted protein thought to be involved in cytokine signaling. TAFA1 is expressed in normal breast tissue, not in MCF10A, and shows differential expression in a range of breast cancer cell lines. The 9p translocation breakpoint results in a deletion of approximately 4 megabases on the derivative chromosome 9, which includes the CDKN2A (p16) gene. Array CGH and FISH analysis demonstrated that BAC 149i22, which contains the CDKN2A/B genes, is also deleted specifically on the apparently normal copy of chromosome 9, making MCF10A null for the p16/p15 genes. The exact extent of gains and losses of chromosome regions resulting from rearrangements involving chromosomes 1q, 5q, and 8q have also been characterized using the BAC arrays.

  14. Effects of clozapine on adipokine secretions/productions and lipid droplets in 3T3-L1 adipocytes.

    Science.gov (United States)

    Tsubai, Tomomi; Yoshimi, Akira; Hamada, Yoji; Nakao, Makoto; Arima, Hiroshi; Oiso, Yutaka; Noda, Yukihiro

    2017-02-01

    Clozapine, a second-generation antipsychotic (SGA), is a cause of side effects related to metabolic syndrome. The participation of serotonin 5-HT 2C and histamine H 1 receptors in the central nervous system has been reported as a mechanism of the weight gain caused by clozapine. In the present study, we investigated the direct pharmacological action of clozapine on the 3T3-L1 adipocytes and compared it to that of blonanserin, an SGA with low affinity for both receptors. Short-term exposure to clozapine decreased secretion and mRNA expression of leptin. Long-term exposure decreased leptin as well as adiponectin secretion, and further increased lipid droplets accumulation. However, short- and long-term exposures to blonanserin did not affect these parameters. A selective serotonin 5-HT 2C , but not a histamine H 1 , receptor antagonist enhanced the decreased secretion of leptin induced by short-term exposure to clozapine, but did not affect the increased accumulation of lipid droplets. Our findings indicate that clozapine, but not blonanserin, strongly and directly affected the secretion of adipokines, such as leptin, in adipocytes and caused adipocyte enlargement. Copyright © 2017 The Authors. Production and hosting by Elsevier B.V. All rights reserved.

  15. Traf2 interacts with Smad4 and regulates BMP signaling pathway in MC3T3-E1 osteoblasts

    International Nuclear Information System (INIS)

    Shimada, Koichi; Ikeda, Kyoko; Ito, Koichi

    2009-01-01

    Bone morphogenetic proteins (BMPs) play important roles in osteoblast differentiation and maturation. In mammals, the BMP-induced receptor-regulated Smads form complexes with Smad4. These complexes translocate and accumulate in the nucleus, where they regulate the transcription of various target genes. However, the function of Smad4 remains unclear. We performed a yeast two-hybrid screen using Smad4 as bait and a cDNA library derived from bone marrow, to indentify the proteins interacting with Smad4. cDNA clones for Tumor necrosis factor (TNF) receptor-associated factor 2 (Traf2) were identified, and the interaction between the endogenous proteins was confirmed in the mouse osteoblast cell line MC3T3-E1. To investigate the function of Traf2, we silenced it with siRNA. The level of BMP-2 protein in the medium, the expression levels of the Bmp2 gene and BMP-induced transcription factor genes, including Runx2, Dlx5, Msx2, and Sp7, and the phosphorylated-Smad1 protein level were increased in cells transfected with Traf2 siRNA. The nuclear accumulation of Smad1 increased with TNF-α stimulation for 30 min at Traf2 silencing. These results suggest that the TNF-α-stimulated nuclear accumulation of Smad1 may be dependent on Traf2. Thus, the interaction between Traf2 and Smad4 may play a role in the cross-talk between TNF-α and BMP signaling pathways.

  16. Rabbit monoclonal antibodies directed at the T3SS effector protein YopM identify human pathogenic Yersinia isolates.

    Science.gov (United States)

    Rüter, Christian; Silva, Mariana Ruiz; Grabowski, Benjamin; Lubos, Marie-Luise; Scharnert, Julia; Poceva, Marija; von Tils, Dominik; Flieger, Antje; Heesemann, Jürgen; Bliska, James B; Schmidt, M Alexander

    2014-05-01

    The Yersinia outer protein M (YopM) is a type 3 secretion system (T3SS)-dependent effector protein of Yersinia enterocolitica, Yersinia pseudotuberculosis and Yersinia pestis. Although YopM is indispensable for full virulence, its molecular functions still remain largely elusive. Recently, we could identify the recombinant YopM (rYopM) protein derived from the Y. enterocolitica strain 8081 (JB580) as a cell-penetrating protein, which down-regulates the expression of various pro-inflammatory cytokines including TNFα. In this study, we have generated rabbit monoclonal anti-YopM antibodies (RabMabs). RabMabs were characterized by SDS-PAGE and Western blotting using various truncated versions of rYopM to identify epitope-containing domains. RabMabs recognizing either the N- or C-terminus of YopM were characterized further and validated using a collection of 61 pathogenic and non-pathogenic Yersinia strains as well as exemplary strains of major intestinal bacterial pathogens such as Salmonella enterica ssp. enterica, Shigella flexneri and intestinal pathogenic Escherichia coli. RabMab 41.3 directed at the N-terminus of YopM of Y. enterocolitica strain 8081 recognized all YopM-expressing pathogenic Yersinia strains analyzed in this study but failed to recognize non-pathogenic isolates. Thus, RabMab 41.3 might be applicable for the detection of pathogenic Yersinia strains. Copyright © 2014 Elsevier GmbH. All rights reserved.

  17. Preparation of Monoclonal Antibodies Against SpiC Protein Secreted by T3SS-2 of Salmonella spp.

    Science.gov (United States)

    Geng, Shizhong; Qian, Shanshan; Pan, Zhiming; Sun, Lin; Chen, Xiang; Jiao, Xinan

    2015-12-01

    SpiC protein, a member of Salmonella spp. type III secretion system (T3SS)-2, is necessary for the survival of Salmonella within macrophages, and it plays a vital role in Salmonella pathogenesis. To develop and test monoclonal antibodies (MAbs) against SpiC protein, two recombinant proteins, rHis-SpiC and rGST-SpiC, were expressed in vitro in the prokaryotic expression vectors pET-30(a) and pGEX-6p-1, respectively, and rHis-SpiC protein used to immunize mice. Hybridomas were generated from the splenocytes of these mice and the monoclonal antibodies produced by these cells were assessed using indirect enzyme-linked immunosorbent assay (ELISA) with rGST-SpiC as the coating antigen. An immunoblotting analysis indicated that all seven of the MAbs developed in this study could specifically recognize the SpiC protein. These MAbs will be very useful in the study of SpiC function and for use in the immunodiagnosis of Salmonella infection.

  18. Effect of hierarchical pore structure on ALP expression of MC3T3-E1 cells on bioglass films.

    Science.gov (United States)

    Yu, Cuixia; Zhuang, Junjun; Dong, Lingqing; Cheng, Kui; Weng, Wenjian

    2017-08-01

    Hierarchical porous bioglass films on the tantalum were designed to enhance osteointegration of metallic implants. The films were prepared by a sol-gel method using P123 as the mesopore template and polystyrene microsphere as the nanopore template. The films with 5.4nm mesopores and 100nm nanopores (MBG-100) elicited an obviously elongated morphology of the cultured MC3T3-E1 cells, as a result, a higher alkaline phosphatase level was expressed. It is suggested that the nanopores play an important role in regulating cellular behavior by initial protein adsorption through nanopore curvatures. The mesopores were proven very effective for loading rhBMP-2, and the rhBMP-2 loaded on MBG-100 films showed a better function of enhancing osteogenic differentiation, which is attributed to that the nanopore structure could expedite rhBMP-2 release and provide a microenvironment for intensifying the interaction of rhBMP-2 with the cells. Hence, the cell osteogenic differentiation can be enhanced by hierarchical porous bioglass films through both the porous structure and rhBMP-2 induction. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Insulin induces an increase in cytosolic glucose levels in 3T3-L1 cells with inhibited glycogen synthase activation.

    Science.gov (United States)

    Chowdhury, Helena H; Kreft, Marko; Jensen, Jørgen; Zorec, Robert

    2014-10-02

    Glucose is an important source of energy for mammalian cells and enters the cytosol via glucose transporters. It has been thought for a long time that glucose entering the cytosol is swiftly phosphorylated in most cell types; hence the levels of free glucose are very low, beyond the detection level. However, the introduction of new fluorescence resonance energy transfer-based glucose nanosensors has made it possible to measure intracellular glucose more accurately. Here, we used the fluorescent indicator protein (FLIPglu-600µ) to monitor cytosolic glucose dynamics in mouse 3T3-L1 cells in which glucose utilization for glycogen synthesis was inhibited. The results show that cells exhibit a low resting cytosolic glucose concentration. However, in cells with inhibited glycogen synthase activation, insulin induced a robust increase in cytosolic free glucose. The insulin-induced increase in cytosolic glucose in these cells is due to an imbalance between the glucose transported into the cytosol and the use of glucose in the cytosol. In untreated cells with sensitive glycogen synthase activation, insulin stimulation did not result in a change in the cytosolic glucose level. This is the first report of dynamic measurements of cytosolic glucose levels in cells devoid of the glycogen synthesis pathway.

  20. Genes Responsive to Low-Intensity Pulsed Ultrasound in MC3T3-E1 Preosteoblast Cells

    Directory of Open Access Journals (Sweden)

    Yoshiaki Tabuchi

    2013-11-01

    Full Text Available Although low-intensity pulsed ultrasound (LIPUS has been shown to enhance bone fracture healing, the underlying mechanism of LIPUS remains to be fully elucidated. Here, to better understand the molecular mechanism underlying cellular responses to LIPUS, we investigated gene expression profiles in mouse MC3T3-E1 preosteoblast cells exposed to LIPUS using high-density oligonucleotide microarrays and computational gene expression analysis tools. Although treatment of the cells with a single 20-min LIPUS (1.5 MHz, 30 mW/cm2 did not affect the cell growth or alkaline phosphatase activity, the treatment significantly increased the mRNA level of Bglap. Microarray analysis demonstrated that 38 genes were upregulated and 37 genes were downregulated by 1.5-fold or more in the cells at 24-h post-treatment. Ingenuity pathway analysis demonstrated that the gene network U (up contained many upregulated genes that were mainly associated with bone morphology in the category of biological functions of skeletal and muscular system development and function. Moreover, the biological function of the gene network D (down, which contained downregulated genes, was associated with gene expression, the cell cycle and connective tissue development and function. These results should help to further clarify the molecular basis of the mechanisms of the LIPUS response in osteoblast cells.

  1. Effects of clozapine on adipokine secretions/productions and lipid droplets in 3T3-L1 adipocytes

    Directory of Open Access Journals (Sweden)

    Tomomi Tsubai

    2017-02-01

    Full Text Available Clozapine, a second-generation antipsychotic (SGA, is a cause of side effects related to metabolic syndrome. The participation of serotonin 5-HT2C and histamine H1 receptors in the central nervous system has been reported as a mechanism of the weight gain caused by clozapine. In the present study, we investigated the direct pharmacological action of clozapine on the 3T3-L1 adipocytes and compared it to that of blonanserin, an SGA with low affinity for both receptors. Short-term exposure to clozapine decreased secretion and mRNA expression of leptin. Long-term exposure decreased leptin as well as adiponectin secretion, and further increased lipid droplets accumulation. However, short- and long-term exposures to blonanserin did not affect these parameters. A selective serotonin 5-HT2C, but not a histamine H1, receptor antagonist enhanced the decreased secretion of leptin induced by short-term exposure to clozapine, but did not affect the increased accumulation of lipid droplets. Our findings indicate that clozapine, but not blonanserin, strongly and directly affected the secretion of adipokines, such as leptin, in adipocytes and caused adipocyte enlargement.

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

    Science.gov (United States)

    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 abolis