Solar power generation system for reducing leakage current

NASA Astrophysics Data System (ADS)

Wu, Jinn-Chang; Jou, Hurng-Liahng; Hung, Chih-Yi

2018-04-01

This paper proposes a transformer-less multi-level solar power generation system. This solar power generation system is composed of a solar cell array, a boost power converter, an isolation switch set and a full-bridge inverter. A unipolar pulse-width modulation (PWM) strategy is used in the full-bridge inverter to attenuate the output ripple current. Circuit isolation is accomplished by integrating the isolation switch set between the solar cell array and the utility, to suppress the leakage current. The isolation switch set also determines the DC bus voltage for the full-bridge inverter connecting to the solar cell array or the output of the boost power converter. Accordingly, the proposed transformer-less multi-level solar power generation system generates a five-level voltage, and the partial power of the solar cell array is also converted to AC power using only the full-bridge inverter, so the power efficiency is increased. A prototype is developed to validate the performance of the proposed transformer-less multi-level solar power generation system.

Development of human cell models for assessing the carcinogenic potential of chemicals

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

Pang Yaqin; Li Wenxue; Ma Rulin

2008-11-01

To develop human cell models for assessing the carcinogenic potential of chemicals, we established transgenic human cell lines and tested the sensitivity of known carcinogens using a cell transformation assay. A retroviral vector encoding an oncogenic allele of H-Ras (HBER) or c-Myc (HBEM) was introduced into human bronchial epithelial cells (HBE) immortalized by SV40 large T (LT) antigen, leading to increased cell proliferation but failing to confer a transformed phenotype characterized by anchorage-independent cell growth and tumor formation of immunodeficient mice. When these pre-transformed cells were treated with nickel sulfate (NiSO{sub 4}), we found that it shortened the latency ofmore » malignant transformation at least by 19 wk in HBER cells or 16 wk in HBEM cells compared to vector control cells. Similarly, the latency of cell transformation was shorter by 15 wk in HBER cells or 9 wk in HBEM cells when cells were treated with benzo(a)pyrenediol epoxide (BPDE). HBER cells appeared to be more sensitive to TPA, NiSO{sub 4} or BPDE-induced cell transformation compared to human embryonic kidney cells expressing H-Ras (HEKR), implying that cell-type specificity is one of important factors determining the effectiveness of the assay. Using AFB{sub 1} and BaP as the representative pro-carcinogens, we also compared the efficiency of three different metabolic conditions in mediating cell transformation. Low dose chemical induction seems to be a prospective system used for metabolic activation of pro-carcinogens. Our findings provided direct evidence that a genetically modified human cell transformation model can be applied to the assessment of potent carcinogens.« less

Molokhia (Corchorus olitorius L.) extract suppresses transformation of the aryl hydrocarbon receptor induced by dioxins.

PubMed

Nishiumi, Shin; Yabushita, Yoshiyuki; Fukuda, Itsuko; Mukai, Rie; Yoshida, Ken-Ichi; Ashida, Hitoshi

2006-02-01

Dioxins enter the body mainly through diet and cause the various toxicological effects by binding to the cytosolic aryl hydrocarbon receptor (AhR) followed by its transformation. In recent reports, it has been shown that certain natural compounds suppress AhR transformation in vitro. In this study, we demonstrated that ethanolic extract from molokhia, known as Egyptian spinach, showed the strongest suppressive effect on AhR transformation induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in cell-free system using rat hepatic cytosol among 41 kinds of extracts from vegetables and fruits. The molokhia extract also suppressed TCDD-induced AhR transformation in mouse hepatoma Hepa-1c1c7 cells and in intestinal permeability system constructed with human colon adenocarcinoma Caco-2 cells and human hepatoma HepG2 cells. Moreover, oral administration of the molokhia extract (100mg/kg body weight) decreased 3-methylcholanthrene-induced AhR transformation to the control level by inhibiting translocation of the AhR from cytosol into the nucleus in the liver of rats. The molokhia extract-administered rat liver showed a tolerance to TCDD-induced AhR transformation by ex vivo experiment. These results indicate that molokhia is an attractive food for isolation and identification of a natural antagonist for the AhR.

M2 polarization of macrophages facilitates arsenic-induced cell transformation of lung epithelial cells

PubMed Central

Li, Hui; Dai, Lu; Frank, Jacqueline A.; Peng, Shaojun; Wang, Siying; Chen, Gang

2017-01-01

The alterations in microenvironment upon chronic arsenic exposure may contribute to arsenic-induced lung carcinogenesis. Immune cells, such as macrophages, play an important role in mediating the microenvironment in the lungs. Macrophages carry out their functions after activation. There are two activation status for macrophages: classical (M1) or alternative (M2); the latter is associated with tumorigenesis. Our previous work showed that long-term arsenic exposure induces transformation of lung epithelial cells. However, the crosstalk between epithelial cells and macrophages upon arsenic exposure has not been investigated. In this study, using a co-culture system in which human lung epithelial cells are cultured with macrophages, we determined that long-term arsenic exposure polarizes macrophages towards M2 status through ROS generation. Co-culture with epithelial cells further enhanced the polarization of macrophages as well as transformation of epithelial cells, while blocking macrophage M2 polarization decreased the transformation. In addition, macrophage M2 polarization decreased autophagy activity, which may account for increased cell transformation of epithelial cells with co-culture of macrophages. PMID:28423485

Rab5-regulated endocytosis plays a crucial role in apical extrusion of transformed cells.

PubMed

Saitoh, Sayaka; Maruyama, Takeshi; Yako, Yuta; Kajita, Mihoko; Fujioka, Yoichiro; Ohba, Yusuke; Kasai, Nobuhiro; Sugama, Natsu; Kon, Shunsuke; Ishikawa, Susumu; Hayashi, Takashi; Yamazaki, Tomohiro; Tada, Masazumi; Fujita, Yasuyuki

2017-03-21

Newly emerging transformed cells are often eliminated from epithelial tissues. Recent studies have revealed that this cancer-preventive process involves the interaction with the surrounding normal epithelial cells; however, the molecular mechanisms underlying this phenomenon remain largely unknown. In this study, using mammalian cell culture and zebrafish embryo systems, we have elucidated the functional involvement of endocytosis in the elimination of RasV12-transformed cells. First, we show that Rab5, a crucial regulator of endocytosis, is accumulated in RasV12-transformed cells that are surrounded by normal epithelial cells, which is accompanied by up-regulation of clathrin-dependent endocytosis. Addition of chlorpromazine or coexpression of a dominant-negative mutant of Rab5 suppresses apical extrusion of RasV12 cells from the epithelium. We also show in zebrafish embryos that Rab5 plays an important role in the elimination of transformed cells from the enveloping layer epithelium. In addition, Rab5-mediated endocytosis of E-cadherin is enhanced at the boundary between normal and RasV12 cells. Rab5 functions upstream of epithelial protein lost in neoplasm (EPLIN), which plays a positive role in apical extrusion of RasV12 cells by regulating protein kinase A. Furthermore, we have revealed that epithelial defense against cancer (EDAC) from normal epithelial cells substantially impacts on Rab5 accumulation in the neighboring transformed cells. This report demonstrates that Rab5-mediated endocytosis is a crucial regulator for the competitive interaction between normal and transformed epithelial cells in mammals.

Does the immune reaction cause malignant transformation by disrupting cell-to-cell or cell-to-matrix communications?

PubMed

Prehn, Richmond T

2007-05-04

TUMOR PROGRESSION: In many (perhaps in all) tumor systems, a malignant cancer is preceded by a benign lesion. Most benign lesions do not transform to malignancy and many regress. The final transformative step to malignancy differs from the preceding steps in, among other things, that it often occurs in the absence of the original carcinogenic stimulus. Relatively low titers of specific immune reactants are known to stimulate, but cell-to-cell or cell-to-matrix interactions appear to be major inhibitors of tumor-growth. Therefore, it seems reasonable to hypothesize that the mechanism of immunostimulation may be an interference with cell-to-cell or cell-to-matrix communication by a sub-lethal immune-reaction. While the above hypothesis remains unproven, some evidence suggests that immunity may have a major facilitating effect on tumor growth especially at the time of malignant transformation. There is even some evidence suggesting that transformation in vivo may seldom occur in the absence of immunostimulation of the premalignant lesion. Positive selection by the immune reaction may be the reason that tumors are immunogenic.

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

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

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

1990-01-01

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

Mechanisms of Radiation Toxicity in Transformed and Non-Transformed Cells

PubMed Central

Panganiban, Ronald-Allan M.; Snow, Andrew L.; Day, Regina M.

2013-01-01

Radiation damage to biological systems is determined by the type of radiation, the total dosage of exposure, the dose rate, and the region of the body exposed. Three modes of cell death—necrosis, apoptosis, and autophagy—as well as accelerated senescence have been demonstrated to occur in vitro and in vivo in response to radiation in cancer cells as well as in normal cells. The basis for cellular selection for each mode depends on various factors including the specific cell type involved, the dose of radiation absorbed by the cell, and whether it is proliferating and/or transformed. Here we review the signaling mechanisms activated by radiation for the induction of toxicity in transformed and normal cells. Understanding the molecular mechanisms of radiation toxicity is critical for the development of radiation countermeasures as well as for the improvement of clinical radiation in cancer treatment. PMID:23912235

Cereal transformation through particle bombardment

NASA Technical Reports Server (NTRS)

Casas, A. M.; Kononowicz, A. K.; Bressan, R. A.; Hasegawa, P. M.; Mitchell, C. A. (Principal Investigator)

1995-01-01

The review focuses on experiments that lead to stable transformation in cereals using microprojectile bombardment. The discussion of biological factors that affect transformation examines target tissues and vector systems for gene transfer. The vector systems include reporter genes, selectable markers, genes of agronomic interest, and vector constructions. Other topics include physical parameters that affect DNA delivery, selection of stably transformed cells and plant regeneration, and analysis of gene expression and transmission to the progeny.

Efficient Transformation of Primary Human Mesenchymal Stromal Cells by Adenovirus Early Region 1 Oncogenes.

PubMed

Speiseder, Thomas; Hofmann-Sieber, Helga; Rodríguez, Estefanía; Schellenberg, Anna; Akyüz, Nuray; Dierlamm, Judith; Spruss, Thilo; Lange, Claudia; Dobner, Thomas

2017-01-01

Previous observations that human amniotic fluid cells (AFC) can be transformed by human adenovirus type 5 (HAdV-5) E1A/E1B oncogenes prompted us to identify the target cells in the AFC population that are susceptible to transformation. Our results demonstrate that one cell type corresponding to mesenchymal stem/stroma cells (hMSCs) can be reproducibly transformed by HAdV-5 E1A/E1B oncogenes as efficiently as primary rodent cultures. HAdV-5 E1-transformed hMSCs exhibit all properties commonly associated with a high grade of oncogenic transformation, including enhanced cell proliferation, anchorage-independent growth, increased growth rate, and high telomerase activity as well as numerical and structural chromosomal aberrations. These data confirm previous work showing that HAdV preferentially transforms cells of mesenchymal origin in rodents. More importantly, they demonstrate for the first time that human cells with stem cell characteristics can be completely transformed by HAdV oncogenes in tissue culture with high efficiency. Our findings strongly support the hypothesis that undifferentiated progenitor cells or cells with stem cell-like properties are highly susceptible targets for HAdV-mediated cell transformation and suggest that virus-associated tumors in humans may originate, at least in part, from infections of these cell types. We expect that primary hMSCs will replace the primary rodent cultures in HAdV viral transformation studies and are confident that these investigations will continue to uncover general principles of viral oncogenesis that can be extended to human DNA tumor viruses as well. It is generally believed that transformation of primary human cells with HAdV-5 E1 oncogenes is very inefficient. However, a few cell lines have been successfully transformed with HAdV-5 E1A and E1B, indicating that there is a certain cell type which is susceptible to HAdV-mediated transformation. Interestingly, all those cell lines have been derived from human embryonic tissue, albeit the exact cell type is not known yet. We show for the first time the successful transformation of primary human mesenchymal stromal cells (hMSCs) by HAdV-5 E1A and E1B. Further, we show upon HAdV-5 E1A and E1B expression that these primary progenitor cells exhibit features of tumor cells and can no longer be differentiated into the adipogenic, chondrogenic, or osteogenic lineage. Hence, primary hMSCs represent a robust and novel model system to elucidate the underlying molecular mechanisms of adenovirus-mediated transformation of multipotent human progenitor cells. Copyright © 2016 American Society for Microbiology.

[Inhibiting target gene expression and controlling growth of Epstein-Barr virus transformed cells by antisense RNA transcripts].

PubMed

Chen, Jian-jing; Raab-Traub, Nancy; Yao, Qing-yun; Zhang, Feng; Huang, Mei-ling; Kuang, Zhu-ji; Zhang, Xiao-shi; Ye, Yan-li; Gu, Li

2002-01-01

The latent membrane protein gene (LMP) of Epstein-Barr virus (EBV) was thought to play an important role in the carcinogenesis of nasopharyngeal carcinoma (NPC). In this study, the authors investigated the effects of antisense RNA (AsRNA) on LMP for down regulating at the target gene over expression in EBV transformed lymphoid cells, and set up an antisense system to inhibit LMP expression. Constructing the single strand antisense transcription system in vitro, the authors have got large amount of AsRNA. Designing and setting up an antisense tracing system in situ (ATSIS), the authors could observe the living particles of AsRNA/sense RNA duplex dimer. With time lapse phase-contrast microscopy, the agglutination phenotype on living cells was easily detected by MTT test, the inhibition rate on EBV transformed cells was calculated. LMP 1.9 fragment ligated into pGEM vector in reverse orientation have been constructed and produced a plentiful amount of AsLMPmRNA which could incorporated into both B95-8 and C1936 cell lines by endophagocytosis and formed the duplex dimer of As/Sense RNA. This particles have been visualized in situ when labelling 35S isotope by ATSIS. When AsLMPmRNA acted as agents for specific inhibition to LMP over expression, the transform phenotype of cell agglutination have been suppressed and MTT particle formatin was apparently reduced both two EBV tansformed cell lines. AsLMPmRNA targets at sense strand have a high effectiveness of down-regulation on EBV-LMP overexpression. This down regulating function of LMP and growth inhibition on transformed cell is demonstrated by the antisenes tracing system in situ (ATSIS). The results provide a clue to overcome the latent EBV infection in human bodies all living long time and to prevent it inducing NPC in high incidence area by antisense strategies.

Cooperative transforming activities of ras, myc, and src viral oncogenes in nonestablished rat adrenocortical cells.

PubMed Central

MacAuley, A; Pawson, T

1988-01-01

Early-passage rat adrenocortical cells were infected with Kirsten murine sarcoma virus and MMCV mouse myc virus, two retroviruses carrying the v-Ki-ras and v-myc oncogenes, respectively. Efficient morphological transformation required coinfection with the two viruses, was dependent on the presence of high serum concentrations, and was not immediately accompanied by growth in soft agar. The doubly infected cells coordinately acquired the capacity for anchorage- and serum-independent growth during passage in culture. The appearance of such highly transformed cells was correlated with the emergence of a dominant clone, as suggested by an analysis of retrovirus integration sites. These results indicate that the concerted expression of v-Ki-ras and v-myc could induce rapid morphological transformation of nonestablished adrenocortical cells but that an additional genetic or epigenetic event was required to permit full transformation by these two oncogenes. In contrast, v-src, introduced by retrovirus infection in conjunction with v-myc, rapidly induced serum- and anchorage-independent growth. Therefore, the p60v-src protein-tyrosine kinase, unlike p21v-ras, is apparently not restricted in the induction of a highly transformed phenotype in adrenocortical cells. This system provides an in vitro model for the progressive transformation of epithelial cells by dominantly acting oncogenes. Images PMID:2846881

Whole-cell fungal transformation of precursors into dyes

PubMed Central

2010-01-01

Background Chemical methods of producing dyes involve extreme temperatures and unsafe toxic compounds. Application of oxidizing enzymes obtained from fungal species, for example laccase, is an alternative to chemical synthesis of dyes. Laccase can be replaced by fungal biomass acting as a whole-cell biocatalyst with properties comparable to the isolated form of the enzyme. The application of the whole-cell system simplifies the transformation process and reduces the time required for its completion. In the present work, four fungal strains with a well-known ability to produce laccase were tested for oxidation of 17 phenolic and non-phenolic precursors into stable and non-toxic dyes. Results An agar-plate screening test of the organic precursors was carried out using four fungal strains: Trametes versicolor, Fomes fomentarius, Abortiporus biennis, and Cerrena unicolor. Out of 17 precursors, nine were transformed into coloured substances in the presence of actively growing fungal mycelium. The immobilized fungal biomass catalyzed the transformation of 1 mM benzene and naphthalene derivatives in liquid cultures yielding stable and non-toxic products with good dyeing properties. The type of fungal strain had a large influence on the absorbance of the coloured products obtained after 48-hour transformation of the selected precursors, and the most effective was Fomes fomentarius (FF25). Whole-cell transformation of AHBS (3-amino-4-hydroxybenzenesulfonic acid) into a phenoxazinone dye was carried out in four different systems: in aqueous media comprising low amounts of carbon and nitrogen source, in buffer, and in distilled water. Conclusions This study demonstrated the ability of four fungal strains belonging to the ecological type of white rot fungi to transform precursors into dyes. This paper highlights the potential of fungal biomass for replacing isolated enzymes as a cheaper industrial-grade biocatalyst for the synthesis of dyes and other commercially important products. The use of immobilized fungal biomass limits free migration of cells and facilitates their reuse in a continuous system for precursor transformation. PMID:20598166

Generation of SV40-transformed rabbit tracheal-epithelial-cell-derived blastocyst by somatic cell nuclear transfer

PubMed Central

de Semir, D.; Maurisse, R.; Du, F.; Xu, J.; Yang, X.; Illek, B.; Gruenert, D. C.

2013-01-01

The prospect of developing large animal models for the study of inherited diseases, such as cystic fibrosis (CF), through somatic cell nuclear transfer (SCNT) has opened up new opportunities for enhancing our understanding of disease pathology and for identifying new therapies. Thus, the development of species-specific in vitro cell systems that will provide broader insight into organ- and cell-type-specific functions relevant to the pathology of the disease is crucial. Studies have been undertaken to establish transformed rabbit airway epithelial cell lines that display differentiated features characteristic of the primary airway epithelium. This study describes the successful establishment and characterization of two SV40-transformed rabbit tracheal epithelial cell lines. These cell lines, 5RTEo- and 9RTEo-, express the CF transmembrane conductance regulator (CFTR) gene, retain epithelial-specific differentiated morphology and show CFTR-based cAMP-dependent Cl− ion transport across the apical membrane of a confluent monolayer. Immunocytochemical analysis indicates the presence of airway cytokeratins and tight-junction proteins in the 9RTEo- cell line after multiple generations. However, the tight junctions appear to diminish in their efficacy in both cell lines after at least 100 generations. Initial SCNT studies with the 9RTEo- cells have revealed that SV40-transformed rabbit airway epithelial donor cells can be used to generate blastocysts. These cell systems provide valuable models for studying the developmental and metabolic modulation of CFTR gene expression and rabbit airway epithelial cell biology. PMID:22234514

Advancing Crop Transformation in the Era of Genome Editing[OPEN

PubMed Central

Blechl, Ann E.; Brutnell, Thomas P.; Conrad, Liza J.; Gelvin, Stanton B.; Jackson, David P.; Kausch, Albert P.; Lemaux, Peggy G.; Medford, June I.; Orozco-Cárdenas, Martha L.; Tricoli, David M.; Van Eck, Joyce; Voytas, Daniel F.

2016-01-01

Plant transformation has enabled fundamental insights into plant biology and revolutionized commercial agriculture. Unfortunately, for most crops, transformation and regeneration remain arduous even after more than 30 years of technological advances. Genome editing provides novel opportunities to enhance crop productivity but relies on genetic transformation and plant regeneration, which are bottlenecks in the process. Here, we review the state of plant transformation and point to innovations needed to enable genome editing in crops. Plant tissue culture methods need optimization and simplification for efficiency and minimization of time in culture. Currently, specialized facilities exist for crop transformation. Single-cell and robotic techniques should be developed for high-throughput genomic screens. Plant genes involved in developmental reprogramming, wound response, and/or homologous recombination should be used to boost the recovery of transformed plants. Engineering universal Agrobacterium tumefaciens strains and recruiting other microbes, such as Ensifer or Rhizobium, could facilitate delivery of DNA and proteins into plant cells. Synthetic biology should be employed for de novo design of transformation systems. Genome editing is a potential game-changer in crop genetics when plant transformation systems are optimized. PMID:27335450

A Phase 1-2 Multi-Center Study Evaluating Axicabtagene Ciloleucel in Subjects With Refractory Aggressive Non-Hodgkin Lymphoma (ZUMA-1)

ClinicalTrials.gov

2018-06-18

Refractory Diffuse Large B Cell Lymphoma; Refractory Primary Mediastinal B Cell Lymphoma; Refractory Transformed Follicular Lymphoma; Relapsed/Refractory Transplant Ineligible Diffuse Large B Cell Lymphoma; Relapsed/Refractory Transplant Ineligible Primary Mediastinal B Cell Lymphoma; Relapsed/Refractory Transplant Ineligible Transformed Follicular Lymphoma; Relapsed/Refractory Large B Cell Lymphoma Including DLBCL, PMBCL, TFL and HGBCL After Two Systemic Lines of Therapy" in Phase 2 Expanded Cohorts

Transformation assay in Bhas 42 cells: a model using initiated cells to study mechanisms of carcinogenesis and predict carcinogenic potential of chemicals.

PubMed

Sasaki, Kiyoshi; Umeda, Makoto; Sakai, Ayako; Yamazaki, Shojiro; Tanaka, Noriho

2015-01-01

Transformation assays using cultured cells have been applied to the study of carcinogenesis. Although various cell systems exist, few cell types such as BALB/c 3T3 subclones and Syrian hamster embryo cells have been used to study chemically induced two-stage carcinogenesis. Bhas 42 cells were established as a clone by the transfection with the v-Ha-ras gene into mouse BALB/c 3T3 A31-1-1 cells and their subsequent selection based on their sensitivity to 12-O-tetradecanoylphorbol-13-acetate. Using Bhas 42 cells, transformed foci were induced by the treatment with nongenotoxic carcinogens, most of which act as tumor promoters. Therefore, Bhas 42 cells were considered to be a model of initiated cells. Subsequently, not only nongenotoxic carcinogens but also genotoxic carcinogens, most of which act as tumor initiators, were found to induce transformed foci by the modification of the protocol. Furthermore, transformation of Bhas 42 cells was induced by the transfection with genes of oncogenic potential. We interpret this high sensitivity of Bhas 42 cells to various types of carcinogenic stimuli to be related to the multistage model of carcinogenesis, as the transfection of v-Ha-ras gene further advances the parental BALB/c 3T3 A31-1-1 cells toward higher transforming potential. Thus, we propose that Bhas 42 cells are a novel and sensitive cell line for the analysis of carcinogenesis and can be used for the detection of not only carcinogenic substances but also gene alterations related to oncogenesis. This review will address characteristics of Bhas 42 cells, the transformation assay protocol, validation studies, and the various chemicals tested in this assay.

Does the immune reaction cause malignant transformation by disrupting cell-to-cell or cell-to-matrix communications?

PubMed Central

Prehn, Richmond T

2007-01-01

Tumor progression In many (perhaps in all) tumor systems, a malignant cancer is preceded by a benign lesion. Most benign lesions do not transform to malignancy and many regress. The final transformative step to malignancy differs from the preceding steps in, among other things, that it often occurs in the absence of the original carcinogenic stimulus. Mechanism of immunostimulation Relatively low titers of specific immune reactants are known to stimulate, but cell-to-cell or cell-to-matrix interactions appear to be major inhibitors of tumor-growth. Therefore, it seems reasonable to hypothesize that the mechanism of immunostimulation may be an interference with cell-to-cell or cell-to-matrix communication by a sub-lethal immune-reaction. Discussion While the above hypothesis remains unproven, some evidence suggests that immunity may have a major facilitating effect on tumor growth especially at the time of malignant transformation. There is even some evidence suggesting that transformation in vivo may seldom occur in the absence of immunostimulation of the premalignant lesion. Positive selection by the immune reaction may be the reason that tumors are immunogenic. PMID:17480231

The human T-lymphotropic virus type I tax gene can cooperate with the ras oncogene to induce neoplastic transformation of cells.

PubMed

Pozzatti, R; Vogel, J; Jay, G

1990-01-01

Epidemiologic studies have linked infection by the human T-lymphotropic virus type I (HTLV-I) with the development of adult T-cell leukemia. The low penetrance of the virus and the long latency for disease manifestation are factors that obscure the role of HTLV-I infection in oncogenesis. We have used an in vitro transformation assay system to determine directly whether the HTLV-I tax gene has transformation potential. Transfection of the tax gene alone into early-passage rat embryo fibroblasts did not induce morphological alterations. However, cotransfection of tax with the selectable marker plasmid pRSVneo gave rise to G418-resistant colonies that could be established as immortalized cell lines. Cotransfection of tax with the ras oncogene into rat embryo fibroblasts gave rise to foci of transformed cells that were highly tumorigenic in nude mice. These data represent a direct demonstration of the oncogenic potential of the tax gene in nonlymphoid cells and establish HTLV-I as a transforming virus.

Genetic transformation system in the archaebacterium Methanobacterium thermoautotrophicum Marburg.

PubMed Central

Worrell, V E; Nagle, D P; McCarthy, D; Eisenbraun, A

1988-01-01

A wild-type strain of Methanobacterium thermoautotrophicum Marburg was transformed by DNA from strains resistant to 5-fluorouracil. Recipient cells were grown without selection on gellan gum (GELRITE) plates with DNA. Drug-resistant cells were recovered by replica plating the resulting colonies onto drug plates. Transformation required high-molecular-weight DNA with appropriate markers and was not observed on agar or in liquid media under a variety of conditions. PMID:3422229

Development of human epithelial cell systems for radiation risk assessment

NASA Technical Reports Server (NTRS)

Yang, C. H.; Craise, L. M.

1994-01-01

The most important health effect of space radiation for astronauts is cancer induction. For radiation risk assessment, an understanding of carcinogenic effect of heavy ions in human cells is most essential. In our laboratory, we have successfully developed a human mammary epithelial cell system for studying the neoplastic transformation in vitro. Growth variants were obtained from heavy ion irradiated immortal mammary cell line. These cloned growth variants can grow in regular tissue culture media and maintain anchorage dependent growth and density inhibition property. Upon further irradiation with high-Linear Energy Transfer (LET) radiation, transformed foci were found. Experimental results from these studies suggest that multiexposure of radiation is required to induce neoplastic tranformation of human epithelial cells. This multihits requirement may be due to high genomic stability of human cells. These growth variants can be useful model systems for space flight experiments to determine the carcinogenic effect of space radiation in human epithelial cells.

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

PubMed

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

1997-04-01

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

Phase equilibrium modeling for high temperature metallization on GaAs solar cells

NASA Technical Reports Server (NTRS)

Chung, M. A.; Davison, J. E.; Smith, S. R.

1991-01-01

Recent trends in performance specifications and functional requirements have brought about the need for high temperature metallization technology to be developed for survivable DOD space systems and to enhance solar cell reliability. The temperature constitution phase diagrams of selected binary and ternary systems were reviewed to determine the temperature and type of phase transformation present in the alloy systems. Of paramount interest are the liquid-solid and solid-solid transformations. Data are being utilized to aid in the selection of electrical contact materials to gallium arsenide solar cells. Published data on the phase diagrams for binary systems is readily available. However, information for ternary systems is limited. A computer model is being developed which will enable the phase equilibrium predictions for ternary systems where experimental data is lacking.

Cadmium-induced malignant transformation of rat liver cells: Potential key role and regulatory mechanism of altered apolipoprotein E expression in enhanced invasiveness.

PubMed

Suzuki, Masayo; Takeda, Shuso; Teraoka-Nishitani, Noriko; Yamagata, Akane; Tanaka, Takahiro; Sasaki, Marika; Yasuda, Natsuki; Oda, Makiko; Okano, Tatsuji; Yamahira, Kazuhiro; Nakamura, Yuta; Kobayashi, Takanobu; Kino, Katsuhito; Miyazawa, Hiroshi; Waalkes, Michael P; Takiguchi, Masufumi

2017-05-01

Cadmium is a transition metal that is classified as human carcinogen by the International Agency for Research on Cancer (IARC) with multiple target sites. Many studies using various model systems provide evidence of cadmium-induced malignancy formation in vivo or malignant cell transformation in vitro. Nonetheless, further studies are needed to completely understand the mechanisms of cadmium carcinogenicity. Our prior studies have utilized a rat liver epithelial cell line (TRL 1215) as a model for cadmium-induced malignant transformation. In the present study, we focused on the molecular mechanisms of this malignant transformation, especially with regard to hyper-invasiveness stimulated by cadmium transformation. By performing a series of biochemical analyses on cadmium transformed cells, it was determined that cadmium had significantly down-regulated the expression of apolipoprotein E (ApoE). ApoE was recently established as a suppressor of cell invasion. A key factor in the suppression of ApoE by cadmium appeared to be that the metal evoked a 5-aza-2'-deoxycytidine-sensitive hypermethylation of the regulatory region of ApoE, coupled with interference of the action of liver X receptor α (LXRα), a transcriptional regulator for ApoE. Furthermore, the expression of LXRα itself was suppressed by cadmium-mediated epigenetic modification. Re-expression of ApoE clearly abrogated the cell invasion stimulated by cadmium-induced malignant transformation. Together, the current results suggest that the cadmium-mediated enhanced cell invasion is linked to down-regulation of ApoE during malignant transformation these liver cells. Copyright © 2017 Elsevier B.V. All rights reserved.

Effect of endocannabinoid signalling on cell fate: life, death, differentiation and proliferation of brain cells.

PubMed

Garcia-Arencibia, Moises; Molina-Holgado, Eduardo; Molina-Holgado, Francisco

2018-05-24

Cell fate events are regulated by different endogenous developmental factors such as the cell micro-environment, external or remote signals and epigenetic factors. Among the many regulatory factors, endocannabinoid-associated signalling pathways are known to conduct several of these events in the developing nervous system and in the adult brain. Interestingly, endocannabinoids exert modulatory actions in both physiological and pathological conditions. Endocannabinoid signalling can promote cell survival by acting on non-transformed brain cells (neurons, astrocytes or oligodendrocytes) and can have either a protumoural or antitumoural effect on transformed cells. Moreover, endocannabinoids are able to attenuate the detrimental effects on neurogenesis and neuroinflammation associated with ageing. Thus, the endocannabinoid system emerges as an important regulator of cell fate, controlling cell survival/cell death decisions depending on the cell type and its environment. © 2018 The British Pharmacological Society.

Intra-hydrogel culture prevents transformation of mesenchymal stem cells induced by monolayer expansion.

PubMed

Jiang, Tongmeng; Liu, Junting; Ouyang, Yiqiang; Wu, Huayu; Zheng, Li; Zhao, Jinmin; Zhang, Xingdong

2018-05-01

In this study, we report that the intra-hydrogel culture system mitigates the transformation of mesenchymal stem cells (MSCs) induced by two-dimensional (2D) expansion. MSCs expanded in monolayer culture prior to encapsulation in collagen hydrogels (group eMSCs-CH) featured impaired stemness in chondrogenesis, comparing with the freshly isolated bone marrow mononuclear cells seeded directly in collagen hydrogels (group fMSCs-CH). The molecular mechanism of the in vitro expansion-triggered damage to MSCs was detected through genome-wide microarray analysis. Results indicated that pathways such as proteoglycans in cancer and pathways in cancer expansion were highly enriched in eMSCs-CH. And multiple up-regulated oncoma-associated genes were verified in eMSCs-CH compared with fMSCs-CH, indicating that expansion in vitro triggered cellular transformation was associated with signaling pathways related to tumorigenicity. Besides, focal adhesion (FA) and mitogen-activated protein kinase (MAPK) signaling pathways were also involved in in vitro expansion, indicating restructuring of the cell architecture. Thus, monolayer expansion in vitro may contribute to vulnerability of MSCs through the regulation of FA and MAPK. This study indicates that intra-hydrogel culture can mitigate the monolayer expansion induced transformation of MSCs and maintain the uniformity of the stem cells, which is a viable in vitro culture system for stem cell therapy.

Advancing Crop Transformation in the Era of Genome Editing.

PubMed

Altpeter, Fredy; Springer, Nathan M; Bartley, Laura E; Blechl, Ann E; Brutnell, Thomas P; Citovsky, Vitaly; Conrad, Liza J; Gelvin, Stanton B; Jackson, David P; Kausch, Albert P; Lemaux, Peggy G; Medford, June I; Orozco-Cárdenas, Martha L; Tricoli, David M; Van Eck, Joyce; Voytas, Daniel F; Walbot, Virginia; Wang, Kan; Zhang, Zhanyuan J; Stewart, C Neal

2016-07-01

Plant transformation has enabled fundamental insights into plant biology and revolutionized commercial agriculture. Unfortunately, for most crops, transformation and regeneration remain arduous even after more than 30 years of technological advances. Genome editing provides novel opportunities to enhance crop productivity but relies on genetic transformation and plant regeneration, which are bottlenecks in the process. Here, we review the state of plant transformation and point to innovations needed to enable genome editing in crops. Plant tissue culture methods need optimization and simplification for efficiency and minimization of time in culture. Currently, specialized facilities exist for crop transformation. Single-cell and robotic techniques should be developed for high-throughput genomic screens. Plant genes involved in developmental reprogramming, wound response, and/or homologous recombination should be used to boost the recovery of transformed plants. Engineering universal Agrobacterium tumefaciens strains and recruiting other microbes, such as Ensifer or Rhizobium, could facilitate delivery of DNA and proteins into plant cells. Synthetic biology should be employed for de novo design of transformation systems. Genome editing is a potential game-changer in crop genetics when plant transformation systems are optimized. © 2016 American Society of Plant Biologists. All rights reserved.

Inhibition of the Hedgehog Signaling Pathway Depresses the Cigarette Smoke-Induced Malignant Transformation of 16HBE Cells on a Microfluidic Chip.

PubMed

Qin, Yong-Xin; Yang, Zhi-Hui; Du, Xiao-Hui; Zhao, Hui; Liu, Yuan-Bin; Guo, Zhe; Wang, Qi

2018-05-20

The hedgehog signaling system (HHS) plays an important role in the regulation of cell proliferation and differentiation during the embryonic phases. However, little is known about the involvement of HHS in the malignant transformation of cells. This study aimed to detect the role of HHS in the malignant transformation of human bronchial epithelial (16HBE) cells. In this study, two microfluidic chips were designed to investigate cigarette smoke extract (CSE)-induced malignant transformation of cells. Chip A contained a concentration gradient generator, while chip B had four cell chambers with a central channel. The 16HBE cells cultured in chip A were used to determine the optimal concentration of CSE for inducing malignant transformation. The 16HBE cells in chip B were cultured with 12.25% CSE (Group A), 12.25% CSE + 5 μmol/L cyclopamine (Group B), or normal complete medium as control for 8 months (Group C), to establish the in vitro lung inflammatory-cancer transformation model. The transformed cells were inoculated into 20 nude mice as cells alone (Group 1) or cells with cyclopamine (Group 2) for tumorigenesis testing. Expression of HHS proteins was detected by Western blot. Data were expressed as mean ± standard deviation. The t-test was used for paired samples, and the difference among groups was analyzed using a one-way analysis of variance. The optimal concentration of CSE was 12.25%. Expression of HHS proteins increased during the process of malignant transformation (Group B vs. Group A, F = 7.65, P < 0.05). After CSE exposure for 8 months, there were significant changes in cellular morphology, which allowed the transformed cells to grow into tumors in 40 days after being inoculated into nude mice. Cyclopamine could effectively depress the expression of HHS proteins (Group C vs. Group B, F = 6.47, P < 0.05) and prevent tumor growth in nude mice (Group 2 vs. Group 1, t = 31.59, P < 0.01). The activity of HHS is upregulated during the CSE-induced malignant transformation of 16HBE cells. Cyclopamine can effectively depress expression of HHS proteins in vitro and prevent tumor growth of the transformed cells in vivo.

Oral Cancer Facts

MedlinePlus

... biophysical processes of aging cells that allows malignant transformation, or perhaps, immune system competence diminishes with age. ... cancer develops We know that all cancers (neoplastic transformations) result from changes (mutations) in genes which control ...

[Transformation of Chlamydomonas reinhardtii CW-15 with the hygromycin phosphotransferase gene as a selective marker].

PubMed

Ladygin, V G; Butanaev, A M

2002-09-01

To transform Chlamydomonas reinhardtii Dang. Cells, plasmid pCTVHyg was constructed with the use of the Escherichia coli hygromycin phosphotransferase gene (hpt) controlled by the SV40 early promoter. Cells of the CW-15 mutant strain were transformed by electroporation, with the yield reaching 10(3) hygromycin-resistant (HygR) clones per 10(6) recipient cells. The exogenous DNA integrated in the Ch. reinhardtii nuclear genome showed stable transmission for approximately 350 cell generations, while hygromycin resistance was expressed as an unstable character. Codon usage was compared for the hpt gene and Ch. reinhardtii nuclear genes. The results testified that codon usage bias, which is characteristic of Ch. reinhardtii, is not the major factor affecting foreign gene expression. The advantages of the selective system for studying Ch. reinhardtii transformation with heterologous genes are discussed.

Battery Fault Detection with Saturating Transformers

NASA Technical Reports Server (NTRS)

Davies, Francis J. (Inventor); Graika, Jason R. (Inventor)

2013-01-01

A battery monitoring system utilizes a plurality of transformers interconnected with a battery having a plurality of battery cells. Windings of the transformers are driven with an excitation waveform whereupon signals are responsively detected, which indicate a health of the battery. In one embodiment, excitation windings and sense windings are separately provided for the plurality of transformers such that the excitation waveform is applied to the excitation windings and the signals are detected on the sense windings. In one embodiment, the number of sense windings and/or excitation windings is varied to permit location of underperforming battery cells utilizing a peak voltage detector.

RB mutation and RAS overexpression induce resistance to NK cell-mediated cytotoxicity in glioma cells.

PubMed

Orozco-Morales, Mario; Sánchez-García, Francisco Javier; Golán-Cancela, Irene; Hernández-Pedro, Norma; Costoya, Jose A; de la Cruz, Verónica Pérez; Moreno-Jiménez, Sergio; Sotelo, Julio; Pineda, Benjamín

2015-01-01

Several theories aim to explain the malignant transformation of cells, including the mutation of tumor suppressors and proto-oncogenes. Deletion of Rb (a tumor suppressor), overexpression of mutated Ras (a proto-oncogene), or both, are sufficient for in vitro gliomagenesis, and these genetic traits are associated with their proliferative capacity. An emerging hallmark of cancer is the ability of tumor cells to evade the immune system. Whether specific mutations are related with this, remains to be analyzed. To address this issue, three transformed glioma cell lines were obtained (Rb(-/-), Ras(V12), and Rb(-/-)/Ras(V12)) by in vitro retroviral transformation of astrocytes, as previously reported. In addition, Ras(V12) and Rb(-/-)/Ras(V12) transformed cells were injected into SCID mice and after tumor growth two stable glioma cell lines were derived. All these cells were characterized in terms of Rb and Ras gene expression, morphology, proliferative capacity, expression of MHC I, Rae1δ, and Rae1αβγδε, mult1, H60a, H60b, H60c, as ligands for NK cell receptors, and their susceptibility to NK cell-mediated cytotoxicity. Our results show that transformation of astrocytes (Rb loss, Ras overexpression, or both) induced phenotypical and functional changes associated with resistance to NK cell-mediated cytotoxicity. Moreover, the transfer of cell lines of transformed astrocytes into SCID mice increased resistance to NK cell-mediated cytotoxicity, thus suggesting that specific changes in a tumor suppressor (Rb) and a proto-oncogene (Ras) are enough to confer resistance to NK cell-mediated cytotoxicity in glioma cells and therefore provide some insight into the ability of tumor cells to evade immune responses.

Characterization of a human MSX-2 cDNA and its fragment isolated as a transformation suppressor gene against v-Ki-ras oncogene.

PubMed

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

1996-05-16

A cDNA (termed CT124) encoding a carboxyl-terminal fragment of the human homeobox protein MSX-2 was found to induce flat reversion when expressed in v-Ki-ras-transformed NIH3T3 cells. Although the expression of endogenous MSX-2 gene is low in most of the normal adult tissues examined, it is frequently activated in carcinoma-derived cell lines. Likewise, the gene is inactive in NIH3T3 cells but is transcriptionally activated after transformation by v-Ki-ras oncogene, suggesting that the intact MSX-2 may play a positive, rather than suppressive, role in cell transformation. To test this possibility, we isolated a near full-length human MSX-2 cDNA and tested its activities in two cell systems, i.e. fibroblast and myoblast. In NIH3T3 fibroblasts, although the gene by itself failed to confer a transformed phenotype, antisense MSX-2 cDNA as well as truncated CT124 cDNA interfered with the transforming activities of v-Ki-ras oncogene. In C2C12 myoblasts, MSX-2 was found to suppress MyoD gene expression, as do activated ras oncogenes, under certain culture conditions, and CT124 was found to inhibit the activities of both MSX-2 and ras in this system as well. Our findings not only suggest that CT124 may act as a dominant suppressor of MSX-2 but also raise the possibility that MSX-2 gene may be an important downstream target for the Ras signaling pathways.

Transformation of somatic embryos of Prunus incisa ‘February Pink’ with a visible reporter gene

USDA-ARS?s Scientific Manuscript database

An Agrobacterium-mediated transformation system was developed for the ornamental cherry species Prunus incisa. This system uses both an antibiotic resistance gene (NPTII) and a visible selectable marker, the green fluorescent protein (GFP), to select plants. Cells from leaf and root explants were tr...

Transcriptional and posttranscriptional regulation of class I major histocompatibility complex genes following transformation with human adenoviruses.

PubMed Central

Shemesh, J; Rotem-Yehudar, R; Ehrlich, R

1991-01-01

Transformation of rodent cells by human adenoviruses is a well-established model system for studying the expression, regulation, and function of class I antigens. In this report, we demonstrate that the highly oncogenic adenovirus type 12 operates at the transcriptional and posttranscriptional levels in regulating the activity of major histocompatibility complex class I genes and products in transformed cells. Adenovirus type 12 suppresses the cell surface expression of class I antigens in most cell lines. Nevertheless, in a number of cell lines suppression is the result of reduction in the amount of stable specific mRNA, while in another group of cell lines suppression involves interference with processing of a posttranscriptional product. The two mechanisms operate both for the endogenous H-2 genes and for a miniature swine class I transgene that is expressed in the cells. Images PMID:1895404

Comparative transfection of DNA into primary and transformed mammalian cells from different lineages

PubMed Central

2010-01-01

Background The delivery of DNA into human cells has been the basis of advances in the understanding of gene function and the development of genetic therapies. Numerous chemical and physical approaches have been used to deliver the DNA, but their efficacy has been variable and is highly dependent on the cell type to be transfected. Results Studies were undertaken to evaluate and compare the transfection efficacy of several chemical reagents to that of the electroporation/nucleofection system using both adherent cells (primary and transformed airway epithelial cells and primary fibroblasts as well as embryonic stem cells) and cells in suspension (primary hematopoietic stem/progenitor cells and lymphoblasts). With the exception of HEK 293 cell transfection, nucleofection proved to be less toxic and more efficient at effectively delivering DNA into the cells as determined by cell proliferation and GFP expression, respectively. Lipofectamine and nucleofection of HEK 293 were essentially equivalent in terms of toxicity and efficiency. Transient transfection efficiency in all the cell systems ranged from 40%-90%, with minimal toxicity and no apparent species specificity. Differences in efficiency and toxicity were cell type/system specific. Conclusions In general, the Amaxa electroporation/nucleofection system appears superior to other chemical systems. However, there are cell-type and species specific differences that need to be evaluated empirically to optimize the conditions for transfection efficiency and cell survival. PMID:20144189

Battery Charge Equalizer with Transformer Array

NASA Technical Reports Server (NTRS)

Davies, Francis

2013-01-01

High-power batteries generally consist of a series connection of many cells or cell banks. In order to maintain high performance over battery life, it is desirable to keep the state of charge of all the cell banks equal. A method provides individual charging for battery cells in a large, high-voltage battery array with a minimum number of transformers while maintaining reasonable efficiency. This is designed to augment a simple highcurrent charger that supplies the main charge energy. The innovation will form part of a larger battery charge system. It consists of a transformer array connected to the battery array through rectification and filtering circuits. The transformer array is connected to a drive circuit and a timing and control circuit that allow individual battery cells or cell banks to be charged. The timing circuit and control circuit connect to a charge controller that uses battery instrumentation to determine which battery bank to charge. It is important to note that the innovation can charge an individual cell bank at the same time that the main battery charger is charging the high-voltage battery. The fact that the battery cell banks are at a non-zero voltage, and that they are all at similar voltages, can be used to allow charging of individual cell banks. A set of transformers can be connected with secondary windings in series to make weighted sums of the voltages on the primaries.

Determination of the transforming activities of adenovirus oncogenes.

PubMed

Speiseder, Thomas; Nevels, Michael; Dobner, Thomas

2014-01-01

The last 50 years of molecular biological investigations into human adenoviruses (Ads) have contributed enormously to our understanding of the basic principles of normal and malignant cell growth. Much of this knowledge stems from analyses of the Ad productive infection cycle in permissive host cells. Also, initial observations concerning the transforming potential of human Ads subsequently revealed decisive insights into the molecular mechanisms of the origins of cancer and established Ads as a model system for explaining virus-mediated transformation processes. Today it is well established that cell transformation by human Ads is a multistep process involving several gene products encoded in early transcription units 1A (E1A) and 1B (E1B). Moreover, a large body of evidence now indicates that alternative or additional mechanisms are engaged in Ad-mediated oncogenic transformation involving gene products encoded in early region 4 (E4) as well as epigenetic changes resulting from viral DNA integration. In particular, studies on the transforming potential of several E4 gene products have now revealed new pathways that point to novel general mechanisms of virus-mediated oncogenesis. In this chapter we describe in vitro and in vivo assays to determine the transforming and oncogenic activities of the E1A, E1B, and E4 oncoproteins in primary baby rat kidney cells, human amniotic fluid cells and athymic nude mice.

Method and system for measurement of mechanical properties of molecules and cells

NASA Technical Reports Server (NTRS)

Fredberg, Jeffrey J. (Inventor); Butler, James P. (Inventor); Ingber, Donald E. (Inventor); Wang, Ning (Inventor)

1996-01-01

Mechanical stresses and deformations are applied directly to cell surface receptors or molecules and measured using a system including a magnetic twisting device in combination with ferromagnetic microbeads coated with ligands for integrins or any other surface receptors. The system can be used diagnostically to characterize cells and molecules and to determine the effect of transformation and compounds, including drugs, on the cells and molecules. The system can also be used to induce cells to grow or alter production of molecules by the cells.

Recombinant glucose uptake system

DOEpatents

Ingrahm, Lonnie O.; Snoep, Jacob L.; Arfman, Nico

1997-01-01

Recombinant organisms are disclosed that contain a pathway for glucose uptake other than the pathway normally utilized by the host cell. In particular, the host cell is one in which glucose transport into the cell normally is coupled to PEP production. This host cell is transformed so that it uses an alternative pathway for glucose transport that is not coupled to PEP production. In a preferred embodiment, the host cell is a bacterium other than Z. mobilis that has been transformed to contain the glf and glk genes of Z. mobilis. By uncoupling glucose transport into the cell from PEP utilization, more PEP is produced for synthesis of products of commercial importance from a given quantity of biomass supplied to the host cells.

Stable transformation via particle bombardment in two different soybean regeneration systems.

PubMed

Sato, S; Newell, C; Kolacz, K; Tredo, L; Finer, J; Hinchee, M

1993-05-01

The Biolistics(®) particle delivery system for the transformation of soybean (Glycine max L. Merr.) was evaluated in two different regeneration systems. The first system was multiple shoot proliferation from shoot tips obtained from immature zygotic embryos of the cultivar Williams 82, and the second was somatic embryogenesis from a long term proliferative suspension culture of the cultivar Fayette. Bombardment of shoot tips with tungsten particles, coated with precipitated DNA containing the gene for β-glucuronidase (GUS), produced GUS-positive sectors in 30% of the regenerated shoots. However, none of the regenerants which developed into plants continued to produce GUS positive tissue. Bombardment of embryogenic suspension cultures produced GUS positive globular somatic embryos which proliferated into GUS positive somatic embryos and plants. An average of 4 independent transgenic lines were generated per bombarded flask of an embryogenic suspension. Particle bombardment delivered particles into the first two cell layers of either shoot tips or somatic embryos. Histological analysis indicated that shoot organogenesis appeared to involve more than the first two superficial cell layers of a shoot tip, while somatic embryo proliferation occurred from the first cell layer of existing somatic embryos. The different transformation results obtained with these two systems appeared to be directly related to differences in the cell types which were responsible for regeneration and their accessibility to particle penetration.

[The liver and the immune system].

PubMed

Jakab, Lajos

2015-07-26

The liver is known to be the metabolic centre of the organism and is under the control of the central nervous system. It has a peculiar tissue structure and its anatomic localisation defines it as part of the immune system having an individual role in the defence of the organism. The determinant of its particular tissue build-up is the sinusoid system. In addition to hepatocytes, one cell row "endothelium", stellate cells close to the external surface, Kupffer cells tightly to its inner surface, as well as dendritic cells and other cell types (T and B lymphocytes, natural killer and natural killer T-cells, mast cells, granulocytes) are present. The multitudes and variety of cells make it possible to carry out the tasks according to the assignment of the organism. The liver is a member of the immune system having immune cells largely in an activated state. Its principal tasks are the assurance of the peripheral immune tolerance of the organism with the help of the haemopoetic cells and transforming growth factor-β. The liver takes part in the determination of the manner of the non-specific immune response of the organism. In addition to acute phase reaction of the organism, the liver has a role in the adaptive/specific immune response. These functions include retardation of the T and B lymphocytes and the defence against harmful pathogens. With the collaboration of transforming growth factor-β, immunoglobulins and their subclasses are inhibited just as the response of the T lymphocytes. The only exception is the undisturbed immunoglobulin A production. Particularly important is the intensive participation of the liver in the acute phase reaction of the organism, which is organised and guided by the coordinated functions of the cortico-hypothalamo-hypophysis-adrenal axis. Beside cellular elements, hormones, adhesion molecules, chemokines and cytokines are also involved in the cooperation with the organs. Acute phase reactants play a central role in these processes. Until recently the α2-macroglobulin was not considered as an acute reactant of the organism, but it is now functionally included in the acute phase reaction presumably due to its close connection with the transforming growth factor-β. Transforming growth factor-β has extraordinarily important roles in all phases of inflammation and in the specific immune response. The peripheral immune tolerance of the organism involves tightly coupled regulation of proliferation, differentiation and survival of lymphocytes.

Quantifying and resolving multiple vector transformants in S. cerevisiae plasmid libraries.

PubMed

Scanlon, Thomas C; Gray, Elizabeth C; Griswold, Karl E

2009-11-20

In addition to providing the molecular machinery for transcription and translation, recombinant microbial expression hosts maintain the critical genotype-phenotype link that is essential for high throughput screening and recovery of proteins encoded by plasmid libraries. It is known that Escherichia coli cells can be simultaneously transformed with multiple unique plasmids and thusly complicate recombinant library screening experiments. As a result of their potential to yield misleading results, bacterial multiple vector transformants have been thoroughly characterized in previous model studies. In contrast to bacterial systems, there is little quantitative information available regarding multiple vector transformants in yeast. Saccharomyces cerevisiae is the most widely used eukaryotic platform for cell surface display, combinatorial protein engineering, and other recombinant library screens. In order to characterize the extent and nature of multiple vector transformants in this important host, plasmid-born gene libraries constructed by yeast homologous recombination were analyzed by DNA sequencing. It was found that up to 90% of clones in yeast homologous recombination libraries may be multiple vector transformants, that on average these clones bear four or more unique mutant genes, and that these multiple vector cells persist as a significant proportion of library populations for greater than 24 hours during liquid outgrowth. Both vector concentration and vector to insert ratio influenced the library proportion of multiple vector transformants, but their population frequency was independent of transformation efficiency. Interestingly, the average number of plasmids born by multiple vector transformants did not vary with their library population proportion. These results highlight the potential for multiple vector transformants to dominate yeast libraries constructed by homologous recombination. The previously unrecognized prevalence and persistence of multiply transformed yeast cells have important implications for yeast library screens. The quantitative information described herein should increase awareness of this issue, and the rapid sequencing approach developed for these studies should be widely useful for identifying multiple vector transformants and avoiding complications associated with cells that have acquired more than one unique plasmid.

[Farnesyl transferase inhibitors (anti-Ras). A new class of anticancer agents].

PubMed

Levy, R

Ras genes are frequently activated in human tumours. The role of their product, the P21 proteins, in the transduction of the mitogenic signal makes them attractive targets for an anti-neoplastic therapy. The p21 ras proteins are linked to the plasma membrane and transformed into an active form for signal transmission. Their effect is to mediate the effects of growth factors. Two drug families, the Benzodiazepine peptidomimetics and the CAAX tetrapeptides which inhibit the farnesylation of P21-Ras proteins abolish the transforming properties of mutated P21. These promising drugs could rapidly have clinical applications. They have been shown to be highly active at precise concentrations on ras-transformed cells but at the same concentrations are not toxic for untransformed cells. They do not effect other similar enzyme systems within the cell, underlining their selective capacity. Theoretically anti-ras therapy could only suspend cell transformation although it might be possible that if given long enough, a lethal threshold could be reached.

Comparison of the genotoxic activities of the K-region dihydrodiol of benzo[a]pyrene with benzo[a]pyrene in mammalian cells: morphological cell transformation; DNA damage; and stable covalent DNA adducts.

PubMed

Nesnow, Stephen; Davis, Christine; Nelson, Garret B; Lambert, Guy; Padgett, William; Pimentel, Maria; Tennant, Alan H; Kligerman, Andrew D; Ross, Jeffrey A

2002-11-26

Benzo[a]pyrene (B[a]P) is the most thoroughly studied polycyclic aromatic hydrocarbon (PAH). Many mechanisms have been suggested to explain its carcinogenic activity, yet many questions still remain. K-region dihydrodiols of PAHs are metabolic intermediates depending on the specific cytochrome P450 and had been thought to be detoxification products. However, K-region dihydrodiols of several PAHs have recently been shown to morphologically transform mouse embryo C3H10T1/2CL8 cells (C3H10T1/2 cells). Because K-region dihydrodiols are not metabolically formed from PAHs by C3H10T1/2 cells, these cells provide a useful tool to independently study the mechanisms of action of PAHs and their K-region dihydrodiols. Here, we compare the morphological cell transforming, DNA damaging, and DNA adducting activities of the K-region dihydrodiol of B[a]P, trans-B[a]P-4,5-diol with B[a]P. Both trans-B[a]P-4,5-diol and B[a]P morphologically transformed C3H10T1/2 cells by producing both Types II and III transformed foci. The morphological cell transforming and cytotoxicity dose response curves for trans-B[a]P-4,5-diol and B[a]P were indistinguishable. Since morphological cell transformation is strongly associated with mutation and/or larger scale DNA damage in C3H10T1/2 cells, the identification of DNA damage induced in these cells by trans-B[a]P-4,5-diol was sought. Both trans-B[a]P-4,5-diol and B[a]P exhibited significant DNA damaging activity without significant concurrent cytotoxicity using the comet assay, but with different dose responses and comet tail distributions. DNA adduct patterns from C3H10T1/2 cells were examined after trans-B[a]P-4,5-diol or B[a]P treatment using 32P-postlabeling techniques and improved TLC elution systems designed to separate polar DNA adducts. While B[a]P treatment produced one major DNA adduct identified as anti-trans-B[a]P-7,8-diol-9,10-epoxide-deoxyguanosine, no stable covalent DNA adducts were detected in the DNA of trans-B[a]P-4,5-diol-treated cells. In summary, this study provides evidence for the DNA damaging and morphological cell transforming activities of the K-region dihydrodiol of B[a]P, in the absence of covalent stable DNA adducts. While trans-B[a]P-4,5-diol and B[a]P both induce morphological cell transformation, their activities as DNA damaging agents differ, both qualitatively and quantitatively. In concert with the morphological cell transformation activities of other K-region dihydrodiols of PAHs, these data suggest a new mechanism/pathway for the morphological cell transforming activities of B[a]P and its metabolites.

Selective effects of quercetin on the cell growth and antioxidant defense system in normal versus transformed mouse hepatic cell lines.

PubMed

Son, Young-Ok; Lee, Kyung-Yeol; Kook, Sung-Ho; Lee, Jeong-Chae; Kim, Jong-Ghee; Jeon, Young-Mi; Jang, Yong-Suk

2004-10-19

Quercetin is a dietary anticancer chemical that is capable of inducing apoptosis in tumor cells. However, little is known about its biological effect on nonmalignant cells, although the effect is one of the critical criteria to evaluate the clinical efficacy of the anticancer agent. In this study, we investigated the effects of quercetin on cell growth and apoptosis using embryonic normal hepatic cell line (BNL CL.2) and its SV40-transformed cell line (BNL SV A.8). We also evaluated the effects of quercetin on the antioxidant defense system in those cells. BNL SV A.8 cells were more sensitive to quercetin-mediated cytotoxicity than BNL CL.2 cells. In addition, the enzyme assays showed that quercetin actively stimulated the antioxidant defense systems including superoxide dismutase, catalase, glutathione, and glutathione reductase only in the BNL CL.2 cells. In particular, quercetin significantly reduced superoxide dismutase activity and increased the malonaldehyde content in BNL SV A.8 cells. These are thought to be closely related to quercetin-mediated apoptosis. Our findings suggest that quercetin is a dietary flavonoid that is capable of inducing selective growth inhibition and apoptosis in hepatic tumor cells, but not in normal cells.

Follicular lymphomas and their transformation: Past and current research.

PubMed

Mendez, Miriam; Torrente, Maria; Provencio, Mariano

2017-06-01

Follicular lymphoma (FL) is the second most common type of non-Hodgkin lymphoma (NHL). Histological transformation (HT) refers to the evolution of a clinically indolent NHL to a clinically aggressive one, defined as those lymphomas in which survival is limited to a few months when untreated. Areas covered: HT is associated with rapid progression of lymphadenopathy, infiltration of extranodal sites, development of systemic symptoms, and elevated serum level of lactate dehydrogenase (LDH). It is frequently related to a poor prognosis, and the median survival after transformation is less than 2 years. Transformation to diffuse large B cell lymphoma (DLBCL) in patients with FL occurs at an annual rate of approximately 3% for the first 15 years, after which the risk of HT falls for reasons that remain unclear. Expert commentary: Although it has long been assumed that transformation reflects the emergence of an aggressive subclone of cells from the primary FL, recent studies suggest that FL transformation might also arise by divergent evolution from a more immature common progenitor cell. Studies on genomic changes and DNA sequencing have shed some light onto the process of transformation. Nowadays, we know that HT is a complex process where several molecular pathways are involved.

The tumor suppressor functions of p27(kip1) include control of the mesenchymal/amoeboid transition.

PubMed

Berton, Stefania; Belletti, Barbara; Wolf, Katarina; Canzonieri, Vincenzo; Lovat, Francesca; Vecchione, Andrea; Colombatti, Alfonso; Friedl, Peter; Baldassarre, Gustavo

2009-09-01

In many human cancers, p27 downregulation correlates with a worse prognosis, suggesting that p27 levels could represent an important determinant in cell transformation and cancer development. Using a mouse model system based on v-src-induced transformation, we show here that p27 absence is always linked to a more aggressive phenotype. When cultured in three-dimensional contexts, v-src-transformed p27-null fibroblasts undergo a morphological switch from an elongated to a rounded cell shape, accompanied by amoeboid-like morphology and motility. Importantly, the acquisition of the amoeboid motility is associated with a greater ability to move and colonize distant sites in vivo. The reintroduction of different p27 mutants in v-src-transformed p27-null cells demonstrates that the control of cell proliferation and motility represents two distinct functions of p27, both necessary for it to fully act as a tumor suppressor. Thus, we highlight here a new p27 function in driving cell plasticity that is associated with its C-terminal portion and does not depend on the control of cyclin-dependent kinase activity.

[Use of the hygromycin phosphotransferase gene as the dominant selective marker for Chlamydomonas reinhardtii transformation].

PubMed

Butanaev, A M

1994-01-01

The hygromycin phosphotransferase gene (hpt) from E. coli under the control of the SV40 early promoter was used as a dominant selectable marker for transformation of Chlamydomonas reinhardtii. Cells were transformed by electroporation (pulse length, 2 ms, field strength, 1 kV/cm). The culture growth phase was a crucial parameter for transformation (optimal density approximately 10(6) cells/ml). It was possible to obtain approximately 10(3) Hyg-resistant colonies under these conditions. Foreign DNA integrated into the Chlamydomonas genome was maintained for at least 8 months but the Hyg-resistant phenotype of the transformed clones was unstable. The frequency of codon usage in the hpt gene was compared with the one in Chlamydomonas nuclear genes. It is supposed that highly biased codon usage in Chlamydomonas does not preclude expression. Advantages of this selection system for studying Chlamydomonas transformation by heterologous genes are discussed.

Cell transformation and mutability of different genetic loci in mammalian cells by metabolically activated carcinogenic polycylic hydrocarbons

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

Huberman, E.

1977-01-01

Treatment of experimental animals with chemical carcinogens, including some polycyclic hydrocarbons, can result in the formation of malignant tumors. The process whereby some chemicals induce malignancy is as yet unknown. However, in a model system using mammalian cells in culture, it was possible to show that the chemical carcinogens induce malignant transformation rather than select for pre-existing tumor cells. In the process of the in vitro cell transformation, the normal cells, which have an oriented pattern of cell growth, a limited life-span in vitro, and are not tumorigenic, are converted into cells that have a hereditary random pattern of cellmore » growth, the ability to grow continuously in culture, and the ability to form tumors in vivo. This stable heritable phenotype of the transformed cells is similar to that of cells derived from spontaneous or experimentally induced tumors. Such stable heritable phenotype changes may arise from alteration in gene expression due to a somatic mutation after interaction of the carcinogen with cellular DNA. In the present experiments we have shown that metabolically activated carcinogenic polycyclic hydrocarbons which have been shown to bind to cellular DNA induce somatic mutations at different genetic loci in mammalian cells and that there is a relationship between the degree of mutant induction and the degree of carcinogenicity of the different hydrocarbons tested.« less

Dual effect of pseudorabies virus growth factor (PRGF) displayed on actin cytoskeleton.

PubMed

Urbancíková, M; Vozárová, G; Lesko, J; Golais, F

1999-10-01

Pseudorabies virus growth factor (PRGF) was shown to possess transforming activity as well as transformation repressing activity in in vitro systems. In order to better understand these phenomena we studied actin cytoskeleton and its alterations induced by PRGF using normal human fibroblasts VH-10 and transformed cell line HeLa. For specific detection of filamentous actin cells were stained with phalloidin conjugated with fluorescein isothiocyanate (FITC)-phalloidin. PRGF was applied to VH-10 cells for various length of time from 10 min up to 48 h. The effect was very fast and changes in actin filament composition could be detected already after 10 min. In comparison to untreated cells the staining of treated cells was more diffuse and a number of actin microfilaments in individual stress fibers became reduced. After 30 min thick short actin bundles appeared in the perinuclear region. A 24-h exposure resulted in a large reduction of actin bundles. After additional 24 h a partial restoration of actin cytoskeleton in cells was observed. In transformed HeLa cells PRGF induced opposite process than in normal cells: the number of filamentous actin structures increased. We hypothesise that PRGF may act as a transcription-like factor and may initiate changes in gene expression which consequently result in actin cytoskeleton alterations.

Versatile Cosmid Vectors for the Isolation, Expression, and Rescue of Gene Sequences: Studies with the Human α -globin Gene Cluster

NASA Astrophysics Data System (ADS)

Lau, Yun-Fai; Kan, Yuet Wai

1983-09-01

We have developed a series of cosmids that can be used as vectors for genomic recombinant DNA library preparations, as expression vectors in mammalian cells for both transient and stable transformations, and as shuttle vectors between bacteria and mammalian cells. These cosmids were constructed by inserting one of the SV2-derived selectable gene markers-SV2-gpt, SV2-DHFR, and SV2-neo-in cosmid pJB8. High efficiency of genomic cloning was obtained with these cosmids and the size of the inserts was 30-42 kilobases. We isolated recombinant cosmids containing the human α -globin gene cluster from these genomic libraries. The simian virus 40 DNA in these selectable gene markers provides the origin of replication and enhancer sequences necessary for replication in permissive cells such as COS 7 cells and thereby allows transient expression of α -globin genes in these cells. These cosmids and their recombinants could also be stably transformed into mammalian cells by using the respective selection systems. Both of the adult α -globin genes were more actively expressed than the embryonic zeta -globin genes in these transformed cell lines. Because of the presence of the cohesive ends of the Charon 4A phage in the cosmids, the transforming DNA sequences could readily be rescued from these stably transformed cells into bacteria by in vitro packaging of total cellular DNA. Thus, these cosmid vectors are potentially useful for direct isolation of structural genes.

Determination of the transforming activities of adenovirus oncogenes.

PubMed

Nevels, Michael; Dobner, Thomas

2007-01-01

The last 50 yr of molecular biological investigations into human adenoviruses (Ads) have contributed enormously to our understanding of the basic principles of normal and malignant cell growth. Much of this knowledge stems from analyses of the Ad productive infection cycle in permissive host cells. Also, initial observations concerning the transforming potential of human Ads subsequently revealed decisive insights into the molecular mechanisms of the origins of cancer and established Ads as a model system for explaining virus-mediated transformation processes. Today it is well established that cell transformation by human Ads is a multistep process involving several gene products encoded in early transcription units 1A (E1A) and 1B (E1B). Moreover, a large body of evidence now indicates that alternative or additional mechanisms are engaged in Ad-mediated oncogenic transformation involving gene products encoded in early region 4 (E4) as well as epigenetic changes resulting from viral DNA integration. In particular, studies on the transforming potential of several E4 gene products have now revealed new pathways that point to novel general mechanisms of virus-mediated oncogenesis. In this chapter we describe in vitro and in vivo assays to determine the transforming and oncogenic activities of the E1A, E1B, and E4 oncoproteins in primary baby rat kidney cells and athymic nude mice.

Role of reactive oxygen species in arsenic-induced transformation of human lung bronchial epithelial (BEAS-2B) cells

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

Zhang, Zhuo, E-mail: zhuo.zhang@uky.edu; Pratheeshkumar, Poyil; Budhraja, Amit

Highlights: • Short term exposure of cells to arsenic causes ROS generation. • Chronical exposure of cells to arsenic causes malignant cell transformation. • Inhibition of ROS generation reduces cell transformation by arsenic. • Arsenic-transformed cells exhibit reduced capacity of generating ROS. • Arsenic-transformed cells exhibit increased levels of antioxidants. - Abstract: Arsenic is an environmental carcinogen, its mechanisms of carcinogenesis remain to be investigated. Reactive oxygen species (ROS) are considered to be important. A previous study (Carpenter et al., 2011) has measured ROS level in human lung bronchial epithelial (BEAS-2B) cells and arsenic-transformed BEAS-2B cells and found that ROSmore » levels were higher in transformed cells than that in parent normal cells. Based on these observations, the authors concluded that cell transformation induced by arsenic is mediated by increased cellular levels of ROS. This conclusion is problematic because this study only measured the basal ROS levels in transformed and parent cells and did not investigate the role of ROS in the process of arsenic-induced cell transformation. The levels of ROS in arsenic-transformed cells represent the result and not the cause of cell transformation. Thus question concerning whether ROS are important in arsenic-induced cell transformation remains to be answered. In the present study, we used expressions of catalase (antioxidant against H{sub 2}O{sub 2}) and superoxide dismutase 2 (SOD2, antioxidant against O{sub 2}{sup ·−}) to decrease ROS level and investigated their role in the process of arsenic-induced cell transformation. Our results show that inhibition of ROS by antioxidant enzymes decreased arsenic-induced cell transformation, demonstrating that ROS are important in this process. We have also shown that in arsenic-transformed cells, ROS generation was lower and levels of antioxidants are higher than those in parent cells, in a disagreement with the previous report. The present study has also shown that the arsenic-transformed cells acquired apoptosis resistance. The inhibition of catalase to increase ROS level restored apoptosis capability of arsenic-transformed BEAS-2B cells, further showing that ROS levels are low in these cells. The apoptosis resistance due to the low ROS levels may increase cells proliferation, providing a favorable environment for tumorigenesis of arsenic-transformed cells.« less

Metformin and phenformin deplete tricarboxylic acid cycle and glycolytic intermediates during cell transformation and NTPs in cancer stem cells

PubMed Central

Janzer, Andreas; German, Natalie J.; Gonzalez-Herrera, Karina N.; Asara, John M.; Haigis, Marcia C.; Struhl, Kevin

2014-01-01

Metformin, a first-line diabetes drug linked to cancer prevention in retrospective clinical analyses, inhibits cellular transformation and selectively kills breast cancer stem cells (CSCs). Although a few metabolic effects of metformin and the related biguanide phenformin have been investigated in established cancer cell lines, the global metabolic impact of biguanides during the process of neoplastic transformation and in CSCs is unknown. Here, we use LC/MS/MS metabolomics (>200 metabolites) to assess metabolic changes induced by metformin and phenformin in an Src-inducible model of cellular transformation and in mammosphere-derived breast CSCs. Although phenformin is the more potent biguanide in both systems, the metabolic profiles of these drugs are remarkably similar, although not identical. During the process of cellular transformation, biguanide treatment prevents the boost in glycolytic intermediates at a specific stage of the pathway and coordinately decreases tricarboxylic acid (TCA) cycle intermediates. In contrast, in breast CSCs, biguanides have a modest effect on glycolytic and TCA cycle intermediates, but they strongly deplete nucleotide triphosphates and may impede nucleotide synthesis. These metabolic profiles are consistent with the idea that biguanides inhibit mitochondrial complex 1, but they indicate that their metabolic effects differ depending on the stage of cellular transformation. PMID:25002509

Metformin and phenformin deplete tricarboxylic acid cycle and glycolytic intermediates during cell transformation and NTPs in cancer stem cells.

PubMed

Janzer, Andreas; German, Natalie J; Gonzalez-Herrera, Karina N; Asara, John M; Haigis, Marcia C; Struhl, Kevin

2014-07-22

Metformin, a first-line diabetes drug linked to cancer prevention in retrospective clinical analyses, inhibits cellular transformation and selectively kills breast cancer stem cells (CSCs). Although a few metabolic effects of metformin and the related biguanide phenformin have been investigated in established cancer cell lines, the global metabolic impact of biguanides during the process of neoplastic transformation and in CSCs is unknown. Here, we use LC/MS/MS metabolomics (>200 metabolites) to assess metabolic changes induced by metformin and phenformin in an Src-inducible model of cellular transformation and in mammosphere-derived breast CSCs. Although phenformin is the more potent biguanide in both systems, the metabolic profiles of these drugs are remarkably similar, although not identical. During the process of cellular transformation, biguanide treatment prevents the boost in glycolytic intermediates at a specific stage of the pathway and coordinately decreases tricarboxylic acid (TCA) cycle intermediates. In contrast, in breast CSCs, biguanides have a modest effect on glycolytic and TCA cycle intermediates, but they strongly deplete nucleotide triphosphates and may impede nucleotide synthesis. These metabolic profiles are consistent with the idea that biguanides inhibit mitochondrial complex 1, but they indicate that their metabolic effects differ depending on the stage of cellular transformation.

Identification of Ras-degrading small molecules that inhibit the transformation of colorectal cancer cells independent of β-catenin signaling.

PubMed

Shin, Wookjin; Lee, Sang-Kyu; Hwang, Jeong-Ha; Park, Jong-Chan; Cho, Yong-Hee; Ro, Eun Ji; Song, Yeonhwa; Seo, Haeng Ran; Choi, Kang-Yell

2018-06-06

Although the development of drugs that control Ras is an emerging topic in cancer therapy, no clinically applicable drug is currently available. We have previously utilized knowledge of the Wnt/β-catenin signaling-dependent mechanism of Ras protein stability regulation to identify small molecules that inhibit the proliferation and transformation of various colorectal cancer (CRC) cells via degradation of both β-catenin and Ras. Due to the absence of Ras degradation in cells expressing a nondegradable mutant form of β-catenin and the need to determine an alternative mechanism of Ras degradation, we designed a cell-based system to screen compounds that degrade Ras independent of the Wnt/β-catenin signaling pathway. A cell-based high-content screening (HCS) system that monitors the levels of EGFP-K-Ras G12V was established using HCT-116 cells harboring a nondegradable mutant CTNNB1 (ΔS45). Through HCS of a chemical library composed of 10,000 compounds and subsequent characterization of hits, we identified several compounds that degrade Ras without affecting the β-catenin levels. KY7749, one of the most effective compounds, inhibited the proliferation and transformation of CRC cells, especially KRAS-mutant cells that are resistant to the EGFR monoclonal antibody cetuximab. Small molecules that degrade Ras independent of β-catenin may able to be used in treatments for cancers caused by aberrant EGFR and Ras.

Paraneoplastic scleroderma-like tissue reactions in the setting of an underlying plasma cell dyscrasia: a report of 10 cases.

PubMed

Magro, Cynthia M; Iwenofu, Hans; Nuovo, Gerard J

2013-07-01

Systemic plasma cell dyscrasias have diverse manifestations in the skin and include an inflammatory paraneoplastic process. We encountered cases of scleroderma and eosinophilic fasciitis in the setting of an underlying plasma cell dyscrasia. Ten cases of scleroderma-like tissue reactions in the setting of an underlying plasma cell dyscrasia were encountered. The biopsies were stained for Transforming growth factor (Transforming growth factor) beta, IgG4, kappa, and lambda. Patients presented with a sclerodermoid reaction represented by eosinophilic fasciitis (5 cases), morphea (3 cases), and systemic scleroderma (2 cases). The mean age of presentation was 70 years with a striking female predominance (4:1). Acral accentuation was noted in 8 cases. In 6 of the cases, the cutaneous sclerosis antedated (4 cases) by weeks to 2 years or occurred concurrently (2 cases) with the initial diagnosis of the plasma cell. The biopsies showed changes typical of eosinophilic fasciitis and/or scleroderma. In 5 cases, light chain-restricted plasma cells were present on the biopsy. There was staining of the plasma cells for Transforming growth factor beta in 3 out of 5 cases tested. In any older patient presenting with a sudden onset of eosinophilic fasciitis or scleroderma especially with acral accentuation, investigations should be conducted in regards to an underlying plasma cell dyscrasia.

Effect of Bromouracil-containing Deoxyribonucleic Acid on Bacillus subtilis

PubMed Central

Gimlin, Dixie M.; Hardman, Sue D.; Kelley, Betty N.; Butler, Grace C.; Leach, Franklin R.

1966-01-01

Gimlin, Dixie M. (Oklahoma State University, Stillwater), Sue D. Hardman, Betty N. Kelley, Grace C. Butler, and Franklin R. Leach. Effect of bromouracil-containing deoxyribonucleic acid on Bacillus subtilis. J. Bacteriol. 92:366–374. 1966.—Replacement of one-half of the thymine with bromouracil in Bacillus subtilis transforming deoxyribonucleic acid (DNA) resulted in a slight decrease in transforming activity, but, when used at high concentrations, this DNA preparation inhibited cell growth. Acid-hydrolyzed DNA, or addition of equivalent concentrations of the free base bromouracil in a transforming mixture, was without effect on cell growth. Treatment of the DNA preparation with deoxyribonuclease completely destroyed transforming activity and killing effect, whereas treatments with ribonuclease and trypsin were without effect on either transformation or killing activity. Growth of competent B. subtilis cells in test tubes was inhibited by high concentrations of both normal and bromouracil-containing DNA, with the bromouracil-containing DNA being significantly more inhibitory. This type of inhibition was also reflected in the time of division of the cells. The inhibitory effect was not due to viscosity, or to mutagenicity. The time course of killing paralleled transformation, and competency was required. These results can be interpreted as being due to uptake of homologous but imperfect DNA (containing bromouracil instead of thymine) by means of the systems involved in transformation, followed by either integration (resulting in lethal transformation, activation of a defective, nonlytic but lethal prophage) or interference with the recombination mechanism. PMID:16562122

Targeted Mutagenesis in Rice Using TALENs and the CRISPR/Cas9 System.

PubMed

Endo, Masaki; Nishizawa-Yokoi, Ayako; Toki, Seiichi

2016-01-01

Sequence-specific nucleases (SSNs), such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspersed short palindromic repeats (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9) system, are powerful tools for understanding gene function and for developing novel traits in plants. In plant species for which transformation and regeneration systems using protoplasts are not yet established, direct delivery to nuclei of SSNs either in the form of RNA or protein is difficult. Thus, Agrobacterium-mediated transformation of SSN expression constructs in cultured cells is a practical means of delivering targeted mutagenesis in some plant species including rice. Because targeted mutagenesis occurs stochastically in transgenic cells and SSN-mediated targeted mutagenesis often leads to no selectable phenotype, identification of highly mutated cell lines is a critical step in obtaining regenerated plants with desired mutations.

Fiber optic gas detection system for health monitoring of oil-filled transformer

NASA Astrophysics Data System (ADS)

Ho, H. L.; Ju, J.; Jin, W.

2009-10-01

This paper reports the development of a fiber-optic gas detection system capable of detecting three types of dissolved fault gases in oil-filled power transformers or equipment. The system is based on absorption spectroscopy and the target gases include acetylene (C2H2), methane (CH4) and ethylene (C2H4). Low-cost multi-pass sensor heads using fiber coupled micro-optic cells are employed for which the interaction length is up to 4m. Also, reference gas cells made of photonic bandgap (PBG) fiber are implemented. The minimum detectable gas concentrations for methane, acetylene and ethylene are 5ppm, 2ppm and 50ppm respectively.

Enhanced proliferation of primary rat type II pneumocytes by Jaagsiekte sheep retrovirus envelope protein

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

Johnson, Chassidy; Jahid, Sohail; Voelker, Dennis R.

Jaagsiekte sheep retrovirus (JSRV) is the causative agent of a contagious lung cancer in sheep. The envelope protein (Env) is the oncogene, as it can transform cell lines in culture and induce tumors in animals, although the mechanisms for transformation are not yet clear because a system to perform transformation assays in differentiated type II pneumocytes does not exist. In this study we report culture of primary rat type II pneumocytes in conditions that favor prolonged expression of markers for type II pneumocytes. Env-expressing cultures formed more colonies that were larger in size and were viable for longer periods ofmore » time compared to vector control samples. The cells that remained in culture longer were confirmed to be derived from type II pneumocytes because they expressed surfactant protein C, cytokeratin, displayed alkaline phosphatase activity and were positive for Nile red. This system will be useful to study JSRV Env in the targets of transformation.« less

Ribosome Profiling Reveals a Cell-Type-Specific Translational Landscape in Brain Tumors

PubMed Central

Gonzalez, Christian; Sims, Jennifer S.; Hornstein, Nicholas; Mela, Angeliki; Garcia, Franklin; Lei, Liang; Gass, David A.; Amendolara, Benjamin; Bruce, Jeffrey N.

2014-01-01

Glioma growth is driven by signaling that ultimately regulates protein synthesis. Gliomas are also complex at the cellular level and involve multiple cell types, including transformed and reactive cells in the brain tumor microenvironment. The distinct functions of the various cell types likely lead to different requirements and regulatory paradigms for protein synthesis. Proneural gliomas can arise from transformation of glial progenitors that are driven to proliferate via mitogenic signaling that affects translation. To investigate translational regulation in this system, we developed a RiboTag glioma mouse model that enables cell-type-specific, genome-wide ribosome profiling of tumor tissue. Infecting glial progenitors with Cre-recombinant retrovirus simultaneously activates expression of tagged ribosomes and delivers a tumor-initiating mutation. Remarkably, we find that although genes specific to transformed cells are highly translated, their translation efficiencies are low compared with normal brain. Ribosome positioning reveals sequence-dependent regulation of ribosomal activity in 5′-leaders upstream of annotated start codons, leading to differential translation in glioma compared with normal brain. Additionally, although transformed cells express a proneural signature, untransformed tumor-associated cells, including reactive astrocytes and microglia, express a mesenchymal signature. Finally, we observe the same phenomena in human disease by combining ribosome profiling of human proneural tumor and non-neoplastic brain tissue with computational deconvolution to assess cell-type-specific translational regulation. PMID:25122893

The type VI secretion system of Vibrio cholerae fosters horizontal gene transfer.

PubMed

Borgeaud, Sandrine; Metzger, Lisa C; Scrignari, Tiziana; Blokesch, Melanie

2015-01-02

Natural competence for transformation is a common mode of horizontal gene transfer and contributes to bacterial evolution. Transformation occurs through the uptake of external DNA and its integration into the genome. Here we show that the type VI secretion system (T6SS), which serves as a predatory killing device, is part of the competence regulon in the naturally transformable pathogen Vibrio cholerae. The T6SS-encoding gene cluster is under the positive control of the competence regulators TfoX and QstR and is induced by growth on chitinous surfaces. Live-cell imaging revealed that deliberate killing of nonimmune cells via competence-mediated induction of T6SS releases DNA and makes it accessible for horizontal gene transfer in V. cholerae. Copyright © 2015, American Association for the Advancement of Science.

Combined effects of space flight factors and radiation on humans

NASA Technical Reports Server (NTRS)

Todd, P.; Pecaut, M. J.; Fleshner, M.; Clarkson, T. W. (Principal Investigator)

1999-01-01

The probability that a dose of ionizing radiation kills a cell is about 10,000 times the probability that the cell will be transformed to malignancy. On the other hand, the number of cells killed required to significantly impact health is about 10,000 times the number that must be transformed to cause a late malignancy. If these two risks, cell killing and malignant transformation, are about equal, then the risk that occurs during a mission is more significant than the risk that occurs after a mission. The latent period for acute irradiation effects (cell killing) is about 2-4 weeks; the latent period for malignancy is 10-20 years. If these statements are approximately true, then the impact of cell killing on health in the low-gravity environment of space flight should be examined to establish an estimate of risk. The objective of this study is to synthesize data and conclusions from three areas of space biology and environmental health to arrive at rational risk assessment for radiations received by spacecraft crews: (1) the increased physiological demands of the space flight environment; (2) the effects of the space flight environment on physiological systems; and (3) the effects of radiation on physiological systems. One physiological system has been chosen: the immune response and its components, consisting of myeloid and lymphoid proliferative cell compartments. Best-case and worst-case scenarios are considered. In the worst case, a doubling of immune-function demand, accompanied by a halving of immune capacity, would reduce the endangering dose to a crew member to around 1 Gy.

Bioluminescence-based system for rapid detection of natural transformation.

PubMed

Santala, Ville; Karp, Matti; Santala, Suvi

2016-07-01

Horizontal gene transfer plays a significant role in bacterial evolution and has major clinical importance. Thus, it is vital to understand the mechanisms and kinetics of genetic transformations. Natural transformation is the driving mechanism for horizontal gene transfer in diverse genera of bacteria. Our study introduces a simple and rapid method for the investigation of natural transformation. This highly sensitive system allows the detection of a transformation event directly from a bacterial population without any separation step or selection of cells. The system is based on the bacterial luciferase operon from Photorhabdus luminescens The studied molecular tools consist of the functional modules luxCDE and luxAB, which involve a replicative plasmid and an integrative gene cassette. A well-established host for bacterial genetic investigations, Acinetobacter baylyi ADP1, is used as the model bacterium. We show that natural transformation followed by homologous recombination or plasmid recircularization can be readily detected in both actively growing and static biofilm-like cultures, including very rare transformation events. The system allows the detection of natural transformation within 1 h of introducing sample DNA into the culture. The introduced method provides a convenient means to study the kinetics of natural transformation under variable conditions and perturbations. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Aggregation of the rhizospheric bacterium Azospirillum brasilense in response to oxygen

NASA Astrophysics Data System (ADS)

Abdoun, Hamid; McMillan, Mary; Pereg, Lily

2016-04-01

Azospirillum brasilense spp. have ecological, scientific and agricultural importance. As model plant growth promoting rhizobacteria they interact with a large variety of plants, including important food and cash crops. Azospirillum strains are known for their production of plant growth hormones that enhance root systems and for their ability to fix nitrogen. Azospirillum cells transform in response to environmental cues. The production of exopolysaccharides and cell aggregation during cellular transformation are important steps in the attachment of Azospirillum to roots. We investigate signals that induce cellular transformation and aggregation in the Azospirillum and report on the importance of oxygen to the process of aggregation in this rhizospheric bacterium.

Enhancing DNA electro-transformation efficiency on a clinical Staphylococcus capitis isolate.

PubMed

Cui, Bintao; Smooker, Peter M; Rouch, Duncan A; Deighton, Margaret A

2015-02-01

Clinical staphylococcus isolates possess a stronger restriction-modification (RM) barrier than laboratory strains. Clinical isolates are therefore more resistant to acceptance of foreign genetic material than laboratory strains, as their restriction systems more readily recognize and destroy foreign DNA. This stronger barrier consequently restricts genetic studies to a small number of domestic strains that are capable of accepting foreign DNA. In this study, an isolate of Staphylococcus capitis, obtained from the blood of a very low birth-weight baby, was transformed with a shuttle vector, pBT2. Optimal conditions for electro-transformation were as follows: cells were harvested at mid-log phase, electro-competent cells were prepared; cells were pre-treated at 55°C for 1min; 3μg of plasmid DNA was mixed with 70-80μL of competent cells (3-4×10(10)cells/mL) at 20°C in 0.5M sucrose, 10% glycerol; and electroporation was conducted using 2.1kV/cm field strength with a 0.1cm gap. Compared to the conventional method, which involves DNA electroporation of Staphylococcus aureus RN4220 as an intermediate strain to overcome the restriction barrier, our proposed approach exhibits a higher level (3 log10 units) of transformation efficiency. Heat treatment was used to temporarily inactivate the recipient RM barrier. Other important parameters contributing to improved electro-transformation efficiency were growth stage for cell harvesting, the quantity of DNA, the transformation temperature and field strength. The approach described here may facilitate genetic manipulations of this opportunistic pathogen. Copyright © 2014 Elsevier B.V. All rights reserved.

The growth transformation of human B cells involves superinduction of hsp70 and hsp90.

PubMed

Cheung, R K; Dosch, H M

1993-04-01

Epstein-Barr virus (EBV) is a latent human herpes virus associated with a range of malignant and non-malignant disorders. EBV binds to CD21 virus receptors on B lymphocytes and growth transforms these cells; in susceptible (e.g., immunodeficient) hosts such cells rapidly expand into fatal lymphomas. Virus binding and infection trigger a cascade of cellular events which are transformation prerequisite and analogous to non-oncogenic cell activation events but which differ in several quantitative or qualitative respects. Unique trans-membrane Ca2+ currents, Na+/H+ exchange, as well as tyrosine phosphorylation and p56lck-gene induction suggest that even early on the transformation process has oncogenic specificity. In this report we describe that two additional cellular gene families, the stress proteins hsp70 and hsp90, are coordinately induced at mRNA and protein levels and, quite different from hsp induction by thermal stress, this induction is dependent on EBV-induced trans-membrane Ca2+ currents. Blockade of hsp induction prevents transformation. The kinetics and induction prerequisites set this response well apart from reported responses to thermal or viral stress protein induction. Like p56lck-, hsp induction is purely a post-receptor binding event and not dependent on expression of any viral gene. The induction kinetics, with a peak at approximately 12-16 hr and subsequent decline to control levels, considerably extend the chronological map of elements in the CD21-dependent branch of the transformation pathway and suggest a specific role of induced hsp different from the cell cycle-related functions observed in other cell systems.

Transforming Lepidopteran Insect Cells for Improved Protein Processing and Expression

USDA-ARS?s Scientific Manuscript database

The lepidopteran insect cells used with the baculovirus expression vector system (BEVS) are capable of synthesizing and accurately processing foreign proteins. However, proteins expressed in baculovirus-infected cells often fail to be completely processed, or are not processed in a manner that meet...

Rosa hybrida orcinol O-methyl transferase-mediated production of pterostilbene in metabolically engineered grapevine cell cultures.

PubMed

Martínez-Márquez, Ascensión; Morante-Carriel, Jaime A; Palazon, Javier; Bru-Martínez, Roque

2018-05-25

Stilbenes are naturally scarce high-added-value plant compounds with chemopreventive, pharmacological and cosmetic properties. Bioproduction strategies include engineering the metabolisms of bacterial, fungal and plant cell systems. Strikingly, one of the most effective strategies consists in the elicitation of wild grapevine cell cultures, which leads to vast stilbene resveratrol accumulation in the extracellular medium. The combination of both cell culture elicitation and metabolic engineering strategies to produce resveratrol analogs proved more efficient for the hydroxylated derivative piceatannol than for the dimethylated derivative pterostilbene, for which human hydroxylase HsCYP1B1- and grapevine O-methyltransferase VvROMT-transformed cell cultures were respectively used. Rose orcinol O-methyltransferase (OOMT) displays enzymatic properties, which makes it an appealing candidate to substitute VvROMT in the combined strategy to enhance the pterostilbene production level by engineered grapevine cells upon elicitation. Here we cloned a Rosa hybrida OOMT gene, and created a genetic construction suitable for Agrobacterium-mediated plant transformation. OOMT's ability to catalyze the conversion of resveratrol into pterostilbene was first assessed in vitro using protein extracts of agroinfiltrated N. benthamiana leaves and transformed grapevine callus. The grapevine cell cultures transformed with RhOOMT produced about 16 mg/L culture of pterostilbene and reached an extracellular distribution of up to 34% of total production at the best, which is by far the highest production reported to date in a plant system. A bonus large resveratrol production of ca. 1500-3000 mg/L was simultaneously obtained. Our results demonstrate a viable successful metabolic engineering strategy to produce pterostilbene, a resveratrol analog with enhanced pharmacological properties. Copyright © 2018 Elsevier B.V. All rights reserved.

Introduction of transformed chloroplasts from tobacco into petunia by asymmetric cell fusion.

PubMed

Sigeno, Asako; Hayashi, Sugane; Terachi, Toru; Yamagishi, Hiroshi

2009-11-01

Plastid engineering technique has been established only in Nicotiana tabacum, and the widespread application is severely limited so far. In order to exploit a method to transfer the genetically transformed plastomes already obtained in tobacco into other plant species, somatic cell fusion was conducted between a plastome transformant of tobacco and a cultivar of petunia (Petunia hybrida). A tobacco strain whose plastids had been transformed with aadA (a streptomycin/spectinomycin adenylyltransferase gene) and mdar [a gene for monodehydroascorbate reductase (MDAR)] and a petunia variety, 'Telstar', were used as cell fusion partners. An efficient regeneration system from the protoplasts of both the parents, and effectiveness of selection for the aadA gene with spectinomycin were established before the cell fusion. In addition, the influence of UV irradiation on the callus development from the protoplasts and shoot regeneration of tobacco was investigated. Protoplasts were cultured after cell fusion treatment with polyethylene glycol, and asymmetric somatic cybrids were selected using the aadA gene as a marker. Although many shoots of tobacco that had escaped the UV irradiation regenerated, several shoots possessing the morphology of petunia and the resistance to spectinomycin were obtained. Molecular analyses of the petunia type regenerants demonstrated that they had the nuclear and mitochondrial genomes derived from petunia besides the chloroplasts of tobacco transformed with aadA and mdar. Furthermore, it was ascertained that mdar was transcribed in the somatic cybrids. The results indicate the success in intergeneric transfer of transformed plastids of tobacco into petunia.

Phospholipase C-mediated hydrolysis of phosphatidylcholine is a target of transforming growth factor beta 1 inhibitory signals.

PubMed Central

Diaz-Meco, M T; Dominguez, I; Sanz, L; Municio, M M; Berra, E; Cornet, M E; Garcia de Herreros, A; Johansen, T; Moscat, J

1992-01-01

Cell growth and tumor transformation can be restrained in certain cell systems by the action of transforming growth factor beta (TGF-beta). It has been established that the mechanism whereby TGF-beta 1 inhibits cell growth does not interfere with the triggering of early mitogenic signal transduction mechanisms. Phospholipase C-catalyzed hydrolysis of phosphatidylcholine (PC) is a relatively late step in the cascade activated by growth factors. Therefore, conceivably activation of phospholipase C-catalyzed hydrolysis of PC could be the target of TGF-beta 1 action. In the study reported here, we demonstrate that TGF-beta 1 inhibits the coupling of ras p21 to the activation of PC hydrolysis, which appears to be critical for the antiproliferative effects of TGF-beta 1. Images PMID:1309592

The biomineralization process of uranium(VI) by Saccharomyces cerevisiae - transformation from amorphous U(VI) to crystalline chernikovite.

PubMed

Shen, Yanghao; Zheng, Xinyan; Wang, Xiaoyu; Wang, Tieshan

2018-05-01

Microorganisms play a significant role in uranium(VI) biogeochemistry and influence U(VI) transformation through biomineralization. In the present work, the process of uranium mineralization was investigated by Saccharomyces cerevisiae. The toxicity experiments showed that the viability of cell was not significantly affected by 100 mg L -1 U(VI) under 4 days of exposure time. The batch experiments showed that the phosphate concentration and pH value increased over time during U(VI) adsorption. Meanwhile, thermodynamic calculations demonstrated that the adsorption system was supersaturated with respect to UO 2 HPO 4 . The X-ray powder diffraction spectroscopy (XRD), field emission scanning electron microscopy (FE-SEM) equipped with energy dispersive spectroscopy (EDX), Fourier transform infrared spectroscopy (FTIR), and X-ray photoelectron spectroscopy (XPS) analyses indicated that the U(VI) was first attached onto the cell surface and reacted with hydroxyl, carboxyl, and phosphate groups through electrostatic interactions and complexation. As the immobilization of U(VI) transformed it from the ionic to the amorphous state, lamellar uranium precipitate was formed on the cell surface. With the prolongation of time, the amorphous uranium compound disappeared, and there were some crystalline substances observed extracellularly, which were well-characterized as tetragonal-chernikovite. Furthermore, the size of chernikovite was regulated at nano-level by cells, and the perfect crystal was formed finally. These findings provided an understanding of the non-reductive transformation process of U(VI) from the amorphous to crystalline state within microbe systems, which would be beneficial for the U(VI) treatment and reuse of nuclides and heavy metals.

Agrobacterium- and Biolistic-Mediated Transformation of Maize B104 Inbred.

PubMed

Raji, Jennifer A; Frame, Bronwyn; Little, Daniel; Santoso, Tri Joko; Wang, Kan

2018-01-01

Genetic transformation of maize inbred genotypes remains non-routine for many laboratories due to variations in cell competency to induce embryogenic callus, as well as the cell's ability to receive and incorporate transgenes into the genome. This chapter describes two transformation protocols using Agrobacterium- and biolistic-mediated methods for gene delivery. Immature zygotic embryos of maize inbred B104, excised from ears harvested 10-14 days post pollination, are used as starting explant material. Disarmed Agrobacterium strains harboring standard binary vectors and the biolistic gun system Bio-Rad PDS-1000/He are used as gene delivery systems. The herbicide resistant bar gene and selection agent bialaphos are used for identifying putative transgenic type I callus events. Using the step-by-step protocols described here, average transformation frequencies (number of bialaphos resistant T 0 callus events per 100 explants infected or bombarded) of 4% and 8% can be achieved using the Agrobacterium- and biolistic-mediated methods, respectively. An estimated duration of 16-21 weeks is needed using either protocol from the start of transformation experiments to obtaining putative transgenic plantlets with established roots. In addition to laboratory in vitro procedures, detailed greenhouse protocols for producing immature ears as transformation starting material and caring for transgenic plants for seed production are also described.

Natural Escherichia coli strains undergo cell-to-cell plasmid transformation.

PubMed

Matsumoto, Akiko; Sekoguchi, Ayuka; Imai, Junko; Kondo, Kumiko; Shibata, Yuka; Maeda, Sumio

2016-12-02

Horizontal gene transfer is a strong tool that allows bacteria to adapt to various environments. Although three conventional mechanisms of horizontal gene transfer (transformation, transduction, and conjugation) are well known, new variations of these mechanisms have also been observed. We recently reported that DNase-sensitive cell-to-cell transfer of nonconjugative plasmids occurs between laboratory strains of Escherichia coli in co-culture. We termed this phenomenon "cell-to-cell transformation." In this report, we found that several combinations of Escherichia coli collection of reference (ECOR) strains, which were co-cultured in liquid media, resulted in DNase-sensitive cell-to-cell transfer of antibiotic resistance genes. Plasmid isolation of these new transformants demonstrated cell-to-cell plasmid transfer between the ECOR strains. Natural transformation experiments, using a combination of purified plasmid DNA and the same ECOR strains, revealed that cell-to-cell transformation occurs much more frequently than natural transformation under the same culture conditions. Thus, cell-to-cell transformation is both unique and effective. In conclusion, this study is the first to demonstrate cell-to-cell plasmid transformation in natural E. coli strains. Copyright © 2016 Elsevier Inc. All rights reserved.

Transforming growth factor-beta controls T helper type 1 cell development through regulation of natural killer cell interferon-gamma.

PubMed

Laouar, Yasmina; Sutterwala, Fayyaz S; Gorelik, Leonid; Flavell, Richard A

2005-06-01

Interferon-gamma and interleukin 12 produced by the innate arm of the immune system are important regulators of T helper type 1 (T(H)1) cell development, but signals that negatively regulate their expression remain controversial. Here we show that transforming growth factor-beta (TGF-beta) controlled T(H)1 differentiation through the regulation of interferon-gamma produced by natural killer (NK) cells. Blockade of TGF-beta signaling in NK cells caused the accumulation of a large pool of NK cells secreting copious interferon-gamma, responsible for T(H)1 differentiation and protection from leishmania infection. In contrast, blockade of TGF-beta signaling in dendritic cells did not affect dendritic cell homeostasis or interleukin 12 production, thus indicating a previously undescribed demarcation of the function of TGF-beta in NK cells versus dendritic cells.

Biolistic transformation of tobacco and maize suspension cells using bacterial cells as microprojectiles.

PubMed

Rasmussen, J L; Kikkert, J R; Roy, M K; Sanford, J C

1994-01-01

We have used both Escherichia coli cells and Agrobacterium tumefaciens cells as microprojectiles to deliver DNA into suspension-cultured tobacco (Nicotiana tabacum L. line NT1) cells using a helium powered biolistic device. In addition, E. coli cells were used as microprojectiles for the transformation of suspension-cultured maize (Zea mays cv. Black Mexican Sweet) cells. Pretreating the bacterial cells with phenol at a concentration of 1.0%, and combining the bacterial cells with tungsten particles increased the rates of transformation. In N. tabacum, we obtained hundreds of transient transformants per bombardment, but were unable to recover any stable transformants. In Z. mays we obtained thousands of transient transformants and an average of six stable transformants per bombardment. This difference is discussed.

Solitary waves in morphogenesis: Determination fronts as strain-cued strain transformations among automatous cells

NASA Astrophysics Data System (ADS)

Cox, Brian N.; Landis, Chad M.

2018-02-01

We present a simple theory of a strain pulse propagating as a solitary wave through a continuous two-dimensional population of cells. A critical strain is assumed to trigger a strain transformation, while, simultaneously, cells move as automata to tend to restore a preferred cell density. We consider systems in which the strain transformation is a shape change, a burst of proliferation, or the commencement of growth (which changes the shape of the population sheet), and demonstrate isomorphism among these cases. Numerical and analytical solutions describe a strain pulse whose height does not depend on how the strain disturbance was first launched, or the rate at which the strain transformation is achieved, or the rate constant in the rule for the restorative cell motion. The strain pulse is therefore very stable, surviving the imposition of strong perturbations: it would serve well as a timing signal in development. The automatous wave formulation is simple, with few model parameters. A strong case exists for the presence of a strain pulse during amelogenesis. Quantitative analysis reveals a simple relationship between the velocity of the leading edge of the pulse in amelogenesis and the known speed of migration of ameloblast cells. This result and energy arguments support the depiction of wave motion as an automatous cell response to strain, rather than as a response to an elastic energy gradient. The theory may also contribute to understanding the determination front in somitogenesis, moving fronts of convergent-extension transformation, and mitotic wavefronts in the syncytial drosophila embryo.

MYCN induces neuroblastoma in primary neural crest cells.

PubMed

Olsen, R R; Otero, J H; García-López, J; Wallace, K; Finkelstein, D; Rehg, J E; Yin, Z; Wang, Y-D; Freeman, K W

2017-08-31

Neuroblastoma (NBL) is an embryonal cancer of the sympathetic nervous system (SNS), which causes 15% of pediatric cancer deaths. High-risk NBL is characterized by N-Myc amplification and segmental chromosomal gains and losses. Owing to limited disease models, the etiology of NBL is largely unknown, including both the cell of origin and the majority of oncogenic drivers. We have established a novel system for studying NBL based on the transformation of neural crest cells (NCCs), the progenitor cells of the SNS, isolated from mouse embryonic day 9.5 trunk neural tube explants. Based on pathology and gene expression analysis, we report the first successful transformation of wild-type NCCs into NBL by enforced expression of N-Myc, to generate phenotypically and molecularly accurate tumors that closely model human MYCN-amplified NBL. Using comparative genomic hybridization, we found that NCC-derived NBL tumors acquired copy number gains and losses that are syntenic to those observed in human MYCN-amplified NBL including 17q gain, 2p gain and loss of 1p36. When p53-compromised NCCs were transformed with N-Myc, we generated primitive neuroectodermal tumors with divergent differentiation including osteosarcoma. These subcutaneous tumors were metastatic to regional lymph nodes, liver and lung. Our novel experimental approach accurately models human NBL and establishes a new system with potential to study early stages of NBL oncogenesis, to functionally assess NBL oncogenic drivers and to characterize NBL metastasis.

ZIP8 expression in human proximal tubule cells, human urothelial cells transformed by Cd+2 and As+3 and in specimens of normal human urothelium and urothelial cancer

PubMed Central

2012-01-01

Background ZIP8 functions endogenously as a Zn+2/HCO3- symporter that can also bring cadmium (Cd+2) into the cell. It has also been proposed that ZIP8 participates in Cd-induced testicular necrosis and renal disease. In this study real-time PCR, western analysis, immunostaining and fluorescent localization were used to define the expression of ZIP8 in human kidney, cultured human proximal tubule (HPT) cells, normal and malignant human urothelium and Cd+2 and arsenite (As+3) transformed urothelial cells. Results It was shown that in the renal system both the non-glycosylated and glycosylated form of ZIP8 was expressed in the proximal tubule cells with localization of ZIP8 to the cytoplasm and cell membrane; findings in line with previous studies on ZIP8. The studies in the bladder were the first to show that ZIP8 was expressed in normal urothelium and that ZIP8 could be localized to the paranuclear region. Studies in the UROtsa cell line confirmed a paranuclear localization of ZIP8, however addition of growth medium to the cells increased the expression of the protein in the UROtsa cells. In archival human samples of the normal urothelium, the expression of ZIP8 was variable in intensity whereas in urothelial cancers ZIP8 was expressed in 13 of 14 samples, with one high grade invasive urothelial cancer showing no expression. The expression of ZIP8 was similar in the Cd+2 and As+3 transformed UROtsa cell lines and their tumor transplants. Conclusion This is the first study which shows that ZIP8 is expressed in the normal urothelium and in bladder cancer. In addition the normal UROtsa cell line and its transformed counterparts show similar expression of ZIP8 compared to the normal urothelium and the urothelial cancers suggesting that the UROtsa cell line could serve as a model system to study the expression of ZIP8 in bladder disease. PMID:22550998

Establishment and characterization of equine fibroblast cell lines transformed in vivo and in vitro by BPV-1: Model systems for equine sarcoids

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

Yuan, Z.Q.; Gault, E.A.; Gobeil, P.

2008-04-10

It is now widely recognized that BPV-1 and less commonly BPV-2 are the causative agents of equine sarcoids. Here we present the generation of equine cell lines harboring BPV-1 genomes and expressing viral genes. These lines have been either explanted from sarcoid biopsies or generated in vitro by transfection of primary fibroblasts with BPV-1 DNA. Previously detected BPV-1 genome variations in equine sarcoids are also found in sarcoid cell lines, and only variant BPV-1 genomes can transform equine cells. These equine cell lines are morphologically transformed, proliferate faster than parental cells, have an extended life span and can grow independentlymore » of substrate. These characteristics are more marked the higher the level of viral E5, E6 and E7 gene expression. These findings confirm that the virus has an active role in the induction of sarcoids and the lines will be invaluable for further studies on the role of BPV-1 in sarcoid pathology.« less

Epithelial to mesenchymal transition in arsenic-transformed cells promotes angiogenesis through activating β-catenin–vascular endothelial growth factor pathway

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

Wang, Zhishan; Humphries, Brock; Xiao, Hua

2013-08-15

Arsenic exposure represents a major health concern increasing cancer risks, yet the mechanism of arsenic carcinogenesis has not been elucidated. We and others recently reported that cell malignant transformation by arsenic is accompanied by epithelial to mesenchymal transition (EMT). However, the role of EMT in arsenic carcinogenesis is not well understood. Although previous studies showed that short term exposure of endothelial cells to arsenic stimulated angiogenesis, it remains to be determined whether cells that were malignantly transformed by long term arsenic exposure have a pro-angiogenic effect. The objective of this study was to investigate the effect of arsenic-transformed human bronchialmore » epithelial cells that underwent EMT on angiogenesis and the underlying mechanism. It was found that the conditioned medium from arsenic-transformed cells strongly stimulated tube formation by human umbilical vein endothelial cells (HUVECs). Moreover, enhanced angiogenesis was detected in mouse xenograft tumor tissues resulting from inoculation of arsenic-transformed cells. Mechanistic studies revealed that β-catenin was activated in arsenic-transformed cells up-regulating its target gene expression including angiogenic-stimulating vascular endothelial growth factor (VEGF). Stably expressing microRNA-200b in arsenic-transformed cells that reversed EMT inhibited β-catenin activation, decreased VEGF expression and reduced tube formation by HUVECs. SiRNA knockdown β-catenin decreased VEGF expression. Adding a VEGF neutralizing antibody into the conditioned medium from arsenic-transformed cells impaired tube formation by HUVECs. Reverse transcriptase-PCR analysis revealed that the mRNA levels of canonical Wnt ligands were not increased in arsenic-transformed cells. These findings suggest that EMT in arsenic-transformed cells promotes angiogenesis through activating β-catenin–VEGF pathway. - Highlights: • Arsenic-transformed cells that underwent EMT displayed a pro-angiogenic effect. • EMT in arsenic-transformed cells activates β-catenin. • β-Catenin activation increases VEGF expression in arsenic-transformed cells. • β-Catenin activation is likely independent of canonical Wnt signaling. • EMT in arsenic-transformed cells promotes angiogenesis via β-catenin–VEGF pathway.« less

Divergent Pseudomonas exotoxin A sensitivity in normal and transformed liver cells is correlated with low-density lipoprotein receptor-related protein expression.

PubMed

Laithwaite, J E; Benn, S J; Marshall, W S; FitzGerald, D J; LaMarre, J

2001-09-01

Pseudomonas exotoxin A (PEA) is an extracellular virulence factor produced by the opportunistic human pathogen Pseudomonas aerguinosa. PEA intoxification begins when PEA binds to the low-density lipoprotein receptor-related protein (LRP). The liver is the primary target of systemic PEA, due largely to the high levels of functional LRP expressed by liver cells. Using a 3H-leucine incorporation assay to measure inhibition of protein synthesis we have demonstrated that normal (BNL CL.2) and transformed (BNL 1ME A7R.1) liver cells exhibit divergent PEA sensitivity; with BNL 1ME A7R.1 cells demonstrating greater PEA sensitivity than their non-transformed counterparts. The receptor-associated protein, a LRP antagonist, decreased PEA toxicity in BNL 1ME A7R.1 cells, confirming the importance of the LRP in PEA intoxification in this cell type. Increased PEA sensitivity in BNL 1ME A7R.1 cells was associated with increased functional cell surface LRP expression, as measured by alpha2-macroglobulin binding and internalization studies, and increased LRP mRNA levels, as determined by Northern blot analysis. Interestingly, BNL CL.2 cells were more sensitive than BNL 1ME A7R.1 cells to conjugate and mutant PEA toxins that do not utilize the LRP for cellular entry. These data demonstrate that increased LRP expression is an important mechanism by which PEA sensitivity is increased in BNL 1ME A7R.1 transformed liver cells.

DNA transformation via local heat shock

NASA Astrophysics Data System (ADS)

Li, Sha; Meadow Anderson, L.; Yang, Jui-Ming; Lin, Liwei; Yang, Haw

2007-07-01

This work describes transformation of foreign DNA into bacterial host cells by local heat shock using a microfluidic system with on-chip, built-in platinum heaters. Plasmid DNA encoding ampicillin resistance and a fluorescent protein can be effectively transformed into the DH5α chemically competent E. coli using this device. Results further demonstrate that only one-thousandth of volume is required to obtain transformation efficiencies as good as or better than conventional practices. As such, this work complements other lab-on-a-chip technologies for potential gene cloning/therapy and protein expression applications.

Product toxicity and cometabolic competitive inhibition modeling of chloroform and trichloroethylene transformation by methanotrophic resting cells.

PubMed Central

Alvarez-Cohen, L; McCarty, P L

1991-01-01

The rate and capacity for chloroform (CF) and trichloroethylene (TCE) transformation by a mixed methanotrophic culture of resting cells (no exogenous energy source) and formate-fed cells were measured. As reported previously for TCE, formate addition resulted in an increased CF transformation rate (0.35 day-1 for resting cells and 1.5 day-1 for formate-fed cells) and transformation capacity (0.0065 mg of CF per mg of cells for resting cells and 0.015 mg of CF per mg of cells for formate-fed cells), suggesting that depletion of energy stores affects transformation behavior. The observed finite transformation capacity, even with an exogenous energy source, suggests that toxicity was also a factor. CF transformation capacity was significantly lower than that for TCE, suggesting a greater toxicity from CF transformation. The toxicity of CF, TCE, and their transformation products to whole cells was evaluated by comparing the formate oxidation activity of acetylene-treated cells to that of non-acetylene-treated cells with and without prior exposure to CF or TCE. Acetylene arrests the activity of methane monooxygenase in CF and TCE oxidation without halting cell activity toward formate. Significantly diminished formate oxidation by cells exposed to either CR or TCE without acetylene compared with that with acetylene suggests that the solvents themselves were not toxic under the experimental conditions but their transformation products were. The concurrent transformation of CF and TCE by resting cells was measured, and results were compared with predictions from a competitive-inhibition cometabolic transformation model. The reasonable fit between model predictions and experimental observations was supportive of model assumptions. PMID:1905516

Demonstration of the oncogenic potential of Herpes simplex viruses and human cytomegalovirus. [UV radiation

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

Rapp, F.; Li, J.L.H.

1975-01-01

The following topics are reviewed: transformation of hamster embryo cells by herpes simplex viruses and human cytomegalovirus; the use of uv radiation and photodynamic action to inactivate virus infectivity while retaining the transformation potential of the virus; detection of virus-specific antigens in transformed cells; oncogenicity of HSV- and CMV-transformed cells in vivo; immunological studies of metastases induced by herpes virus-transformed cells; resistance of transformed cells to superinfection; maintenance of the virus genome in the transformed state; and stimulation of cellular DNA synthesis by human cytomegalovirus. (HLW)

Therapeutic effect of mesenchymal multipotent stromal cells on memory in animals with Alzheimer-type neurodegeneration.

PubMed

Bobkova, N V; Poltavtseva, R A; Samokhin, A N; Sukhikh, G T

2013-11-01

Transplantation of human mesenchymal multipotent stromal cells improved spatial memory in bulbectomized mice with Alzheimer-type neurodegeneration. The positive effect was observed in 1 month after intracerebral transplantation and in 3 months after systemic injection of mesenchymal multipotent stromal cells. No cases of malignant transformation were noted. These findings indicate prospects of using mesenchymal multipotent stromal cells for the therapy of Alzheimer disease and the possibility of their systemic administration for attaining the therapeutic effect.

Increased concentration of an apparently identical cellular protein in cells transformed by either Abelson murine leukemia virus or other transforming agents.

PubMed

Rotter, V; Boss, M A; Baltimore, D

1981-04-01

Abelson murine leukemia virus (A-MuLV)-transformed cells, simian virus 40 (SV40)-transformed cells, and chemically transformed cells all have increased levels of a 50,000-molecular-weight host cell protein. The protein was detected with sera raised to the A-MuLV-transformed and chemically transformed cells and was tightly bound to T-antigen in extracts of SV40-transformed cells. Partial protease digests showed that the proteins from all three sources were indistinguishable. The three proteins were phosphorylated in cells, and the linkage of phosphate to the A-MuLV-associated P50 was to a serine residue. By immunofluorescence methods, P50-related protein was found on the surface of both normal lymphoid cells and A-MuLV-transformed lymphoid cells, but cell fractionation showed that the majority of P50 was free in the cytoplasm of the transformed cells. Immunofluorescence also showed that P50 was found in granules in the cytoplasm of both untransformed and SV40-transformed fibroblasts. Other cells gave indistinct patterns. Cocapping experiments showed that the A-MuLV-specified P120 protein is weakly associated with the surface P50-related protein of lymphoid cells, but no association of P120 and P50 could be demonstrated by immunoprecipitation methods. Although a monoclonal antiserum to P50 was used in many of these studies, the identity of the bulk P50 protein with the molecules that are reactive at the cell surface requires further study.

Immunization with SV40-transformed cells yields mainly MHC-restricted monoclonal antibodies

PubMed Central

1986-01-01

Recognition of antigens on cell surfaces only in the context of the MHC- encoded alloantigens of the presenting cell (self + X) has classically been considered the province of T cells. However, evidence from several sources has indicated that B cells and antibodies can exhibit self + X- restricted recognition as well. This report concerns the mAb response to SV40-transformed H-2b fibroblast cell lines. The specificities of the antibodies obtained have been analyzed for binding to a panel of SV40-transformed H-2-syngeneic, H-2-allogeneic, and H-2b mutant fibroblast cell lines, as well as cell lines not bearing cell surface SV40 transformation-associated antigens. A large proportion of primary C57BL/6 (71%) and BALB/c (68%) splenic B cells responding to in vitro stimulation with SV40-transformed H-2b cells recognize cell surface antigens associated with SV40 transformation only when coexpressed with MHC antigens of the immunizing cell, particularly the Kb molecule, on transformed cells. To extensively define the nature of antigen recognition by these antibodies, we have generated and characterized nine hybridoma antibodies specific for SV40-transformed H-2-syngeneic cell lines. Seven of these hybridoma antibodies recognize SV40- associated transformation antigens in the context of H-2b molecules. Six of these are restricted by the Kb molecule and discriminate among a panel of SV40-transformed Kb mutant cell lines, thus confirming the participation of class I MHC-encoded molecules in the recognition by B cells of cell surface antigens. PMID:3014034

Selective antiproliferative and apoptotic effects of flavonoids purified from Rhus verniciflua Stokes on normal versus transformed hepatic cell lines.

PubMed

Son, Young-Ok; Lee, Kyung-Yeol; Lee, Jeong-Chae; Jang, Hyon-Seok; Kim, Jong-Ghee; Jeon, Young-Mi; Jang, Yong-Suk

2005-01-15

Considerable attention is being concentrated on dietary flavonoids in developing novel cancer-preventive approaches due to their potential ability to induce selective apoptosis of cancer cells. In this study, we prepared a flavonoid-containing fraction from a crude acetone extract of Rhus verniciflua Stokes (RVS), traditionally used as a food additive and as an herbal medicine, and named RVS chloroform-methanol fraction (RCMF). We evaluated the effects of RCMF on proliferation and apoptosis using mouse embryonic primary hepatic cells (MPHC), embryonic normal hepatic cell line (BNL CL.2), and its SV40-mediated transformed cell line (BNL SV A.8). We also investigated the effects of RCMF on the antioxidant defense system in those cells. This study demonstrated that RCMF exhibited a selective growth inhibition and apoptosis induction on transformed cells. BNL SV A.8 cells were more sensitive to RCMF-mediated cytotoxicity than were MPHC or BNL CL.2. RCMF-mediated reduction of MnSOD activity and glutathione (GSH) content in BNL SV A.8 cells is thought to be associated with RCMF-induced apoptosis. Our findings suggest that RCMF is an agent which may be capable of inducing growth inhibition and apoptosis of hepatic tumor cells.

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

PubMed Central

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

2010-01-01

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

A fully automated cell segmentation and morphometric parameter system for quantifying corneal endothelial cell morphology.

PubMed

Al-Fahdawi, Shumoos; Qahwaji, Rami; Al-Waisy, Alaa S; Ipson, Stanley; Ferdousi, Maryam; Malik, Rayaz A; Brahma, Arun

2018-07-01

Corneal endothelial cell abnormalities may be associated with a number of corneal and systemic diseases. Damage to the endothelial cells can significantly affect corneal transparency by altering hydration of the corneal stroma, which can lead to irreversible endothelial cell pathology requiring corneal transplantation. To date, quantitative analysis of endothelial cell abnormalities has been manually performed by ophthalmologists using time consuming and highly subjective semi-automatic tools, which require an operator interaction. We developed and applied a fully-automated and real-time system, termed the Corneal Endothelium Analysis System (CEAS) for the segmentation and computation of endothelial cells in images of the human cornea obtained by in vivo corneal confocal microscopy. First, a Fast Fourier Transform (FFT) Band-pass filter is applied to reduce noise and enhance the image quality to make the cells more visible. Secondly, endothelial cell boundaries are detected using watershed transformations and Voronoi tessellations to accurately quantify the morphological parameters of the human corneal endothelial cells. The performance of the automated segmentation system was tested against manually traced ground-truth images based on a database consisting of 40 corneal confocal endothelial cell images in terms of segmentation accuracy and obtained clinical features. In addition, the robustness and efficiency of the proposed CEAS system were compared with manually obtained cell densities using a separate database of 40 images from controls (n = 11), obese subjects (n = 16) and patients with diabetes (n = 13). The Pearson correlation coefficient between automated and manual endothelial cell densities is 0.9 (p < 0.0001) and a Bland-Altman plot shows that 95% of the data are between the 2SD agreement lines. We demonstrate the effectiveness and robustness of the CEAS system, and the possibility of utilizing it in a real world clinical setting to enable rapid diagnosis and for patient follow-up, with an execution time of only 6 seconds per image. Copyright © 2018 Elsevier B.V. All rights reserved.

Progress in corneal wound healing

PubMed Central

Ljubimov, Alexander V.; Saghizadeh, Mehrnoosh

2015-01-01

Corneal wound healing is a complex process involving cell death, migration, proliferation, differentiation, and extracellular matrix remodeling. Many similarities are observed in the healing processes of corneal epithelial, stromal and endothelial cells, as well as cell-specific differences. Corneal epithelial healing largely depends on limbal stem cells and remodeling of the basement membrane. During stromal healing, keratocytes get transformed to motile and contractile myofibroblasts largely due to activation of transforming growth factor-β system. Endothelial cells heal mostly by migration and spreading, with cell proliferation playing a secondary role. In the last decade, many aspects of wound healing process in different parts of the cornea have been elucidated, and some new therapeutic approaches have emerged. The concept of limbal stem cells received rigorous experimental corroboration, with new markers uncovered and new treatment options including gene and microRNA therapy tested in experimental systems. Transplantation of limbal stem cell-enriched cultures for efficient re-epithelialization in stem cell deficiency and corneal injuries has become reality in clinical setting. Mediators and course of events during stromal healing have been detailed, and new treatment regimens including gene (decorin) and stem cell therapy for excessive healing have been designed. This is a very important advance given the popularity of various refractive surgeries entailing stromal wound healing. Successful surgical ways of replacing the diseased endothelium have been clinically tested, and new approaches to accelerate endothelial healing and suppress endothelial-mesenchymal transformation have been proposed including Rho kinase (ROCK) inhibitor eye drops and gene therapy to activate TGF-β inhibitor SMAD7. Promising new technologies with potential for corneal wound healing manipulation including microRNA, induced pluripotent stem cells to generate corneal epithelium, and nanocarriers for corneal drug delivery are discussed. Attention is also paid to problems in wound healing understanding and treatment, such as lack of specific epithelial stem cell markers, reliable identification of stem cells, efficient prevention of haze and stromal scar formation, lack of data on wound regulating microRNAs in keratocytes and endothelial cells, as well as virtual lack of targeted systems for drug and gene delivery to select corneal cells. PMID:26197361

Progress in corneal wound healing.

PubMed

Ljubimov, Alexander V; Saghizadeh, Mehrnoosh

2015-11-01

Corneal wound healing is a complex process involving cell death, migration, proliferation, differentiation, and extracellular matrix remodeling. Many similarities are observed in the healing processes of corneal epithelial, stromal and endothelial cells, as well as cell-specific differences. Corneal epithelial healing largely depends on limbal stem cells and remodeling of the basement membrane. During stromal healing, keratocytes get transformed to motile and contractile myofibroblasts largely due to activation of transforming growth factor-β (TGF-β) system. Endothelial cells heal mostly by migration and spreading, with cell proliferation playing a secondary role. In the last decade, many aspects of wound healing process in different parts of the cornea have been elucidated, and some new therapeutic approaches have emerged. The concept of limbal stem cells received rigorous experimental corroboration, with new markers uncovered and new treatment options including gene and microRNA therapy tested in experimental systems. Transplantation of limbal stem cell-enriched cultures for efficient re-epithelialization in stem cell deficiency and corneal injuries has become reality in clinical setting. Mediators and course of events during stromal healing have been detailed, and new treatment regimens including gene (decorin) and stem cell therapy for excessive healing have been designed. This is a very important advance given the popularity of various refractive surgeries entailing stromal wound healing. Successful surgical ways of replacing the diseased endothelium have been clinically tested, and new approaches to accelerate endothelial healing and suppress endothelial-mesenchymal transformation have been proposed including Rho kinase (ROCK) inhibitor eye drops and gene therapy to activate TGF-β inhibitor SMAD7. Promising new technologies with potential for corneal wound healing manipulation including microRNA, induced pluripotent stem cells to generate corneal epithelium, and nanocarriers for corneal drug delivery are discussed. Attention is also paid to problems in wound healing understanding and treatment, such as lack of specific epithelial stem cell markers, reliable identification of stem cells, efficient prevention of haze and stromal scar formation, lack of data on wound regulating microRNAs in keratocytes and endothelial cells, as well as virtual lack of targeted systems for drug and gene delivery to select corneal cells. Copyright © 2015 Elsevier Ltd. All rights reserved.

2015 Annual Progress Report: DOE Hydrogen and Fuel Cells Program

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

None, None

The 2015 Annual Progress Report summarizes fiscal year 2015 activities and accomplishments by projects funded by the DOE Hydrogen and Fuel Cells Program. It covers the program areas of hydrogen production; hydrogen delivery; hydrogen storage; fuel cells; manufacturing R&D; technology validation; safety, codes and standards; systems analysis; and market transformation.

Radiogenic transformation of human mammary epithelial cells in vitro

NASA Technical Reports Server (NTRS)

Yang, T. C.; Georgy, K. A.; Tavakoli, A.; Craise, L. M.; Durante, M.

1996-01-01

Cancer induction by space radiations is a major concern for manned space exploration. Accurate assessment of radiation risk at low doses requires basic understanding of mechanism(s) of radiation carcinogenesis. For determining the oncogenic effects of ionizing radiation in human epithelial cells, we transformed a mammary epithelial cell line (185B5), which was immortalized by benzo(a)pyrene, with energetic heavy ions and obtained several transformed clones. These transformed cells showed growth properties on Matrigel similar to human mammary tumor cells. To better understand the mechanisms of radiogenic transformation of human cells, we systematically examined the alterations in chromosomes and cancer genes. Among 16 autosomes examined for translocations, by using fluorescence in situ hybridization (FISH) technique, chromosomes 3, 12, 13, 15, 16, and 18 appeared to be normal in transformed cells. Chromosomes 1, 4, 6, 8, and 17 in transformed cells, however, showed patterns different from those in nontransformed cells. Southern blot analyses indicated no detectable alterations in myc, ras, Rb, or p53 genes. Further studies of chromosome 17 by using in situ hybridization with unique sequence p53 gene probe and a centromere probe showed no loss of p53 gene in transformed cells. Experimental results from cell fusion studies indicated that the transforming gene(s) is recessive. The role of genomic instability and tumor suppressor gene(s) in radiogenic transformation of human breast cells remains to be identified.

P01.29 Mutant (R132H) IDH1-driven cellular transformation makes cells dependent on continued wild type IDH1 expression in a model of in vitro gliomagenesis

PubMed Central

Johannessen, T.; Mukherjee, J.; Wood, M.; Viswanath, P.; Ohba, S.; Ronen, S.; Berkvig, R.; Pieper, R.

2017-01-01

Abstract Introduction: Missense R132H mutations in the active site of isocitrate dehydrogenase 1 (IDH1) biologically and diagnostically distinguish low-grade gliomas and secondary glioblastomas from primary glioblastomas. IDH1 mutations lead to the formation of the oncometabolite 2-hydroxyglutarate (2-HG) from the reduction of α-ketoglutarate (α-KG), which in turn facilitates tumorigenesis by modifying DNA and histone methylation as well blocking differentiation processes. We recently showed (Mol Cancer Res 14: 976–983, 2016) that although mutant IDH1 expression in hTERT-immortalized, p53/pRb-deficient astrocytes can drive cellular transformation and gliomagenesis, selective pharmacologic inhibition and elimination of 2-HG by the mutant IDH1 inhibitor AGI-5198 has little effect on the growth or clonagenicity of these transformed cells. To address the possible role of WT IDH1 in the growth of mutant IDH-driven tumor cells, we used a slightly different gliomagenesis model in which the transformation of TERT-deficient, p53/pRb-deficient astrocytes (pre-crisis cells) occurs only after prolonged expression of mutant IDH and passage through cellular crisis (post-crisis cells, Cancer Res 76:6680–6689, 2016). METHODS AND MATERIALS: Using this system we introduced AGI-5198, or siRNA targeting both WT and mutant forms of IDH1 into p53/pRb-deficient, mutant IDH1-expressing human astrocytes prior to or following their transformation, and compared the effects on cell growth and clonagenicity. Results: AGI-5198 exposure decreased levels of 2HG by greater than 90%, and as previously reported had no effect on the growth of either the pre-or post-crisis cell populations. A one-day exposure to a pan IDH1 siRNA resulted in a similar, prolonged (greater than 6 day), 80% inhibition of both WT and mutant IDH1 protein levels and 2HG in both cell groups. While the growth of the mutant IDH-expressing, non-transformed cells was similar to that of scramble siRNA controls, the growth of the mutant IDH-transformed cells was significantly reduced. This growth suppression was also accompanied by a four-fold increase in annexin V-positive apoptotic cells. Furthermore, the growth suppression in the cells transformed by mutant IDH1 expression could not be reversed by addition of a cell-permeable form of 2-HG. Conclusions: These results show that the in vitro transformative events driven by expression of mutant IDH1 make cells dependent not on continued mutant IDH1 expression, but rather on continued WT IDH1 expression. The data also support the development and testing of agents that can inhibit both the WT and mutant forms of IDH1.

The shape modulation of osteoblast-osteocyte transformation and its correlation with the fibrillar organization in secondary osteons: a SEM study employing the graded osmic maceration technique.

PubMed

Pazzaglia, Ugo E; Congiu, Terenzio; Marchese, Marcella; Dell'Orbo, Carlo

2010-06-01

Cortex fractured surface and graded osmic maceration techniques were used to study the secretory activity of osteoblasts, the transformation of osteoblast to osteocytes, and the structural organization of the matrix around the cells with scanning electron microscopy (SEM). A specialized membrane differentiation at the base of the cell was observed with finger-like, flattened processes which formed a diffuse meshwork. These findings suggested that this membrane differentiation below the cells had not only functioned in transporting collagen through the membrane but also in orienting the fibrils once assembled. Thin ramifications arose from the large and flat membrane foldings oriented perpendicular to the plane of the osteoblasts. This meshwork of fine filaments could not be visualized with SEM because they were obscured within the matrix substance. Their 3-D structure, however, should be similar to the canalicular system. The meshwork of large, flattened processes was no more evident in the cells which had completed their transformation into osteocytes.

Tumor suppressors Sav/Scrib and oncogene Ras regulate stem cell transformation in adult Drosophila Malpighian Tubules

PubMed Central

Zeng, Xiankun; Singh, Shree Ram; Hou, David; Hou, Steven X.

2012-01-01

An increasing body of evidence suggests that tumors might originate from a few transformed cells that share many properties with normal stem cells. However, it remains unclear how normal stem cells are transformed into cancer stem cells. Here, we demonstrated that mutations causing the loss of tumor suppressor Sav or Scrib or activation of the oncogene Ras transform normal stem cells into cancer stem cells through a multistep process in the adult Drosophila Malpighian Tubules (MTs). In wild-type MTs, each stem cell generates one self-renewing and one differentiating daughter cell. However, in flies with loss-of-function sav or scrib or gain-of-function Ras mutations, both daughter cells grew and behaved like stem cells, leading to the formation of tumors in MTs. Ras functioned downstream of Sav and Scrib in regulating the stem cell transformation. The Ras-transformed stem cells exhibited many of the hallmarks of cancer, such as increased proliferation, reduced cell death, and failure to differentiate. We further demonstrated that several signal transduction pathways (including MEK/MAPK, RhoA, PKA, and TOR) mediate Rasṕ function in the stem cell transformation. Therefore, we have identified a molecular mechanism that regulates stem cell transformation, and this finding may lead to strategies for preventing tumor formation in certain organs. PMID:20432470

ptxD gene in combination with phosphite serves as a highly effective selection system to generate transgenic cotton (Gossypium hirsutum L.).

PubMed

Pandeya, Devendra; Campbell, LeAnne M; Nunes, Eugenia; Lopez-Arredondo, Damar L; Janga, Madhusudhana R; Herrera-Estrella, Luis; Rathore, Keerti S

2017-12-01

This report demonstrates the usefulness of ptxD/phosphite as a selection system that not only provides a highly efficient and simple means to generate transgenic cotton plants, but also helps address many of the concerns related to the use of antibiotic and herbicide resistance genes in the production of transgenic crops. Two of the most popular dominant selectable marker systems for plant transformation are based on either antibiotic or herbicide resistance genes. Due to concerns regarding their safety and in order to stack multiple traits in a single plant, there is a need for alternative selectable marker genes. The ptxD gene, derived from Pseudomonas stutzeri WM88, that confers to cells the ability to convert phosphite (Phi) into orthophosphate (Pi) offers an alternative selectable marker gene as demonstrated for tobacco and maize. Here, we show that the ptxD gene in combination with a protocol based on selection medium containing Phi, as the sole source of phosphorus (P), can serve as an effective and efficient system to select for transformed cells and generate transgenic cotton plants. Fluorescence microscopy examination of the cultures under selection and molecular analyses on the regenerated plants demonstrate the efficacy of the system in recovering cotton transformants following Agrobacterium-mediated transformation. Under the ptxD/Phi selection, an average of 3.43 transgenic events per 100 infected explants were recovered as opposed to only 0.41% recovery when bar/phosphinothricin (PPT) selection was used. The event recovery rates for nptII/kanamycin and hpt/hygromycin systems were 2.88 and 2.47%, respectively. Molecular analysis on regenerated events showed a selection efficiency of ~ 97% under the ptxD/Phi system. Thus, ptxD/Phi has proven to be a very efficient, positive selection system for the generation of transgenic cotton plants with equal or higher transformation efficiencies compared to the commonly used, negative selection systems.

Visuomotor Transformation in the Fly Gaze Stabilization System

PubMed Central

Huston, Stephen J; Krapp, Holger G

2008-01-01

For sensory signals to control an animal's behavior, they must first be transformed into a format appropriate for use by its motor systems. This fundamental problem is faced by all animals, including humans. Beyond simple reflexes, little is known about how such sensorimotor transformations take place. Here we describe how the outputs of a well-characterized population of fly visual interneurons, lobula plate tangential cells (LPTCs), are used by the animal's gaze-stabilizing neck motor system. The LPTCs respond to visual input arising from both self-rotations and translations of the fly. The neck motor system however is involved in gaze stabilization and thus mainly controls compensatory head rotations. We investigated how the neck motor system is able to selectively extract rotation information from the mixed responses of the LPTCs. We recorded extracellularly from fly neck motor neurons (NMNs) and mapped the directional preferences across their extended visual receptive fields. Our results suggest that—like the tangential cells—NMNs are tuned to panoramic retinal image shifts, or optic flow fields, which occur when the fly rotates about particular body axes. In many cases, tangential cells and motor neurons appear to be tuned to similar axes of rotation, resulting in a correlation between the coordinate systems the two neural populations employ. However, in contrast to the primarily monocular receptive fields of the tangential cells, most NMNs are sensitive to visual motion presented to either eye. This results in the NMNs being more selective for rotation than the LPTCs. Thus, the neck motor system increases its rotation selectivity by a comparatively simple mechanism: the integration of binocular visual motion information. PMID:18651791

Prediction of lung cells oncogenic transformation for induced radon progeny alpha particles using sugarscape cellular automata.

PubMed

Baradaran, Samaneh; Maleknasr, Niaz; Setayeshi, Saeed; Akbari, Mohammad Esmaeil

2014-01-01

Alpha particle irradiation from radon progeny is one of the major natural sources of effective dose in the public population. Oncogenic transformation is a biological effectiveness of radon progeny alpha particle hits. The biological effects which has caused by exposure to radon, were the main result of a complex series of physical, chemical, biological and physiological interactions. The cellular and molecular mechanisms for radon-induced carcinogenesis have not been clear yet. Various biological models, including cultured cells and animals, have been found useful for studying the carcinogenesis effects of radon progeny alpha particles. In this paper, sugars cape cellular automata have been presented for computational study of complex biological effect of radon progeny alpha particles in lung bronchial airways. The model has included mechanism of DNA damage, which has been induced alpha particles hits, and then formation of transformation in the lung cells. Biomarkers were an objective measure or evaluation of normal or abnormal biological processes. In the model, the metabolism rate of infected cell has been induced alpha particles traversals, as a biomarker, has been followed to reach oncogenic transformation. The model results have successfully validated in comparison with "in vitro oncogenic transformation data" for C3H 10T1/2 cells. This model has provided an opportunity to study the cellular and molecular changes, at the various stages in radiation carcinogenesis, involving human cells. It has become well known that simulation could be used to investigate complex biomedical systems, in situations where traditional methodologies were difficult or too costly to employ.

Embryo-specific expression of a visual reporter gene as a selection system for citrus transformation

PubMed Central

Zambon, Flavia T.; Erpen, Lígia; Soriano, Leonardo; Grosser, Jude

2018-01-01

The embryo-specific Dc3 gene promoter driving the VvMybA1 anthocyanin regulatory gene was used to develop a visual selection system for the genetic transformation of citrus. Agrobacterium-mediated transformation of cell suspension cultures resulted in the production of purple transgenic somatic embryos that could be easily separated from the green non-transgenic embryos. The somatic embryos produced phenotypically normal plants devoid of any visual purple coloration. These results were also confirmed using protoplast transformation. There was minimal gene expression in unstressed one-year-old transgenic lines. Cold and drought stress did not have any effect on gene expression, while exogenous ABA and NaCl application resulted in a minor change in gene expression in several transgenic lines. When gas exchange was measured in intact leaves, the transgenic lines were similar to controls under the same environment. Our results provide conclusive evidence for the utilization of a plant-derived, embryo-specific visual reporter system for the genetic transformation of citrus. Such a system could aid in the development of an all-plant, consumer-friendly GM citrus tree. PMID:29293649

A Model System to Investigate the Effect of BRCA1 and/or p53 Inactivation in the Ovarian Stroma on Growth and Transformation Potential of the Ovarian Epithelium

DTIC Science & Technology

2009-10-01

Effect of BRCA1 and/or p53 Inactivation in the Ovarian Stroma on Growth and Transformation Potential of the Ovarian Epithelium PRINCIPAL...AND SUBTITLE 5a. CONTRACT NUMBER A Model System To Investigate the Effect of BRCA1 and/or p53 Inactivation in the Ovarian Stroma on Growth and... effects of loss of function of BRCA1 or BRCA1 and Trp53 in the stroma on the growth and neoplastic transformation of epithelial cells using 2D and 3D in

Microtubule reorganization in tobacco BY-2 cells stably expressing GFP-MBD

NASA Technical Reports Server (NTRS)

Granger, C. L.; Cyr, R. J.

2000-01-01

Microtubule organization plays an important role in plant morphogenesis; however, little is known about how microtubule arrays transit from one organized state to another. The use of a genetically incorporated fluorescent marker would allow long-term observation of microtubule behavior in living cells. Here, we have characterized a Nicotiana tabacum L. cv. Bright Yellow 2 (BY-2) cell line that had been stably transformed with a gfp-mbd construct previously demonstrated to label microtubules (J. Marc et al., 1998, Plant Cell 10: 1927-1939). Fluorescence levels were low, but interphase and mitotic microtubule arrays, as well as the transitions between these arrays, could be observed in individual gfp-mbd-transformed cells. By comparing several attributes of transformed and untransformed cells it was concluded that the transgenic cells are not adversely affected by low-level expression of the transgene and that these cells will serve as a useful and accurate model system for observing microtubule reorganization in vivo. Indeed, some initial observations were made that are consistent with the involvement of motor proteins in the transition between the spindle and phragmoplast arrays. Our observations also support the role of the perinuclear region in nucleating microtubules at the end of cell division with a progressive shift of these microtubules and/or nucleating activity to the cortex to form the interphase cortical array.

Establishment of prostate cancer spheres from a prostate cancer cell line after phenethyl isothiocyanate treatment and discovery of androgen-dependent reversible differentiation between sphere and neuroendocrine cells.

PubMed

Chen, Yamei; Cang, Shundong; Han, Liying; Liu, Christina; Yang, Patrick; Solangi, Zeeshan; Lu, Quanyi; Liu, Delong; Chiao, J W

2016-05-03

Prostate cancer can transform from androgen-responsive to an androgen-independent phenotype. The mechanism responsible for the transformation remains unclear. We studied the effects of an epigenetic modulator, phenethyl isothiocyanate (PEITC), on the androgen-responsive LNCaP cells. After treatment with PEITC, floating spheres were formed with characteristics of prostate cancer stem cells (PCSC). These spheres were capable of self-renewal in media with and without androgen. They have been maintained in both types of media as long term cultures. Upon androgen deprivation, the adherent spheres differentiated to neuroendocrine cells (NEC) with decreased proliferation, expression of androgen receptor, and PSA. NEC reverse differentiated to spheres when androgen was replenished. The sphere cells expressed surface marker CD44 and had enhanced histone H3K4 acetylation, DNMT1 down-regulation and GSTP1 activation. We hypothesize that PEITC-mediated alteration in epigenomics of LNCaP cells may give rise to sphere cells, whereas reversible androgenomic alterations govern the shuttling between sphere PCSC and progeny NEC. Our findings identify unrecognized properties of prostate cancer sphere cells with multi-potential plasticity. This system will facilitate development of novel therapeutic agents and allow further exploration into epigenomics and androgenomics governing the transformation to hormone refractory prostate cancer.

Closing the Phenotypic Gap between Transformed Neuronal Cell Lines in Culture and Untransformed Neurons

NASA Technical Reports Server (NTRS)

Myers, Tereance A.; Nickerson, Cheryl A.; Kaushal, Deepak; Ott, C. Mark; HonerzuBentrup, Kerstin; Ramamurthy, Rajee; Nelman-Gonzales, Mayra; Pierson, Duane L.; Philipp, Mario T.

2008-01-01

Studies of neuronal dysfunction in the central nervous system (CNS) are frequently limited by the failure of primary neurons to propagate in vitro. Neuronal cell lines can be substituted for primary cells but they often misrepresent normal conditions. We hypothesized that a dimensional (3-D) cell culture system would drive the phenotype of transformed neurons closer to that of untransformed cells. In our studies comparing 3-D versus 2-dimensional (2-D) culture, neuronal SH-SY5Y (SY) cells underwent distinct morphological changes combined with a significant drop in their rate of cell division. Expression of the proto-oncogene N-myc and the RNA binding protein HuD was decreased in 3-D culture as compared to standard 2-D conditions. We observed a decline in the anti-apoptotic protein Bcl-2 in 3-D culture, coupled with increased expression of the pro-apoptotic proteins Bax and Bak. Moreover, thapsigargin (TG)-induced apoptosis was enhanced in the 3-D cells. Microarray analysis demonstrated significantly differing mRNA levels for over 700 genes in the cells of each culture type. These results indicate that a 3-D culture approach narrows the phenotypic gap between neuronal cell lines and primary neurons. The resulting cells may readily be used for in vitro research of neuronal pathogenesis.

Spontaneous transformation of adult mesenchymal stem cells from cynomolgus macaques in vitro

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

Ren, Zhenhua; Key Laboratory of Neurodegeneration, Ministry of Education, Beijing; Department of Anatomy, Anhui Medical University, Hefei, 230032

2011-12-10

Mesenchymal stem cells (MSCs) have shown potential clinical utility in cell therapy and tissue engineering, due to their ability to proliferate as well as to differentiate into multiple lineages, including osteogenic, adipogenic, and chondrogenic specifications. Therefore, it is crucial to assess the safety of MSCs while extensive expansion ex vivo is a prerequisite to obtain the cell numbers for cell transplantation. Here we show that MSCs derived from adult cynomolgus monkey can undergo spontaneous transformation following in vitro culture. In comparison with MSCs, the spontaneously transformed mesenchymal cells (TMCs) display significantly different growth pattern and morphology, reminiscent of the characteristicsmore » of tumor cells. Importantly, TMCs are highly tumorigenic, causing subcutaneous tumors when injected into NOD/SCID mice. Moreover, no multiple differentiation potential of TMCs is observed in vitro or in vivo, suggesting that spontaneously transformed adult stem cells may not necessarily turn into cancer stem cells. These data indicate a direct transformation of cynomolgus monkey MSCs into tumor cells following long-term expansion in vitro. The spontaneous transformation of the cultured cynomolgus monkey MSCs may have important implications for ongoing clinical trials and for models of oncogenesis, thus warranting a more strict assessment of MSCs prior to cell therapy. -- Highlights: Black-Right-Pointing-Pointer Spontaneous transformation of cynomolgus monkey MSCs in vitro. Black-Right-Pointing-Pointer Transformed mesenchymal cells lack multipotency. Black-Right-Pointing-Pointer Transformed mesenchymal cells are highly tumorigenic. Black-Right-Pointing-Pointer Transformed mesenchymal cells do not have the characteristics of cancer stem cells.« less

A novel approach for in vitro studies applying electrical fields to cell cultures by transformer-like coupling.

PubMed

Hess, R; Neubert, H; Seifert, A; Bierbaum, S; Hart, D A; Scharnweber, D

2012-12-01

The purpose of this study was to develop a new apparatus for in vitro studies applying low frequency electrical fields to cells without interfering side effects like biochemical reactions or magnetic fields which occur in currently available systems. We developed a non-invasive method by means of the principle of transformer-like coupling where the magnetic field is concentrated in a toroid and, therefore, does not affect the cell culture. Next to an extensive characterization of the electrical field parameters, initial cell culture studies have focused on examining the response of bone marrow-derived human mesenchymal stem cells (MSCs) to pulsed electrical fields. While no significant differences in the proliferation of human MSCs could be detected, significant increases in ALP activity as well as in gene expression of other osteogenic markers were observed. The results indicate that transformer-like coupled electrical fields can be used to influence osteogenic differentiation of human MSCs in vitro and can pose a useful tool in understanding the influence of electrical fields on the cellular and molecular level.

Adenomatous Polyposis Coli-Mediated Accumulation of Abasic DNA Lesions Lead to Cigarette Smoke Condensate-Induced Neoplastic Transformation of Normal Breast Epithelial Cells1

PubMed Central

Jaiswal, Aruna S; Panda, Harekrushna; Pampo, Christine A; Siemann, Dietmar W; Gairola, C Gary; Hromas, Robert; Narayan, Satya

2013-01-01

Adenomatous polyposis coli (APC) is a multifunctional protein having diverse cellular functions including cell migration, cell-cell adhesion, cell cycle control, chromosomal segregation, and apoptosis. Recently, we found a new role of APC in base excision repair (BER) and showed that it interacts with DNA polymerase β and 5′-flap endonuclease 1 and interferes in BER. Previously, we have also reported that cigarette smoke condensate (CSC) increases expression of APC and enhances the growth of normal human breast epithelial (MCF10A) cells in vitro. In the present study, using APC overexpression and knockdown systems, we have examined the molecular mechanisms by which CSC and its major component, Benzo[α]pyrene, enhances APC-mediated accumulation of abasic DNA lesions, which is cytotoxic and mutagenic in nature, leading to enhanced neoplastic transformation of MCF10A cells in an orthotopic xenograft model. PMID:23555190

Construction of transformed, cultured silkworm cells and transgenic silkworm using the site-specific integrase system from phage φC31.

PubMed

Yin, Yajuan; Cao, Guangli; Xue, Renyu; Gong, Chengliang

2014-10-01

The Streptomyces bacteriophage, φC31, uses a site-specific integrase enzyme to perform efficient recombination. The recombination system uses specific sequences to integrate exogenous DNA from the phage into a host. The sequences are known as the attP site in the phage and the attB site in the host. The system can be used as a genetic manipulation tool. In this study it has been applied to the transformation of cultured BmN cells and the construction of transgenic Bombyx mori individuals. A plasmid, pSK-attB/Pie1-EGFP/Zeo-PASV40, containing a cassette designed to express a egfp-zeocin fusion gene, was co-transfected into cultured BmN cells with a helper plasmid, pSK-Pie1/NLS-Int/NSL. Expression of the egfp-zeocin fusion gene was driven by an ie-1 promoter, downstream of a φC31 attB site. The helper plasmid encoded the φC31 integrase enzyme, which was flanked by two nuclear localization signals. Expression of the egfp-zeocin fusion gene could be observed in transformed cells. The two plasmids were also transferred into silkworm eggs to obtain transgenic silkworms. Successful integration of the fusion gene was indicated by the detection of green fluorescence, which was emitted by the silkworms. Nucleotide sequence analysis demonstrated that the attB site had been cut, to allow recombination between the attB and endogenous pseudo attP sites in the cultured silkworm cells and silkworm individuals.

Malignant transformation of guinea pig cells after exposure to ultraviolet-irradiated guinea pig cytomegalovirus

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

Isom, H.C.; Mummaw, J.; Kreider, J.W.

1983-04-30

Guinea pig cells were malignantly transformed in vitro by ultraviolet (uv)-irradiated guinea pig cytomegalovirus (GPCMV). When guinea pig hepatocyte monolayers were infected with uv-irradiated GPCMV, three continuous epithelioid cell lines which grew in soft agarose were established. Two independently derived GPCMV-transformed liver cells and a cell line derived from a soft agarose clone of one of these lines induced invasive tumors when inoculated subcutaneously or intraperitoneally into nude mice. The tumors were sarcomas possibly derived from hepatic stroma or sinusoid. Transformed cell lines were also established after infection of guinea pig hepatocyte monolayers with human cytomegalovirus (HCMV) or simian virusmore » 40 (SV40). These cell lines also formed colonies in soft agarose and induced sarcomas in nude mice. It is concluded that (i) GPCMV can malignantly transform guinea pig cells; (ii) cloning of GPCMV-transformed cells in soft agarose produced cells that induced tumors with a shorter latency period but with no alteration in growth rate or final tumor size; and (iii) the tumors produced by GPCMV-and HCMV-transformed guinea pig cells were more similar to each other in growth rate than to those induced by SV40-transformed guinea pig cells.« less

Novel compounds that enhance Agrobacterium-mediated plant transformation by mitigating oxidative stress.

PubMed

Dan, Yinghui; Zhang, Song; Zhong, Heng; Yi, Hochul; Sainz, Manuel B

2015-02-01

Agrobacterium tumefaciens caused tissue browning leading to subsequent cell death in plant transformation and novel anti-oxidative compounds enhanced Agrobacterium -mediated plant transformation by mitigating oxidative stress. Browning and death of cells transformed with Agrobacterium tumefaciens is a long-standing and high impact problem in plant transformation and the agricultural biotechnology industry, severely limiting the production of transgenic plants. Using our tomato cv. MicroTom transformation system, we demonstrated that Agrobacterium caused tissue browning (TB) leading to subsequent cell death by our correlation study. Without an antioxidant (lipoic acid, LA) TB was severe and associated with high levels of GUS transient expression and low stable transformation frequency (STF). LA addition shifted the curve in that most TB was intermediate and associated with the highest levels of GUS transient expression and STF. We evaluated 18 novel anti-oxidative compounds for their potential to enhance Agrobacterium-mediated transformation, by screening for TB reduction and monitoring GUS transient expression. Promising compounds were further evaluated for their effect on MicroTom and soybean STF. Among twelve non-antioxidant compounds, seven and five significantly (P < 0.05) reduced TB and increased STF, respectively. Among six antioxidants four of them significantly reduced TB and five of them significantly increased STF. The most efficient compound found to increase STF was melatonin (MEL, an antioxidant). Optimal concentrations and stages to use MEL in transformation were determined, and Southern blot analysis showed that T-DNA integration was not affected by MEL. The ability of diverse compounds with different anti-oxidative mechanisms can reduce Agrobacterium-mediated TB and increase STF, strongly supporting that oxidative stress is an important limiting factor in Agrobacterium-mediated transformation and the limiting factor can be controlled by these compounds at different levels.

Characteristics of Mitochondrial Transformation into Human Cells

PubMed Central

Kesner, E. E.; Saada-Reich, A.; Lorberboum-Galski, H.

2016-01-01

Mitochondria can be incorporated into mammalian cells by simple co-incubation of isolated mitochondria with cells, without the need of transfection reagents or any other type of intervention. This phenomenon was termed mitochondrial transformation, and although it was discovered in 1982, currently little is known regarding its mechanism(s). Here we demonstrate that mitochondria can be transformed into recipient cells very quickly, and co-localize with endogenous mitochondria. The isolated mitochondria interact directly with cells, which engulf the mitochondria with cellular extensions in a way, which may suggest the involvement of macropinocytosis or macropinocytosis-like mechanisms in mitochondrial transformation. Indeed, macropinocytosis inhibitors but not clathrin-mediated endocytosis inhibition-treatments, blocks mitochondria transformation. The integrity of the mitochondrial outer membrane and its proteins is essential for the transformation of the mitochondria into cells; cells can distinguish mitochondria from similar particles and transform only intact mitochondria. Mitochondrial transformation is blocked in the presence of the heparan sulfate molecules pentosan polysulfate and heparin, which indicate crucial involvement of cellular heparan sulfate proteoglycans in the mitochondrial transformation process. PMID:27184109

Multiresolution motion planning for autonomous agents via wavelet-based cell decompositions.

PubMed

Cowlagi, Raghvendra V; Tsiotras, Panagiotis

2012-10-01

We present a path- and motion-planning scheme that is "multiresolution" both in the sense of representing the environment with high accuracy only locally and in the sense of addressing the vehicle kinematic and dynamic constraints only locally. The proposed scheme uses rectangular multiresolution cell decompositions, efficiently generated using the wavelet transform. The wavelet transform is widely used in signal and image processing, with emerging applications in autonomous sensing and perception systems. The proposed motion planner enables the simultaneous use of the wavelet transform in both the perception and in the motion-planning layers of vehicle autonomy, thus potentially reducing online computations. We rigorously prove the completeness of the proposed path-planning scheme, and we provide numerical simulation results to illustrate its efficacy.

Cell transformation by human adenoviruses.

PubMed

Endter, C; Dobner, T

2004-01-01

The last 40 years of molecular biological investigations into human adenoviruses have contributed enormously to our understanding of the basic principles of normal and malignant cell growth. Much of this knowledge stems from analyses of their productive infection cycle in permissive host cells. Also, initial observations concerning the carcinogenic potential of human adenoviruses subsequently revealed decisive insights into the molecular mechanisms of the origins of cancer, and established adenoviruses as a model system for explaining virus-mediated transformation processes. Today it is well established that cell transformation by human adenoviruses is a multistep process involving several gene products encoded in early transcription units 1A (E1A) and 1B (E1B). Moreover, a large body of evidence now indicates that alternative or additional mechanisms are engaged in adenovirus-mediated oncogenic transformation involving gene products encoded in early region 4 (E4) as well as epigenetic changes resulting from viral DNA integration. In particular, detailed studies on the tumorigenic potential of subgroup D adenovirus type 9 (Ad9) E4 have now revealed a new pathway that points to a novel, general mechanism of virus-mediated oncogenesis. In this chapter, we summarize the current state of knowledge about the oncogenes and oncogene products of human adenoviruses, focusing particularly on recent findings concerning the transforming and oncogenic properties of viral proteins encoded in the E1B and E4 transcription units.

Construction of brewing-wine Aspergillus oryzae pyrG- mutant by pyrG gene deletion and its application in homology transformation.

PubMed

Du, Yu; Xie, Guizhen; Yang, Chunfa; Fang, Baishan; Chen, Hongwen

2014-06-01

pyrG(-) host cells are indispensable for pyrG(-) based transformation system. Isolations of pyrG(-) host cells by random mutations are limited by time-consuming, unclear genetic background and potential interferences of homogenous recombination. The purpose of this study was to construct brewing-wine Aspergillus oryzae pyrG(-) mutant by site-directed mutation of pyrG gene deletion which would be used as a host for further transformation. pMD-pyrGAB, a vector carrying pyrG deletion cassette, was used to construct pyrG(-) mutant of A. oryzae. Three stable pyrG deletion mutants of A. oryzae were isolated by resistant to 5-fluoroorotic acid and confirmed by polymerase chain reaction analysis, indicating that pyrG was completely excised. The ΔpyrG mutants were applied as pyrG(-) host cells to disrupt xdh gene encoding xylitol dehydrogenase, which involves in xylitol production of A. oryzae. The xdh disruption mutants were efficiently constructed by transforming a pMD-pyrG-xdh disruption plasmid carrying pyrG, and the produced xylitol concentration of the Δxdh mutant was three times as much as that of the ΔpyrG recipient. Site-directed pyrG gene deletion is thus an effective way for the isolation of pyrG(-) host cells, and the established host-vector system could be applied in further functional genomics analysis and molecular breeding of A. oryzae. © The Author 2014. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

Agrobacterium tumefaciens-mediated transformation of Lasiodiplodia theobromae, the causal agent of gummosis in cashew nut plants.

PubMed

Muniz, C R; da Silva, G F; Souza, M T; Freire, F C O; Kema, G H J; Guedes, M I F

2014-02-21

Lasiodiplodia theobromae is a major pathogen of many different crop cultures, including cashew nut plants. This paper describes an efficient Agrobacterium tumefaciens-mediated transformation (ATMT) system for the successful delivery of T-DNA, transferring the genes of green fluorescent protein (gfp) and hygromycin B phosphotransferase (hph) to L. theobromae. When the fungal pycnidiospores were co-cultured with A. tumefaciens harboring the binary vector with hph-gfp gene, hygromycin-resistant fungus only developed with acetosyringone supplementation. The cashew plants inoculated with the fungus expressing GFP revealed characteristic pathogen colonization by epifluorescence microscopy. Intense and bright green hyphae were observed for transformants in all extensions of mycelium cultures. The penetration of parenchyma cells near to the inoculation site, beneath the epicuticle surface, was observed prior to 25 dpi. Penetration was followed by the development of hyphae within invaded host cells. These findings provide a rapid and reproducible ATMT method for L. theobromae transformation.

Development of a high temperature microbial fermentation process for butanol

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

Jeor, Jeffery D. St.; Reed, David W.; Daubaras, Dayna L.

2015-08-01

Transforming renewable biomass into cost-competitive high-performance biofuels and bioproducts is key to the U.S. future energy and chemical needs. Butanol production by microbial fermentation for chemical conversion to polyolefins, elastomers, drop-in jet or diesel fuel, and other chemicals is a promising solution. A high temperature fermentation process could decrease energy costs, capital cost, give higher butanol production, and allow for continuous fermentation. In this paper, we describe our approach to genetically transform Geobacillus caldoxylosiliticus, using a pUCG18 plasmid, for potential insertion of a butanol production pathway. Transformation methods tested were electroporation of electrocompetent cells, ternary conjugation with E. coli donormore » and helper strains, and protoplast fusion. These methods have not been successful using the current plasmid. Growth controls show cells survive the various methods tested, suggesting the possibility of transformation inhibition from a DNA restriction modification system in G. caldoxylosiliticus, as reported in the literature.« less

Cell Fusion as a Cause of Prostate Cancer Metastasis

DTIC Science & Technology

2009-03-01

PC-3 cells? Does XRMV2 transform normal human cells? Does XRMV2 affect cell proliferation or viability? Is XRMV2 present in other prostate cancer...retroviral transduction. pathways regulated by tetraploidy in premalignant cells (Figure 1). In this experimental system, normal diploid human ...or findings contained in this report are those of the author(s) and should not be construed as an official Department of the Army position, policy

MYCN induces neuroblastoma in primary neural crest cells

PubMed Central

Olsen, R R; Otero, J H; García-López, J; Wallace, K; Finkelstein, D; Rehg, J E; Yin, Z; Wang, Y-D; Freeman, K W

2017-01-01

Neuroblastoma (NBL) is an embryonal cancer of the sympathetic nervous system (SNS), which causes 15% of pediatric cancer deaths. High-risk NBL is characterized by N-Myc amplification and segmental chromosomal gains and losses. Owing to limited disease models, the etiology of NBL is largely unknown, including both the cell of origin and the majority of oncogenic drivers. We have established a novel system for studying NBL based on the transformation of neural crest cells (NCCs), the progenitor cells of the SNS, isolated from mouse embryonic day 9.5 trunk neural tube explants. Based on pathology and gene expression analysis, we report the first successful transformation of wild-type NCCs into NBL by enforced expression of N-Myc, to generate phenotypically and molecularly accurate tumors that closely model human MYCN-amplified NBL. Using comparative genomic hybridization, we found that NCC-derived NBL tumors acquired copy number gains and losses that are syntenic to those observed in human MYCN-amplified NBL including 17q gain, 2p gain and loss of 1p36. When p53-compromised NCCs were transformed with N-Myc, we generated primitive neuroectodermal tumors with divergent differentiation including osteosarcoma. These subcutaneous tumors were metastatic to regional lymph nodes, liver and lung. Our novel experimental approach accurately models human NBL and establishes a new system with potential to study early stages of NBL oncogenesis, to functionally assess NBL oncogenic drivers and to characterize NBL metastasis. PMID:28459463

Cell competition with normal epithelial cells promotes apical extrusion of transformed cells through metabolic changes.

PubMed

Kon, Shunsuke; Ishibashi, Kojiro; Katoh, Hiroto; Kitamoto, Sho; Shirai, Takanobu; Tanaka, Shinya; Kajita, Mihoko; Ishikawa, Susumu; Yamauchi, Hajime; Yako, Yuta; Kamasaki, Tomoko; Matsumoto, Tomohiro; Watanabe, Hirotaka; Egami, Riku; Sasaki, Ayana; Nishikawa, Atsuko; Kameda, Ikumi; Maruyama, Takeshi; Narumi, Rika; Morita, Tomoko; Sasaki, Yoshiteru; Enoki, Ryosuke; Honma, Sato; Imamura, Hiromi; Oshima, Masanobu; Soga, Tomoyoshi; Miyazaki, Jun-Ichi; Duchen, Michael R; Nam, Jin-Min; Onodera, Yasuhito; Yoshioka, Shingo; Kikuta, Junichi; Ishii, Masaru; Imajo, Masamichi; Nishida, Eisuke; Fujioka, Yoichiro; Ohba, Yusuke; Sato, Toshiro; Fujita, Yasuyuki

2017-05-01

Recent studies have revealed that newly emerging transformed cells are often apically extruded from epithelial tissues. During this process, normal epithelial cells can recognize and actively eliminate transformed cells, a process called epithelial defence against cancer (EDAC). Here, we show that mitochondrial membrane potential is diminished in RasV12-transformed cells when they are surrounded by normal cells. In addition, glucose uptake is elevated, leading to higher lactate production. The mitochondrial dysfunction is driven by upregulation of pyruvate dehydrogenase kinase 4 (PDK4), which positively regulates elimination of RasV12-transformed cells. Furthermore, EDAC from the surrounding normal cells, involving filamin, drives the Warburg-effect-like metabolic alteration. Moreover, using a cell-competition mouse model, we demonstrate that PDK-mediated metabolic changes promote the elimination of RasV12-transformed cells from intestinal epithelia. These data indicate that non-cell-autonomous metabolic modulation is a crucial regulator for cell competition, shedding light on the unexplored events at the initial stage of carcinogenesis.

Prokaryotic Information Games: How and When to Take up and Secrete DNA.

PubMed

Stingl, Kerstin; Koraimann, Günther

2017-01-01

Besides transduction via bacteriophages natural transformation and bacterial conjugation are the most important mechanisms driving bacterial evolution and horizontal gene spread. Conjugation systems have evolved in eubacteria and archaea. In Gram-positive and Gram-negative bacteria, cell-to-cell DNA transport is typically facilitated by a type IV secretion system (T4SS). T4SSs also mediate uptake of free DNA in Helicobacter pylori, while most transformable bacteria use a type II secretion/type IV pilus system. In this chapter, we focus on how and when bacteria "decide" that such a DNA transport apparatus is to be expressed and assembled in a cell that becomes competent. Development of DNA uptake competence and DNA transfer competence is driven by a variety of stimuli and often involves intricate regulatory networks leading to dramatic changes in gene expression patterns and bacterial physiology. In both cases, genetically homogeneous populations generate a distinct subpopulation that is competent for DNA uptake or DNA transfer or might uniformly switch into competent state. Phenotypic conversion from one state to the other can rely on bistable genetic networks that are activated stochastically with the integration of external signaling molecules. In addition, we discuss principles of DNA uptake processes in naturally transformable bacteria and intend to understand the exceptional use of a T4SS for DNA import in the gastric pathogen H. pylori. Realizing the events that trigger developmental transformation into competence within a bacterial population will eventually help to create novel and effective therapies against the transmission of antibiotic resistances among pathogens.

2016 Annual Progress Report: DOE Hydrogen and Fuel Cells Program

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

None, None

The 2016 Annual Progress Report summarizes fiscal year 2016 activities and accomplishments by projects funded by the DOE Hydrogen and Fuel Cells Program. It covers the program areas of hydrogen production; hydrogen delivery; hydrogen storage; fuel cells; manufacturing R&D; technology validation; safety, codes and standards; systems analysis; market transformation; and Small Business Innovation Research projects.

Transforming Growth Factor-Beta and Oxidative Stress Interplay: Implications in Tumorigenesis and Cancer Progression

PubMed Central

Krstić, Jelena; Trivanović, Drenka; Mojsilović, Slavko; Santibanez, Juan F.

2015-01-01

Transforming growth factor-beta (TGF-β) and oxidative stress/Reactive Oxygen Species (ROS) both have pivotal roles in health and disease. In this review we are analyzing the interplay between TGF-β and ROS in tumorigenesis and cancer progression. They have contradictory roles in cancer progression since both can have antitumor effects, through the induction of cell death, senescence and cell cycle arrest, and protumor effects by contributing to cancer cell spreading, proliferation, survival, and metastasis. TGF-β can control ROS production directly or by downregulating antioxidative systems. Meanwhile, ROS can influence TGF-β signaling and increase its expression as well as its activation from the latent complex. This way, both are building a strong interplay which can be taken as an advantage by cancer cells in order to increment their malignancy. In addition, both TGF-β and ROS are able to induce cell senescence, which in one way protects damaged cells from neoplastic transformation but also may collaborate in cancer progression. The mutual collaboration of TGF-β and ROS in tumorigenesis is highly complex, and, due to their differential roles in tumor progression, careful consideration should be taken when thinking of combinatorial targeting in cancer therapies. PMID:26078812

Note: Modification of an FTIR spectrometer for optoelectronic characterizations.

PubMed

Puspitosari, N; Longeaud, C

2017-08-01

We propose a very simple system to be adapted to a Fourier Transform Infra-Red (FTIR) spectrometer with which three different types of characterizations can be done: the Fourier transform photocurrent spectroscopy, the recording of reflection-transmission spectra of thin film semiconductors, and the acquisition of spectral responses of solar cells. In addition to gather three techniques into a single apparatus, this FTIR-based system also significantly reduces the recording time and largely improves the resolution of the measured spectra compared to standard equipments.

Note: Modification of an FTIR spectrometer for optoelectronic characterizations

NASA Astrophysics Data System (ADS)

Puspitosari, N.; Longeaud, C.

2017-08-01

We propose a very simple system to be adapted to a Fourier Transform Infra-Red (FTIR) spectrometer with which three different types of characterizations can be done: the Fourier transform photocurrent spectroscopy, the recording of reflection-transmission spectra of thin film semiconductors, and the acquisition of spectral responses of solar cells. In addition to gather three techniques into a single apparatus, this FTIR-based system also significantly reduces the recording time and largely improves the resolution of the measured spectra compared to standard equipments.

Simian virus 40 small t antigen is not required for the maintenance of transformation but may act as a promoter (cocarcinogen) during establishment of transformation in resting rat cells.

PubMed Central

Seif, R; Martin, R G

1979-01-01

Simian virus 40 deletion mutants affecting the 20,000-dalton (20K) t antigen and tsA mutants rendering the 90K T antigen temperature sensitive, as well as double mutants containing both mutations, induced host DNA synthesis in resting rat cells at the restrictive temperature. Nonetheless, the deletion mutants and double mutants did not induce transformation in resting cells even at the permissive temperature. On the other hand, the deletion mutants did induce full transformants when actively growing rat cells were infected; the transformants grew efficiently in agar and to high saturation densities on platic. The double mutants did not induce T-antigen-independent (temperature-insensitive) transformants which were shown previously to arise preferentially from resting cells. Thus, small t antigen was dispensable for the maintenance of the transformed phenotype in T-antigen-dependent rat transformants (transformants derived from growing cells) and may play a role in the establishment of T-antigen-independent transformants. We attempt to establish a parallel between transformation induced by chemical carcinogens and simian virus 40-induced transformation. Images PMID:229274

Simian virus 40 small t antigen is not required for the maintenance of transformation but may act as a promoter (cocarcinogen) during establishment of transformation in resting rat cells.

PubMed

Seif, R; Martin, R G

1979-12-01

Simian virus 40 deletion mutants affecting the 20,000-dalton (20K) t antigen and tsA mutants rendering the 90K T antigen temperature sensitive, as well as double mutants containing both mutations, induced host DNA synthesis in resting rat cells at the restrictive temperature. Nonetheless, the deletion mutants and double mutants did not induce transformation in resting cells even at the permissive temperature. On the other hand, the deletion mutants did induce full transformants when actively growing rat cells were infected; the transformants grew efficiently in agar and to high saturation densities on platic. The double mutants did not induce T-antigen-independent (temperature-insensitive) transformants which were shown previously to arise preferentially from resting cells. Thus, small t antigen was dispensable for the maintenance of the transformed phenotype in T-antigen-dependent rat transformants (transformants derived from growing cells) and may play a role in the establishment of T-antigen-independent transformants. We attempt to establish a parallel between transformation induced by chemical carcinogens and simian virus 40-induced transformation.

Agrobacterium tumefaciens Integrates Transfer DNA into Single Chromosomal Sites of Dimorphic Fungi and Yields Homokaryotic Progeny from Multinucleate Yeast

PubMed Central

Sullivan, Thomas D.; Rooney, Peggy J.; Klein, Bruce S.

2002-01-01

The dimorphic fungi Blastomyces dermatitidis and Histoplasma capsulatum cause systemic mycoses in humans and other animals. Forward genetic approaches to generating and screening mutants for biologically important phenotypes have been underutilized for these pathogens. The plant-transforming bacterium Agrobacterium tumefaciens was tested to determine whether it could transform these fungi and if the fate of transforming DNA was suited for use as an insertional mutagen. Yeast cells from both fungi and germinating conidia from B. dermatitidis were transformed via A. tumefaciens by using hygromycin resistance for selection. Transformation frequencies up to 1 per 100 yeast cells were obtained at high effector-to-target ratios of 3,000:1. B. dermatitidis and H. capsulatum ura5 lines were complemented with transfer DNA vectors expressing URA5 at efficiencies 5 to 10 times greater than those obtained using hygromycin selection. Southern blot analyses indicated that in 80% of transformants the transferred DNA was integrated into chromosomal DNA at single, unique sites in the genome. Progeny of B. dermatitidis transformants unexpectedly showed that a single round of colony growth under hygromycin selection or visible selection of transformants by lacZ expression generated homokaryotic progeny from multinucleate yeast. Theoretical analysis of random organelle sorting suggests that the majority of B. dermatitidis cells would be homokaryons after the ca. 20 generations necessary for colony formation. Taken together, the results demonstrate that A. tumefaciens efficiently transfers DNA into B. dermatitidis and H. capsulatum and has the properties necessary for use as an insertional mutagen in these fungi. PMID:12477790

Model system for plant cell biology: GFP imaging in living onion epidermal cells

NASA Technical Reports Server (NTRS)

Scott, A.; Wyatt, S.; Tsou, P. L.; Robertson, D.; Allen, N. S.

1999-01-01

The ability to visualize organelle localization and dynamics is very useful in studying cellular physiological events. Until recently, this has been accomplished using a variety of staining methods. However, staining can give inaccurate information due to nonspecific staining, diffusion of the stain or through toxic effects. The ability to target green fluorescent protein (GFP) to various organelles allows for specific labeling of organelles in vivo. The disadvantages of GFP thus far have been the time and money involved in developing stable transformants or maintaining cell cultures for transient expression. In this paper, we present a rapid transient expression system using onion epidermal peels. We have localized GFP to various cellular compartments (including the cell wall) to illustrate the utility of this method and to visualize dynamics of these compartments. The onion epidermis has large, living, transparent cells in a monolayer, making them ideal for visualizing GFP. This method is easy and inexpensive, and it allows for testing of new GFP fusion proteins in a living tissue to determine deleterious effects and the ability to express before stable transformants are attempted.

“Agrolistic” transformation of plant cells: Integration of T-strands generated in planta

PubMed Central

Hansen, Geneviève; Chilton, Mary-Dell

1996-01-01

We describe a novel plant transformation technique, termed “agrolistic,” that combines the advantages of the Agrobacterium transformation system with the high efficiency of biolistic DNA delivery. Agrolistic transformation allows integration of the gene of interest without undesired vector sequence. The virulence genes virD1 and virD2 from Agrobacterium tumefaciens that are required in bacteria for excision of T-strands from the tumor-inducing plasmid were placed under the control of the CaMV35S promoter and codelivered with a target plasmid containing border sequences flanking the gene of interest. Transient expression assays in tobacco and in maize cells indicated that vir gene products caused strand-specific nicking in planta at the right border sequence, similar to VirD1/VirD2-catalyzed T-strand excision observed in Agrobacterium. Agrolistically transformed tobacco calli were obtained after codelivery of virD1 and virD2 genes together with a selectable marker flanked by border sequences. Some inserts exhibited right junctions with plant DNA that corresponded precisely to the sequence expected for T-DNA (portion of the tumor-inducing plasmid that is transferred to plant cells) insertion events. We designate these as “agrolistic” inserts, as distinguished from “biolistic” inserts. Both types of inserts were found in some transformed lines. The frequency of agrolistic inserts was 20% that of biolistic inserts. PMID:8962167

Progression from productive infection to integration and oncogenic transformation in human papillomavirus type 59-immortalized foreskin keratinocytes.

PubMed

Spartz, Helena; Lehr, Elizabeth; Zhang, Benyue; Roman, Ann; Brown, Darron R

2005-05-25

Studies of changes in the virus and host cell upon progression from human papillomavirus (HPV) episomal infection to integration are critical to understanding HPV-related malignant transformation. However, there exist only a few in vitro models of both productive HPV infection and neoplastic progression on the same host background. We recently described a unique foreskin keratinocyte cell line (ERIN 59) that contains HPV 59 (a close relative of HPV 18). Early passages of ERIN 59 cells (passages 9-13) contained approximately 50 copies of episomes/cell, were feeder cell-dependent, and could be induced to differentiate and produce infectious virus in a simple culture system. We now report that late passage cells (passages greater than 50) were morphologically different from early passage cells, were feeder cell independent, and did not differentiate or produce virus. These late passage cells contained HPV in an integrated form. An integration-derived oncogene transcript was expressed in late passage cells. The E2 open reading frame was interrupted in this transcript at nucleotide 3351. Despite a lower viral genome copy number in late passage ERIN 59 cells, expression of E6/E7 oncogene transcripts was similar to early passage cells. We conclude that ERIN 59 cells are a valuable cell line representing a model of progression from HPV 59 episomal infection and virus production to HPV 59 integration and associated oncogenic transformation on the same host background.

Herpesvirus Saimiri Transforms Human T-Cell Clones to Stable Growth without Inducing Resistance to Apoptosis

PubMed Central

Kraft, Michael S.; Henning, Golo; Fickenscher, Helmut; Lengenfelder, Doris; Tschopp, Jürg; Fleckenstein, Bernhard; Meinl, Edgar

1998-01-01

Herpesvirus saimiri (HVS) transforms human T cells to stable growth in vitro. Since HVS codes for two different antiapoptotic proteins, growth transformation by HVS might be expected to confer resistance to apoptosis. We found that the expression of both viral antiapoptotic genes was restricted to cultures with viral replication and absent in growth-transformed human T cells. A comparative examination of HVS-transformed T-cell clones and their native parental clones revealed that the expression of Bcl-2, Bcl-XL, Bax, and members of the tumor necrosis factor receptor (TNF-R) superfamily with a death domain, namely, TNF-RI, CD95, and TRAMP, were not modulated by HVS. Expression of CD30 was induced in HVS-transformed T cells, and these cells also expressed the CD30 ligand. Uninfected and transformed T cells were sensitive to CD95 ligation but resistant to apoptosis mediated by TRAIL or soluble TNF-α. CD95 ligand was constitutively expressed on transformed but not uninfected parental T cells. Both cell types showed similar sensitivity to cell death induction or inhibition of T-cell activation mediated by irradiation, oxygen radicals, dexamethasone, cyclosporine, and prostaglandin E2. Altogether, this study strongly suggests that growth transformation by HVS is based not on resistance to apoptosis but, rather, on utilization of normal cellular activation pathways. PMID:9525639

Barriers to horizontal cell transformation by extracellular vesicles containing oncogenic H-ras.

PubMed

Lee, Tae Hoon; Chennakrishnaiah, Shilpa; Meehan, Brian; Montermini, Laura; Garnier, Delphine; D'Asti, Esterina; Hou, Wenyang; Magnus, Nathalie; Gayden, Tenzin; Jabado, Nada; Eppert, Kolja; Majewska, Loydie; Rak, Janusz

2016-08-09

Extracellular vesicles (EVs) enable the exit of regulatory, mutant and oncogenic macromolecules (proteins, RNA and DNA) from their parental tumor cells and uptake of this material by unrelated cellular populations. Among the resulting biological effects of interest is the notion that cancer-derived EVs may mediate horizontal transformation of normal cells through transfer of mutant genes, including mutant ras. Here, we report that H-ras-mediated transformation of intestinal epithelial cells (IEC-18) results in the emission of exosome-like EVs containing genomic DNA, HRAS oncoprotein and transcript. However, EV-mediated horizontal transformation of non-transformed cells (epithelial, astrocytic, fibroblastic and endothelial) is transient, limited or absent due to barrier mechanisms that curtail the uptake, retention and function of oncogenic H-ras in recipient cells. Thus, epithelial cells and astrocytes are resistant to EV uptake, unless they undergo malignant transformation. In contrast, primary and immortalized fibroblasts are susceptible to the EV uptake, retention of H-ras DNA and phenotypic transformation, but these effects are transient and fail to produce a permanent tumorigenic conversion of these cells in vitro and in vivo, even after several months of observation. Increased exposure to EVs isolated from H-ras-transformed cancer cells, but not to those from their indolent counterparts, triggers demise of recipient fibroblasts. Uptake of H-ras-containing EVs stimulates but fails to transform primary endothelial cells. Thus, we suggest that intercellular transfer of oncogenes exerts regulatory rather than transforming influence on recipient cells, while cancer cells may often act as preferential EV recipients.

Barriers to horizontal cell transformation by extracellular vesicles containing oncogenic H-ras

PubMed Central

Lee, Tae Hoon; Chennakrishnaiah, Shilpa; Meehan, Brian; Montermini, Laura; Garnier, Delphine; D'Asti, Esterina; Hou, Wenyang; Magnus, Nathalie; Gayden, Tenzin; Jabado, Nada; Eppert, Kolja; Majewska, Loydie; Rak, Janusz

2016-01-01

Extracellular vesicles (EVs) enable the exit of regulatory, mutant and oncogenic macromolecules (proteins, RNA and DNA) from their parental tumor cells and uptake of this material by unrelated cellular populations. Among the resulting biological effects of interest is the notion that cancer-derived EVs may mediate horizontal transformation of normal cells through transfer of mutant genes, including mutant ras. Here, we report that H-ras-mediated transformation of intestinal epithelial cells (IEC-18) results in the emission of exosome-like EVs containing genomic DNA, HRAS oncoprotein and transcript. However, EV-mediated horizontal transformation of non-transformed cells (epithelial, astrocytic, fibroblastic and endothelial) is transient, limited or absent due to barrier mechanisms that curtail the uptake, retention and function of oncogenic H-ras in recipient cells. Thus, epithelial cells and astrocytes are resistant to EV uptake, unless they undergo malignant transformation. In contrast, primary and immortalized fibroblasts are susceptible to the EV uptake, retention of H-ras DNA and phenotypic transformation, but these effects are transient and fail to produce a permanent tumorigenic conversion of these cells in vitro and in vivo, even after several months of observation. Increased exposure to EVs isolated from H-ras-transformed cancer cells, but not to those from their indolent counterparts, triggers demise of recipient fibroblasts. Uptake of H-ras-containing EVs stimulates but fails to transform primary endothelial cells. Thus, we suggest that intercellular transfer of oncogenes exerts regulatory rather than transforming influence on recipient cells, while cancer cells may often act as preferential EV recipients. PMID:27437771

Plants having modified response to ethylene by transformation with an ETR nucleic acid

DOEpatents

Meyerowitz, Elliott M.; Chang, Caren; Bleecker, Anthony B.

2001-01-01

The invention includes transformed plants having at least one cell transformed with a modified ETR nucleic acid. Such plants have a phenotype characterized by a decrease in the response of at least one transformed plant cell to ethylene as compared to a plant not containing the transformed plant cell. Tissue and/or temporal specificity for expression of the modified ETR nucleic acid is controlled by selecting appropriate expression regulation sequences to target the location and/or time of expression of the transformed nucleic acid. The plants are made by transforming at least one plant cell with an appropriate modified ETR nucleic acid, regenerating plants from one or more of the transformed plant cells and selecting at least one plant having the desired phenotype.

Cell-of-Origin of Cancer versus Cancer Stem Cells: Assays and Interpretations.

PubMed

Rycaj, Kiera; Tang, Dean G

2015-10-01

A tumor originates from a normal cell that has undergone tumorigenic transformation as a result of genetic mutations. This transformed cell is the cell-of-origin for the tumor. In contrast, an established clinical tumor is sustained by subpopulations of self-renewing cancer cells operationally called cancer stem cells (CSC) that can generate, intraclonally, both tumorigenic and nontumorigenic cells. Identifying and characterizing tumor cell-of-origin and CSCs should help elucidate tumor cell heterogeneity, which, in turn, should help understand tumor cell responses to clinical treatments, drug resistance, tumor relapse, and metastatic spread. Both tumor transplantation and lineage-tracing assays have been helpful in characterizing these cancer cell populations, although each system has its strengths and caveats. In this article, we briefly review and summarize advantages and limitations of both assays in support of a combinatorial approach to accurately define the roles of both cancer-initiating and cancer-propagating cells. As an aside, we also wish to clarify the definitions of cancer cell-of-origin and CSCs, which are often interchangeably used by mistake. ©2015 American Association for Cancer Research.

Ha-ras(val12) induces HSP70b transcription via the HSE/HSF1 system, but HSP70b expression is suppressed in Ha-ras(val12)-transformed cells.

PubMed

Stanhill, A; Levin, V; Hendel, A; Shachar, I; Kazanov, D; Arber, N; Kaminski, N; Engelberg, D

2006-03-09

Heat shock proteins (Hsps) are overexpressed in many tumors, but are downregulated in some tumors. To check for a direct effect of Ha-Ras(val12) on HSP70 transcription, we transiently expressed the oncoprotein in Rat1 fibroblasts and monitored its effect on HSP70b promoter-driven reporter gene. We show that expression of Ha-Ras(val12) induced this promoter. Promoter analysis via systematic deletions and point mutations revealed that Ha-Ras(val12) induces HSP70b transcription via heat shock elements (HSEs). Also, Ha-Ras(val12) induction of HSE-mediated transcription was dramatically reduced in HSF1-/- cells. Yet, residual effect of Ha-Ras(val12) that was still measured in HSF1-/- cells suggests that some of the Ha-Ras(val12) effect is Hsf1-independent. When HSF1-/- cells, stably expressing Ha-Ras(val12), were grown on soft agar only small colonies were formed suggesting a role for heat shock factor 1 (Hsf1) in Ha-Ras(val12)-mediated transformation. Although Ha-ras(Val12) seems to be an inducer of HSP70's expression, we found that in Ha-ras(Val12-)transformed fibroblasts expression of this gene is suppressed. This suppression is correlated with higher sensitivity of Ha-ras(val12)-transformed cells to heat shock. We suggest that Ha-ras(Val12) is involved in Hsf1 activation, thereby inducing the cellular protective response. Cells that repress this response are perhaps those that acquire the capability to further proliferate and become transformed clones.

Assessment of the Relative Toxicity of N,N-Dipropylcyclohexanecarboxamide, AI3-36326.

DTIC Science & Technology

1983-04-01

cells with or without an in vitro metabolic activation system. The in vitro metabolic activation system was composed of rat liver enzymes and an energy...producing system. The enzymes were contained in a preparation of liver microsomes (S9 fraction)JI fron rats treated with an alkylating agent, Aroclor...to induce enzymes capable of transforming chemicals to more active forms. Cells were examined 10 to 12 hours following treatment when entering mitosis

PKA-regulated VASP phosphorylation promotes extrusion of transformed cells from the epithelium

PubMed Central

Anton, Katarzyna A.; Sinclair, John; Ohoka, Atsuko; Kajita, Mihoko; Ishikawa, Susumu; Benz, Peter M.; Renne, Thomas; Balda, Maria; Matter, Karl; Fujita, Yasuyuki

2014-01-01

ABSTRACT At the early stages of carcinogenesis, transformation occurs in single cells within tissues. In an epithelial monolayer, such mutated cells are recognized by their normal neighbors and are often apically extruded. The apical extrusion requires cytoskeletal reorganization and changes in cell shape, but the molecular switches involved in the regulation of these processes are poorly understood. Here, using stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative mass spectrometry, we have identified proteins that are modulated in transformed cells upon their interaction with normal cells. Phosphorylation of VASP at serine 239 is specifically upregulated in RasV12-transformed cells when they are surrounded by normal cells. VASP phosphorylation is required for the cell shape changes and apical extrusion of Ras-transformed cells. Furthermore, PKA is activated in Ras-transformed cells that are surrounded by normal cells, leading to VASP phosphorylation. These results indicate that the PKA–VASP pathway is a crucial regulator of tumor cell extrusion from the epithelium, and they shed light on the events occurring at the early stage of carcinogenesis. PMID:24963131

Improving Papanicolaou test quality and reducing medical errors by using Toyota production system methods.

PubMed

Raab, Stephen S; Andrew-Jaja, Carey; Condel, Jennifer L; Dabbs, David J

2006-01-01

The objective of the study was to determine whether the Toyota production system process improves Papanicolaou test quality and patient safety. An 8-month nonconcurrent cohort study that included 464 case and 639 control women who had a Papanicolaou test was performed. Office workflow was redesigned using Toyota production system methods by introducing a 1-by-1 continuous flow process. We measured the frequency of Papanicolaou tests without a transformation zone component, follow-up and Bethesda System diagnostic frequency of atypical squamous cells of undetermined significance, and diagnostic error frequency. After the intervention, the percentage of Papanicolaou tests lacking a transformation zone component decreased from 9.9% to 4.7% (P = .001). The percentage of Papanicolaou tests with a diagnosis of atypical squamous cells of undetermined significance decreased from 7.8% to 3.9% (P = .007). The frequency of error per correlating cytologic-histologic specimen pair decreased from 9.52% to 7.84%. The introduction of the Toyota production system process resulted in improved Papanicolaou test quality.

STAT3-regulated exosomal miR-21 promotes angiogenesis and is involved in neoplastic processes of transformed human bronchial epithelial cells.

PubMed

Liu, Yi; Luo, Fei; Wang, Bairu; Li, Huiqiao; Xu, Yuan; Liu, Xinlu; Shi, Le; Lu, Xiaolin; Xu, Wenchao; Lu, Lu; Qin, Yu; Xiang, Quanyong; Liu, Qizhan

2016-01-01

Although microRNA (miRNA) enclosed in exosomes can mediate intercellular communication, the roles of exosomal miRNA and angiogenesis in lung cancer remain unclear. We investigated functions of STAT3-regulated exosomal miR-21 derived from cigarette smoke extract (CSE)-transformed human bronchial epithelial (HBE) cells in the angiogenesis of CSE-induced carcinogenesis. miR-21 levels in serum were higher in smokers than those in non-smokers. The medium from transformed HBE cells promoted miR-21 levels in normal HBE cells and angiogenesis of human umbilical vein endothelial cells (HUVEC). Transformed cells transferred miR-21 into normal HBE cells via exosomes. Knockdown of STAT3 reduced miR-21 levels in exosomes derived from transformed HBE cells, which blocked the angiogenesis. Exosomes derived from transformed HBE cells elevated levels of vascular endothelial growth factor (VEGF) in HBE cells and thereby promoted angiogenesis in HUVEC cells. Inhibition of exosomal miR-21, however, decreased VEGF levels in recipient cells, which blocked exosome-induced angiogenesis. Thus, miR-21 in exosomes leads to STAT3 activation, which increases VEGF levels in recipient cells, a process involved in angiogenesis and malignant transformation of HBE cells. These results, demonstrating the function of exosomal miR-21 from transformed HBE cells, provide a new perspective for intervention strategies to prevent carcinogenesis of lung cancer. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Transformation of proanthocyanidin A2 to its isomers under different physiological pH conditions and common cell culture medium.

PubMed

Lu, Wen-Chien; Huang, Wei-Ting; Kumaran, Alaganandam; Ho, Chi-Tang; Hwang, Lucy Sun

2011-06-08

Proanthocyanidins constitute an important class of polyphenols ubiquitously found in plants. They have been extensively studied for their antioxidant capacity and bioactivity in vitro and in animal models. However, their stability under different pH conditions and in cell culture medium has not been well documented. In the present study, it was observed that proanthocyanidin A2 (PA2) was relatively more stable in acidic condition than in weak alkaline condition. PA2 was also quite unstable in basal-Dulbecco's Modified Eagle medium (b-DMEM medium) at 37 °C. The addition of PA2 to the cell culture medium accelerated its epimerization with a half-life of <15 min, and ethylenediaminetetraacetic acid (EDTA) could not stop the reaction. The results also demonstrated that the major isomers transformed in the weak alkaline condition or cell culture medium at 37 °C were identified as epicatechin-(4β→8; 2β→O→7)-ent-catechin (proanthocyanidin A4) and epicatechin-(4β→6; 2β→O→7)-ent-catechin. The rates of transformation were dependent on the pH or the components of the medium. Therefore, the results obtained for PA2 in the cell culture bioassays, which were usually carried out for 24 h, might not represent the true activity of the original PA2. The stability and transformation of PA2 should be considered when the bioactivity of PA2 is evaluated in a given cell culture system.

Mitochondrial clearance by the STK38 kinase supports oncogenic Ras-induced cell transformation

PubMed Central

Bettoun, Audrey; Surdez, Didier; Vallerand, David; Gundogdu, Ramazan; Sharif, Ahmad A.D.; Gomez, Marta; Cascone, Ilaria; Meunier, Brigitte; White, Michael A.; Codogno, Patrice; Parrini, Maria Carla; Camonis, Jacques H.; Hergovich, Alexander

2016-01-01

Oncogenic Ras signalling occurs frequently in many human cancers. However, no effective targeted therapies are currently available to treat patients suffering from Ras-driven tumours. Therefore, it is imperative to identify downstream effectors of Ras signalling that potentially represent promising new therapeutic options. Particularly, considering that autophagy inhibition can impair the survival of Ras-transformed cells in tissue culture and mouse models, an understanding of factors regulating the balance between autophagy and apoptosis in Ras-transformed human cells is needed. Here, we report critical roles of the STK38 protein kinase in oncogenic Ras transformation. STK38 knockdown impaired anoikis resistance, anchorage-independent soft agar growth, and in vivo xenograft growth of Ras-transformed human cells. Mechanistically, STK38 supports Ras-driven transformation through promoting detachment-induced autophagy. Even more importantly, upon cell detachment STK38 is required to sustain the removal of damaged mitochondria by mitophagy, a selective autophagic process, to prevent excessive mitochondrial reactive oxygen species production that can negatively affect cancer cell survival. Significantly, knockdown of PINK1 or Parkin, two positive regulators of mitophagy, also impaired anoikis resistance and anchorage-independent growth of Ras-transformed human cells, while knockdown of USP30, a negative regulator of PINK1/Parkin-mediated mitophagy, restored anchorage-independent growth of STK38-depleted Ras-transformed human cells. Therefore, our findings collectively reveal novel molecular players that determine whether Ras-transformed human cells die or survive upon cell detachment, which potentially could be exploited for the development of novel strategies to target Ras-transformed cells. PMID:27283898

Plants having modified response to ethylene

DOEpatents

Meyerowitz, Elliott M.; Chang, Caren; Bleecker, Anthony B.

1997-01-01

The invention includes transformed plants having at least one cell transformed with a modified ETR nucleic acid. Such plants have a phenotype characterized by a decrease in the response of at least one transformed plant cell to ethylene as compared to a plant not containing the transformed plant cell. Tissue and/or temporal specificity for expression of the modified ETR nucleic acid is controlled by selecting appropriate expression regulation sequences to target the location and/or time of expression of the transformed nucleic acid. The plants are made by transforming at least one plant cell with an appropriate modified ETR nucleic acid, regenerating plants from one or more of the transformed plant cells and selecting at least one plant having the desired phenotype.

Plants having modified response to ethylene

DOEpatents

Meyerowitz, E.M.; Chang, C.; Bleecker, A.B.

1998-10-20

The invention includes transformed plants having at least one cell transformed with a modified ETR nucleic acid. Such plants have a phenotype characterized by a decrease in the response of at least one transformed plant cell to ethylene as compared to a plant not containing the transformed plant cell. Tissue and/or temporal specificity for expression of the modified ETR nucleic acid is controlled by selecting appropriate expression regulation sequences to target the location and/or time of expression of the transformed nucleic acid. The plants are made by transforming at least one plant cell with an appropriate modified ETR nucleic acid, regenerating plants from one or more of the transformed plant cells and selecting at least one plant having the desired phenotype. 67 figs.

Plants having modified response to ethylene

DOEpatents

Meyerowitz, Elliot M.; Chang, Caren; Bleecker, Anthony B.

1998-01-01

The invention includes transformed plants having at least one cell transformed with a modified ETR nucleic acid. Such plants have a phenotype characterized by a decrease in the response of at least one transformed plant cell to ethylene as compared to a plant not containing the transformed plant cell. Tissue and/or temporal specificity for expression of the modified ETR nucleic acid is controlled by selecting appropriate expression regulation sequences to target the location and/or time of expression of the transformed nucleic acid. The plants are made by transforming at least one plant cell with an appropriate modified ETR nucleic acid, regenerating plants from one or more of the transformed plant cells and selecting at least one plant having the desired phenotype.

Plants having modified response to ethylene

DOEpatents

Meyerowitz, E.M.; Chang, C.; Bleecker, A.B.

1997-11-18

The invention includes transformed plants having at least one cell transformed with a modified ETR nucleic acid. Such plants have a phenotype characterized by a decrease in the response of at least one transformed plant cell to ethylene as compared to a plant not containing the transformed plant cell. Tissue and/or temporal specificity for expression of the modified ETR nucleic acid is controlled by selecting appropriate expression regulation sequences to target the location and/or time of expression of the transformed nucleic acid. The plants are made by transforming at least one plant cell with an appropriate modified ETR nucleic acid, regenerating plants from one or more of the transformed plant cells and selecting at least one plant having the desired phenotype. 31 figs.

Analysis and test for space shuttle propellant dynamics (1/10th scale model test results). Volume 2: 1/10th scale model test data

NASA Technical Reports Server (NTRS)

Berry, R. L.; Tegart, J. R.; Demchak, L. J.

1979-01-01

Thirty sets of test data selected from the 89 low-g aircraft tests flown by NASA KC-135 zero-g aircraft are listed in tables with their accompanying test conditions. The data for each test consists of the time history plots of digitalized data (in engineering units) and the time history plots of the load cell data transformed to the tank axis system. The transformed load cell data was developed for future analytical comparisons; therefore, these data were transformed and plotted from the time at which the aircraft Z axis acceleration passed through l-g. There are 14 time history plots per test condition. The contents of each plot is shown in a table.

INDUCIBLE CELL LYSIS SYSTEM IN BACILLUS SUBTILIS AND TRANSFORMATION BY DNA RELEASED FROM LYSED CELLS. (R826107)

EPA Science Inventory

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

2014 Annual Progress Report: DOE Hydrogen and Fuel Cells Program

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

none,

2014-11-01

The 2014 Annual Progress Report summarizes fiscal year 2014 activities and accomplishments by projects funded by the DOE Hydrogen Program. It covers the program areas of hydrogen production and delivery; hydrogen storage; fuel cells; manufacturing; technology validation; safety, codes and standards; market transformation; and systems analysis.

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

none,

This report summarizes comments from the Peer Review Panel at the 2013 DOE Hydrogen and Fuel Cells Program Annual Merit Review, held on May 13-17, 2013, in Arlington, Virginia. It covers the program areas of hydrogen production and delivery; hydrogen storage; fuel cells; manufacturing R&D; technology validation; safety, codes, and standards; market transformation; and systems analysis.

IL-3 induces apoptosis in a ras-transformed myeloid cell line.

PubMed

Ahmed, N; Anderson, S M; Berridge, M V

1999-04-01

Growth factors promote cell survival and proliferation. Homeostasis is maintained by programmed cell death which occurs when the growth stimulus is withdrawn, in response to negative growth regulators such as interferons, TNF-alpha and CD95 ligand, or following differentiation. Although acutely-transforming oncogenes often overcome the need for growth factors, growth regulatory cytokines can influence proliferative responses of transformed cells. In this study we investigated the effects of IL-3 on the proliferative responses of parental bone marrow-derived 32D cells and cells transformed with ras and abl oncogenes. We show that treatment of ras-transformed 32D cells with IL-3 reduced proliferative responses and decreased colony-forming ability. These effects were exacerbated in the absence of serum and associated with inhibition of tyrosine kinase activity, down-regulation of RAS and MYC expression, and induction of apoptosis as indicated by DNA fragmentation. In contrast, treatment of parental 32D cells with IL-3, which is obligatory for cell survival and proliferation, increased tyrosine kinase activity, upregulated MYC and RAS expression and maintained DNA integrity. With abl-transformed cells, proliferation and colony-forming ability were also inhibited by IL-3. Tyrosine kinase activity and MYC expression were reduced, but early apoptosis was not evident. Calcium uptake however, was stimulated by IL-3 in both parental and oncogene-transformed cells. These results suggest that threshold levels of tyrosine kinase activity are necessary for cell survival and proliferation and that with ras-transformed cells, IL-3 treatment may result in this threshold being breached. We conclude that in some situations, growth-promoting cytokines can inhibit proliferation of transformed cells and induce cell death by apoptosis.

Regulation of early gene expression from the bovine papillomavirus genome in transiently transfected C127 cells.

PubMed Central

Szymanski, P; Stenlund, A

1991-01-01

Expression of bovine papillomavirus (BPV) early gene products is required for viral DNA replication and establishment of the transformed phenotype. By the use of a highly efficient electroporation system, we have examined for the first time the transcriptional activity of BPV promoters in their natural genomic context in a replication-permissive cell line. We have determined that a qualitatively distinct stage of transcription is not detectable prior to DNA replication in transiently transfected cells. This suggests that the transcriptional activity of the BPV genome in stably transformed cells represents the early stage of BPV gene expression. Quantitative differences in promoter activity between transiently transfected and stably transformed cells suggest that subtle changes in gene expression may control progression of the viral life cycle. Deletion analysis demonstrated that the E2 transactivator protein stimulates all of the early promoters through sequences located in the upstream regulatory region. This E2-dependent enhancer was found to be highly redundant, and particular E2 binding sites did not display a preference for particular promoters. Despite this dependence on a common cis-acting sequence, the various promoters displayed different sensitivities to the E2 transactivator. The findings that E2 regulates all promoters and, with the exception of the E2 repressors, that no other known viral gene product appears to affect transcription indicate that the E2 system functions as the master regulator of BPV early gene expression. Images PMID:1656065

Genetic changes in mammalian cells transformed by helium ions

NASA Astrophysics Data System (ADS)

Durante, M.; Grossi, G.; Yang, T. C.; Roots, R.

Midterm Syrian Hamster embryo (SHE) cells were employed to study high LET-radiation induced tumorigenesis. Normal SHE cells (secondary passage) were irradiated with accelerated helium ions at an incident energy of 22 MeV/u (9-10 keV/μm). Transformed clones were isolated after growth in soft agar of cells obtained from the foci of the initial monolayer plated postirradiation. To study the progression process of malignant transformation, the transformed clones were followed by monolayer subculturing for prolonged periods of time. Subsequently, neoplasia tests in nude mice were done. In this work, however, we have focused on karyotypic changes in the banding patterns of the chromosomes during the early part of the progressive process of cell transformation for helium ion-induced transformed cells.

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

NASA Technical Reports Server (NTRS)

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

2000-01-01

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

A Model for the Epigenetic Switch Linking Inflammation to Cell Transformation: Deterministic and Stochastic Approaches

PubMed Central

Gérard, Claude; Gonze, Didier; Lemaigre, Frédéric; Novák, Béla

2014-01-01

Recently, a molecular pathway linking inflammation to cell transformation has been discovered. This molecular pathway rests on a positive inflammatory feedback loop between NF-κB, Lin28, Let-7 microRNA and IL6, which leads to an epigenetic switch allowing cell transformation. A transient activation of an inflammatory signal, mediated by the oncoprotein Src, activates NF-κB, which elicits the expression of Lin28. Lin28 decreases the expression of Let-7 microRNA, which results in higher level of IL6 than achieved directly by NF-κB. In turn, IL6 can promote NF-κB activation. Finally, IL6 also elicits the synthesis of STAT3, which is a crucial activator for cell transformation. Here, we propose a computational model to account for the dynamical behavior of this positive inflammatory feedback loop. By means of a deterministic model, we show that an irreversible bistable switch between a transformed and a non-transformed state of the cell is at the core of the dynamical behavior of the positive feedback loop linking inflammation to cell transformation. The model indicates that inhibitors (tumor suppressors) or activators (oncogenes) of this positive feedback loop regulate the occurrence of the epigenetic switch by modulating the threshold of inflammatory signal (Src) needed to promote cell transformation. Both stochastic simulations and deterministic simulations of a heterogeneous cell population suggest that random fluctuations (due to molecular noise or cell-to-cell variability) are able to trigger cell transformation. Moreover, the model predicts that oncogenes/tumor suppressors respectively decrease/increase the robustness of the non-transformed state of the cell towards random fluctuations. Finally, the model accounts for the potential effect of competing endogenous RNAs, ceRNAs, on the dynamics of the epigenetic switch. Depending on their microRNA targets, the model predicts that ceRNAs could act as oncogenes or tumor suppressors by regulating the occurrence of cell transformation. PMID:24499937

Changes in cell surface structure by viral transformation studied by binding of lectins differing in sugar specificity.

PubMed

Tsuda, M; Kurokawa, T; Takeuchi, M; Sugino, Y

1975-10-01

Changes in cell surface structure by viral transformation were studied by examining changes in the binding of various lectins differing in carbohydrate specificities. Binding of lectins was assayed directly using cells grown in coverslips. The following 125I-lectins were used: Concanavalin-A (specific for glucose and mannose), wheat germ agglutinin (specific for N-acetylglucosamine), castor bean agglutinin (specific for galactose), Wistaria floribunda agglutinin (specific for N-acetylgalactosamine), and soybean agglutinin (specific for N-acetyl-galactosamine). Cells for a clone, SS7, transformed by bovine adenovirus type-3, were found to bind 5 to 6 times more Wistaria floribunda agglutinin than the normal counterpart cells (clone C31, from C3H mouse kidney). In contrast, the binding of soybean agglutinin, which has a sugar specificity similar to Wistaria floribunda agglutinin, to normal and transformed cells was similar. The binding of wheat germ agglutinin and castor bean agglutinin, respectively, to normal and transformed cells was also similar. However, normal cells bound twice as much concanavalin-A as transformed cells. Only half as much Wistaria floribunda agglutinin was bound to transformed cells when they had been dispersed with EDTA. These changes in the number of lectin binding sites on transformation are thought to reflect alteration of the cell surface structure. The amount of lectins bound per cell decreased with increase in cell density, especially in the case of binding of Wistaria floribunda agglutinin to normal cells.

SPARC Expression Is Selectively Suppressed in Tumor Initiating Urospheres Isolated from As+3- and Cd+2-Transformed Human Urothelial Cells (UROtsa) Stably Transfected with SPARC.

PubMed

Slusser-Nore, Andrea; Larson-Casey, Jennifer L; Zhang, Ruowen; Zhou, Xu Dong; Somji, Seema; Garrett, Scott H; Sens, Donald A; Dunlevy, Jane R

2016-01-01

This laboratory previously analyzed the expression of SPARC in the parental UROtsa cells, their arsenite (As(+3)) and cadmium (Cd(+2))-transformed cell lines, and tumor transplants generated from the transformed cells. It was demonstrated that SPARC expression was down-regulated to background levels in Cd(+2)-and As(+3)-transformed UROtsa cells and tumor transplants compared to parental cells. In the present study, the transformed cell lines were stably transfected with a SPARC expression vector to determine the effect of SPARC expression on the ability of the cells to form tumors in immune-compromised mice. Real time PCR, western blotting, immunohistochemistry, and immunofluorescence were used to define the expression of SPARC in the As(+3)-and Cd(+2)-transformed cell lines, and urospheres isolated from these cell lines, following their stable transfection with an expression vector containing the SPARC open reading frame (ORF). Transplantation of the cultured cells into immune-compromised mice by subcutaneous injection was used to assess the effect of SPARC expression on tumors generated from the above cell lines and urospheres. It was shown that the As(+3)-and Cd(+2)-transformed UROtsa cells could undergo stable transfection with a SPARC expression vector and that the transfected cells expressed both SPARC mRNA and secreted protein. Tumors formed from these SPARC-transfected cells were shown to have no expression of SPARC. Urospheres isolated from cultures of the SPARC-transfected As(+3)-and Cd(+2)-transformed cell lines were shown to have only background expression of SPARC. Urospheres from both the non-transfected and SPARC-transfected cell lines were tumorigenic and thus fit the definition for a population of tumor initiating cells. Tumor initiating cells isolated from SPARC-transfected As(+3)-and Cd(+2)-transformed cell lines have an inherent mechanism to suppress the expression of SPARC mRNA.

SPARC Expression Is Selectively Suppressed in Tumor Initiating Urospheres Isolated from As+3- and Cd+2-Transformed Human Urothelial Cells (UROtsa) Stably Transfected with SPARC

PubMed Central

Slusser-Nore, Andrea; Larson-Casey, Jennifer L.; Zhang, Ruowen; Zhou, Xu Dong; Somji, Seema; Garrett, Scott H.; Sens, Donald A.; Dunlevy, Jane R.

2016-01-01

Background This laboratory previously analyzed the expression of SPARC in the parental UROtsa cells, their arsenite (As+3) and cadmium (Cd+2)-transformed cell lines, and tumor transplants generated from the transformed cells. It was demonstrated that SPARC expression was down-regulated to background levels in Cd+2-and As+3-transformed UROtsa cells and tumor transplants compared to parental cells. In the present study, the transformed cell lines were stably transfected with a SPARC expression vector to determine the effect of SPARC expression on the ability of the cells to form tumors in immune-compromised mice. Methods Real time PCR, western blotting, immunohistochemistry, and immunofluorescence were used to define the expression of SPARC in the As+3-and Cd+2-transformed cell lines, and urospheres isolated from these cell lines, following their stable transfection with an expression vector containing the SPARC open reading frame (ORF). Transplantation of the cultured cells into immune-compromised mice by subcutaneous injection was used to assess the effect of SPARC expression on tumors generated from the above cell lines and urospheres. Results It was shown that the As+3-and Cd+2-transformed UROtsa cells could undergo stable transfection with a SPARC expression vector and that the transfected cells expressed both SPARC mRNA and secreted protein. Tumors formed from these SPARC-transfected cells were shown to have no expression of SPARC. Urospheres isolated from cultures of the SPARC-transfected As+3-and Cd+2-transformed cell lines were shown to have only background expression of SPARC. Urospheres from both the non-transfected and SPARC-transfected cell lines were tumorigenic and thus fit the definition for a population of tumor initiating cells. Conclusions Tumor initiating cells isolated from SPARC-transfected As+3-and Cd+2-transformed cell lines have an inherent mechanism to suppress the expression of SPARC mRNA. PMID:26783756

Atezolizumab, Gemcitabine, Oxaliplatin, and Rituximab in Treating Patients With Relapsed or Refractory Transformed Diffuse Large B-Cell Lymphoma

ClinicalTrials.gov

2018-06-06

Recurrent Diffuse Large B-Cell Lymphoma; Refractory Diffuse Large B-Cell Lymphoma; Refractory Transformed Indolent Non-Hodgkin Lymphoma; Transformed Follicular Lymphoma to Diffuse Large B-Cell Lymphoma

Mechanical phenotype of cancer cells: cell softening and loss of stiffness sensing.

PubMed

Lin, Hsi-Hui; Lin, Hsiu-Kuan; Lin, I-Hsuan; Chiou, Yu-Wei; Chen, Horn-Wei; Liu, Ching-Yi; Harn, Hans I-Chen; Chiu, Wen-Tai; Wang, Yang-Kao; Shen, Meng-Ru; Tang, Ming-Jer

2015-08-28

The stiffness sensing ability is required to respond to the stiffness of the matrix. Here we determined whether normal cells and cancer cells display distinct mechanical phenotypes. Cancer cells were softer than their normal counterparts, regardless of the type of cancer (breast, bladder, cervix, pancreas, or Ha-RasV12-transformed cells). When cultured on matrices of varying stiffness, low stiffness decreased proliferation in normal cells, while cancer cells and transformed cells lost this response. Thus, cancer cells undergo a change in their mechanical phenotype that includes cell softening and loss of stiffness sensing. Caveolin-1, which is suppressed in many tumor cells and in oncogene-transformed cells, regulates the mechanical phenotype. Caveolin-1-upregulated RhoA activity and Y397FAK phosphorylation directed actin cap formation, which was positively correlated with cell elasticity and stiffness sensing in fibroblasts. Ha-RasV12-induced transformation and changes in the mechanical phenotypes were reversed by re-expression of caveolin-1 and mimicked by the suppression of caveolin-1 in normal fibroblasts. This is the first study to describe this novel role for caveolin-1, linking mechanical phenotype to cell transformation. Furthermore, mechanical characteristics may serve as biomarkers for cell transformation.

Analysis of simian virus 40 small t antigen-induced progression of rat F111 cells minimally transformed by large T antigen.

PubMed Central

Zerrahn, J; Deppert, W

1993-01-01

Minimal transformants of rat F111 fibroblasts were established after infection with the large T antigen (large T)-encoding retroviral expression vector pZIPTEX (M. Brown, M. McCormack, K. Zinn, M. Farrell, I. Bikel, and D. Livingston, J. Virol. 60:290-293, 1986). Coexpression of small t antigen (small t) in these cells efficiently led to their progression toward a significantly enhanced transformed phenotype. Small t forms a complex with phosphatase 2A and thereby might influence cellular phosphorylation processes, including the phosphorylation of large T. Since phosphorylation can modulate the transforming activity of large T, we asked whether the phosphorylation status of large T in minimally transformed cells might differ from that of large T in maximally transformed FR(wt648) cells and whether it might be altered by coexpression of small t. We found the phosphate turnover on large T in minimally transformed cells significantly different from that in fully transformed cells. This resulted in underphosphorylation of large T in minimally transformed cells at phosphorylation sites previously shown to be involved in the regulation of the transforming activity of large T. However, coexpression of small t in the minimally transformed cells did not alter the phosphate turnover on large T during progression; i.e., it did not induce a change in the steady-state phosphorylation of large T. This suggests that the helper function of small t during the progression of these cells was not mediated by modulating phosphatase 2A activity toward large T. Images PMID:8382310

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

PubMed

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

2015-10-23

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

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

PubMed Central

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

2015-01-01

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

Complete transformation of ZnO and CuO nanoparticles in culture medium and lymphocyte cells during toxicity testing.

PubMed

Ivask, Angela; Scheckel, Kirk G; Kapruwan, Pankaj; Stone, Vicki; Yin, Hong; Voelcker, Nicolas H; Lombi, Enzo

2017-03-01

Here, we present evidence on complete transformation of ZnO and CuO nanoparticles, which are among the most heavily studied metal oxide particles, during 24 h in vitro toxicological testing with human T-lymphocytes. Synchrotron radiation-based X-ray absorption near edge structure (XANES) spectroscopy results revealed that Zn speciation profiles of 30 nm and 80 nm ZnO nanoparticles, and ZnSO 4 - exposed cells were almost identical with the prevailing species being Zn-cysteine. This suggests that ZnO nanoparticles are rapidly transformed during a standard in vitro toxicological assay, and are sequestered intracellularly, analogously to soluble Zn. Complete transformation of ZnO in the test conditions was further supported by almost identical Zn spectra in medium to which ZnO nanoparticles or ZnSO 4 was added. Likewise, Cu XANES spectra for CuO and CuSO 4 -exposed cells and cell culture media were similar. These results together with our observation on similar toxicological profiles of ZnO and soluble Zn, and CuO and soluble Cu, underline the importance of dissolution and subsequent transformation of ZnO and CuO nanoparticles during toxicological testing and provide evidence that the nano-specific effect of ZnO and CuO nanoparticles is negligible in this system. We strongly suggest to account for this aspect when interpreting the toxicological results of ZnO and CuO nanoparticles.

Method for hull-less barley transformation and manipulation of grain mixed-linkage beta-glucan.

PubMed

Lim, Wai Li; Collins, Helen M; Singh, Rohan R; Kibble, Natalie A J; Yap, Kuok; Taylor, Jillian; Fincher, Geoffrey B; Burton, Rachel A

2018-05-01

Hull-less barley is increasingly offering scope for breeding grains with improved characteristics for human nutrition; however, recalcitrance of hull-less cultivars to transformation has limited the use of these varieties. To overcome this limitation, we sought to develop an effective transformation system for hull-less barley using the cultivar Torrens. Torrens yielded a transformation efficiency of 1.8%, using a modified Agrobacterium transformation method. This method was used to over-express genes encoding synthases for the important dietary fiber component, (1,3;1,4)-β-glucan (mixed-linkage glucan), primarily present in starchy endosperm cell walls. Over-expression of the HvCslF6 gene, driven by an endosperm-specific promoter, produced lines where mixed-linkage glucan content increased on average by 45%, peaking at 70% in some lines, with smaller increases in transgenic HvCslH1 grain. Transgenic HvCslF6 lines displayed alterations where grain had a darker color, were more easily crushed than wild type and were smaller. This was associated with an enlarged cavity in the central endosperm and changes in cell morphology, including aleurone and sub-aleurone cells. This work provides proof-of-concept evidence that mixed-linkage glucan content in hull-less barley grain can be increased by over-expression of the HvCslF6 gene, but also indicates that hull-less cultivars may be more sensitive to attempts to modify cell wall composition. © 2017 Institute of Botany, Chinese Academy of Sciences.

Method for distinguishing normal and transformed cells using G1 kinase inhibitors

DOEpatents

Crissman, Harry A.; Gadbois, Donna M.; Tobey, Robert A.; Bradbury, E. Morton

1993-01-01

A G.sub.1 phase kinase inhibitor is applied in a low concentration to a population of normal and transformed mammalian cells. The concentration of G.sub.1 phase kinase inhibitor is selected to reversibly arrest normal mammalian cells in the G.sub.1 cell cycle without arresting growth of transformed cells. The transformed cells may then be selectively identified and/or cloned for research or diagnostic purposes. The transformed cells may also be selectively killed by therapeutic agents that do not affect normal cells in the G.sub.1 phase, suggesting that such G.sub.1 phase kinase inhibitors may form an effective adjuvant for use with chemotherapeutic agents in cancer therapy for optimizing the killing dose of chemotherapeutic agents while minimizing undesirable side effects on normal cells.

Method for distinguishing normal and transformed cells using G1 kinase inhibitors

DOEpatents

Crissman, H.A.; Gadbois, D.M.; Tobey, R.A.; Bradbury, E.M.

1993-02-09

A G[sub 1] phase kinase inhibitor is applied in a low concentration to a population of normal and transformed mammalian cells. The concentration of G[sub 1] phase kinase inhibitor is selected to reversibly arrest normal mammalian cells in the G[sub 1] cell cycle without arresting growth of transformed cells. The transformed cells may then be selectively identified and/or cloned for research or diagnostic purposes. The transformed cells may also be selectively killed by therapeutic agents that do not affect normal cells in the G[sub 1] phase, suggesting that such G[sub 1] phase kinase inhibitors may form an effective adjuvant for use with chemotherapeutic agents in cancer therapy for optimizing the killing dose of chemotherapeutic agents while minimizing undesirable side effects on normal cells.

Hybridization and Polyploidization of Saccharomyces cerevisiae Strains by Transformation-Associated Cell Fusion

PubMed Central

Takagi, Atsuko; Harashima, Satoshi; Oshima, Yasuji

1985-01-01

Hybrid or polyploid clones of Saccharomyces cerevisiae produced by protoplast fusion were easily isolated by selecting transformants with the plasmid phenotype because the transformation was directly associated with cell fusion. When haploid cells were used as the original strain, the transformants were mostly diploids with a significant fraction of polyploids (triploids or tetraploids). Repeated transformation after curing the plasmid gave rise to clones with higher ploidy, but the frequency of cell fusion was severely reduced as ploidy increased. Images PMID:16346702

Wavelet transform fast inverse light scattering analysis for size determination of spherical scatterers

PubMed Central

Ho, Derek; Kim, Sanghoon; Drake, Tyler K.; Eldridge, Will J.; Wax, Adam

2014-01-01

We present a fast approach for size determination of spherical scatterers using the continuous wavelet transform of the angular light scattering profile to address the computational limitations of previously developed sizing techniques. The potential accuracy, speed, and robustness of the algorithm were determined in simulated models of scattering by polystyrene beads and cells. The algorithm was tested experimentally on angular light scattering data from polystyrene bead phantoms and MCF-7 breast cancer cells using a 2D a/LCI system. Theoretical sizing of simulated profiles of beads and cells produced strong fits between calculated and actual size (r2 = 0.9969 and r2 = 0.9979 respectively), and experimental size determinations were accurate to within one micron. PMID:25360350

A novel rat fibrosarcoma cell line from transformed bone marrow-derived mesenchymal stem cells with maintained in vitro and in vivo stemness properties.

PubMed

Wang, Meng-Yu; Nestvold, Janne; Rekdal, Øystein; Kvalheim, Gunnar; Fodstad, Øystein

2017-03-15

Increasing evidence suggests a possible relationship between mesenchymal stem cells (MSCs) and sarcoma. MSCs are hypothesized to be the cells initiating sarcomagenesis, and cancer stem cells (CSCs) sharing features of MSCs have been identified in sarcomas. Here, we report on the characteristics of a bone marrow-derived rat mesenchymal stem cell line that spontaneously transformed in long-term culture. The rat transformed mesenchymal stem cells (rTMSCs) produced soft-tissue fibrosarcomas in immunocompromised mice and immunocompetent rats. In vitro, the rTMSCs displayed increased proliferation capacity compared to the untransformed cell line. The transformed MSCs maintained the mesenchymal phenotype by expression of the stem cell marker CD 90 and the lack of hematopoietic and endothelial markers. Cytogenetic analysis detected trisomy 6 in the rTMSCs. Side population (SP) isolation and tumorsphere cultivation of the transformed cells confirmed the presence of CSCs among the rTMSCs. Importantly, the rTMSCs retained their differentiation capacity towards osteogenic and adipogenic lineages. This transformed MSC-based cell line may be valuable in examining the balance in a mixed cell population between cancer stem cell properties and the ability to differentiate to specific non-transformed cell populations. Moreover, it may also be a useful tool to evaluate the efficacy of novel targeted immunotherapies in vivo. Copyright © 2017 Elsevier Inc. All rights reserved.

A review of integration strategies for solid oxide fuel cells

NASA Astrophysics Data System (ADS)

Zhang, Xiongwen; Chan, S. H.; Li, Guojun; Ho, H. K.; Li, Jun; Feng, Zhenping

Due to increasing oil and gas demand, the depletion of fossil resources, serious global warming, efficient energy systems and new energy conversion processes are urgently needed. Fuel cells and hybrid systems have emerged as advanced thermodynamic systems with great promise in achieving high energy/power efficiency with reduced environmental loads. In particular, due to the synergistic effect of using integrated solid oxide fuel cell (SOFC) and classical thermodynamic cycle technologies, the efficiency of the integrated system can be significantly improved. This paper reviews different concepts/strategies for SOFC-based integration systems, which are timely transformational energy-related technologies available to overcome the threats posed by climate change and energy security.

Disruption of adherens junction and alterations in YAP-related proliferation behavior as part of the underlying cell transformation process of alcohol-induced oral carcinogenesis.

PubMed

Husari, Ayman; Hülter-Hassler, Diana; Steinberg, Thorsten; Schulz, Simon Daniel; Tomakidi, Pascal

2018-01-01

Accumulating evidences indicate that alcohol might play a causative in oral cancer. Unfortunately, in vitro cell systems, uncovering the molecular background of the underlying cell transformation process, are rare. Therefore, this study was conducted, to identify molecular changes and characterize their putative cell behavioral consequences in epitheloid (EPI) and fibroblastoid (FIB) oral keratinocyte phenotypes, arising from chronical alcohol treatment. Concerning adherens junctions (AJs), both EPI and FIB showed membrane-bound β-catenin, but exhibited differences for E-cadherin and zyxin. While EPI revealed E-cadherin/β-catenin membrane co-localization, which in parts also applied for zyxin, FIB membranes were devoid of E-cadherin and exhibited marginal zyxin expression. Fetal calf serum (FCS) administration in starved cells promoted proliferation in both keratinocyte phenotypes, whereat EPI and FIB yielded a strikingly modified FCS sensitivity on the temporal scale. Impedance measurement-based cell index detection yielded proliferation stimulation occurring much earlier in FIB (<20h) compared to EPI (>45h). Nuclear preference of the proliferation-associated YAP co-transcription factor in FIB was FCS independent, while it required FCS in EPI. Taken together, the lack of membrane-inherent E-cadherin/β-catenin co-localization together with low zyxin - reveals perturbation of AJ integrity in FIB. Regarding cell behavior, perturbed AJs in FIB correlate with temporal proliferation sensitivity towards FCS. CYF of 5.6 strongly suggests involvement of chromatin-bound YAP in FIB's proliferation temperosensitivity. These molecular differences detected for EPI and FIB are part of the underlying cell transformation process of alcohol-induced oral carcinogenesis, and indicate FIB being in a more advanced transformation stage. Copyright © 2017 Elsevier B.V. All rights reserved.

High Efficiency Transformation of Cultured Tobacco Cells 1

PubMed Central

An, Gynheung

1985-01-01

Tobacco calli were transformed at levels up to 50% by cocultivation of tobacco cultured cells with Agrobacterium tumefaciens harboring the binary transfer-DNA vector, pGA472, containing a kanamycin resistance marker. Transformation frequency was dependent on the physiological state of the tobacco cells, the nature of Agrobacterium strain and, less so, on the expression of the vir genes of the tumor-inducing plasmid. Maximum transformation frequency was obtained with exponentially growing plant cells, suggesting that rapid growth of plant cells is an essental factor for efficient transformation of higher plants. Images Fig. 1 PMID:16664453

A simple Gateway-assisted construction system of TALEN genes for plant genome editing.

PubMed

Kusano, Hiroaki; Onodera, Hitomi; Kihira, Miho; Aoki, Hiromi; Matsuzaki, Hikaru; Shimada, Hiroaki

2016-07-25

TALEN is an artificial nuclease being applied for sequence-specific genome editing. For the plant genome editing, a pair of TALEN genes is expressed in the cells, and a binary plasmid for Agrobacterium-mediated transformation should be assembled. We developed a novel procedure using the Gateway-assisted plasmids, named Emerald-Gateway TALEN system. We constructed entry vectors, pPlat plasmids, for construction of a desired TALEN gene using Platinum Gate TALEN kit. We also created destination plasmid, pDual35SGw1301, which allowed two TALEN genes to both DNA strands to recruit using Gateway technology. Resultant TALEN genes were evaluated by the single-strand annealing (SSA) assay in E. coli cells. By this assay, the TALENs recognized the corresponding targets in the divided luciferase gene, and induced a specific recombination to generate an active luciferase gene. Using the TALEN genes constructed, we created a transformant potato cells in which a site-specific mutation occurred at the target site of the GBSS gene. This suggested that our system worked effectively and was applicable as a convenient tool for the plant genome editing.

A novel rat fibrosarcoma cell line from transformed bone marrow-derived mesenchymal stem cells with maintained in vitro and in vivo stemness properties

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

Wang, Meng-Yu; Nestvold, Janne, E-mail: j.m.nestvold@medisin.uio.no; Rekdal, Øystein

Increasing evidence suggests a possible relationship between mesenchymal stem cells (MSCs) and sarcoma. MSCs are hypothesized to be the cells initiating sarcomagenesis, and cancer stem cells (CSCs) sharing features of MSCs have been identified in sarcomas. Here, we report on the characteristics of a bone marrow-derived rat mesenchymal stem cell line that spontaneously transformed in long-term culture. The rat transformed mesenchymal stem cells (rTMSCs) produced soft-tissue fibrosarcomas in immunocompromised mice and immunocompetent rats. In vitro, the rTMSCs displayed increased proliferation capacity compared to the untransformed cell line. The transformed MSCs maintained the mesenchymal phenotype by expression of the stem cellmore » marker CD 90 and the lack of hematopoietic and endothelial markers. Cytogenetic analysis detected trisomy 6 in the rTMSCs. Side population (SP) isolation and tumorsphere cultivation of the transformed cells confirmed the presence of CSCs among the rTMSCs. Importantly, the rTMSCs retained their differentiation capacity towards osteogenic and adipogenic lineages. This transformed MSC-based cell line may be valuable in examining the balance in a mixed cell population between cancer stem cell properties and the ability to differentiate to specific non-transformed cell populations. Moreover, it may also be a useful tool to evaluate the efficacy of novel targeted immunotherapies in vivo. - Highlights: • Spontaneously transformed rat MSCs (rTMSCs) share characteristics with normal MSCs. • rTMSCs possess a side population, enriched with tumorigenic cells. • rTMSCs model fibrosarcoma in vivo.« less

Alpha lipoic acid selectively inhibits proliferation and adhesion to fibronectin of v-H-ras-transformed 3Y1 cells.

PubMed

Yamasaki, Masao; Iwase, Masahiro; Kawano, Kazuo; Sakakibara, Yoichi; Suiko, Masahito; Nishiyama, Kazuo

2012-05-01

Here, we focused on the effects of racemic α-lipoic acid on proliferation and adhesion properties of 3Y1 rat fibroblasts and the v-H-ras-transformed derivative, HR-3Y1-2 cells. Racemic α-lipoic acid inhibited proliferation of HR-3Y1-2 but not 3Y1 cells at 0.3 and 1.0 mM. R-(+)-α-lipoic acid also inhibited proliferation of HR-3Y1-2 cells equivalent to that of racemic α-lipoic acid. In addition, racemic α-lipoic acid decreased intracellular reactive oxygen species levels in HR-3Y1 cells but not 3Y1 cells. Next, we evaluated the effects of racemic α-lipoic acid on cell adhesion to fibronectin. The results indicated that racemic α-lipoic acid decreased adhesive ability of HR-3Y1-2 cells to fibronectin-coated plates. As blocking antibody experiment revealed that β1-integrin plays a key role in cell adhesion in this experimental system, the effects of racemic α-lipoic acid on the expression of β1-integrin were examined. The results indicated that racemic α-lipoic acid selectively downregulated the expression of cell surface β1-integrin expression in HR-3Y1-2 cells. Intriguingly, exogenous hydrogen peroxide upregulated cell surface β1-integrin expression in 3Y1 cells. Taken together, these data suggest that reduction of intracellular reactive oxygen species levels by α-lipoic acid could be an effective means of ameliorating abnormal growth and adhesive properties in v-H-ras transformed cells.

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

PubMed

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

2010-09-07

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

The viability of MCM-41 as separator in secondary alkaline cells

NASA Astrophysics Data System (ADS)

Meskon, S. R.; Othman, R.; Ani, M. H.

2018-01-01

The viability of MCM-41 membrane as a separator material in secondary alkaline cell is investigated. The inorganic membrane was employed in an alkaline nickel-zinc system. MCM-41 mesoporous material consists of arrays of hexagonal nano-pore channels. The membrane was synthesized using sol-gel route from parent solution comprising of quarternary ammonium surfactant, cethyltrimethylammonium bromide C16H33(CH3)3NBr (CTAB), hydrochloric acid (HCl), deionized water (H2O), ethanol (C2H5OH), and tetraethylortosilicate (TEOS). Both the anodic zinc/zinc oxide and cathodic nickel hydroxide electrodeposited film were coated with MCM-41 membrane. The Ni/MCM-41/Zn alkaline cell was then subjected to 100-cycle durability test and the structural stability of MCM-41 separator throughout the progression of the charge-discharge cycles is studied. X-ray diffraction (XRD) analysis on the dismantled cell shows that MCM-41 began to transform to lamellar MCM-50 on the 5th cycle and transformed almost completely on the 25th cycle. The phase transformation of MCM-41 hexagonal structure into gel-like MCM-50 prevents the mesoporous cell separator from diminished in the caustic alkaline surround. This work has hence demonstrated MCM-41 membrane is viable to be employed in secondary alkaline cells.

Mitochondrial NDUFS3 regulates the ROS-mediated onset of metabolic switch in transformed cells

PubMed Central

Suhane, Sonal; Kanzaki, Hirotaka; Arumugaswami, Vaithilingaraja; Murali, Ramachandran; Ramanujan, V. Krishnan

2013-01-01

Summary Aerobic glycolysis in transformed cells is an unique metabolic phenotype characterized by a hyperactivated glycolytic pathway even in the presence of oxygen. It is not clear if the onset of aerobic glycolysis is regulated by mitochondrial dysfunction and, if so, what the metabolic windows of opportunity available to control this metabolic switch (mitochondrial to glycolytic) landscape are in transformed cells. Here we report a genetically-defined model system based on the gene-silencing of a mitochondrial complex I subunit, NDUFS3, where we demonstrate the onset of metabolic switch in isogenic human embryonic kidney cells by differential expression of NDUFS3. By means of extensive metabolic characterization, we demonstrate that NDUFS3 gene silencing systematically introduces mitochondrial dysfunction thereby leading to the onset of aerobic glycolysis in a manner dependent on NDUFS3 protein levels. Furthermore, we show that the sustained imbalance in free radical dynamics is a necessary condition to sustain the observed metabolic switch in cell lines with the most severe NDUFS3 suppression. Together, our data reveal a novel role for mitochondrial complex I subunit NDUFS3 in regulating the degree of mitochondrial dysfunction in living cells, thereby setting a “metabolic threshold” for the observation of aerobic glycolysis phenotype within the confines of mitochondrial dysfunction. PMID:23519235

Initiation of oncogenic transformation in human mammary epithelial cells by charged particles

NASA Technical Reports Server (NTRS)

Yang, T. C.; Georgy, K. A.; Craise, L. M.; Durante, M.

1997-01-01

Experimental studies have shown that high linear-energy transfer (LET) charged particles can be more effective than x-rays and gamma-rays in inducing oncogenic transformation in cultured cells and tumors in animals. Based on these results, experiments were designed and performed with an immortal human mammary epithelial cell line (H184B5), and several clones transformed by heavy ions were obtained. Cell fusion experiments were subsequently done, and results indicate that the transforming gene(s) is recessive. Chromosome analysis with fluorescence in situ hybridization (FISH) techniques also showed additional translocations in transformed human mammary epithelial cells. In addition, studies with these cell lines indicate that heavy ions can effectively induce deletion, break, and dicentrics. Deletion of tumor suppressor gene(s) and/or formation of translocation through DNA double strand breaks is a likely mechanism for the initiation of oncogenic transformation in human mammary epithelial cells.

Biomechanical cell regulatory networks as complex adaptive systems in relation to cancer.

PubMed

Feller, Liviu; Khammissa, Razia Abdool Gafaar; Lemmer, Johan

2017-01-01

Physiological structure and function of cells are maintained by ongoing complex dynamic adaptive processes in the intracellular molecular pathways controlling the overall profile of gene expression, and by genes in cellular gene regulatory circuits. Cytogenetic mutations and non-genetic factors such as chronic inflammation or repetitive trauma, intrinsic mechanical stresses within extracellular matrix may induce redirection of gene regulatory circuits with abnormal reactivation of embryonic developmental programmes which can now drive cell transformation and cancer initiation, and later cancer progression and metastasis. Some of the non-genetic factors that may also favour cancerization are dysregulation in epithelial-mesenchymal interactions, in cell-to-cell communication, in extracellular matrix turnover, in extracellular matrix-to-cell interactions and in mechanotransduction pathways. Persistent increase in extracellular matrix stiffness, for whatever reason, has been shown to play an important role in cell transformation, and later in cancer cell invasion. In this article we review certain cell regulatory networks driving carcinogenesis, focussing on the role of mechanical stresses modulating structure and function of cells and their extracellular matrices.

Plant genetic transformation efficiency of selected Malaysian rice based on selectable marker gene (hptII).

PubMed

Htwe, Nwe Nwe; Ling, Ho Chai; Zaman, Faridah Qamaruz; Maziah, Mahmood

2014-04-01

Rice is one of the most important cereal crops with great potential for biotechnology progress. In transformation method, antibiotic resistance genes are routinely used as powerful markers for selecting transformed cells from surrounding non-transformed cells. In this study, the toxicity level of hygromycin was optimized for two selected mutant rice lines, MR219 line 4 and line 9. The mature embryos were isolated and cultured on an MS medium with different hygromycin concentrations (0, 20, 40, 60, 80 and 100 mg L(-1)). Evidently, above 60 mg L(-1) was effective for callus formation and observed completely dead. Further there were tested for specific concentration (0-60). Although, 21.28% calli survived on the medium containing 45 mg L(-1) hygromycin, it seemed suitable for the identification of putative transformants. These findings indicated that a system for rice transformation in a relatively high frequency and the transgenes are stably expressed in the transgenic plants. Green shoots were regenerated from the explant under hygromycin stress. RT-PCR using hptII and gus sequence specific primer and Southern blot analysis were used to confirm the presence of the transgene and to determine the transformation efficiency for their stable integration in regenerated plants. This study demonstrated that the hygromycin resistance can be used as an effective marker for rice transformation.

Highly efficient Agrobacterium-mediated transformation of embryogenic cell suspensions of Musa acuminata cv. Mas (AA) via a liquid co-cultivation system.

PubMed

Huang, Xia; Huang, Xue-Lin; Xiao, Wang; Zhao, Jie-Tang; Dai, Xue-Mei; Chen, Yun-Feng; Li, Xiao-Ju

2007-10-01

A high efficient protocol of Agrobacterium-mediated transformation of Musa acuminata cv. Mas (AA), a major banana variety of the South East Asia region, was developed in this study. Male-flower-derived embryogenic cell suspensions (ECS) were co-cultivated in liquid medium with Agrobacterium strain EHA105 harboring a binary vector pCAMBIA2301 carrying nptII and gusA gene in the T-DNA. Depending upon conditions and duration of co-cultivation in liquid medium, 0-490 transgenic plants per 0.5 ml packed cell volume (PCV) of ECS were obtained. The optimum duration of inoculation was 2 h, and the highest transformation frequency was achieved when infected ECS were co-cultivated in liquid medium first for 12 h at 40 rpm and then for 156 h at 100 rpm on a rotary shaker. Co-cultivation for a shorter duration (72 h) or shaking constantly at 100 rpm at the same duration gave 1.6 and 1.8 folds lower transformation efficiency, respectively. No transgenic plants were obtained in parallel experiments carried on semi-solid media. Histochemical GUS assay and molecular analysis in several tissues of the transgenic plants demonstrated that foreign genes were stably integrated into the banana genome. Compared to semi-solid co-cultivation transformation in other banana species, it is remarkable that liquid co-cultivation was much more efficient for transformation of the Mas cultivar, and was at least 1 month faster for regenerating transgenic plants.

Complete transformation of ZnO and CuO nanoparticles in ...

EPA Pesticide Factsheets

Here, we present evidence on complete transformation of ZnO and CuO nanoparticles, which are among the most heavily studied metal oxide particles, during 24 h in vitro toxicological testing with human T-lymphocytes. Synchrotron radiation-based X-ray absorption near edge structure (XANES) spectroscopy results revealed that Zn speciation profiles of 30 nm and 80 nm ZnO nanoparticles, and ZnSO4- exposed cells were almost identical with the prevailing species being Zn-cysteine. This suggests that ZnO nanoparticles are rapidly transformed during a standard in vitro toxicological assay, and are sequestered intracellularly, analogously to soluble Zn. Complete transformation of ZnO in the test conditions was further supported by almost identical Zn spectra in medium to which ZnO nanoparticles or ZnSO4 was added. Likewise, Cu XANES spectra for CuO and CuSO4-exposed cells and cell culture media were similar. These results together with our observation on similar toxicological profiles of ZnO and soluble Zn, and CuO and soluble Cu, underline the importance of dissolution and subsequent transformation of ZnO and CuO nanoparticles during toxicological testing and provide evidence that the nano-specific effect of ZnO and CuO nanoparticulates is negligible in this system. We strongly suggest to account for this aspect when interpreting the toxicological results of ZnO and CuO nanoparticles. Although a number of studies have discussed the transformation of nanoparticles during

Wound Healing and Cancer Stem Cells: Inflammation as a Driver of Treatment Resistance in Breast Cancer

PubMed Central

Arnold, Kimberly M; Opdenaker, Lynn M; Flynn, Daniel; Sims-Mourtada, Jennifer

2015-01-01

The relationship between wound healing and cancer has long been recognized. The mechanisms that regulate wound healing have been shown to promote transformation and growth of malignant cells. In addition, chronic inflammation has been associated with malignant transformation in many tissues. Recently, pathways involved in inflammation and wound healing have been reported to enhance cancer stem cell (CSC) populations. These cells, which are highly resistant to current treatments, are capable of repopulating the tumor after treatment, causing local and systemic recurrences. In this review, we highlight proinflammatory cytokines and developmental pathways involved in tissue repair, whose deregulation in the tumor microenvironment may promote growth and survival of CSCs. We propose that the addition of anti-inflammatory agents to current treatment regimens may slow the growth of CSCs and improve therapeutic outcomes. PMID:25674014

Nrf2/p62 signaling in apoptosis resistance and its role in cadmium-induced carcinogenesis.

PubMed

Son, Young-Ok; Pratheeshkumar, Poyil; Roy, Ram Vinod; Hitron, John Andrew; Wang, Lei; Zhang, Zhuo; Shi, Xianglin

2014-10-10

The cadmium-transformed human lung bronchial epithelial BEAS-2B cells exhibit a property of apoptosis resistance as compared with normal non-transformed BEAS-2B cells. The level of basal reactive oxygen species (ROS) is extremely low in transformed cells in correlation with elevated expressions of both antioxidant enzymes (catalase, SOD1, and SOD2) and antiapoptotic proteins (Bcl-2/Bcl-xL). Moreover, Nrf2 and p62 are highly expressed in these transformed cells. The knockdown of Nrf2 or p62 by siRNA enhances ROS levels and cadmium-induced apoptosis. The binding activities of Nrf2 on the antioxidant response element promoter regions of p62/Bcl-2/Bcl-xL were dramatically increased in the cadmium-exposed transformed cells. Cadmium exposure increased the formation of LC3-II and the frequency of GFP-LC3 punctal cells in non-transformed BEAS-2B cells, whereas these increases are not shown in transformed cells, an indication of autophagy deficiency of transformed cells. Furthermore, the expression levels of Nrf2 and p62 are dramatically increased during chronic long term exposure to cadmium in the BEAS-2B cells as well as antiapoptotic proteins and antioxidant enzymes. These proteins are overexpressed in the tumor tissues derived from xenograft mouse models. Moreover, the colony growth is significantly attenuated in the transformed cells by siRNA transfection specific for Nrf2 or p62. Taken together, this study demonstrates that cadmium-transformed cells have acquired autophagy deficiency, leading to constitutive p62 and Nrf2 overexpression. These overexpressions up-regulate the antioxidant proteins catalase and SOD and the antiapoptotic proteins Bcl-2 and Bcl-xL. The final consequences are decrease in ROS generation, apoptotic resistance, and increased cell survival, proliferation, and tumorigenesis. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Nrf2/p62 Signaling in Apoptosis Resistance and Its Role in Cadmium-induced Carcinogenesis*

PubMed Central

Son, Young-Ok; Pratheeshkumar, Poyil; Roy, Ram Vinod; Hitron, John Andrew; Wang, Lei; Zhang, Zhuo; Shi, Xianglin

2014-01-01

The cadmium-transformed human lung bronchial epithelial BEAS-2B cells exhibit a property of apoptosis resistance as compared with normal non-transformed BEAS-2B cells. The level of basal reactive oxygen species (ROS) is extremely low in transformed cells in correlation with elevated expressions of both antioxidant enzymes (catalase, SOD1, and SOD2) and antiapoptotic proteins (Bcl-2/Bcl-xL). Moreover, Nrf2 and p62 are highly expressed in these transformed cells. The knockdown of Nrf2 or p62 by siRNA enhances ROS levels and cadmium-induced apoptosis. The binding activities of Nrf2 on the antioxidant response element promoter regions of p62/Bcl-2/Bcl-xL were dramatically increased in the cadmium-exposed transformed cells. Cadmium exposure increased the formation of LC3-II and the frequency of GFP-LC3 punctal cells in non-transformed BEAS-2B cells, whereas these increases are not shown in transformed cells, an indication of autophagy deficiency of transformed cells. Furthermore, the expression levels of Nrf2 and p62 are dramatically increased during chronic long term exposure to cadmium in the BEAS-2B cells as well as antiapoptotic proteins and antioxidant enzymes. These proteins are overexpressed in the tumor tissues derived from xenograft mouse models. Moreover, the colony growth is significantly attenuated in the transformed cells by siRNA transfection specific for Nrf2 or p62. Taken together, this study demonstrates that cadmium-transformed cells have acquired autophagy deficiency, leading to constitutive p62 and Nrf2 overexpression. These overexpressions up-regulate the antioxidant proteins catalase and SOD and the antiapoptotic proteins Bcl-2 and Bcl-xL. The final consequences are decrease in ROS generation, apoptotic resistance, and increased cell survival, proliferation, and tumorigenesis. PMID:25157103

Obesity Suppresses Cell-Competition-Mediated Apical Elimination of RasV12-Transformed Cells from Epithelial Tissues.

PubMed

Sasaki, Ayana; Nagatake, Takahiro; Egami, Riku; Gu, Guoqiang; Takigawa, Ichigaku; Ikeda, Wataru; Nakatani, Tomoya; Kunisawa, Jun; Fujita, Yasuyuki

2018-04-24

Recent studies have revealed that newly emerging transformed cells are often eliminated from epithelial tissues via cell competition with the surrounding normal epithelial cells. This cancer preventive phenomenon is termed epithelial defense against cancer (EDAC). However, it remains largely unknown whether and how EDAC is diminished during carcinogenesis. In this study, using a cell competition mouse model, we show that high-fat diet (HFD) feeding substantially attenuates the frequency of apical elimination of RasV12-transformed cells from intestinal and pancreatic epithelia. This process involves both lipid metabolism and chronic inflammation. Furthermore, aspirin treatment significantly facilitates eradication of transformed cells from the epithelial tissues in HFD-fed mice. Thus, our work demonstrates that obesity can profoundly influence competitive interaction between normal and transformed cells, providing insights into cell competition and cancer preventive medicine. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

2014 DOE Hydrogen and Fuel Cells Program Annual Merit Review and Peer Evaluation Report

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

none,

2014-10-01

This report summarizes comments from the Peer Review Panel at the 2014 DOE Hydrogen and Fuel Cells Program Annual Merit Review, held on June 16-20, 2014, in Washington, DC. It covers the program areas of hydrogen production and delivery; hydrogen storage; fuel cells; manufacturing R&D; technology validation; safety, codes, and standards; market transformation; and systems analysis.

2015 DOE Hydrogen and Fuel Cells Program Annual Merit Review and Peer Evaluation Report

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

none,

This report summarizes comments from the Peer Review Panel at the 2015 DOE Hydrogen and Fuel Cells Program Annual Merit Review, held on June 8-12, 2015, in Arlington, Virginia. It covers the program areas of hydrogen production and delivery; hydrogen storage; fuel cells; manufacturing R&D; technology validation; safety, codes, and standards; market transformation; and systems analysis.

2012 DOE Hydrogen and Fuel Cells Program Annual Merit Review and Peer Evaluation Report

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

none,

2012-09-01

This report summarizes comments from the Peer Review Panel at the 2012 DOE Hydrogen and Fuel Cells Program Annual Merit Review, held on May 14-18, 2012, in Arlington, Virginia. It covers the program areas of hydrogen production and delivery; hydrogen storage; fuel cells; manufacturing R&D; technology validation; safety, codes, and standards; education; market transformation; and systems analysis.

2011 DOE Hydrogen and Fuel Cells Program Annual Merit Review and Peer Evaluation Report

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

none,

2011-09-01

This report summarizes comments from the Peer Review Panel at the 2011 DOE Hydrogen and Fuel Cells Program Annual Merit Review, held on May 9-13, 2011, in Arlington, Virginia. It covers the program areas of hydrogen production and delivery; hydrogen storage; fuel cells; manufacturing R&D; technology validation; safety, codes, and standards; education; market transformation; and systems analysis.

High-frequency transformation of a methylotrophic yeast, Candida boidinii, with autonomously replicating plasmids which are also functional in Saccharomyces cerevisiae.

PubMed

Sakai, Y; Goh, T K; Tani, Y

1993-06-01

We have developed a transformation system which uses autonomous replicating plasmids for a methylotrophic yeast, Candida boidinii. Two autonomous replication sequences, CARS1 and CARS2, were newly cloned from the genome of C. boidinii. Plasmids having both a CARS fragment and the C. boidinii URA3 gene transformed C. boidinii ura3 cells to Ura+ phenotype at frequencies of up to 10(4) CFU/micrograms of DNA. From Southern blot analysis, CARS plasmids seemed to exist in polymeric forms as well as in monomeric forms in C. boidinii cells. The C. boidinii URA3 gene was overexpressed in C. boidinii on these CARS vectors. CARS1 and CARS2 were found to function as an autonomous replicating element in Saccharomyces cerevisiae as well. Different portions of the CARS1 sequence were needed for autonomous replicating activity in C. boidinii and S. cerevisiae. C. boidinii could also be transformed with vectors harboring a CARS fragment and the S. cerevisiae URA3 gene.

SV40-IMMORTALIZED NON-TUMORIGENIC AND TUMORIGENIC CELL LINES DIFFER IN EXPRESSION OF HALLMARK VIRAL RESPONSE MRNAS

EPA Science Inventory

SV40-Immortalized Non-Tumorigenic and Tumorigenic Cell Lines Differ in Expression of Hallmark Viral Response mRNAs.

Prior to the use of an in vitra/in viva transformation system to examine the tumorigenic activity of environmental contaminants, in vitra gene expression pa...

Early events in Agrobacterium-mediated genetic transformation of citrus explants.

PubMed

Peña, Leandro; Pérez, Rosa M; Cervera, Magdalena; Juárez, José A; Navarro, Luis

2004-07-01

Genetic transformation of plants relies on two independent but concurrent processes: integration of foreign DNA into plant cells and regeneration of whole plants from these transformed cells. Cell competence for regeneration and for transformation does not always fall into the same cell type/developmental stage, and this is one of the main causes of the so-called recalcitrance for transformation of certain plant species. In this study, a detailed examination of the first steps of morphogenesis from citrus explants after co-cultivation with Agrobacterium tumefaciens was performed, and an investigation into which cells and tissues are competent for regeneration and transformation was carried out. Moreover, the role of phytohormones in the co-cultivation medium as possible enhancers of gene transfer was also studied. A highly responsive citrus genotype and well-established culture conditions were used to perform a histological analysis of morphogenesis and cell competence for transformation after co-cultivation of citrus epicotyl segments with A. tumefaciens. In addition, the role of phytohormones as transformation enhancers was investigated by flow cytometry. It is demonstrated that cells competent for transformation are located in the newly formed callus growing from the cambial ring. Conditions conducive to further development of this callus, such as treatment of explants in a medium rich in auxins, resulted in a more pronounced formation of cambial callus and a slower shoot regeneration process, both in Agrobacterium-inoculated and non-inoculated explants. Furthermore, co- cultivation in a medium rich in auxins caused a significant increase in the rate of actively dividing cells in S-phase, the stage in which cells are more prone to integrate foreign DNA. Use of proper co-cultivation medium and conditions led to a higher number of stably transformed cells and to an increase in the final number of regenerated transgenic plants.

Cell-free protein synthesis: the state of the art.

PubMed

Whittaker, James W

2013-02-01

Cell-free protein synthesis harnesses the synthetic power of biology, programming the ribosomal translational machinery of the cell to create macromolecular products. Like PCR, which uses cellular replication machinery to create a DNA amplifier, cell-free protein synthesis is emerging as a transformative technology with broad applications in protein engineering, biopharmaceutical development, and post-genomic research. By breaking free from the constraints of cell-based systems, it takes the next step towards synthetic biology. Recent advances in reconstituted cell-free protein synthesis (Protein synthesis Using Recombinant Elements expression systems) are creating new opportunities to tailor the reactions for specialized applications including in vitro protein evolution, printing protein microarrays, isotopic labeling, and incorporating nonnatural amino acids.

Origin and early evolution of neural circuits for the control of ciliary locomotion.

PubMed

Jékely, Gáspár

2011-03-22

Behaviour evolved before nervous systems. Various single-celled eukaryotes (protists) and the ciliated larvae of sponges devoid of neurons can display sophisticated behaviours, including phototaxis, gravitaxis or chemotaxis. In single-celled eukaryotes, sensory inputs directly influence the motor behaviour of the cell. In swimming sponge larvae, sensory cells influence the activity of cilia on the same cell, thereby steering the multicellular larva. In these organisms, the efficiency of sensory-to-motor transformation (defined as the ratio of sensory cells to total cell number) is low. With the advent of neurons, signal amplification and fast, long-range communication between sensory and motor cells became possible. This may have first occurred in a ciliated swimming stage of the first eumetazoans. The first axons may have had en passant synaptic contacts to several ciliated cells to improve the efficiency of sensory-to-motor transformation, thereby allowing a reduction in the number of sensory cells tuned for the same input. This could have allowed the diversification of sensory modalities and of the behavioural repertoire. I propose that the first nervous systems consisted of combined sensory-motor neurons, directly translating sensory input into motor output on locomotor ciliated cells and steering muscle cells. Neuronal circuitry with low levels of integration has been retained in cnidarians and in the ciliated larvae of some marine invertebrates. This parallel processing stage could have been the starting point for the evolution of more integrated circuits performing the first complex computations such as persistence or coincidence detection. The sensory-motor nervous systems of cnidarians and ciliated larvae of diverse phyla show that brains, like all biological structures, are not irreducibly complex.

Onion cell imaging by using Talbot/self-imaging effect

NASA Astrophysics Data System (ADS)

Agarwal, Shilpi; Kumar, Varun; Shakher, Chandra

2017-08-01

This paper presents the amplitude and phase imaging of onion epidermis cell using the self-imaging capabilities of a grating (Talbot effect) in visible light region. In proposed method, the Fresnel diffraction pattern from the first grating and object is recorded at self-image plane. Fast Fourier Transform (FFT) is used for extracting the 3D amplitude and phase image of onion epidermis cell. The stability of the proposed system, from environmental perturbation as well as its compactness and portability give the proposed system a high potential for several clinical applications.

Single-cell technologies to study the immune system.

PubMed

Proserpio, Valentina; Mahata, Bidesh

2016-02-01

The immune system is composed of a variety of cells that act in a coordinated fashion to protect the organism against a multitude of different pathogens. The great variability of existing pathogens corresponds to a similar high heterogeneity of the immune cells. The study of individual immune cells, the fundamental unit of immunity, has recently transformed from a qualitative microscopic imaging to a nearly complete quantitative transcriptomic analysis. This shift has been driven by the rapid development of multiple single-cell technologies. These new advances are expected to boost the detection of less frequent cell types and transient or intermediate cell states. They will highlight the individuality of each single cell and greatly expand the resolution of current available classifications and differentiation trajectories. In this review we discuss the recent advancement and application of single-cell technologies, their limitations and future applications to study the immune system. © 2015 The Authors. Immunology Published by John Wiley & Sons Ltd.

CELL SHAPE AND HEXOSE TRANSPORT IN NORMAL AND VIRUS-TRANSFORMED CELLS IN CULTURE

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

Bissell, M.J.; Farson, D.; Tung, A.S.C.

1976-07-01

The rate of hexose transport was compared in normal and virus-transformed cells on a monolayer and in suspension. It was shown that: (1) Both trypsin-removed cells and those suspended for an additional day in methyl cellulose had decreased rates of transport and lower available water space when compared with cells on a monolayer. Thus, cell shape affects the overall rate of hexose transport, especially at higher sugar concentrations. (2) Even in suspension, the initial transport rates remained higher in transformed cells with reference to normal cells. Scanning electron micrographs of normal and transformed chick cells revealed morphological differences only inmore » the flat state. This indicates that the increased rate of hexose transport after transformation is not due to a difference in the shape of these cells on a monolayer. The relation between the geometry of cells, transport rates, and growth regulation is undoubtedly very complex, and our knowledge of these relationships is still very elementary. In a recent review on the influence of geometry on control of cell growth, Folkman and Greenspan (1) pointed out that the permeability of cells in a flat versus a spherical state may indeed be very different. The growth properties of cells on a surface and in suspension have been compared often (1-5). However, with one exception. little is known about the changes in transport properties when cell shape is changed. Foster and Pardee (6) demonstrated that the active transport of a-aminoisobutyric acid was reduced 2.5 times in suspension cultures of Chinese hamster cells with respect to the cells grown on a coverslip. They attributed this to the smaller surface area of suspended cells. While it is not clear why active transport should be dependent on the surface area available, it is possible that once the cells assume a spherical configuration, the carrier proteins are redistributed in such a way as to make them less accessible to the substrate. What happens to facilitated and nonmediated diffusion when cells are placed in suspension has not been determined. The transport of hexoses into animal cells in culture has been shown to occur by facilitated diffusion (7). In chick embryo fibroblasts in culture, there is more than one component to this transport system: a saturable carrier-mediated transport with a Km for 2-deoxy-D-glucose (2DG) of about 1-5 mM (8-1 1) and a nonsaturable component which may include a low affinity transport site and/or nonmediated diffusion (8, 10, 11). It was shown previously (1 1) that growth rate, cell density, glucose deprivation, and virus transformation may alter not only the overall transport rate but also the rate of transport of one mode relative to the other. The rate of the nonmediated uptake, at least, is dependent upon the surface area available, and in addition total level of transport is dependent on the available water space (which in turn is roughly related to cell volume). Since it is estimated that the surface area may change as much as tenfold when cells go from a flat configuration to a spherical one (I), it is apparent that the cell shape could play a role in overall transport characteristics of cultured cells. Using glucose analogues, 2DG and 3-0-methylglucose (3MG), we compared the transport properties of normal and virus-transformed cells in suspension and on monolayers. It was found that both the rates as well as the total levels of transport were decreased after the cells were placed in suspension, with the nonsaturable component being most affected. Nevertheless, a difference in the initial rates of transport between normal and transformed cells remained, indicating that the difference is independent of cell shape.« less

Breast-feeding regulates immune system development via transforming growth factor-β in mice pups.

PubMed

Sakaguchi, Keita; Koyanagi, Akemi; Kamachi, Fumitaka; Harauma, Akiko; Chiba, Asako; Hisata, Ken; Moriguchi, Toru; Shimizu, Toshiaki; Miyake, Sachiko

2018-03-01

Breast milk contains important nutrients and immunoregulatory factors that are essential for newborn infants. Recently, epidemiological studies suggested that breast-feeding prevents a wide range of infectious diseases and lowers the incidence of infant allergic diseases. To examine the effects of breast milk on immunological development in infancy, we established an artificial rearing system for hand-feeding mice and compared mouse pups fed with either breast milk or milk substitute. All mice were killed at 14 days of age and immune cells in the thymus, spleen, and small intestine were examined on flow cytometry. The number of thymocytes was higher whereas that of total immune cells of peripheral lymphoid tissues was lower in mice fed breast milk compared with milk substitute-fed mice. In peripheral lymphoid tissues, the proportion of B cells was higher and that of CD8 + T cells, macrophages, dendritic cells, and granulocytes was significantly lower in breast milk-fed mice. The same alteration in immune cells of the thymus and peripheral lymphoid tissues in milk substitute-fed mice was also observed in pups reared by mother mice treated with anti-transforming growth factor-β (anti-TGF-β) monoclonal antibody. Breast milk regulates the differentiation and expansion of innate and adaptive immune cells partly due to TGF-β. Hence, TGF-β in breast milk may be a new therapeutic target for innate immune system-mediated diseases of infancy. © 2017 Japan Pediatric Society.

Recombinant Protein Production and Insect Cell Culture and Process

NASA Technical Reports Server (NTRS)

Spaulding, Glenn F. (Inventor); Goodwin, Thomas J. (Inventor); OConnor, Kim C. (Inventor); Francis, Karen M. (Inventor); Andrews, Angela D. (Inventor); Prewett, Tracey L. (Inventor)

1997-01-01

A process has been developed for recombinant production of selected polypeptides using transformed insect cells cultured in a horizontally rotating culture vessel modulated to create low shear conditions. A metabolically transformed insect cell line is produced using the culture procedure regardless of genetic transformation. The recombinant polypeptide can be produced by an alternative process using virtually infected or stably transformed insect cells containing a gene encoding the described polypeptide. The insect cells can also be a host for viral production.

GROWTH REGULATION IN RSV INFECTED CHECKEN EMBRYO FIBROBLASTS: THE ROLE OF THE src GENE

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

Parry, G.; Bartholomew, J.C.; Bissell, M.J.

1980-03-01

The relationship between growth regulation and cell transformation has been studied in many cultured cell lines transformed by a range of oncogenic agents. The main conclusion derived from these investigations is that the nature of the growth regulatory lesion in transformed cells is a function of the agent used to induce transformation. For example, when 3T3 fibroblasts are rendered stationary by serum deprivation, normal cells accumulate in G{sub 1} but SV40 transformed cells are arrested at all stages of the cell cycle. In contrast, 3T3 cells transformed with Rous sarcoma virus B77, accumulate in G{sub 1} upon serum deprivation. Thismore » is also true when mouse sarcoma virus (MSV) is used as the transforming agent. MSV-transformed cells accumulate in G{sub 1}, just as do normal cells. In this letter we report a detailed study of the mechanisms leading to loss of growth control in chicken embryo fibroblasts transformed by Rous sarcoma virus (RSV). We have been particularly concerned with the role of the src gene in the process, and have used RSV mutants temperature sensitive (ts) for transformation to investigate the nature of the growth regulatory lesion. Two principal findings have emerged: (a) the stationary phase of the cell cycle (G{sub 1}) in chick embryo fibroblasts has two distinct compartments, (for simplicity referred to as G{sub 1} and G{sub 0} states), (b) when rendered stationary at 41.5{sup o} by serum deprivation, normal cells enter a G{sub 0}-like state, but cells infected with the ts-mutant occupy a G{sub 1} state, even though a known src gene product, a kinase, should be inactive at this temperature. The possibility is discussed that viral factors other than the active src protein kinase influence growth control.« less

Global Impact of Oncogenic Src on a Phosphotyrosine Proteome

PubMed Central

Luo, Weifeng; Slebos, Robbert J.; Hill, Salisha; Li, Ming; Brábek, Jan; Amanchy, Ramars; Chaerkady, Raghothama; Pandey, Akhilesh; Ham, Amy-Joan L.; Hanks, Steven K.

2008-01-01

Elevated activity of Src, the first characterized protein-tyrosine kinase, is associated with progression of many human cancers, and Src has attracted interest as a therapeutic target. Src is known to act in various receptor signaling systems to impact cell behavior, yet it remains likely that the spectrum of Src protein substrates relevant to cancer is incompletely understood. To better understand the cellular impact of deregulated Src kinase activity, we extensively applied a mass spectrometry shotgun phosphotyrosine (pTyr) proteomics strategy to obtain global pTyr profiles of Src-transformed mouse fibroblasts as well as their nontransformed counterparts. A total of 867 peptides representing 563 distinct pTyr sites on 374 different proteins were identified from the Src-transformed cells, while 514 peptides representing 275 pTyr sites on 167 proteins were identified from nontransformed cells. Distinct characteristics of the two profiles were revealed by spectral counting, indicative of pTyr site relative abundance, and by complementary quantitative analysis using stable isotope labeling with amino acids in cell culture (SILAC). While both pTyr profiles are replete with sites on signaling and adhesion/cytoskeletal regulatory proteins, the Src-transformed profile is more diverse with enrichment in sites on metabolic enzymes and RNA and protein synthesis and processing machinery. Forty-three pTyr sites (32 proteins) are predicted as major biologically relevant Src targets on the basis of frequent identification in both cell populations. This select group, of particular interest as diagnostic biomarkers, includes well-established Src sites on signaling/adhesion/cytoskeletal proteins, but also uncharacterized sites of potential relevance to the transformed cell phenotype. PMID:18563927

Neoplastic transformation of human thyroid epithelial cells by ionizing radiation

NASA Astrophysics Data System (ADS)

Herceg, Zdenko

Neoplastic transformation of human thyroid epithelial cells has been investigated following exposure to ionizing radiation in vitro. The effects of radiation type, irradiation regime, and postirradiation passaging were examined using a human thyroid epithelial cell line, designated HToriS, which was previously immortalized with SV40 genome. Exponentially growing HToriS cells were irradiated with graded doses of 137 Cs gamma- and 238pu alpha-irradiation. Cells were irradiated with either a single or multiple doses of 0.5, 1, 2, 3, or 4 Gy gamma-radiation, or single doses of 0.125, 0.25, 0.5, 1, or 1.5 Gy gamma-radiation. Following passaging, the cells were transplanted into the athymic nude mice, and the animals were screened for tumour formation. Statistically significant increases in tumour incidence were obtained with both gamma- and alpha-irradiation and with both single and multiple irradiation regimes as compared with the un-irradiated group. Regardless of radiation type and or radiation regime there appears to be a trend, with increasing doses of radiation, in which tumour incidence increases and reaches a maximum, after which the tumour incidence decreases. Tumours were characterized by histopathological examination as undifferentiated carcinomas. Investigation of expression time following irradiation demonstrated that post-irradiation passaging, generally regarded as a critical step for expression of radiation-induced DNA damage, was not a prerequisite for the neoplastic conversion of irradiated cells with this system. Cell lines were established from the tumours and their identification and characterization carried out. All cell lines established were determined to be derived from the parent HTori3 cells by DNA fingerprinting, karyotype analysis, cytokeratin staining, and SV40 large T-antigen staining. Tumorigenicity of the cell lines was confirmed by retransplantation. Comparison of the morphology in vitro showed that the tumour cell lines retained the basic epithelial morphology of the parent HToriS cells. Investigation of radiosensitivity showed that none of the 6 tumour cell lines examined had a higher radiosensitivity compared to the parent HToriS cells. This excludes the possibility that the observed transformation was the result of the selection of a pre-existing transformed subpopulation of the parent cells but that radiation-induced transformants were being induced de novo. The tumour cell lines were screened for mutations in H- and K-ras oncogenes using restriction enzyme analysis of PCR amplified DNA. No mutations were detected in 26 tumour cell lines suggesting that mutations in these two genes do not appear to be involved in radiation- induced neoplastic transformation in human thyroid epithelial cells. Screening for mutations in p53 protein using immunoprecipitation method detected no mutations in 6 tumour cell lines. This human thyroid epithelial cell line may thus be useful for the in vitro study of cellular and molecular mechanisms that are involved in human epithelial cell carcinogenesis.

A model for size- and rotation-invariant pattern processing in the visual system.

PubMed

Reitboeck, H J; Altmann, J

1984-01-01

The mapping of retinal space onto the striate cortex of some mammals can be approximated by a log-polar function. It has been proposed that this mapping is of functional importance for scale- and rotation-invariant pattern recognition in the visual system. An exact log-polar transform converts centered scaling and rotation into translations. A subsequent translation-invariant transform, such as the absolute value of the Fourier transform, thus generates overall size- and rotation-invariance. In our model, the translation-invariance is realized via the R-transform. This transform can be executed by simple neural networks, and it does not require the complex computations of the Fourier transform, used in Mellin-transform size-invariance models. The logarithmic space distortion and differentiation in the first processing stage of the model is realized via "Mexican hat" filters whose diameter increases linearly with eccentricity, similar to the characteristics of the receptive fields of retinal ganglion cells. Except for some special cases, the model can explain object recognition independent of size, orientation and position. Some general problems of Mellin-type size-invariance models-that also apply to our model-are discussed.

How connected vehicles work.

DOT National Transportation Integrated Search

2011-01-01

Connected vehicles have the potential to transform the way Americans travel through the creation of a safe, interoperable wireless communications networka system that includes cars, buses, trucks, trains, traffic signals, cell phones, and other de...

Enrichment of cardiac pacemaker-like cells: neuregulin-1 and cyclic AMP increase I(f)-current density and connexin 40 mRNA levels in fetal cardiomyocytes.

PubMed

Ruhparwar, Arjang; Er, Fikret; Martin, Ulrich; Radke, Kristin; Gruh, Ina; Niehaus, Michael; Karck, Matthias; Haverich, Axel; Hoppe, Uta C

2007-02-01

Generation of a large number of cells belonging to the cardiac pacemaker system would constitute an important step towards their utilization as a biological cardiac pacemaker system. The aim of the present study was to identify factors, which might induce transformation of a heterogenous population of fetal cardiomyocytes into cells with a pacemaker-like phenotype. Neuregulin-1 (alpha- and beta-isoform) or the cAMP was added to fresh cell cultures of murine embryonic cardiomyocytes. Quantitative northern blot analysis and flowcytometry were performed to detect the expression of connexins 40, 43 and 45. Patch clamp recordings in the whole cell configuration were performed to determine current density of I (f), a characteristic ion current of pacemaker cells. Fetal cardiomyocytes without supplement of neuregulin or cAMP served as control group. Neuregulin and cAMP significantly increased mRNA levels of connexin 40 (Cx-40), a marker of the early differentiating conduction system in mice. On the protein level, flowcytometry revealed no significant differences between treated and untreated groups with regard to the expression of connexins 40, 43 and 45. Treatment with cAMP (11.2 +/- 2.24 pA/pF; P < 0.001) and neuregulin-1-beta (6.23 +/- 1.07 pA/pF; P < 0.001) significantly increased the pacemaker current density compared to control cardiomyocytes (1.76 +/- 0.49 pA/pF). Our results indicate that neuregulin-1 and cAMP possess the capacity to cause significant transformation of a mixed population of fetal cardiomyocytes into cardiac pacemaker-like cells as shown by electrophysiology and increase of Cx-40 mRNA. This method may allow the development of a biological cardiac pacemaker system when applied to adult or embryonic stem cells.

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

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

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

2007-01-26

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

Nitric oxide prodrug JS-K inhibits ubiquitin E1 and kills tumor cells retaining wild-type p53.

PubMed

Kitagaki, J; Yang, Y; Saavedra, J E; Colburn, N H; Keefer, L K; Perantoni, A O

2009-01-29

Nitric oxide (NO) is a major effector molecule in cancer prevention. A number of studies have shown that NO prodrug JS-K (O(2)-(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate) induces apoptotic cell death in vitro and in vivo, indicating that it is a promising new therapeutic for cancer. However, the mechanism of its tumor-killing activity remains unclear. Ubiquitin plays an important role in the regulation of tumorigenesis and cell apoptosis. Our earlier report has shown that inactivation of the ubiquitin system through blocking E1 (ubiquitin-activating enzyme) activity preferentially induces apoptosis in p53-expressing transformed cells. As E1 has an active cysteine residue that could potentially interact with NO, we hypothesized that JS-K could inactivate E1 activity. E1 activity was evaluated by detecting ubiquitin-E1 conjugates through immunoblotting. JS-K strikingly inhibits the ubiquitin-E1 thioester formation in cells in a dose-dependent manner with an IC(50) of approximately 2 microM, whereas a JS-K analog that cannot release NO did not affect these levels in cells. Moreover, JS-K decreases total ubiquitylated proteins and increases p53 levels, which is mainly regulated by ubiquitin and proteasomal degradation. Furthermore, JS-K preferentially induces cell apoptosis in p53-expressing transformed cells. These findings indicate that JS-K inhibits E1 activity and kills transformed cells harboring wild-type p53.

Radar cross-section reduction based on an iterative fast Fourier transform optimized metasurface

NASA Astrophysics Data System (ADS)

Song, Yi-Chuan; Ding, Jun; Guo, Chen-Jiang; Ren, Yu-Hui; Zhang, Jia-Kai

2016-07-01

A novel polarization insensitive metasurface with over 25 dB monostatic radar cross-section (RCS) reduction is introduced. The proposed metasurface is comprised of carefully arranged unit cells with spatially varied dimension, which enables approximate uniform diffusion of incoming electromagnetic (EM) energy and reduces the threat from bistatic radar system. An iterative fast Fourier transform (FFT) method for conventional antenna array pattern synthesis is innovatively applied to find the best unit cell geometry parameter arrangement. Finally, a metasurface sample is fabricated and tested to validate RCS reduction behavior predicted by full wave simulation software Ansys HFSSTM and marvelous agreement is observed.

Chemo-mechanical modeling of tumor growth in elastic epithelial tissue

NASA Astrophysics Data System (ADS)

Bratsun, Dmitry A.; Zakharov, Andrey P.; Pismen, Len

2016-08-01

We propose a multiscale chemo-mechanical model of the cancer tumor development in the epithelial tissue. The epithelium is represented by an elastic 2D array of polygonal cells with its own gene regulation dynamics. The model allows the simulation of the evolution of multiple cells interacting via the chemical signaling or mechanically induced strain. The algorithm includes the division and intercalation of cells as well as the transformation of normal cells into a cancerous state triggered by a local failure of the spatial synchronization of the cellular rhythms driven by transcription/translation processes. Both deterministic and stochastic descriptions of the system are given for chemical signaling. The transformation of cells means the modification of their respective parameters responsible for chemo-mechanical interactions. The simulations reproduce a distinct behavior of invasive and localized carcinoma. Generally, the model is designed in such a way that it can be readily modified to take account of any newly understood gene regulation processes and feedback mechanisms affecting chemo-mechanical properties of cells.

Charging system and method for multicell storage batteries

DOEpatents

Cox, Jay A.

1978-01-01

A battery-charging system includes a first charging circuit connected in series with a plurality of battery cells for controlled current charging. A second charging circuit applies a controlled voltage across each individual cell for equalization of the cells to the fully charged condition. This controlled voltage is determined at a level above the fully charged open-circuit voltage but at a sufficiently low level to prevent corrosion of cell components by electrochemical reaction. In this second circuit for cell equalization, a transformer primary receives closely regulated, square-wave voltage which is coupled to a plurality of equal secondary coil windings. Each secondary winding is connected in parallel to each cell of a series-connected pair of cells through half-wave rectifiers and a shared, intermediate conductor.

Transformation of human mesenchymal cells and skin fibroblasts into hematopoietic cells.

PubMed

Harris, David M; Hazan-Haley, Inbal; Coombes, Kevin; Bueso-Ramos, Carlos; Liu, Jie; Liu, Zhiming; Li, Ping; Ravoori, Murali; Abruzzo, Lynne; Han, Lin; Singh, Sheela; Sun, Michael; Kundra, Vikas; Kurzrock, Razelle; Estrov, Zeev

2011-01-01

Patients with prolonged myelosuppression require frequent platelet and occasional granulocyte transfusions. Multi-donor transfusions induce alloimmunization, thereby increasing morbidity and mortality. Therefore, an autologous or HLA-matched allogeneic source of platelets and granulocytes is needed. To determine whether nonhematopoietic cells can be reprogrammed into hematopoietic cells, human mesenchymal stromal cells (MSCs) and skin fibroblasts were incubated with the demethylating agent 5-azacytidine (Aza) and the growth factors (GF) granulocyte-macrophage colony-stimulating factor and stem cell factor. This treatment transformed MSCs to round, non-adherent cells expressing T-, B-, myeloid-, or stem/progenitor-cell markers. The transformed cells engrafted as hematopoietic cells in bone marrow of immunodeficient mice. DNA methylation and mRNA array analysis suggested that Aza and GF treatment demethylated and activated HOXB genes. Indeed, transfection of MSCs or skin fibroblasts with HOXB4, HOXB5, and HOXB2 genes transformed them into hematopoietic cells. Further studies are needed to determine whether transformed MSCs or skin fibroblasts are suitable for therapy.

Transformation of Human Mesenchymal Cells and Skin Fibroblasts into Hematopoietic Cells

PubMed Central

Harris, David M.; Hazan-Haley, Inbal; Coombes, Kevin; Bueso-Ramos, Carlos; Liu, Jie; Liu, Zhiming; Li, Ping; Ravoori, Murali; Abruzzo, Lynne; Han, Lin; Singh, Sheela; Sun, Michael; Kundra, Vikas; Kurzrock, Razelle; Estrov, Zeev

2011-01-01

Patients with prolonged myelosuppression require frequent platelet and occasional granulocyte transfusions. Multi-donor transfusions induce alloimmunization, thereby increasing morbidity and mortality. Therefore, an autologous or HLA-matched allogeneic source of platelets and granulocytes is needed. To determine whether nonhematopoietic cells can be reprogrammed into hematopoietic cells, human mesenchymal stromal cells (MSCs) and skin fibroblasts were incubated with the demethylating agent 5-azacytidine (Aza) and the growth factors (GF) granulocyte-macrophage colony-stimulating factor and stem cell factor. This treatment transformed MSCs to round, non-adherent cells expressing T-, B-, myeloid-, or stem/progenitor-cell markers. The transformed cells engrafted as hematopoietic cells in bone marrow of immunodeficient mice. DNA methylation and mRNA array analysis suggested that Aza and GF treatment demethylated and activated HOXB genes. Indeed, transfection of MSCs or skin fibroblasts with HOXB4, HOXB5, and HOXB2 genes transformed them into hematopoietic cells. Further studies are needed to determine whether transformed MSCs or skin fibroblasts are suitable for therapy. PMID:21731684

Transformed yeast (Schizosaccharomyces pombe) overexpressing rice Tau class glutathione S-transferase (OsGSTU30 and OsGSTU41) shows enhanced resistance to hexavalent chromium.

PubMed

Tripathi, Ankita; Indoliya, Yuvraj; Tiwari, Madhu; Tiwari, Poonam; Srivastava, Dipali; Verma, Pankaj kumar; Verma, Shikha; Gautam, Neelam; Chakrabarty, Debasis

2014-08-01

Extensive use of hexavalent chromium [Cr(VI)] in leather tanning, stainless-steel production, wood preservatives and electroplating industries has resulted in widespread environmental pollution and poses a serious threat to human health. A plant's response to Cr(VI) stress results in growth inhibition and toxicity leading to changes in components of antioxidant systems. In a previous study, we observed that a large number of glutathione S-transferase (GST) genes were up-regulated under Cr(VI) stress in rice. In this study, two rice root-specific Tau class GST genes (OsGSTU30 and OsGSTU41) were introduced into yeast (Schizosaccharomyces pombe). Transformed yeast cells overexpressing OsGSTU30 and OsGSTU41 had normal growth, but had much higher levels of GST activities and showed enhanced resistance to Cr(VI) as compared to control cells (transformed with empty vector). Also, a higher accumulation of chromium was found in the transformed yeast cells as compared to the control cells. Manipulation of glutathione biosynthesis by exogenous application of buthionine sulfoximine abolishes the protective effect of OsGSTs against Cr(VI) stress. These results suggest that Tau class OsGSTs play a significant role in detoxification of Cr(VI), probably by chelating and sequestrating glutathione-Cr(VI) complexes into vacuoles.

Serpin Facilitates Tumor-Suppressive Cell Competition by Blocking Toll-Mediated Yki Activation in Drosophila.

PubMed

Katsukawa, Mitsuko; Ohsawa, Shizue; Zhang, Lina; Yan, Yan; Igaki, Tatsushi

2018-06-04

Normal epithelial tissue exerts an intrinsic tumor-suppressive effect against oncogenically transformed cells. In Drosophila imaginal epithelium, clones of oncogenic polarity-deficient cells mutant for scribble (scrib) or discs large (dlg) are eliminated by cell competition when surrounded by wild-type cells. Here, through a genetic screen in Drosophila, we identify Serpin5 (Spn5), a secreted negative regulator of Toll signaling, as a crucial factor for epithelial cells to eliminate scrib mutant clones from epithelium. Downregulation of Spn5 in wild-type cells leads to elevation of Toll signaling in neighboring scrib cells. Strikingly, forced activation of Toll signaling or Toll-related receptor (TRR) signaling in scrib clones transforms scrib cells from losers to supercompetitors, resulting in tumorous overgrowth of mutant clones. Mechanistically, Toll activation in scrib clones leads to c-Jun N-terminal kinase (JNK) activation and F-actin accumulation, which cause strong activation of the Hippo pathway effector Yorkie that blocks cell death and promotes cell proliferation. Our data suggest that Spn5 secreted from normal epithelial cells acts as a component of the extracellular surveillance system that facilitates elimination of pre-malignant cells from epithelium. Copyright © 2018 Elsevier Ltd. All rights reserved.

Potential complications when developing gene deletion clones in Xylella fastidiosa.

PubMed

Johnson, Kameka L; Cursino, Luciana; Athinuwat, Dusit; Burr, Thomas J; Mowery, Patricia

2015-04-16

The Gram-negative xylem-limited bacterium, Xylella fastidiosa, is an important plant pathogen that infects a number of high value crops. The Temecula 1 strain infects grapevines and induces Pierce's disease, which causes symptoms such as scorching on leaves, cluster collapse, and eventual plant death. In order to understand the pathogenesis of X. fastidiosa, researchers routinely perform gene deletion studies and select mutants via antibiotic markers. Site-directed pilJ mutant of X. fastidiosa were generated and selected on antibiotic media. Mutant cultures were assessed by PCR to determine if they were composed of purely transformant cells or included mixtures of non-transformants cells. Then pure pilJ mutant and wildtype cells were mixed in PD2 medium and following incubation and exposure to kanamycin were assessed by PCR for presence of mutant and wildtype populations. We have discovered that when creating clones of targeted mutants of X. fastidiosa Temecula 1 with selection on antibiotic plates, X. fastidiosa lacking the gene deletion often persist in association with targeted mutant cells. We believe this phenomenon is due to spontaneous antibiotic resistance and/or X. fastidiosa characteristically forming aggregates that can be comprised of transformed and non-transformed cells. A combined population was confirmed by PCR, which showed that targeted mutant clones were mixed with non-transformed cells. After repeated transfer and storage the non-transformed cells became the dominant clone present. We have discovered that special precautions are warranted when developing a targeted gene mutation in X. fastidiosa because colonies that arise following transformation and selection are often comprised of transformed and non-transformed cells. Following transfer and storage the cells can consist primarily of the non-transformed strain. As a result, careful monitoring of targeted mutant strains must be performed to avoid mixed populations and confounding results.

Production of Japanese encephalitis virus-like particles in insect cells.

PubMed

Yamaji, Hideki; Konishi, Eiji

2013-01-01

Virus-like particles (VLPs) are composed of one or several recombinant viral surface proteins that spontaneously assemble into particulate structures without the incorporation of virus DNA or RNA. The baculovirus-insect cell system has been used extensively for the production of recombinant virus proteins including VLPs. While the baculovirus-insect cell system directs the transient expression of recombinant proteins in a batch culture, stably transformed insect cells allow constitutive production. In our recent study, a secretory form of Japanese encephalitis (JE) VLPs was successfully produced by Trichoplusia ni BTI-TN-5B1-4 (High Five) cells engineered to coexpress the JE virus (JEV) premembrane (prM) and envelope (E) proteins. A higher yield of E protein was attained with recombinant High Five cells than with the baculovirus-insect cell system. This study demonstrated that recombinant insect cells offer a promising approach to the high-level production of VLPs for use as vaccines and diagnostic antigens.

T Cells Prevent Hemorrhagic Transformation in Ischemic Stroke by P-Selectin Binding.

PubMed

Salas-Perdomo, Angélica; Miró-Mur, Francesc; Urra, Xabier; Justicia, Carles; Gallizioli, Mattia; Zhao, Yashu; Brait, Vanessa H; Laredo, Carlos; Tudela, Raúl; Hidalgo, Andrés; Chamorro, Ángel; Planas, Anna M

2018-06-14

Hemorrhagic transformation is a serious complication of ischemic stroke after recanalization therapies. This study aims to identify mechanisms underlying hemorrhagic transformation after cerebral ischemia/reperfusion. We used wild-type mice and Selplg -/- and Fut7 -/- mice defective in P-selectin binding and lymphopenic Rag2 -/- mice. We induced 30-minute or 45-minute ischemia by intraluminal occlusion of the middle cerebral artery and assessed hemorrhagic transformation at 48 hours with a hemorrhage grading score, histological means, brain hemoglobin content, or magnetic resonance imaging. We depleted platelets and adoptively transferred T cells of the different genotypes to lymphopenic mice. Interactions of T cells with platelets in blood were studied by flow cytometry and image stream technology. We show that platelet depletion increased the bleeding risk only after large infarcts. Lymphopenia predisposed to hemorrhagic transformation after severe stroke, and adoptive transfer of T cells prevented hemorrhagic transformation in lymphopenic mice. CD4 + memory T cells were the subset of T cells binding P-selectin and platelets through functional P-selectin glycoprotein ligand-1. Mice defective in P-selectin binding had a higher hemorrhagic score than wild-type mice. Adoptive transfer of T cells defective in P-selectin binding into lymphopenic mice did not prevent hemorrhagic transformation. The study identifies lymphopenia as a previously unrecognized risk factor for secondary hemorrhagic transformation in mice after severe ischemic stroke. T cells prevent hemorrhagic transformation by their capacity to bind platelets through P-selectin. The results highlight the role of T cells in bridging immunity and hemostasis in ischemic stroke. © 2018 American Heart Association, Inc.

Tracing Acetylene Dissolved in Transformer Oil by Tunable Diode Laser Absorption Spectrum.

PubMed

Ma, Guo-Ming; Zhao, Shu-Jing; Jiang, Jun; Song, Hong-Tu; Li, Cheng-Rong; Luo, Ying-Ting; Wu, Hao

2017-11-02

Dissolved gas analysis (DGA) is widely used in monitoring and diagnosing of power transformer, since the insulation material in the power transformer decomposes gases under abnormal operation condition. Among the gases, acetylene, as a symbol of low energy spark discharge and high energy electrical faults (arc discharge) of power transformer, is an important monitoring parameter. The current gas detection method used by the online DGA equipment suffers from problems such as cross sensitivity, electromagnetic compatibility and reliability. In this paper, an optical gas detection system based on TDLAS technology is proposed to detect acetylene dissolved in transformer oil. We selected a 1530.370 nm laser in the near infrared wavelength range to correspond to the absorption peak of acetylene, while using the wavelength modulation strategy and Herriott cell to improve the detection precision. Results show that the limit of detection reaches 0.49 ppm. The detection system responds quickly to changes of gas concentration and is easily to maintenance while has no electromagnetic interference, cross-sensitivity, or carrier gas. In addition, a complete detection process of the system takes only 8 minutes, implying a practical prospect of online monitoring technology.

Spectroscopy detection of green and red fluorescent proteins in genetically modified plants using a fiber optics system

NASA Astrophysics Data System (ADS)

Liew, Oi Wah; Asundi, Anand K.; Chen, Jun-Wei; Chew, Yiwen; Yu, Shangjuan; Yeo, Gare H.

2001-05-01

In this paper, fiber optic spectroscopy is developed to detect and quantify recombinant green (EGFP) and red (DsRED) fluorescent proteins in vitro and in vivo. The bacterial expression vectors carrying the coding regions of EGFP and DsRED were introduced into Escherichia coli host cells and fluorescent proteins were produced following induction with IPTG. Soluble EGFP and DsRED proteins were isolated from lysed bacterial cells and serially diluted for quantitative analysis by fiber optic spectroscopy. Fluorescence at the appropriate emission wavelengths could be detected up to 64X dilution for EGFP and 40X dilution for DsRED. To determine the capability of spectroscopy detection in vivo, transgenic potato hairy roots expressing EGFP and DsRED were regenerated. This was achieved by cloning the EGFP and DsRED genes into the plant binary vector, pTMV35S, to create the recombinant vectors pGLOWGreen and pGLOWRed. These latter binary vectors were introduced into Agrobacterium rhizogenes strain A4T. Infection of potato cells with transformed agrobacteria was used to insert the fluorescent protein genes into the potato genome. Genetically modified potato cells were then regenerated into hairy roots. A panel of transformed hairy roots expressing varying levels of fluorescent proteins was selected by fluorescence microscopy. We are now assessing the capability of spectroscopic detection system for in vivo quantification of green and red fluorescence levels in transformed roots.

A single-cell correlative nanoelectromechanosensing approach to detect cancerous transformation: monitoring the function of F-actin microfilaments in the modulation of the ion channel activity

NASA Astrophysics Data System (ADS)

AbdolahadThe Authors With Same Contributions., Mohammad; Saeidi, Ali; Janmaleki, Mohsen; Mashinchian, Omid; Taghinejad, Mohammad; Taghinejad, Hossein; Azimi, Soheil; Mahmoudi, Morteza; Mohajerzadeh, Shams

2015-01-01

Cancerous transformation may be dependent on correlation between electrical disruptions in the cell membrane and mechanical disruptions of cytoskeleton structures. Silicon nanotube (SiNT)-based electrical probes, as ultra-accurate signal recorders with subcellular resolution, may create many opportunities for fundamental biological research and biomedical applications. Here, we used this technology to electrically monitor cellular mechanosensing. The SiNT probe was combined with an electrically activated glass micropipette aspiration system to achieve a new cancer diagnostic technique that is based on real-time correlation between mechanical and electrical behaviour of single cells. Our studies demonstrated marked changes in the electrical response following increases in the mechanical aspiration force in healthy cells. In contrast, such responses were extremely weak for malignant cells. Confocal microscopy results showed the impact of actin microfilament remodelling on the reduction of the electrical response for aspirated cancer cells due to the significant role of actin in modulating the ion channel activity in the cell membrane.Cancerous transformation may be dependent on correlation between electrical disruptions in the cell membrane and mechanical disruptions of cytoskeleton structures. Silicon nanotube (SiNT)-based electrical probes, as ultra-accurate signal recorders with subcellular resolution, may create many opportunities for fundamental biological research and biomedical applications. Here, we used this technology to electrically monitor cellular mechanosensing. The SiNT probe was combined with an electrically activated glass micropipette aspiration system to achieve a new cancer diagnostic technique that is based on real-time correlation between mechanical and electrical behaviour of single cells. Our studies demonstrated marked changes in the electrical response following increases in the mechanical aspiration force in healthy cells. In contrast, such responses were extremely weak for malignant cells. Confocal microscopy results showed the impact of actin microfilament remodelling on the reduction of the electrical response for aspirated cancer cells due to the significant role of actin in modulating the ion channel activity in the cell membrane. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr06102k

Regulation and dysregulation of Epstein-Barr virus latency: implications for the development of autoimmune diseases.

PubMed

Niller, Hans Helmut; Wolf, Hans; Minarovits, Janos

2008-05-01

Epstein-Barr virus (EBV) is a human herpesvirus hiding in a latent form in memory B cells in the majority of the world population. Although, primary EBV infection is asymptomatic or causes a self-limiting disease, infectious mononucleosis, the virus is associated with a wide variety of neoplasms developing in immunosuppressed or immunodeficient individuals, but also in patients with an apparently intact immune system. In memory B cells, tumor cells, and lymphoblastoid cell lines (LCLs, transformed by EBV in vitro) the expression of the viral genes is highly restricted. There is no virus production (lytic viral replication associated with the expression of all viral genes) in tight latency. The expression of latent viral oncogenes and RNAs is under a strict epigenetic control via DNA methylation and histone modifications that results either in a complete silencing of the EBV genome in memory B cells, or in a cell-type dependent usage of latent promoters in tumor cells, germinal center B cells, and LCLs. Both the latent and lytic EBV proteins are potent immunogens and elicit vigorous B- and T-cell responses. In immunosuppressed and immunodeficient patients, or in individuals with a functional defect of EBV-specific T cells, lytic EBV replication is regularly activated and an increased viral load can be detected in the blood. Enhanced lytic replication results in new infection events and EBV-associated transformation events, and seems to be a risk factor both for malignant transformation and the development of autoimmune diseases. One may speculate that an increased load or altered presentation of a limited set of lytic or latent EBV proteins that cross-react with cellular antigens triggers and perpetuates the pathogenic processes that result in multiple sclerosis, systemic lupus erythematosus (SLE), and rheumatoid arthritis. In addition, in SLE patients EBV may cause defects of B-cell tolerance checkpoints because latent membrane protein 1, an EBV-encoded viral oncoprotein can induce BAFF, a B-cell activating factor that rescues self-reactive B cells and induces a lupus-like autoimmune disease in transgenic mice.

Integrable nonlinear Schrödinger system on a lattice with three structural elements in the unit cell

NASA Astrophysics Data System (ADS)

Vakhnenko, Oleksiy O.

2018-05-01

Developing the idea of increasing the number of structural elements in the unit cell of a quasi-one-dimensional lattice as applied to the semi-discrete integrable systems of nonlinear Schrödinger type, we construct the zero-curvature representation for the general integrable nonlinear system on a lattice with three structural elements in the unit cell. The integrability of the obtained general system permits to find explicitly a number of local conservation laws responsible for the main features of system dynamics and in particular for the so-called natural constraints separating the field variables into the basic and the concomitant ones. Thus, considering the reduction to the semi-discrete integrable system of nonlinear Schrödinger type, we revealed the essentially nontrivial impact of concomitant fields on the Poisson structure and on the whole Hamiltonian formulation of system dynamics caused by the nonzero background values of these fields. On the other hand, the zero-curvature representation of a general nonlinear system serves as an indispensable key to the dressing procedure of system integration based upon the Darboux transformation of the auxiliary linear problem and the implicit Bäcklund transformation of field variables. Due to the symmetries inherent to the six-component semi-discrete integrable nonlinear Schrödinger system with attractive-type nonlinearities, the Darboux-Bäcklund dressing scheme is shown to be simplified considerably, giving rise to the appropriately parameterized multi-component soliton solution consisting of six basic and four concomitant components.

Chapter 15. transforming lepidopteran insect cells for continuous recombinant protein expression

USDA-ARS?s Scientific Manuscript database

The baculovirus expression vector system (BEVS) is widely used to produce large quantities of recombinant proteins. However, yields of extracellular and membrane-bound proteins obtained with this system often are very low, possibly due to the adverse effects of baculovirus infection on the host ins...

Hydrogen-driven asymmetric reduction of hydroxyacetone to (R)-1,2-propanediol by Ralstonia eutropha transformant expressing alcohol dehydrogenase from Kluyveromyces lactis.

PubMed

Oda, Takahiro; Oda, Koji; Yamamoto, Hiroaki; Matsuyama, Akinobu; Ishii, Masaharu; Igarashi, Yasuo; Nishihara, Hirofumi

2013-01-10

Conversion of industrial processes to more nature-friendly modes is a crucial subject for achieving sustainable development. Utilization of hydrogen-oxidation reactions by hydrogenase as a driving force of bioprocess reaction can be an environmentally ideal method because the reaction creates no pollutants. We expressed NAD-dependent alcohol dehydrogenase from Kluyveromyces lactis in a hydrogen-oxidizing bacterium: Ralstonia eutropha. This is the first report of hydrogen-driven in vivo coupling reaction of the alcohol dehydrogenase and indigenous soluble NAD-reducing hydrogenase. Asymmetric reduction of hydroxyacetone to (R)-1,2-propanediol, which is a commercial building block for antibacterial agents, was performed using the transformant as the microbial cell catalyst. The two enzymes coupled in vitro in vials without a marked decrease of reactivity during the 20 hr reaction because of the hydrogenase reaction, which generates no by-product that affects enzymes. Alcohol dehydrogenase was expressed functionally in R. eutropha in an activity level equivalent to that of indigenous NAD-reducing hydrogenase under the hydrogenase promoter. The hydrogen-driven in vivo coupling reaction proceeded only by the transformant cell without exogenous addition of a cofactor. The decrease of reaction velocity at higher concentration of hydroxyacetone was markedly reduced by application of an in vivo coupling system. Production of (R)-1,2-propanediol (99.8% e.e.) reached 67.7 g/l in 76 hr with almost a constant rate using a jar fermenter. The reaction velocity under 10% PH2 was almost equivalent to that under 100% hydrogen, indicating the availability of crude hydrogen gas from various sources. The in vivo coupling system enabled cell-recycling as catalysts. Asymmetric reduction of hydroxyacetone by a coupling reaction of the two enzymes continued in both in vitro and in vivo systems in the presence of hydrogen. The in vivo reaction system using R. eutropha transformant expressing heterologous alcohol dehydrogenase showed advantages for practical usage relative to the in vitro coupling system. The results suggest a hopeful perspective of the hydrogen-driven bioprocess as an environmentally outstanding method to achieve industrial green innovation. Hydrogen-oxidizing bacteria can be useful hosts for the development of hydrogen-driven microbial cell factories.

Oximidine III, a new antitumor antibiotic against transformed cells from Pseudomonas sp. I. Taxonomy, fermentation, isolation, physico-chemical properties and biological activity.

PubMed

Hayakawa, Yoichi; Tomikawa, Taijiro; Shin-ya, Kazuo; Arao, Nakako; Nagai, Koji; Suzuki, Ken-ichi

2003-11-01

Our screening for antitumor antibiotics against transformed cells resulted in the isolation of a new active metabolite, oximidine III, from Pseudomonas sp. QN05727. This substance selectively inhibited the growth of rat 3Y1 fibroblasts transformed with various oncogenes. In ras- or src-transformed cells, oximidine III arrested the cell cycle at G1 phase and increased the expression of p21WAF1.

Escape from the competence state in Streptococcus mutans is governed by the bacterial population density.

PubMed

Dufour, D; Villemin, C; Perry, J A; Lévesque, C M

2016-12-01

Horizontal gene transfer through natural DNA transformation is an important evolutionary mechanism among bacteria. Transformation requires that the bacteria are physiologically competent to take and incorporate free DNA directly from the environment. Although natural genetic transformation is a remarkable feature of many naturally competent bacteria, the process is energetically expensive for the cells. Consequently, a tight control of the competence state is necessary. The objective of the present work was to help decipher the molecular mechanisms regulating the escape from the competence state in Streptococcus mutans, the principal etiological agent responsible for tooth decay in humans. Our results showed that the cessation of competence in S. mutans was abrupt, and did not involve the accumulation of a competence inhibitor nor the depletion of a competence activator in the extracellular environment. The competence state was repressed at high cell population density via concomitant repression of sigX gene encoding the master regulator of the competence regulon. Co-culture experiments performed with oral and non-oral bacteria showed that S. mutans assesses its own population density and also the microbial density of its surroundings to regulate its competence escape. Interestingly, neither the intra-species and extra-species quorum-sensing systems nor the other 13 two-component regulatory systems identified in S. mutans were involved in the cell-density-dependent escape of the competence state. Altogether, our results suggest a complex mechanism regulating the competence shut-off involving cell-density-dependent repression of sigX through an as yet undefined system, and possibly SigX protein stability. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Salvia Hispanica Seed in Reducing Risk of Disease Recurrence in Patients With Non-Hodgkin Lymphoma

ClinicalTrials.gov

2018-02-05

Adult Nasal Type Extranodal NK/T-Cell Lymphoma; Adult T-Cell Leukemia/Lymphoma; Anaplastic Large Cell Lymphoma; Angioimmunoblastic T-Cell Lymphoma; B Lymphoblastic Leukemia/Lymphoma; Blastic Plasmacytoid Dendritic Cell Neoplasm; Burkitt Leukemia; Central Nervous System Lymphoma; Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma; Diffuse Large B-Cell Lymphoma; Enteropathy-Associated T-Cell Lymphoma; Extranodal Marginal Zone Lymphoma of Mucosa-Associated Lymphoid Tissue; Grade 1 Follicular Lymphoma; Grade 2 Follicular Lymphoma; Grade 3 Follicular Lymphoma; Hepatosplenic T-Cell Lymphoma; Lymphoplasmacytic Lymphoma; Mantle Cell Lymphoma; Mediastinal (Thymic) Large B-Cell Lymphoma; Mycosis Fungoides; Nasal Type Extranodal NK/T-Cell Lymphoma; Nodal Marginal Zone Lymphoma; Peripheral T-Cell Lymphoma, Not Otherwise Specified; Post-Transplant Lymphoproliferative Disorder; Primary Cutaneous Anaplastic Large Cell Lymphoma; Primary Effusion Lymphoma; Sezary Syndrome; Splenic Marginal Zone Lymphoma; Subcutaneous Panniculitis-Like T-Cell Lymphoma; Systemic Anaplastic Large Cell Lymphoma; T Lymphoblastic Leukemia/Lymphoma; Transformed Recurrent Non-Hodgkin Lymphoma

Development of a Candida glabrata dominant nutritional transformation marker utilizing the Aspergillus nidulans acetamidase gene (amdS).

PubMed

Fu, Jianmin; Blaylock, Morganne; Wickes, Cameron F; Welte, William; Mehrtash, Adrian; Wiederhold, Nathan; Wickes, Brian L

2016-05-01

The gene encoding Aspergillus nidulans acetamidase (amdS) was placed under control of Candida albicans ACT1 promoter and terminator sequences and then cloned into a plasmid containing C. glabrata ARS10,CEN8 or ARS10+CEN8 sequences. All plasmids transformed C. glabrata wild-type cells to acetamide+, with the ARS-only containing plasmid transforming cells at the highest frequencies (>1.0 × 10(4) transformants μg(-1)). Plasmids were rapidly lost under non-selective conditions with the frequency dependent on chromosomal element, thus recycling the acetamide- phenotype. The amdS plasmid was used to transform a set of clinical isolates resistant to a variety of antifungal drugs. All strains were successfully transformed to the acetamide+ phenotype at high frequency, confirming that this plasmid construct could be used as a simple dominant marker on virtually any strain. Gap repair experiments demonstrated that just as in Saccharomyces cerevisiae, gap repair functions efficiently inC. glabrata, suggesting that C. glabrata has numerous similarities toS. cerevisiae with regard to ease of molecular manipulation. The amdS system is inexpensive and efficient, and combined with existing C. glabrata plasmid elements, confers a high transformation frequency for C. glabrata with a phenotype that can be easily recycled. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

In vitro analysis of multistage carcinogenesis

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

Nettesheim, P.; Fitzgerald, D.J.; Kitamura, H.

1987-11-01

Several key events in the multistep process of neoplastic transformation of rat tracheal epithelium (RTE) are described. Whether tracheal epithelium is exposed in vivo to carcinogenic agents or whether primary tracheal epithelial cells are exposed in vitro to carcinogens, initiated stem cells can be detected soon after the exposure by their ability to grow under selective conditions in culture. These initiated stem cells differ fundamentally from normal stem cells in their response to factors normally constraining proliferation and self-renewal. Thus, disruption of inhibitory control mechanisms of stem cell replication appears to be the first event in RTE cell transformation. Whilemore » the probability of self-renewal (PSR) is clearly increased in initiated stem cells, most of the descendants derived form such stem cells differentiate and become terminal and do not express transformed characteristics. Progression from the first to the second stage of RTE cell transformation, the stage of the immortal growth variant (IGV), is characterized by loss of responsiveness to the growth-restraining effects of retinoic acid. In the third stage of neoplastic transformation, the stage during which neoplastic growth variants (NGV) appear, a growth factor receptor gene is inappropriately expressed in some of the transformants. Thus, it appears that loss of growth-restraining mechanisms may be an early event, and activation of a growth stimulatory mechanism a late event, in neoplastic transformation of RTE cells.« less

A Theileria parva Isolate of Low Virulence Infects a Subpopulation of Lymphocytes

PubMed Central

Geysen, Dirk; Goddeeris, Bruno M.; Awino, Elias; Dobbelaere, Dirk A. E.; Naessens, Jan

2012-01-01

Theileria parva is a tick-transmitted protozoan parasite that infects and transforms bovine lymphocytes. We have previously shown that Theileria parva Chitongo is an isolate with a lower virulence than that of T. parva Muguga. Lower virulence appeared to be correlated with a delayed onset of the logarithmic growth phase of T. parva Chitongo-transformed peripheral blood mononuclear cells after in vitro infection. In the current study, infection experiments with WC1+ γδ T cells revealed that only T. parva Muguga could infect these cells and that no transformed cells could be obtained with T. parva Chitongo sporozoites. Subsequent analysis of the susceptibility of different cell lines and purified populations of lymphocytes to infection and transformation by both isolates showed that T. parva Muguga sporozoites could attach to and infect CD4+, CD8+, and WC1+ T lymphocytes, but T. parva Chitongo sporozoites were observed to bind only to the CD8+ T cell population. Flow cytometry analysis of established, transformed clones confirmed this bias in target cells. T. parva Muguga-transformed clones consisted of different cell surface phenotypes, suggesting that they were derived from either host CD4+, CD8+, or WC1+ T cells. In contrast, all in vitro and in vivo T. parva Chitongo-transformed clones expressed CD8 but not CD4 or WC1, suggesting that the T. parva Chitongo-transformed target cells were exclusively infected CD8+ lymphocytes. Thus, a role of cell tropism in virulence is likely. Since the adhesion molecule p67 is 100% identical between the two strains, a second, high-affinity adhesin that determines target cell specificity appears to exist. PMID:22202119

Identification of p53 unbound to T-antigen in human cells transformed by simian virus 40 T-antigen.

PubMed

O'Neill, F J; Hu, Y; Chen, T; Carney, H

1997-02-27

In several clones of SV40-transformed human cells, we investigated the relative amounts of large T-Antigen (T-Ag) and p53 proteins, both unbound and associated within complexes, with the goal of identifying changes associated with transformation and immortalization. Cells were transformed by wild type (wt) T-Ag, a functionally temperature sensitive T-Ag (tsA58) and other T-Ag variants. Western analysis showed that while most of the T-Ag was ultimately bound by p53, most of the p53 remained unbound to T-Ag. Unbound p53 remained in the supernatant after a T-Ag immunoprecipitation and p53 was present in two to fourfold excess of T-Ag. In one transformant there was five to tenfold more p53 than T-Ag. p53 was present in transformants in amounts at least 200-fold greater than in untransformed human cells. In wt and variant T-Ag transformants, including those generated with tsA58 T-Ag, large amounts of unbound p53 were present in both pre-crisis and immortal cells and when the cells were grown at permissive or non-permissive temperatures. We also found that in transformants produced by tsA58, an SV40/JCV chimeric T-Ag and other variants, T-Ag appeared to form a complex with p53 slowly perhaps because one or both proteins matured slowly. The presence in transformed human cells of large amounts of unbound p53 and in excess of T-Ag suggests that sequestration of p53 by T-Ag, resulting from complex formation, is required neither for morphological transformation nor immortalization of human cells. Rather, these results support the proposal that high levels of p53, the T-Ag/p53 complexes, or other biochemical event(s), lead to transformation and immortalization of human cells by T-Ag.

Synthesis of some potent immunomodulatory and anti-inflammatory metabolites by fungal transformation of anabolic steroid oxymetholone

PubMed Central

2012-01-01

Background Biotransformation of organic compounds by using microbial whole cells provides an efficient approach to obtain novel analogues which are often difficult to synthesize chemically. In this manuscript, we report for the first time the microbial transformation of a synthetic anabolic steroidal drug, oxymetholone, by fungal cell cultures. Results Incubation of oxymetholone (1) with Macrophomina phaseolina, Aspergillus niger, Rhizopus stolonifer, and Fusarium lini produced 17β-hydroxy-2-(hydroxy-methyl)-17α-methyl-5α-androstan-1-en-3-one (2), 2α,17α-di(hydroxyl-methyl)-5α-androstan-3β,17β-diol (3), 17α-methyl-5α-androstan-2α,3β,17β-triol (4), 17β-hydroxy-2-(hydroxymethyl)-17α-methyl-androst-1,4-dien-3-one (5), 17β-hydroxy-2α-(hydroxy-methyl)-17α-methyl-5α-androstan-3-one (6), and 2α-(hydroxymethyl)-17α-methyl-5α-androstan-3β-17β-diol (7). Their structures were deduced by spectral analyses, as well as single-crystal X-ray diffraction studies. Compounds 2–5 were identified as the new metabolites of 1. The immunomodulatory, and anti-inflammatory activities and cytotoxicity of compounds 1–7 were evaluated by observing their effects on T-cell proliferation, reactive oxygen species (ROS) production, and normal cell growth in MTT assays, respectively. These compounds showed immunosuppressant effect in the T-cell proliferation assay with IC50 values between 31.2 to 2.7 μg/mL, while the IC50 values for ROS inhibition, representing anti-inflammatory effect, were in the range of 25.6 to 2.0 μg/mL. All the compounds were found to be non-toxic in a cell-based cytotoxicity assay. Conclusion Microbial transformation of oxymetholone (1) provides an efficient method for structural transformation of 1. The transformed products were obtained as a result of de novo stereoselective reduction of the enone system, isomerization of double bond, insertion of double bond and hydroxylation. The transformed products, which showed significant immunosuppressant and anti-inflammatory activities, can be further studied for their potential as novel drugs. PMID:23237028

HTLV-1 Tax protects against CD95-mediated apoptosis by induction of the cellular FLICE-inhibitory protein (c-FLIP).

PubMed

Krueger, Andreas; Fas, Stefanie C; Giaisi, Marco; Bleumink, Marc; Merling, Anette; Stumpf, Christine; Baumann, Sven; Holtkotte, Denise; Bosch, Valerie; Krammer, Peter H; Li-Weber, Min

2006-05-15

The HTLV-1 transactivator protein Tax is essential for malignant transformation of CD4 T cells, ultimately leading to adult T-cell leukemia/lymphoma (ATL). Malignant transformation may involve development of apoptosis resistance. In this study we investigated the molecular mechanisms by which HTLV-1 Tax confers resistance toward CD95-mediated apoptosis. We show that Tax-expressing T-cell lines derived from HTLV-1-infected patients express elevated levels of c-FLIP(L) and c-FLIP(S). The levels of c-FLIP correlated with resistance toward CD95-mediated apoptosis. Using an inducible system we demonstrated that both resistance toward CD95-mediated apoptosis and induction of c-FLIP are dependent on Tax. In addition, analysis of early cleavage of the BH3-only Bcl-2 family member Bid, a direct caspase-8 substrate, revealed that apoptosis is inhibited at a CD95 death receptor proximal level in Tax-expressing cells. Finally, using siRNA we directly showed that c-FLIP confers Tax-mediated resistance toward CD95-mediated apoptosis. In conclusion, our data suggest an important mechanism by which expression of HTLV-1 Tax may lead to immune escape of infected T cells and, thus, to persistent infection and transformation.

Morphological feature detection for cervical cancer screening

NASA Astrophysics Data System (ADS)

Narayanswamy, Ramkumar; Sharpe, John P.; Duke, Heather J.; Stewart, Rosemary J.; Johnson, Kristina M.

1995-03-01

An optoelectronic system has been designed to pre-screen pap-smear slides and detect the suspicious cells using the hit/miss transform. Computer simulation of the algorithm tested on 184 pap-smear images detected 95% of the suspicious region as suspect while tagging just 5% of the normal regions as suspect. An optoelectronic implementation of the hit/miss transform using a 4f Vander-Lugt correlator architecture is proposed and demonstrated with experimental results.

Downregulation of peptide transporter genes in cell lines transformed with the highly oncogenic adenovirus 12

PubMed Central

1994-01-01

The expression of class I major histocompatibility complex antigens on the surface of cells transformed by adenovirus 12 (Ad12) is generally very low, and correlates with the high oncogenicity of this virus. In primary embryonal fibroblasts from transgenic mice that express both endogenous H-2 genes and a miniature swine class I gene (PD1), Ad12- mediated transformation results in suppression of cell surface expression of all class I antigens. Although class I mRNA levels of PD1 and H-2Db are similar to those in nonvirally transformed cells, recognition of newly synthesized class I molecules by a panel of monoclonal antibodies is impaired, presumably as a result of inefficient assembly and transport of the class I molecules. Class I expression can be partially induced by culturing cells at 26 degrees C, or by coculture of cells with class I binding peptides at 37 degrees C. Analysis of steady state mRNA levels of the TAP1 and TAP2 transporter genes for Ad12-transformed cell lines revealed that they both are significantly reduced, TAP2 by about 100-fold and TAP1 by 5-10-fold. Reconstitution of PD1 and H-2Db, but not H-2Kb, expression is achieved in an Ad12-transformed cell line by stable transfection with a TAP2, but not a TAP1, expression construct. From these data it may be concluded that suppressed expression of peptide transporter genes, especially TAP2, in Ad12-transformed cells inhibits cell surface expression of class I molecules. The failure to fully reconstitute H- 2Db and H-2Kb expression indicates that additional factors are involved in controlling class I gene expression in Ad12-transformed cells. Nevertheless, these results suggest that suppression of peptide transporter genes might be an important mechanism whereby virus- transformed cells escape immune recognition in vivo. PMID:7519239

Human mesothelial cells are unusually susceptible to simian virus 40-mediated transformation and asbestos cocarcinogenicity

PubMed Central

Bocchetta, Maurizio; Di Resta, Ilaria; Powers, Amy; Fresco, Raoul; Tosolini, Alessandra; Testa, Joseph R.; Pass, Harvey I.; Rizzo, Paola; Carbone, Michele

2000-01-01

Mesothelioma, a malignancy associated with asbestos, has been recently linked to simian virus 40 (SV40). We found that infection of human mesothelial cells by SV40 is very different from the semipermissive infection thought to be characteristic of human cells. Mesothelial cells are uniformly infected but not lysed by SV40, a mechanism related to p53, and undergo cell transformation at an extremely high rate. Exposure of mesothelial cells to asbestos complemented SV40 mutants in transformation. Our data provide a mechanistic explanation for the ability of SV40 to transform mesothelial cells preferentially and indicate that asbestos and SV40 may be cocarcinogens. PMID:10954737

Nuclear Factor kappa B is central to Marek’s Disease herpesvirus induced neoplastic transformation of CD30 expressing lymphocytes in-vivo

PubMed Central

2012-01-01

Background Marek’s Disease (MD) is a hyperproliferative, lymphomatous, neoplastic disease of chickens caused by the oncogenic Gallid herpesvirus type 2 (GaHV-2; MDV). Like several human lymphomas the neoplastic MD lymphoma cells overexpress the CD30 antigen (CD30hi) and are in minority, while the non-neoplastic cells (CD30lo) form the majority of population. MD is a unique natural in-vivo model of human CD30hi lymphomas with both natural CD30hi lymphomagenesis and spontaneous regression. The exact mechanism of neoplastic transformation from CD30lo expressing phenotype to CD30hi expressing neoplastic phenotype is unknown. Here, using microarray, proteomics and Systems Biology modeling; we compare the global gene expression of CD30lo and CD30hi cells to identify key pathways of neoplastic transformation. We propose and test a specific mechanism of neoplastic transformation, and genetic resistance, involving the MDV oncogene Meq, host gene products of the Nuclear Factor Kappa B (NF-κB) family and CD30; we also identify a novel Meq protein interactome. Results Our results show that a) CD30lo lymphocytes are pre-neoplastic precursors and not merely reactive lymphocytes; b) multiple transformation mechanisms exist and are potentially controlled by Meq; c) Meq can drive a feed-forward cycle that induces CD30 transcription, increases CD30 signaling which activates NF-κB, and, in turn, increases Meq transcription; d) Meq transcriptional repression or activation of the CD30 promoter generally correlates with polymorphisms in the CD30 promoter distinguishing MD-lymphoma resistant and susceptible chicken genotypes e) MDV oncoprotein Meq interacts with proteins involved in physiological processes central to lymphomagenesis. Conclusions In the context of the MD lymphoma microenvironment (and potentially in other CD30hi lymphomas as well), our results show that the neoplastic transformation is a continuum and the non-neoplastic cells are actually pre-neoplastic precursor cells and not merely immune bystanders. We also show that NF-κB is a central player in MDV induced neoplastic transformation of CD30-expressing lymphocytes in vivo. Our results provide insights into molecular mechanisms of neoplastic transformation in MD specifically and also herpesvirus induced lymphoma in general. PMID:22979947

Pattern of CD14+ follicular dendritic cells and PD1+ T cells independently predicts time to transformation in follicular lymphoma.

PubMed

Smeltzer, Jacob P; Jones, Jason M; Ziesmer, Steven C; Grote, Deanna M; Xiu, Bing; Ristow, Kay M; Yang, Zhi Zhang; Nowakowski, Grzegorz S; Feldman, Andrew L; Cerhan, James R; Novak, Anne J; Ansell, Stephen M

2014-06-01

Transformation of follicular lymphoma is a critical event associated with a poor prognosis. The role of the tumor microenvironment in previous transformation studies has yielded conflicting results. To define cell subtypes associated with transformation, we examined tissue specimens at diagnosis from patients with follicular lymphoma that later transformed and, using immunohistochemistry (IHC), stained for CD68, CD11c, CD21, CXCL13, FOXP3, PD1, and CD14. Cell content and the pattern of expression were evaluated. Those identified as significantly associated with time to transformation (TTT) and overall survival (OS) were further characterized by flow cytometry and multicolor IHC. Of note, 58 patients were analyzed with median TTT of 4.7 years. The pattern of PD1(+) and CD14(+) cells rather than the quantity of cells was predictive of clinical outcomes. On multivariate analysis, including the follicular lymphoma international prognostic index score, CD14(+) cells localized in the follicle were associated with a shorter TTT (HR, 3.0; P = 0.004). PD1(+) cells with diffuse staining were associated with a shorter TTT (HR, 1.9; P = 0.045) and inferior OS (HR, 2.5; P = 0.012). Multicolor IHC and flow cytometry identified CD14(+) cells as follicular dendritic cells (FDC), whereas PD1(+) cells represented two separate populations, TFH and exhausted T cells. These results identify the presence of PD1(+) T cells and CD14(+) FDC as independent predictors of transformation in follicular lymphoma. Clin Cancer Res; 20(11); 2862-72. ©2014 AACR. ©2014 American Association for Cancer Research.

Glioma Invasiveness Responds Variably to Irradiation in a Co-Culture Model

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

Nakamura, Jean L.; Haas-Kogan, Daphne A.; Department of Neurological Surgery, University of California-San Francisco, San Francisco, CA

2007-11-01

Purpose: We developed a co-culture system to quantitate the growth and invasion of human malignant gliomas into a background of confluent normal human astrocytes, then used this assay to assess independently the effects of irradiating both cell types on glioma invasion. Methods and Materials: Enhanced green fluorescent protein (EGFP)-labeled immortalized human astrocytes, human malignant glioma cells, or transformed human astrocytes were focally plated onto a confluent layer of normal human astrocytes, and the invasiveness of EGFP-labeled cells was scored after 96 h. To address the consequences of irradiation on glioma invasion, the invasiveness of irradiated glioma cell lines and irradiatedmore » astrocytic backgrounds was assessed. Fluorescence-activated cell sorting was used to quantitate the total number of EGFP-labeled cells. Results: Growth in the co-culture assay consistently reflected transformation states of the plated cells. Immortalized, but untransformed human astrocytes failed even to establish growth on confluent normal human astrocytes. In contrast, all malignant human glioma cell lines and transformed human astrocytes demonstrated various degrees of infiltration into the astrocytic bed. Irradiation failed to alter the invasiveness of U87, A172, and U373. A 1-Gy dose slightly reduced the invasiveness of U251 MG by 75% (p < 0.05 by one-way analysis of variance and post hoc Neuman-Keuls), without reducing total cell numbers. Independently irradiating the human astrocytic bed did not alter the invasiveness of nonirradiated U251, whereas the matrix metalloproteinase (MMP) inhibitor GM6001 reduced U251 invasiveness in the co-culture assay. Conclusions: Growth in the co-culture assay reflects the transformation status and provides a useful in vitro model for assessing invasiveness. Human glioma invasiveness in the co-culture model responds variably to single low-dose fractions. MMP activity promotes invasiveness in the co-culture model. Reduced invasiveness in irradiated U251 appears to be mediated by MMP-independent mechanisms.« less

Plant Cell Division Analyzed by Transient Agrobacterium-Mediated Transformation of Tobacco BY-2 Cells.

PubMed

Buschmann, Henrik

2016-01-01

The continuing analysis of plant cell division will require additional protein localization studies. This is greatly aided by GFP-technology, but plant transformation and the maintenance of transgenic lines can present a significant technical bottleneck. In this chapter I describe a method for the Agrobacterium-mediated genetic transformation of tobacco BY-2 cells. The method allows for the microscopic analysis of fluorescence-tagged proteins in dividing cells in within 2 days after starting a coculture. This transient transformation procedure requires only standard laboratory equipment. It is hoped that this rapid method would aid researchers conducting live-cell localization studies in plant mitosis and cytokinesis.

Establishment of rat pancreatic endocrine cell lines by infection with simian virus 40.

PubMed Central

Niesor, E J; Wollheim, C B; Mintz, D H; Blondel, B; Renold, A E; Weil, R

1979-01-01

The feasibility of infection and transformation by SV40 (simian virus 40) of primary cell cultures derived from newborn-rat pancreas was investigated. As judged by the presence of intranuclear SV40 T-antigen, exposure to the virus resulted specifically in infection and transformation of epithelioid (predominantly endocrine) cells. The transformed cells were subcultured (more than 64 passages) and cloned. Culture medium and acid/ethanol extracts of the cells did not contain detectable amounts of immunoreactive insulin after the third subculture. However, inoculation of such SV40-transformed pancreatic cells into immunodeficient rats results in tumours in which insulin production was partially restored through the passage in vivo, since the tumour cells contained and synthesized small amounts of immunoreactive insulin which co-migrated with an insulin marker on gel chromatography. Interestingly, the transformed cells maintained under tissue-culture conditions produced a protein immunologically related to insulin, soluble in aqueous buffer but insoluble in acid/ethanol. This 3000-dalton protein is too large to be a translation product of the rat preproinsulin 9S mRNA. SV40-transformed pancreatic cells might prove useful in the investigation of the factors controlling and maintaining insulin biosynthesis. Images Fig. 1. PMID:222255

Differences in the epigenetic regulation of MT-3 gene expression between parental and Cd+2 or As+3 transformed human urothelial cells

PubMed Central

2011-01-01

Background Studies have shown that metallothionein 3 (MT-3) is not expressed in normal urothelium or in the UROtsa cell line, but is expressed in urothelial cancer and in tumors generated from the UROtsa cells that have been transformed by cadmium (Cd+2) or arsenite (As+3).The present study had two major goals. One, to determine if epigenetic modifications control urothelial MT-3 gene expression and if regulation is altered by malignant transformation by Cd+2 or As+3. Two, to determine if MT-3 expression might translate clinically as a biomarker for malignant urothelial cells released into the urine. Results The histone deacetylase inhibitor MS-275 induced MT-3 mRNA expression in both parental UROtsa cells and their transformed counterparts. The demethylating agent, 5-Aza-2'-deoxycytidine (5-AZC) had no effect on MT-3 mRNA expression. ChIP analysis showed that metal-responsive transformation factor-1 (MTF-1) binding to metal response elements (MRE) elements of the MT-3 promoter was restricted in parental UROtsa cells, but MTF-1 binding to the MREs was unrestricted in the transformed cell lines. Histone modifications at acetyl H4, trimethyl H3K4, trimethyl H3K27, and trimethyl H3K9 were compared between the parental and transformed cell lines in the presence and absence of MS-275. The pattern of histone modifications suggested that the MT-3 promoter in the Cd+2 and As+3 transformed cells has gained bivalent chromatin structure, having elements of being "transcriptionally repressed" and "transcription ready", when compared to parental cells. An analysis of MT-3 staining in urinary cytologies showed that a subset of both active and non-active patients with urothelial cancer shed positive cells in their urine, but that control patients only rarely shed MT-3 positive cells. Conclusion The MT-3 gene is silenced in non-transformed urothelial cells by a mechanism involving histone modification of the MT-3 promoter. In contrast, transformation of the urothelial cells with either Cd+2 or As+3 modified the chromatin of the MT-3 promoter to a bivalent state of promoter readiness. Urinary cytology for MT-3 positive cells would not improve the diagnosis of urothelial cancer, but might have potential as a biomarker for tumor progression. PMID:21303554

Transformation of ATLA-negative leukocytes by blood components from anti-ATLA-positive donors in vitro.

PubMed

Miyamoto, K; Tomita, N; Ishii, A; Nishizaki, T; Kitajima, K; Tanaka, T; Nakamura, T; Watanabe, S; Oda, T

1984-06-15

Anti-ATLA-positive blood components transformed healthy human leukocytes in vitro. Blood components examined were packed red cells, whole blood, platelet concentrate and fresh frozen plasma. Leukocytes present in anti-ATLA-positive blood components such as packed red cells, whole blood and platelet concentrate easily transformed anti-ATLA-negative leukocytes. Co-culture in fresh frozen plasma, however, did not transform recipient leukocytes, and leukocytes of anti-ATLA-positive recipients proved refractory to transformation. The transformed cells were morphologically lymphoid, grew in suspension, and possessed normal recipient karyotypes except in the case of three platelet concentrates. A high proportion of all the transformed populations formed E-rosettes with neuraminidase-treated sheep erythrocytes. The cytoplasm of over 90% of each recipient was stained brilliantly with antibodies against ATLV-determined antigens. Electron microscopy of these transformed cells revealed many C-type virus particles in the extracellular space. Blood components, such as packed red cells, whole blood and platelet concentrate, containing leukocytes from anti-ATLA-positive donors, should be used cautiously to prevent the transmission on ATLV to anti-ATLA-negative recipients.

The modulation of apoptosis by oncogenic viruses

PubMed Central

2013-01-01

Transforming viruses can change a normal cell into a cancer cell during their normal life cycle. Persistent infections with these viruses have been recognized to cause some types of cancer. These viruses have been implicated in the modulation of various biological processes, such as proliferation, differentiation and apoptosis. The study of infections caused by oncogenic viruses had helped in our understanding of several mechanisms that regulate cell growth, as well as the molecular alterations leading to cancer. Therefore, transforming viruses provide models of study that have enabled the advances in cancer research. Viruses with transforming abilities, include different members of the Human Papillomavirus (HPV) family, Hepatitis C virus (HCV), Human T-cell Leukemia virus (HTLV-1), Epstein Barr virus (EBV) and Kaposi’s Sarcoma Herpesvirus (KSHV). Apoptosis, or programmed cell death, is a tightly regulated process that plays an important role in development and homeostasis. Additionally, it functions as an antiviral defense mechanism. The deregulation of apoptosis has been implicated in the etiology of diverse diseases, including cancer. Oncogenic viruses employ different mechanisms to inhibit the apoptotic process, allowing the propagation of infected and damaged cells. During this process, some viral proteins are able to evade the immune system, while others can directly interact with the caspases involved in apoptotic signaling. In some instances, viral proteins can also promote apoptosis, which may be necessary for an accurate regulation of the initial stages of infection. PMID:23741982

Epigenetic dysregulation of epstein-barr virus latency and development of autoimmune disease.

PubMed

Niller, Hans Helmut; Wolf, Hans; Ay, Eva; Minarovits, Janos

2011-01-01

Epstein-Barr virus (EBV) is ahumanherpesvirus thatpersists in the memory B-cells of the majority of the world population in a latent form. Primary EBV infection is asymptomatic or causes a self-limiting disease, infectious mononucleosis. Virus latency is associated with a wide variety of neoplasms whereof some occur in immune suppressed individuals. Virus production does not occur in strict latency. The expression of latent viral oncoproteins and nontranslated RNAs is under epigenetic control via DNA methylation and histone modifications that results either in a complete silencing of the EBV genome in memory B cells, or in a cell-type dependent usage of a couple of latency promoters in tumor cells, germinal center B cells and lymphoblastoid cells (LCL, transformed by EBV in vitro). Both, latent and lytic EBV proteins elicit a strong immune response. In immune suppressed and infectious mononucleosis patients, an increased viral load can be detected in the blood. Enhanced lytic replication may result in new infection- and transformation-events and thus is a risk factor both for malignant transformation and the development of autoimmune diseases. An increased viral load or a changed presentation of a subset of lytic or latent EBV proteins that cross-react with cellular antigens may trigger pathogenic processes through molecular mimicry that result in multiple sclerosis (MS), systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA).

Transformation with TT8 and HB12 RNAi Constructs in Model Forage (Medicago sativa, Alfalfa) Affects Carbohydrate Structure and Metabolic Characteristics in Ruminant Livestock Systems.

PubMed

Li, Xinxin; Zhang, Yonggen; Hannoufa, Abdelali; Yu, Peiqiang

2015-11-04

Lignin, a phenylpropanoid polymer present in secondary cell walls, has a negative impact on feed digestibility. TT8 and HB12 genes were shown to have low expression levels in low-lignin tissues of alfalfa, but to date, there has been no study on the effect of down-regulation of these two genes in alfalfa on nutrient chemical profiles and availability in ruminant livestock systems. The objectives of this study were to investigate the effect of transformation of alfalfa with TT8 and HB12 RNAi constructs on carbohydrate (CHO) structure and CHO nutritive value in ruminant livestock systems. The results showed that transformation with TT8 and HB12 RNAi constructs reduced rumen, rapidly degraded CHO fractions (RDCA4, P = 0.06; RDCB1, P < 0.01) and totally degraded CHO fraction (TRDCHO, P = 0.08). Both HB12 and TT8 populations had significantly higher in vitro digestibility of neutral detergent fiber (NDF) at 30 h of incubation (ivNDF30) compared to the control (P < 0.01). The TT8 populations had highest ivDM30 and ivNDF240. Transformation of alfalfa with TT8 and HB12 RNAi constructs induced molecular structure changes. Different CHO functional groups had different sensitivities and different responses to the transformation. The CHO molecular structure changes induced by the transformation were associated with predicted CHO availability. Compared with HB12 RNAi, transformation with TT8 RNAi could improve forage quality by increasing the availability of both NDF and DM. Further study is needed on the relationship between the transformation-induced structure changes at a molecular level and nutrient utilization in ruminant livestock systems when lignification is much higher.

2D-DIGE and MALDI TOF/TOF MS analysis reveal that small GTPase signaling pathways may play an important role in cadmium-induced colon cell malignant transformation

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

Lu, Jian, E-mail: lujian@ujs.edu.cn; Institute of Life Sciences, Jiangsu University, Zhenjiang 212013; Zhou, Zhongping

Cadmium is a toxic heavy metal present in the environment and in industrial materials. Cadmium has demonstrated carcinogenic activity that induces cell transformation, but how this occurs is unclear. We used 2D-DIGE and MALDI TOF/TOF MS combined with bioinformatics and immunoblotting to investigate the molecular mechanism of cadmium transformation. We found that small GTPases were critical for transformation. Additionally, proteins involved in mitochondrial transcription, DNA repair, and translation also had altered expression patterns in cadmium treated cells. Collectively, our results suggest that activation of small GTPases contributes to cadmium-induced transformation of colon cells. - Highlights: • Colon epithelial cell linemore » is firstly successfully transformed by cadmium. • 2D-DIGE is applied to visualize the differentially expressed proteins. • RhoA plays an important role in cadmium induced malignant transformation. • Bioinformatic and experimental methods are combined to explore new mechanisms.« less

Neoplastic transformation of human cells

NASA Technical Reports Server (NTRS)

Goth-Goldstein, Regine

1995-01-01

The goal of this project was to gain a better understanding of the cellular mechanisms of cancer induction by ionizing radiation as a risk assessment for workers subjected to high LET irradiation such as that found in space. The following ions were used for irradiation: Iron, Argon, Neon, and Lanthanum. Two tests were performed: growth in low serum and growth in agar were used as indicators of cell transformation. The specific aims of this project were to: (1) compare the effectiveness of various ions on degree of transformation of a single dose of the same RBE; (2) determine if successive irradiations with the same ion (Ge 600 MeV/u) increases the degree of transformation; (3) test if clones with the greatest degree of transformation produce tumors in nude mice; and (4) construct a cell hybrid of a transformed and control (non-transformed) clone. The cells used for this work are human mammary epithelial cells with an extended lifespan and selected for growth in MEM + 10% serum.

Arsenite induces cell transformation by reactive oxygen species, AKT, ERK1/2, and p70S6K1

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

Carpenter, Richard L.; Jiang, Yue; Jing, Yi

2011-10-28

Highlights: Black-Right-Pointing-Pointer Chronic exposure to arsenite induces cell proliferation and transformation. Black-Right-Pointing-Pointer Arsenite-induced transformation increases ROS production and downstream signalings. Black-Right-Pointing-Pointer Inhibition of ROS levels via catalase reduces arsenite-induced cell transformation. Black-Right-Pointing-Pointer Interruption of AKT, ERK, or p70S6K1 inhibits arsenite-induced cell transformation. -- Abstract: Arsenic is naturally occurring element that exists in both organic and inorganic formulations. The inorganic form arsenite has a positive association with development of multiple cancer types. There are significant populations throughout the world with high exposure to arsenite via drinking water. Thus, human exposure to arsenic has become a significant public health problem. Recent evidencemore » suggests that reactive oxygen species (ROS) mediate multiple changes to cell behavior after acute arsenic exposure, including activation of proliferative signaling and angiogenesis. However, the role of ROS in mediating cell transformation by chronic arsenic exposure is unknown. We found that cells chronically exposed to sodium arsenite increased proliferation and gained anchorage-independent growth. This cell transformation phenotype required constitutive activation of AKT, ERK1/2, mTOR, and p70S6K1. We also observed these cells constitutively produce ROS, which was required for the constitutive activation of AKT, ERK1/2, mTOR, and p70S6K1. Suppression of ROS levels by forced expression of catalase also reduced cell proliferation and anchorage-independent growth. These results indicate cell transformation induced by chronic arsenic exposure is mediated by increased cellular levels of ROS, which mediates activation of AKT, ERK1/2, and p70S6K1.« less

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

PubMed Central

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

2012-01-01

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

In Vitro Transformation of Rat and Mouse Cells by DNA from Simian Virus 40

PubMed Central

Abrahams, P. J.; van der Eb, A. J.

1975-01-01

Primary rat kidney cells and mouse 3T3 cells can be transformed by DNA of simian virus 40 when use is made of the calcium technique (Graham and van der Eb, 1973). The transformation assay in primary rat cells is reproducible, but the dose response is not linear. PMID:166204

[Study on high accuracy detection of multi-component gas in oil-immerse power transformer].

PubMed

Fan, Jie; Chen, Xiao; Huang, Qi-Feng; Zhou, Yu; Chen, Gang

2013-12-01

In order to solve the problem of low accuracy and mutual interference in multi-component gas detection, a kind of multi-component gas detection network with high accuracy was designed. A semiconductor laser with narrow bandwidth was utilized as light source and a novel long-path gas cell was also used in this system. By taking the single sine signal to modulate the spectrum of laser and using space division multiplexing (SDM) and time division multiplexing (TDM) technique, the detection of multi-component gas was achieved. The experiments indicate that the linearity relevance coefficient is 0. 99 and the measurement relative error is less than 4%. The system dynamic response time is less than 15 s, by filling a volume of multi-component gas into the gas cell gradually. The system has advantages of high accuracy and quick response, which can be used in the fault gas on-line monitoring for power transformers in real time.

Altered receptor trafficking in Huntingtin Interacting Protein 1-transformed cells.

PubMed

Rao, Dinesh S; Bradley, Sarah V; Kumar, Priti D; Hyun, Teresa S; Saint-Dic, Djenann; Oravecz-Wilson, Katherine; Kleer, Celina G; Ross, Theodora S

2003-05-01

The clathrin-associated protein, Huntingtin Interacting Protein 1 (HIP1), is overexpressed in multiple human epithelial tumors. Here, we report that HIP1 is a novel oncoprotein that transforms cells. HIP1-transformed cells, in contrast to RasV12-transformed cells, have dysregulation of multiple receptors involved in clathrin trafficking. Examples include upregulation of the epidermal growth factor receptor (EGFR) and the transferrin receptor. Furthermore, accumulation of transferrin and EGF in the HIP1-transformed cells was increased, and breast tumors that had EGFR expressed also had HIP1 upregulated. Thus, HIP1 overexpression promotes tumor formation and is associated with a general alteration in receptor trafficking. HIP1 is the first endocytic protein to be directly implicated in tumor formation.

Generation and CRISPR/Cas9 editing of transformed progenitor B cells as a pseudo-physiological system to study DNA repair gene function in V(D)J recombination.

PubMed

Lenden Hasse, Hélène; Lescale, Chloé; Bianchi, Joy J; Yu, Wei; Bedora-Faure, Marie; Deriano, Ludovic

2017-12-01

Antigen receptor gene assembly is accomplished in developing lymphocytes by the V(D)J recombination reaction, which can be separated into two steps: DNA cleavage by the recombination-activating gene (RAG) nuclease and joining of DNA double strand breaks (DSBs) by components of the nonhomologous end joining (NHEJ) pathway. Deficiencies for NHEJ factors can result in immunodeficiency and a propensity to accumulate genomic instability, thus highlighting the importance of identifying all players in this process and deciphering their functions. Bcl2 transgenic v-Abl kinase-transformed pro-B cells provide a pseudo-physiological cellular system to study V(D)J recombination. Treatment of v-Abl/Bcl2 pro-B cells with the Abl kinase inhibitor Imatinib leads to G1 cell cycle arrest, the rapid induction of Rag1/2 gene expression and V(D)J recombination. In this system, the Bcl2 transgene alleviates Imatinib-induced apoptosis enabling the analysis of induced V(D)J recombination. Although powerful, the use of mouse models carrying the Bcl2 transgene for the generation of v-Abl pro-B cell lines is time and money consuming. Here, we describe a method for generating v-Abl/Bcl2 pro-B cell lines from wild type mice and for performing gene knock-out using episomal CRISPR/Cas9 targeting vectors. Using this approach, we generated distinct NHEJ-deficient pro-B cell lines and quantified V(D)J recombination levels in these cells. Furthermore, this methodology can be adapted to generate pro-B cell lines deficient for any gene suspected to play a role in V(D)J recombination, and more generally DSB repair. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Human hybrid hybridoma

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

Tiebout, R.F.; van Boxtel-Oosterhof, F.; Stricker, E.A.M.

1987-11-15

Hybrid hybridomas are obtained by fusion of two cells, each producing its own antibody. Several authors have reported the construction of murine hybrid hybridomas with the aim to obtain bispecific monoclonal antibodies. The authors have investigated, in a model system, the feasibility of constructing a human hybrid hybridoma. They fused two monoclonal cell lines: an ouabain-sensitive and azaserine/hypoxanthine-resistant Epstein-Barr virus-transformed human cell line that produces an IgG1kappa antibody directed against tetanus toxiod and an azaserine/hypoxanthine-sensitive and ouabain-resistant human-mouse xenohybrid cell line that produces a human IgG1lambda antibody directed against hepatitis-B surface antigen. Hybrid hybridoma cells were selected in culture mediummore » containing azaserine/hypoxanthine and ouabain. The hybrid nature of the secreted antibodies was analyzed by means of two antigen-specific immunoassay. The results show that it is possible, with the combined use of transformation and xenohybridization techniques, to construct human hybrid hybridomas that produce bispecific antibodies. Bispecific antibodies activity was measured by means of two radioimmunoassays.« less

Oncogene-like induction of cellular invasion from centrosome amplification

PubMed Central

Godinho, Susana A.; Picone, Remigio; Burute, Mithila; Dagher, Regina; Su, Ying; Leung, Cheuk T.; Polyak, Kornelia; Brugge, Joan S.; Thery, Manuel; Pellman, David

2014-01-01

Centrosome amplification has long been recognized as a feature of human tumors, however its role in tumorigenesis remains unclear1. Centrosome amplification is poorly tolerated by non-transformed cells, and, in the absence of selection, extra centrosomes are spontaneously lost2. Thus, the high frequency of centrosome amplification, particularly in more aggressive tumors3, raises the possibility that extra centrosomes could, in some contexts, confer advantageous characteristics that promote tumor progression. Using a three-dimensional model system and other approaches to culture human mammary epithelial cells, we find that centrosome amplification triggers cell invasion. This invasive behavior is similar to that induced by overexpression of the breast cancer oncogene ErbB24 and indeed enhances invasiveness triggered by ErbB2. We show that, through increased centrosomal microtubule nucleation, centrosome amplification increases Rac1 activity, which disrupts normal cell-cell adhesion and promotes invasion. These findings demonstrate that centrosome amplification, a structural alteration of the cytoskeleton, can promote features of malignant transformation. PMID:24739973

Dissection of Drosophila Visual Circuits Implicative in Figure Motion

NASA Astrophysics Data System (ADS)

Kelley, Ross G.

The Drosophila visual system offers a model to study the foundations of how motion signals are computed from raw visual input and transformed into behavioral output. My studies focus on how specific cells in the Drosophila nervous system implement this input-output transformation. The individual cell types are known from classical studies using Golgi impregnations, but the assembly of motion processing circuits and the behavioral outputs remain poorly understood. Using an electronic flight simulator for flies and a white-noise analysis developed by Aptekar et al., I screen specific neurons in the optic lobes for behavioral ramifications. This approach produces wing responses to both the spatial and temporal dynamics of motion signals. The results of these experiments give Spatiotemporal Action Fields (STAFs) across the entire visual panorama. Genetically inactivating a distinct grouping of cells in the third optic ganglion, the Lobula Plate, the Horizontal System (HS) cell group, produced a robust phenotype through STAF analysis. Using the Gal4-UAS transgene expression system, we selectively inactivated the HS cells by expressing in their membrane inward rectifying potassium channels (Kir2.1) to hyperpolarize these cells, preventing their role in synaptic signaling. The results of the experiments show mutants lose steering responses to several distinct categories of figure motion and reduced behavioral responses to figure motion set against a contrasting moving background, highlighting their role in figure tracking behavior. Finally, a synapse inactivating protein, tetanus toxin (TNT), expressed in the HS cell group, produces a different behavioral phenotype than overexpressing inward rectifier. TNT, a bacterial neurotoxin, cleaves SNARE proteins resulting in loss of synaptic output of the cell, but the dendrites are intact and signal normally, preserving dendro-dendritic interactions known to sculpt the visual receptive fields of these cells. The two distinct phenotypes to each genetically targeted silencer differentiate the functional role of dendritic integration versus axonal output in this important cell group.

Caveolin-1 is a negative regulator of caveolae-mediated endocytosis to the endoplasmic reticulum.

PubMed

Le, Phuong U; Guay, Ginette; Altschuler, Yoram; Nabi, Ivan R

2002-02-01

Caveolae are flask-shaped invaginations at the plasma membrane that constitute a subclass of detergent-resistant membrane domains enriched in cholesterol and sphingolipids and that express caveolin, a caveolar coat protein. Autocrine motility factor receptor (AMF-R) is stably localized to caveolae, and the cholesterol extracting reagent, methyl-beta-cyclodextrin, inhibits its internalization to the endoplasmic reticulum implicating caveolae in this distinct receptor-mediated endocytic pathway. Curiously, the rate of methyl-beta-cyclodextrin-sensitive endocytosis of AMF-R to the endoplasmic reticulum is increased in ras- and abl-transformed NIH-3T3 cells that express significantly reduced levels of caveolin and few caveolae. Overexpression of the dynamin K44A dominant negative mutant via an adenovirus expression system induces caveolar invaginations sensitive to methyl-beta-cyclodextrin extraction in the transformed cells without increasing caveolin expression. Dynamin K44A expression further inhibits AMF-R-mediated endocytosis to the endoplasmic reticulum in untransformed and transformed NIH-3T3 cells. Adenoviral expression of caveolin-1 also induces caveolae in the transformed NIH-3T3 cells and reduces AMF-R-mediated endocytosis to the endoplasmic reticulum to levels observed in untransformed NIH-3T3 cells. Cholesterol-rich detergent-resistant membrane domains or glycolipid rafts therefore invaginate independently of caveolin-1 expression to form endocytosis-competent caveolar vesicles via rapid dynamin-dependent detachment from the plasma membrane. Caveolin-1 stabilizes the plasma membrane association of caveolae and thereby acts as a negative regulator of the caveolae-mediated endocytosis of AMF-R to the endoplasmic reticulum.

Epithelial–mesenchymal transition during oncogenic transformation induced by hexavalent chromium involves reactive oxygen species-dependent mechanism in lung epithelial cells

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

Ding, Song-Ze, E-mail: dingsongze@hotmail.com; Graduate Center for Toxicology, College of Medicine, University of Kentucky, Lexington, KY 40536; Yang, Yu-Xiu

2013-05-15

Hexavalent chromium [Cr(VI)] is an important human carcinogen associated with pulmonary diseases and lung cancer. Exposure to Cr(VI) induces DNA damage, cell morphological change and malignant transformation in human lung epithelial cells. Despite extensive studies, the molecular mechanisms remain elusive, it is also not known if Cr(VI)-induced transformation might accompany with invasive properties to facilitate metastasis. We aimed to study Cr(VI)-induced epithelial–mesenchymal transition (EMT) and invasion during oncogenic transformation in lung epithelial cells. The results showed that Cr(VI) at low doses represses E-cadherin mRNA and protein expression, enhances mesenchymal marker vimentin expression and transforms the epithelial cell into fibroblastoid morphology.more » Cr(VI) also increases cell invasion and promotes colony formation. Further studies indicated that Cr(VI) uses multiple mechanisms to repress E-cadherin expression, including activation of E-cadherin repressors such as Slug, ZEB1, KLF8 and enhancement the binding of HDAC1 in E-cadherin gene promoter, but DNA methylation is not responsible for the loss of E-cadherin. Catalase reduces Cr(VI)-induced E-cadherin and vimentin protein expression, attenuates cell invasion in matrigel and colony formation on soft agar. These results demonstrate that exposure to a common human carcinogen, Cr(VI), induces EMT and invasion during oncogenic transformation in lung epithelial cells and implicate in cancer metastasis and prevention. - Graphical abstract: Epithelial–mesenchymal transition during oncogenic transformation induced by hexavalent chromium involves reactive oxygen species-dependent mechanisms in lung epithelial cells. - Highlights: • We study if Cr(VI) might induce EMT and invasion in epithelial cells. • Cr(VI) induces EMT by altering E-cadherin and vimentin expression. • It also increases cell invasion and promotes oncogenic transformation. • Catalase reduces Cr(VI)-induced EMT, invasion and transformation.« less

Development of an optimized tetracycline-inducible expression system to increase the accumulation of interleukin-10 in tobacco BY-2 suspension cells.

PubMed

Bortesi, Luisa; Rademacher, Thomas; Schiermeyer, Andreas; Schuster, Flora; Pezzotti, Mario; Schillberg, Stefan

2012-07-11

Plant cell suspension cultures can be used for the production of valuable pharmaceutical and industrial proteins. When the recombinant protein is secreted into the culture medium, restricting expression to a defined growth phase can improve both the quality and quantity of the recovered product by minimizing proteolytic activity. Temporal restriction is also useful for recombinant proteins whose constitutive expression affects cell growth and viability, such as viral interleukin-10 (vIL-10). We have developed a novel, tetracycline-inducible system suitable for tobacco BY-2 suspension cells which increases the yields of vIL-10. The new system is based on a binary vector that is easier to handle than conventional vectors, contains an enhanced inducible promoter and 5'-UTR to improve yields, and incorporates a constitutively-expressed visible marker gene to allow the rapid and straightforward selection of the most promising transformed clones. Stable transformation of BY-2 cells with this vector, without extensive optimization of the induction conditions, led to a 3.5 fold increase in vIL-10 levels compared to constitutive expression in the same host. We have developed an effective and straightforward molecular farming platform technology that improves both the quality and the quantity of recombinant proteins produced in plant cells, particularly those whose constitutive expression has a negative impact on plant growth and development. Although we tested the platform using vIL-10 produced in BY-2 cells, it can be applied to other host/product combinations and is also useful for basic research requiring strictly controlled transgene expression.

Cell transformation mediated by chromosomal deoxyribonucleic acid of polyoma virus-transformed cells.

PubMed Central

Della Valle, G; Fenton, R G; Basilico, C

1981-01-01

To study the mechanism of deoxyribonucleic acid (DNA)-mediated gene transfer, normal rat cells were transfected with total cellular DNA extracted from polyoma virus-transformed cells. This resulted in the appearance of the transformed phenotype in 1 X 10(-6) to 3 X 10(-6) of the transfected cells. Transformation was invariably associated with the acquisition of integrated viral DNA sequences characteristic of the donor DNA. This was caused not by the integration of free DNA molecules, but by the transfer of large DNA fragments (10 to 20 kilobases) containing linked cellular and viral sequences. Although Southern blot analysis showed that integration did not appear to occur in a homologous region of the recipient chromosome, the frequency of transformation was rather high when compared with that of purified polyoma DNA, perhaps due to "position" effects or to the high efficiency of recombination of large DNA fragments. Images PMID:6100965

Neoplastic cell transformation by high-LET radiation - Molecular mechanisms

NASA Technical Reports Server (NTRS)

Yang, Tracy Chui-Hsu; Craise, Laurie M.; Tobias, Cornelius A.; Mei, Man-Tong

1989-01-01

Quantitative data were collected on dose-response curves of cultured mouse-embryo cells (C3H10T1/2) irradiated with heavy ions of various charges and energies. Results suggests that two breaks formed on DNA within 80 A may cause cell transformation and that two DNA breaks formed within 20 A may be lethal. From results of experiments with restriction enzymes which produce DNA damages at specific sites, it was found that DNA double strand breaks are important primary lesions for radiogenic cell transformation and that blunt-ended double-strand breaks can form lethal as well as transformational damages due to misrepair or incomplete repair in the cell. The RBE-LET relationship for high-LET radiation is similar to that for HGPRT locus mutation, chromosomal deletion, and cell transformation, indicating that common lesions may be involved in these radiation effects.

A High-Throughput Regeneration and Transformation Platform for Production of Genetically Modified Banana.

PubMed

Tripathi, Jaindra N; Oduor, Richard O; Tripathi, Leena

2015-01-01

Banana (Musa spp.) is an important staple food as well as cash crop in tropical and subtropical countries. Various bacterial, fungal, and viral diseases and pests such as nematodes are major constraints in its production and are currently destabilizing the banana production in sub-Saharan Africa. Genetic engineering is a complementary option used for incorporating useful traits in banana to bypass the long generation time, polyploidy, and sterility of most of the cultivated varieties. A robust transformation protocol for farmer preferred varieties is crucial for banana genomics and improvement. A robust and reproducible system for genetic transformation of banana using embryogenic cell suspensions (ECS) has been developed in this study. Two different types of explants (immature male flowers and multiple buds) were tested for their ability to develop ECS in several varieties of banana locally grown in Africa. ECS of banana varieties "Cavendish Williams" and "Gros Michel" were developed using multiple buds, whereas ECS of "Sukali Ndiizi" was developed using immature male flowers. Regeneration efficiency of ECS was about 20,000-50,000 plantlets per ml of settled cell volume (SCV) depending on variety. ECS of three different varieties were transformed through Agrobacterium-mediated transformation using gusA reporter gene and 20-70 independent transgenic events per ml SCV of ECS were regenerated on selective medium. The presence and integration of gusA gene in transgenic plants was confirmed by PCR, dot blot, and Southern blot analysis and expression by histochemical GUS assays. The robust transformation platform was successfully used to generate hundreds of transgenic lines with disease resistance. Such a platform will facilitate the transfer of technologies to national agricultural research systems (NARS) in Africa.

A High-Throughput Regeneration and Transformation Platform for Production of Genetically Modified Banana

PubMed Central

Tripathi, Jaindra N.; Oduor, Richard O.; Tripathi, Leena

2015-01-01

Banana (Musa spp.) is an important staple food as well as cash crop in tropical and subtropical countries. Various bacterial, fungal, and viral diseases and pests such as nematodes are major constraints in its production and are currently destabilizing the banana production in sub-Saharan Africa. Genetic engineering is a complementary option used for incorporating useful traits in banana to bypass the long generation time, polyploidy, and sterility of most of the cultivated varieties. A robust transformation protocol for farmer preferred varieties is crucial for banana genomics and improvement. A robust and reproducible system for genetic transformation of banana using embryogenic cell suspensions (ECS) has been developed in this study. Two different types of explants (immature male flowers and multiple buds) were tested for their ability to develop ECS in several varieties of banana locally grown in Africa. ECS of banana varieties “Cavendish Williams” and “Gros Michel” were developed using multiple buds, whereas ECS of “Sukali Ndiizi” was developed using immature male flowers. Regeneration efficiency of ECS was about 20,000–50,000 plantlets per ml of settled cell volume (SCV) depending on variety. ECS of three different varieties were transformed through Agrobacterium-mediated transformation using gusA reporter gene and 20–70 independent transgenic events per ml SCV of ECS were regenerated on selective medium. The presence and integration of gusA gene in transgenic plants was confirmed by PCR, dot blot, and Southern blot analysis and expression by histochemical GUS assays. The robust transformation platform was successfully used to generate hundreds of transgenic lines with disease resistance. Such a platform will facilitate the transfer of technologies to national agricultural research systems (NARS) in Africa. PMID:26635849

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

EPA Science Inventory

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

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

The Impact of Ethnicity-Dependent Differences in Breast Epithelial Hierarchy on Tumor Incidence and Characteristics

DTIC Science & Technology

2016-10-01

TNBC) is significantly higher in African American than Caucasian women suggesting that the biology of normal breast epithelial cells between these two...we have generated immortalized cell lines from healthy breast tissues of African American and Caucasian women and transformed these cells with...progenitor phenotype. Transformed cells are being characterized for signal transduction pathway activation. Transformed cells from African American women

Genomic instability and tumorigenic induction in immortalized human bronchial epithelial cells by heavy ions

NASA Astrophysics Data System (ADS)

Hei, T. K.; Piao, C. Q.; Wu, L. J.; Willey, J. C.; Hall, E. J.

1998-11-01

Carcinogenesis is postulated to be a progressive multistage process characterized by an increase in genomic instability and clonal selection with each mutational event endowing a selective growth advantage. Genomic instability as manifested by the amplification of specific gene fragments is common among tumor and transformed cells. In the present study, immortalized human bronchial (BEP2D) cells were irradiated with graded doses of either 1GeV/nucleon 56Fe ions or 150 keV/μm alpha particles. Transformed cells developed through a series of successive steps before becoming tumorigenic in nude mice. Tumorigenic cells showed neither ras mutations nor deletion in the p16 tumor suppressor gene. In contrast, they harbored mutations in the p53 gene and over-expressed cyclin D1. Genomic instability among transformed cells at various stage of the carcinogenic process was examined based on frequencies of PALA resistance. Incidence of genomic instability was highest among established tumor cell lines relative to transformed, non-tumorigenic and control cell lines. Treatment of BEP2D cells with a 4 mM dose of the aminothiol WR-1065 significantly reduced their neoplastic transforming response to 56Fe particles. This model provides an opportunity to study the cellular and molecular mechanisms involved in malignant transformation of human epithelial cells by heavy ions.

Simian virus 40 T-antigen-related cell surface antigen: serological demonstration on simian virus 40-transformed monolayer cells in situ.

PubMed Central

Deppert, W; Hanke, K; Henning, R

1980-01-01

Simian virus 40 (SV40)-transformed monolayer cells were analyzed in situ by indirect immunofluorescence microscopy for the postulated cell surface location of SV40 T-antigen-related molecules. With antisera prepared against purified, sodium dodecyl sulfate-denatured SV40 T-antigen, positive surface staining was obtained when the cells had been treated with formaldehyde before immunofluorescence analysis. In contrast, living SV40-transformed cells analyzed in monolayer were surface fluorescence negative. The fixation procedure developed in this study combined with a double staining immunofluorescence technique allowed the simultaneous analysis of the same cells for the expression of both SV40 T-antigen-related surface antigen and nuclear T-antigen. The localization of SV40 T-antigen-related surface antigen on the outer surface of the plasma membrane of formaldehyde-fixed SV40-transformed cells was demonstrated directly by the protein A-mediated binding of Staphylococcus aureus bacteria on formaldehyde-fixed SV40-transformed cells precoated with antiserum against sodium dodecyl sulfate-denatured T-antigen. Both cell surface staining and S. aureus binding were found to be highly specific for SV40 T-antigen-related binding sites. These results indicate that T-antigen-related molecules in a cryptic form are located on the surface of SV40-transformed monolayer cells and can be detected in situ after modification of the cell surface architecture. Images PMID:6255189

E2 Proteins from High- and Low-Risk Human Papillomavirus Types Differ in Their Ability To Bind p53 and Induce Apoptotic Cell Death

PubMed Central

Parish, Joanna L.; Kowalczyk, Anna; Chen, Hsin-Tien; Roeder, Geraldine E.; Sessions, Richard; Buckle, Malcolm; Gaston, Kevin

2006-01-01

The E2 proteins from oncogenic (high-risk) human papillomaviruses (HPVs) can induce apoptotic cell death in both HPV-transformed and non-HPV-transformed cells. Here we show that the E2 proteins from HPV type 6 (HPV6) and HPV11, two nononcogenic (low-risk) HPV types, fail to induce apoptosis. Unlike the high-risk HPV16 E2 protein, these low-risk E2 proteins fail to bind p53 and fail to induce p53-dependent transcription activation. Interestingly, neither the ability of p53 to activate transcription nor the ability of p53 to bind DNA, are required for HPV16 E2-induced apoptosis in non-HPV-transformed cells. However, mutations that reduce the binding of the HPV16 E2 protein to p53 inhibit E2-induced apoptosis in non-HPV-transformed cells. In contrast, the interaction between HPV16 E2 and p53 is not required for this E2 protein to induce apoptosis in HPV-transformed cells. Thus, our data suggest that this high-risk HPV E2 protein induces apoptosis via two pathways. One pathway involves the binding of E2 to p53 and can operate in both HPV-transformed and non-HPV-transformed cells. The second pathway requires the binding of E2 to the viral genome and can only operate in HPV-transformed cells. PMID:16611918

Transformation of Primary Hamster Brain Cells with JC Virus and Its DNA

PubMed Central

Frisque, R. J.; Rifkin, D. B.; Walker, D. L.

1980-01-01

We transformed primary hamster brain cells with four isolates of JC virus and JC virus DNA. Several properties of these transformants were characterized and compared to those of simian virus 40 transformants isolated under identical conditions. Images PMID:6251275

Analysis of plasma membrane Ca(2+)-ATPase expression in control and SV40-transformed human fibroblasts.

PubMed

Reisner, P D; Brandt, P C; Vanaman, T C

1997-01-01

It has been long known that neoplastic transformation is accompanied by a lowered requirement for extracellular Ca2+ for growth. The studies presented here demonstrate that human fibroblastic cell lines produce the two commonly found 'housekeeping' isoforms of the plasma membrane Ca(2+)-ATPase (PMCA), PMCA1b and 4b, and at the expression of both is demonstrably lower in cell lines neoplastically transformed by SV40 than in the corresponding parental cell lines. Western blot analyses of lysates from control (GM00037) and SV40-transformed (GM00637) skin fibroblasts revealed a 138 kDa PMCA whose level was significantly lower in the SV40-transformed cells relative to either total cellular protein or alpha-tubulin. Similar analyses of plasma membrane preparations from control WI-38) and SV40-transformed (WI-38VA13) lung fibroblasts revealed 3-4-fold lower levels of PMCA in the SV40-transformed cells. Competitive ELISAs performed on detergent solubilized plasma membrane preparations indicated at least 3-4-fold lower levels of PMCA in the SV40-transformed cell lines compared to controls. Reverse transcriptase coupled-PCR analyses showed that PMCA1b and PMCA4b were the only isoforms expressed in all four cell lines. The PMCA4b mRNA level detected by Northern analysis also was substantially lower in SV40 transformed skin fibroblasts than in non-transformed fibroblasts. Quantitative RT-PCR analyses showed levels of PMCA1b and 4b mRNAs to be 5 and 10-fold lower, respectively, in GM00637 than in GM00037 when the levels of PCR products were normalized to glyceraldehyde-3-phosphate dehydrogenase (G3PDH) mRNA. These results demonstrate that the expression of these distinct PMCA genes is substantially lower in SV40 transformed human skin and lung fibroblasts and may be coordinately regulated in these cells.

Transforming activity of E5a protein of human papillomavirus type 6 in NIH 3T3 and C127 cells.

PubMed

Chen, S L; Mounts, P

1990-07-01

Human papillomavirus type 6 (HPV-6) is the etiologic agent of genital warts and recurrent respiratory papillomatosis. We are investigating the mechanism by which this virus stimulates cell proliferation during infection. In this paper, we report that the E5a gene of HPV-6c, an independent isolate of HPV-11, is capable of transforming NIH 3T3 cells. The E5a open reading frame (ORF) was expressed under the control of the mouse metallothionein promoter in the expression vector pMt.neo.1, which also contains the gene for G418 resistance. Transfected cells were selected for G418 resistance and analyzed for a transformed phenotype. The transformed NIH 3T3 cells overgrew a confluent monolayer, had an accelerated generation time, and were anchorage independent. In contrast, E5a did not induce foci in C127 cells, but C127 cells expressing E5a did form small colonies in suspension. The presence of the 12-kilodalton E5a gene product in the transformed NIH 3T3 cells was shown by immunoprecipitation and was localized predominantly to nuclei by an immunoperoxidase assay. A mutation in the E5a ORF was engineered to terminate translation. This mutant was defective for transformation, demonstrating that translation of the E5a ORF is required for biological activity. This is the first demonstration of a transforming oncogene in HPV-6, and the differential activity of E5a in these two cell lines should facilitate future investigations on the mechanism of transformation.

Depleted uranium induces neoplastic transformation in human lung epithelial cells.

PubMed

Xie, Hong; LaCerte, Carolyne; Thompson, W Douglas; Wise, John Pierce

2010-02-15

Depleted uranium (DU) is commonly used in military armor and munitions, and thus, exposure of soldiers and noncombatants is frequent and widespread. Previous studies have shown that DU has both chemical and radiological toxicity and that the primary route of exposure of DU to humans is through inhalation and ingestion. However, there is limited research information on the potential carcinogenicity of DU in human bronchial cells. Accordingly, we determined the neoplastic transforming ability of particulate DU to human bronchial epithelial cells (BEP2D). We observed the loss of contact inhibition and anchorage independent growth in cells exposed to DU after 24 h. We also characterized these DU-induced transformed cell lines and found that 40% of the cell lines exhibit alterations in plating efficiency and no significant changes in the cytotoxic response to DU. Cytogenetic analyses showed that 53% of the DU-transformed cell lines possess a hypodiploid phenotype. These data indicate that human bronchial cells are transformed by DU and exhibit significant chromosome instability consistent with a neoplastic phenotype.

Intra-species bacterial quorum sensing studied at single cell level in a double droplet trapping system.

PubMed

Bai, Yunpeng; Patil, Santoshkumar N; Bowden, Steven D; Poulter, Simon; Pan, Jie; Salmond, George P C; Welch, Martin; Huck, Wilhelm T S; Abell, Chris

2013-05-21

In this paper, we investigated the intra-species bacterial quorum sensing at the single cell level using a double droplet trapping system. Escherichia coli transformed to express the quorum sensing receptor protein, LasR, were encapsulated in microdroplets that were positioned adjacent to microdroplets containing the autoinducer, N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL). Functional activation of the LasR protein by diffusion of the OdDHL across the droplet interface was measured by monitoring the expression of green fluorescent protein (GFP) from a LasR-dependent promoter. A threshold concentration of OdDHL was found to induce production of quorum-sensing associated GFP by E. coli. Additionally, we demonstrated that LasR-dependent activation of GFP expression was also initiated when the adjacent droplets contained single E. coli transformed with the OdDHL synthase gene, LasI, representing a simple quorum sensing circuit between two droplets.

Maize, tropical (Zea mays L.).

PubMed

Assem, Shireen K

2015-01-01

Maize (Zea mays L.) is the third most important food crop globally after wheat and rice. In sub-Saharan Africa, tropical maize has traditionally been the main staple of the diet; 95 % of the maize grown is consumed directly as human food and as an important source of income for the resource-poor rural population. The biotechnological approach to engineer biotic and abiotic traits implies the availability of an efficient plant transformation method. The production of genetically transformed plants depends both on the ability to integrate foreign genes into target cells and the efficiency with which plants are regenerated. Maize transformation and regeneration through immature embryo culture is the most efficient system to regenerate normal transgenic plants. However, this system is highly genotype dependent. Genotypes adapted to tropic areas are difficult to regenerate. Therefore, transformation methods used with model genotypes adapted to temperate areas are not necessarily efficient with tropical lines. Agrobacterium-mediated transformation is the method of choice since it has been first achieved in 1996. In this report, we describe a transformation method used successfully with several tropical maize lines. All the steps of transformation and regeneration are described in details. This protocol can be used with a wide variety of tropical lines. However, some modifications may be needed with recalcitrant lines.

Amplification of bovine papillomavirus DNA by N-methyl-N'-nitro-N-nitrosoguanidine, ultraviolet irradiation, or infection with herpes simplex virus

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

Schmitt, J.; Schlehofer, J.R.; Mergener, K.

1989-09-01

Treatment with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or irradiation with ultraviolet light (uv254 nm) induces amplification of integrated as well as episomal sequences of bovine papillomavirus (BPV) type 1 DNA in BPV-1-transformed mouse C127 cells (i.e., ID13 cells). This is shown by filter in situ hybridization and Southern blot analysis of cellular DNA. Similarly, infection of ID13 cells with herpes simplex virus (HSV) type 1 which has been shown to be mutagenic for host cell DNA leads to amplification of BPV DNA sequences. In contrast to this induction of DNA amplification by initiators, treatment of ID13 cells with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA)more » does not result in increased synthesis of BPV DNA nor does TPA treatment modulate the initiator-induced DNA amplification. Similar to other cell systems infection with adeno-associated virus (AAV) type 2 inhibits BPV-1 DNA amplification irrespective of the inducing agent. In contrast to initiator-induced DNA amplification, treatment with carcinogen (MNNG) or tumor promoters or combination of MNNG and promoter of C127 cells prior to transformation by BPV-1 does not lead to an increase in the number of transformed foci. The induction of amplification of papillomavirus DNA by initiating agents possibly represents one of the mechanisms by which the observed synergism between papillomavirus infection and initiators in tumorigenesis might occur.« less

Comprehensive Thinking and Defense Analysis Transformation

DTIC Science & Technology

2011-06-01

that has taken two distinct forms: the biological evolution of cells , organs, animals and man; and the sociological evolution of groups...human systems learn and adapt as they deal with their Group Organization Community Societal Supranational Organism Organ Cell OOOOOOO oooooooo...better adapt on the fly, executing self synchronized movement towards a common goal. It requires stamina and endurance, in addition to multiple ball

Iron deposits in the chronically inflamed central nervous system and contributes to neurodegeneration.

PubMed

Andersen, Hjalte Holm; Johnsen, Kasper Bendix; Moos, Torben

2014-05-01

Neurodegenerative disorders are characterized by the presence of inflammation in areas with neuronal cell death and a regional increase in iron that exceeds what occurs during normal aging. The inflammatory process accompanying the neuronal degeneration involves glial cells of the central nervous system (CNS) and monocytes of the circulation that migrate into the CNS while transforming into phagocytic macrophages. This review outlines the possible mechanisms responsible for deposition of iron in neurodegenerative disorders with a main emphasis on how iron-containing monocytes may migrate into the CNS, transform into macrophages, and die out subsequently to their phagocytosis of damaged and dying neuronal cells. The dying macrophages may in turn release their iron, which enters the pool of labile iron to catalytically promote formation of free-radical-mediated stress and oxidative damage to adjacent cells, including neurons. Healthy neurons may also chronically acquire iron from the extracellular space as another principle mechanism for oxidative stress-mediated damage. Pharmacological handling of monocyte migration into the CNS combined with chelators that neutralize the effects of extracellular iron occurring due to the release from dying macrophages as well as intraneuronal chelation may denote good possibilities for reducing the deleterious consequences of iron deposition in the CNS.

The c-Jun N-terminal kinase pathway is critical for cell transformation by the latent membrane protein 1 of Epstein-Barr virus

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

Kutz, Helmut; Reisbach, Gilbert; Schultheiss, Ute

The latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) transforms cells activating signal transduction pathways such as NF-{kappa}B, PI3-kinase, or c-Jun N-terminal kinase (JNK). Here, we investigated the functional role of the LMP1-induced JNK pathway in cell transformation. Expression of a novel dominant-negative JNK1 allele caused a block of proliferation in LMP1-transformed Rat1 fibroblasts. The JNK-specific inhibitor SP600125 reproduced this effect in Rat1-LMP1 cells and efficiently interfered with proliferation of EBV-transformed lymphoblastoid cells (LCLs). Inhibition of the LMP1-induced JNK pathway in LCLs caused the downregulation of c-Jun and Cdc2, the essential G2/M cell cycle kinase, which was accompanied bymore » a cell cycle arrest of LCLs at G2/M phase transition. Moreover, SP600125 retarded tumor growth of LCLs in a xenograft model in SCID mice. Our data support a critical role of the LMP1-induced JNK pathway for proliferation of LMP1-transformed cells and characterize JNK as a potential target for intervention against EBV-induced malignancies.« less

Straw blood cell count, growth, inhibition and comparison to apoptotic bodies.

PubMed

Wu, Yonnie; Henry, David C; Heim, Kyle; Tomkins, Jeffrey P; Kuan, Cheng-Yi

2008-05-20

Mammalian cells transform into individual tubular straw cells naturally in tissues and in response to desiccation related stress in vitro. The transformation event is characterized by a dramatic cellular deformation process which includes: condensation of certain cellular materials into a much smaller tubular structure, synthesis of a tubular wall and growth of filamentous extensions. This study continues the characterization of straw cells in blood, as well as the mechanisms of tubular transformation in response to stress; with specific emphasis placed on investigating whether tubular transformation shares the same signaling pathway as apoptosis. There are approximately 100 billion, unconventional, tubular straw cells in human blood at any given time. The straw blood cell count (SBC) is 45 million/ml, which accounts for 6.9% of the bloods dry weight. Straw cells originating from the lungs, liver and lymphocytes have varying nodules, hairiness and dimensions. Lipid profiling reveals severe disruption of the plasma membrane in CACO cells during transformation. The growth rates for the elongation of filaments and enlargement of rabbit straw cells is 0.6 approximately 1.1 (microm/hr) and 3.8 (microm(3)/hr), respectively. Studies using apoptosis inhibitors and a tubular transformation inhibitor in CACO2 cells and in mice suggested apoptosis produced apoptotic bodies are mediated differently than tubular transformation produced straw cells. A single dose of 0.01 mg/kg/day of p38 MAPK inhibitor in wild type mice results in a 30% reduction in the SBC. In 9 domestic animals SBC appears to correlate inversely with an animal's average lifespan (R2 = 0.7). Straw cells are observed residing in the mammalian blood with large quantities. Production of SBC appears to be constant for a given animal and may involve a stress-inducible protein kinase (P38 MAPK). Tubular transformation is a programmed cell survival process that diverges from apoptosis. SBCs may be an important indicator of intrinsic aging-related stress.

Systems and methods for the secretion of recombinant proteins in gram negative bacteria

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

Withers, III, Sydnor T.; Dominguez, Miguel A.; DeLisa, Matthew P.

Disclosed herein are systems and methods for producing recombinant proteins utilizing mutant E. coli strains containing expression vectors carrying nucleic acids encoding the proteins, and secretory signal sequences to direct the secretion of the proteins to the culture medium. Host cells transformed with the expression vectors are also provided.

Systems and methods for the secretion of recombinant proteins in gram negative bacteria

DOEpatents

Withers, III, Sydnor T.; Dominguez, Miguel A; DeLisa, Matthew P.; Haitjema, Charles H.

2016-08-09

Disclosed herein are systems and methods for producing recombinant proteins utilizing mutant E. coli strains containing expression vectors carrying nucleic acids encoding the proteins, and secretory signal sequences to direct the secretion of the proteins to the culture medium. Host cells transformed with the expression vectors are also provided.

Generation of Osteosarcomas from a Combination of Rb Silencing and c‐Myc Overexpression in Human Mesenchymal Stem Cells

PubMed Central

Wang, Jir‐You; Wu, Po‐Kuei; Chen, Paul Chih‐Hsueh; Lee, Chia‐Wen

2016-01-01

Abstract Osteosarcoma (OS) was a malignant tumor occurring with unknown etiology that made prevention and early diagnosis difficult. Mesenchymal stem cells (MSCs), which were found in bone marrow, were claimed to be a possible origin of OS but with little direct evidence. We aimed to characterize OS cells transformed from human MSCs (hMSCs) and identify their association with human primary OS cells and patient survival. Genetic modification with p53 or retinoblastoma (Rb) knockdown and c‐Myc or Ras overexpression was applied for hMSC transformation. Transformed cells were assayed for proliferation, differentiation, tumorigenecity, and gene expression profile. Only the combination of Rb knockdown and c‐Myc overexpression successfully transformed hMSCs derived from four individual donors, with increasing cell proliferation, decreasing cell senescence rate, and increasing ability to form colonies and spheres in serum‐free medium. These transformed cells lost the expression of certain surface markers, increased in osteogenic potential, and decreased in adipogenic potential. After injection in immunodeficient mice, these cells formed OS‐like tumors, as evidenced by radiographic analyses and immunohistochemistry of various OS markers. Microarray with cluster analysis revealed that these transformed cells have gene profiles more similar to patient‐derived primary OS cells than their normal MSC counterparts. Most importantly, comparison of OS patient tumor samples revealed that a combination of Rb loss and c‐Myc overexpression correlated with a decrease in patient survival. This study successfully transformed human MSCs to OS‐like cells by Rb knockdown and c‐Myc overexpression that may be a useful platform for further investigation of preventive and target therapy for human OS. Stem Cells Translational Medicine 2017;6:512–526 PMID:28191765

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

PubMed

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

1995-12-11

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

Downregulation of proapoptotic Bim augments IL-2-independent T-cell transformation by human T-cell leukemia virus type-1 Tax

PubMed Central

Higuchi, Masaya; Takahashi, Masahiko; Tanaka, Yuetsu; Fujii, Masahiro

2014-01-01

Human T-cell leukemia virus type 1 (HTLV-1), an etiological agent of adult T-cell leukemia, immortalizes and transforms primary human T cells in vitro in both an interleukin (IL)-2-dependent and IL-2-independent manner. Expression of the HTLV-1 oncoprotein Tax transforms the growth of the mouse T-cell line CTLL-2 from being IL-2-dependent to IL-2-independent. Withdrawal of IL-2 from normal activated T cells induces apoptosis, which is mediated through the inducible expression of several proapoptotic proteins, including Bim. In this study, we found that Tax protects IL-2-depleted T cells against Bim-induced apoptosis. Withdrawal of IL-2 from CTLL-2 cells induced a prominent increase in the level of Bim protein in CTLL-2 cells, but not in Tax-transformed CTLL-2 cells. This inhibition of Bim in Tax-transformed CTLL-2 cells was mediated by two mechanisms: downregulation of Bim mRNA and posttranscriptional reduction of Bim protein. Transient expression of Tax in CTLL-2 cells also inhibited IL-2 depletion–induced expression of Bim, however, this decrease in Bim protein expression was not due to downregulation of Bim mRNA, thus indicating that Bim mRNA downregulation in Tax-transformed CTLL-2 occurs only after long-term expression of Tax. Transient expression of Tax in CTLL-2 cells also induced Erk activation, however, this was not involved in the reduction of Bim protein. Knockdown of Bim expression in CTLL-2 cells augmented Tax-induced IL-2-independent transformation. HTLV-1 infection of human T cells also reduced their levels of Bim protein, and restoring Bim expression in HTLV-1-infected cells reduced their proliferation by inducing apoptosis. Taken together, these results indicate that Tax-induced downregulation of Bim in HTLV-1-infected T cells promotes their IL-2-independent growth, thereby supporting the persistence of HTLV-1 infection in vivo. PMID:25175936

Separation of cell survival, growth, migration, and mesenchymal transdifferentiation effects of fibroblast secretome on tumor cells of head and neck squamous cell carcinoma.

PubMed

Metzler, Veronika Maria; Pritz, Christian; Riml, Anna; Romani, Angela; Tuertscher, Raphaela; Steinbichler, Teresa; Dejaco, Daniel; Riechelmann, Herbert; Dudás, József

2017-11-01

Fibroblasts play a central role in tumor invasion, recurrence, and metastasis in head and neck squamous cell carcinoma. The aim of this study was to investigate the influence of tumor cell self-produced factors and paracrine fibroblast-secreted factors in comparison to indirect co-culture on cancer cell survival, growth, migration, and epithelial-mesenchymal transition using the cell lines SCC-25 and human gingival fibroblasts. Thereby, we particularly focused on the participation of the fibroblast-secreted transforming growth factor beta-1.Tumor cell self-produced factors were sufficient to ensure tumor cell survival and basic cell growth, but fibroblast-secreted paracrine factors significantly increased cell proliferation, migration, and epithelial-mesenchymal transition-related phenotype changes in tumor cells. Transforming growth factor beta-1 generated individually migrating disseminating tumor cell groups or single cells separated from the tumor cell nest, which were characterized by reduced E-cadherin expression. At the same time, transforming growth factor beta-1 inhibited tumor cell proliferation under serum-starved conditions. Neutralizing transforming growth factor beta antibody reduced the cell migration support of fibroblast-conditioned medium. Transforming growth factor beta-1 as a single factor was sufficient for generation of disseminating tumor cells from epithelial tumor cell nests, while other fibroblast paracrine factors supported tumor nest outgrowth. Different fibroblast-released factors might support tumor cell proliferation and invasion, as two separate effects.

Development of oriC-Based Plasmids for Mesoplasma florum.

PubMed

Matteau, Dominick; Pepin, Marie-Eve; Baby, Vincent; Gauthier, Samuel; Arango Giraldo, Mélissa; Knight, Thomas F; Rodrigue, Sébastien

2017-04-01

The near-minimal bacterium Mesoplasma florum constitutes an attractive model for systems biology and for the development of a simplified cell chassis in synthetic biology. However, the lack of genetic engineering tools for this microorganism has limited our capacity to understand its basic biology and modify its genome. To address this issue, we have evaluated the susceptibility of M. florum to common antibiotics and developed the first generation of artificial plasmids able to replicate in this bacterium. Selected regions of the predicted M. florum chromosomal origin of replication ( oriC ) were used to create different plasmid versions that were tested for their transformation frequency and stability. Using polyethylene glycol-mediated transformation, we observed that plasmids harboring both rpmH-dnaA and dnaA-dnaN intergenic regions, interspaced or not with a copy of the dnaA gene, resulted in a frequency of ∼4.1 × 10 -6 transformants per viable cell and were stably maintained throughout multiple generations. In contrast, plasmids containing only one M. florum oriC intergenic region or the heterologous oriC region of Mycoplasma capricolum , Mycoplasma mycoides , or Spiroplasma citri failed to produce any detectable transformants. We also developed alternative transformation procedures based on electroporation and conjugation from Escherichia coli , reaching frequencies up to 7.87 × 10 -6 and 8.44 × 10 -7 transformants per viable cell, respectively. Finally, we demonstrated the functionality of antibiotic resistance genes active against tetracycline, puromycin, and spectinomycin/streptomycin in M. florum Taken together, these valuable genetic tools will facilitate efforts toward building an M. florum -based near-minimal cellular chassis for synthetic biology. IMPORTANCE Mesoplasma florum constitutes an attractive model for systems biology and for the development of a simplified cell chassis in synthetic biology. M. florum is closely related to the mycoides cluster of mycoplasmas, which has become a model for whole-genome cloning, genome transplantation, and genome minimization. However, M. florum shows higher growth rates than other Mollicutes , has no known pathogenic potential, and possesses a significantly smaller genome that positions this species among some of the simplest free-living organisms. So far, the lack of genetic engineering tools has limited our capacity to understand the basic biology of M. florum in order to modify its genome. To address this issue, we have evaluated the susceptibility of M. florum to common antibiotics and developed the first artificial plasmids and transformation methods for this bacterium. This represents a strong basis for ongoing genome engineering efforts using this near-minimal microorganism. Copyright © 2017 American Society for Microbiology.

Uncovering the intrinsic size dependence of hydriding phase transformations in nanocrystals.

PubMed

Bardhan, Rizia; Hedges, Lester O; Pint, Cary L; Javey, Ali; Whitelam, Stephen; Urban, Jeffrey J

2013-10-01

A quantitative understanding of nanocrystal phase transformations would enable more efficient energy conversion and catalysis, but has been hindered by difficulties in directly monitoring well-characterized nanoscale systems in reactive environments. We present a new in situ luminescence-based probe enabling direct quantification of nanocrystal phase transformations, applied here to the hydriding transformation of palladium nanocrystals. Our approach reveals the intrinsic kinetics and thermodynamics of nanocrystal phase transformations, eliminating complications of substrate strain, ligand effects and external signal transducers. Clear size-dependent trends emerge in nanocrystals long accepted to be bulk-like in behaviour. Statistical mechanical simulations show these trends to be a consequence of nanoconfinement of a thermally driven, first-order phase transition: near the phase boundary, critical nuclei of the new phase are comparable in size to the nanocrystal itself. Transformation rates are then unavoidably governed by nanocrystal dimensions. Our results provide a general framework for understanding how nanoconfinement fundamentally impacts broad classes of thermally driven solid-state phase transformations relevant to hydrogen storage, catalysis, batteries and fuel cells.

Perforin and human diseases.

PubMed

Naneh, Omar; Avčin, Tadej; Bedina Zavec, Apolonija

2014-01-01

Natural killer (NK) cells and cytotoxic T lymphocytes (CTL) use a highly toxic pore-forming protein perforin (PFN) to destroy cells infected with intracellular pathogens and cells with pre-cancerous transformations. However, mutations of PFN and defects in its expression can cause an abnormal function of the immune system and difficulties in elimination of altered cells. As discussed in this chapter, deficiency of PFN due to the mutations of its gene, PFN1, can be associated with malignancies and severe immune disorders such as familial hemophagocytic lymphohistiocytosis (FHL) and macrophage activation syndrome. On the other hand, overactivity of PFN can turn the immune system against autologous cells resulting in other diseases such as systemic lupus erythematosus, polymyositis, rheumatoid arthritis and cutaneous inflammation. PFN also has a crucial role in the cellular rejection of solid organ allografts and destruction of pancreatic β-cells resulting in type 1 diabetes. These facts highlight the importance of understanding the biochemical characteristics of PFN.

Constitutive expression of thymidylate synthase from LCDV-C induces a transformed phenotype in fish cells.

PubMed

Zhao, Zhe; Shi, Yan; Ke, Fei; Wei, Sun; Gui, Jianfang; Zhang, Qiya

2008-03-01

Thymidylate synthase (TS), an essential enzyme in DNA synthesis and repair, plays a key role in the events of cell cycle regulation and tumor formation. Here, an investigation was presented about subcellular location and biological function of viral TS from lymphocystis disease virus from China (LCDV-C) in fish cells. Fluorescence microscopy revealed that LCDV-C TS was predominantly localized in the cytoplasm in fish cells. Cell cycle analysis demonstrated that LCDV-C TS promoted cell cycle progression into S and G2/M phase in the constitutive expressed cells. As a result, the cells have a faster growth rate compared with the control cells as revealed by cell growth curves. For foci assay, the TS-expressed cells gave rise to foci 4-5 weeks after incubation. Microscopic examination of the TS-induced foci revealed multilayered growth and crisscross morphology characteristic of transformed cells. Moreover, LCDV-C TS predisposed the transfected cells to acquire an anchorage-independent phenotype and could grow in 0.3% soft agar. So the data reveal LCDV-C TS is sufficient to induce a transformed phenotype in fish cells in vitro and exhibits its potential ability in cell transformation. To our knowledge, it is the first report on viral TS sequences associated with transforming activity.

Repair-dependent cell radiation survival and transformation: an integrated theory.

PubMed

Sutherland, John C

2014-09-07

The repair-dependent model of cell radiation survival is extended to include radiation-induced transformations. The probability of transformation is presumed to scale with the number of potentially lethal damages that are repaired in a surviving cell or the interactions of such damages. The theory predicts that at doses corresponding to high survival, the transformation frequency is the sum of simple polynomial functions of dose; linear, quadratic, etc, essentially as described in widely used linear-quadratic expressions. At high doses, corresponding to low survival, the ratio of transformed to surviving cells asymptotically approaches an upper limit. The low dose fundamental- and high dose plateau domains are separated by a downwardly concave transition region. Published transformation data for mammalian cells show the high-dose plateaus predicted by the repair-dependent model for both ultraviolet and ionizing radiation. For the neoplastic transformation experiments that were analyzed, the data can be fit with only the repair-dependent quadratic function. At low doses, the transformation frequency is strictly quadratic, but becomes sigmodial over a wider range of doses. Inclusion of data from the transition region in a traditional linear-quadratic analysis of neoplastic transformation frequency data can exaggerate the magnitude of, or create the appearance of, a linear component. Quantitative analysis of survival and transformation data shows good agreement for ultraviolet radiation; the shapes of the transformation components can be predicted from survival data. For ionizing radiations, both neutrons and x-rays, survival data overestimate the transforming ability for low to moderate doses. The presumed cause of this difference is that, unlike UV photons, a single x-ray or neutron may generate more than one lethal damage in a cell, so the distribution of such damages in the population is not accurately described by Poisson statistics. However, the complete sigmodial dose-response data for neoplastic transformations can be fit using the repair-dependent functions with all parameters determined only from transformation frequency data.

Apigenin suppresses migration and invasion of transformed cells through down-regulation of C-X-C chemokine receptor 4 expression

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

Wang, Lei; Kuang, Lisha; Hitron, John Andrew

Environmental exposure to arsenic is known to cause various cancers. There are some potential relationships between cell malignant transformation and C-X-C chemokine receptor type 4 (CXCR4) expressions. Metastasis, one of the major characteristics of malignantly transformed cells, contributes to the high mortality of cells. CXCR4 and its natural chemokine ligand C-X-C motif ligand 12 (CXCL12) play a critical role in metastasis. Therefore, identification of nutritional factors which are able to inhibit CXCR4 is important for protection from environmental arsenic-induced carcinogenesis and for abolishing metastasis of malignantly transformed cells. The present study demonstrates that apigenin (4′,5,7-trihydroxyflavone), a natural dietary flavonoid, suppressedmore » CXCR4 expression in arsenic-transformed Beas-2B cells (B-AsT) and several other types of transformed/cancer cells in a dose- and time-dependent manner. Neither proteasome nor lysosome inhibitor had any effect in reducing the apigenin-induced down-regulation of CXCR4, indicating that apigenin-induced down-regulation of CXCR4 is not due to proteolytic degradation. The down-regulation of CXCR4 is mainly due to the inhibition of nuclear factor κB (NF-κB) transcriptional activity. Apigenin also abolished migration and invasion of transformed cells induced by CXCL12. In a xenograft mouse model, apigenin down-regulated CXCR4 expression and suppressed tumor growth. Taken together, our results show that apigenin is a novel inhibitor of CXCR4 expression. This dietary flavonoid has the potential to suppress migration and invasion of transformed cells and prevent environmental arsenic-induced carcinogenesis. - Highlights: • Apigenin has a potential in preventing environmental arsenic induced carcinogenesis. • Apigenin suppresses CXCR4 in malignant transformed cells in vitro and in vivo. • The down-regulation of CXCR4 is mainly due to inhibition of NF-κB activity.« less

Reverse Transcriptase-Containing Particles Induced in Rous Sarcoma Virus-Transformed Rat Cells by Arginine Deprivation

PubMed Central

Kotler, Moshe; Weinberg, Eynat; Haspel, Osnat; Becker, Yechiel

1972-01-01

Incubation of rat cells transformed by Rous sarcoma virus (RSV) in an arginine-deficient medium resulted in accumulation of particles in the culture medium. Such particles did not appear when the transformed rat cells were incubated in a complete medium nor in the medium of primary rat cells which were incubated either in arginine-deficient or complete media. The particles which were released from the arginine-deprived transformed rat cells resemble C-type particles in their properties. These particles band in sucrose gradients at a density of 1.16 g/ml and contain 35S ribonucleic acid (RNA) molecules and a reverse transcriptase activity. Analysis of the cytoplasm of transformed and primary rat cells, deprived and undeprived of arginine, revealed the presence of reverse transcriptase-containing particles which banded in sucrose gradients at a density of 1.14 g/ml. These particles differed from the particles released into the medium by the arginine-deprived RSV-transformed rat cells. The deoxyribonucleic acid (DNA) molecules synthesized in vitro by the reverse transcriptase present in the particles isolated from the medium of arginine-deprived cells hybridized to RSV RNA, whereas the DNA synthesized by the cell-bound enzyme had no homology to RSV RNA. PMID:4116137

c-Jun/AP-1 pathway-mediated cyclin D1 expression participates in low dose arsenite-induced transformation in mouse epidermal JB6 Cl41 cells

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

Zhang Dongyun; Li Jingxia; Gao Jimin

2009-02-15

Arsenic is a well-documented human carcinogen associated with skin carcinogenesis. Our previous work reveals that arsenite exposure is able to induce cell transformation in mouse epidermal cell JB6 Cl41 through the activation of ERK, rather than JNK pathway. Our current studies further evaluate downstream pathway in low dose arsenite-induced cell transformation in JB6 Cl41 cells. Our results showed that treatment of cells with low dose arsenite induced activation of c-Jun/AP-1 pathway, and ectopic expression of dominant negative mutant of c-Jun (TAM67) blocked arsenite-induced transformation. Furthermore, our data indicated that cyclin D1 was an important downstream molecule involved in c-Jun/AP-1-mediated cellmore » transformation upon low dose arsenite exposure, because inhibition of cyclin D1 expression by its specific siRNA in the JB6 Cl41 cells resulted in impairment of anchorage-independent growth of cells induced by low dose arsenite. Collectively, our results demonstrate that c-Jun/AP-1-mediated cyclin D1 expression is at least one of the key events implicated in cell transformation upon low dose arsenite exposure.« less

The use of spectral methods in bidomain studies.

PubMed

Trayanova, N; Pilkington, T

1992-01-01

A Fourier transform method is developed for solving the bidomain coupled differential equations governing the intracellular and extracellular potentials on a finite sheet of cardiac cells undergoing stimulation. The spectral formulation converts the system of differential equations into a "diagonal" system of algebraic equations. Solving the algebraic equations directly and taking the inverse transform of the potentials proved numerically less expensive than solving the coupled differential equations by means of traditional numerical techniques, such as finite differences; the comparison between the computer execution times showed that the Fourier transform method was about 40 times faster than the finite difference method. By application of the Fourier transform method, transmembrane potential distributions in the two-dimensional myocardial slice were calculated. For a tissue characterized by a ratio of the intra- to extracellular conductivities that is different in all principal directions, the transmembrane potential distribution exhibits a rather complicated geometrical pattern. The influence of the different anisotropy ratios, the finite tissue size, and the stimuli configuration on the pattern of membrane polarization is investigated.

Spontaneous Transformation of Murine Epithelial Cells Requires the Early Acquisition of Specific Chromosomal Aneuploidies and Genomic Imbalances

PubMed Central

Padilla-Nash, Hesed M.; Hathcock, Karen; McNeil, Nicole E.; Mack, David; Hoeppner, Daniel; Ravin, Rea; Knutsen, Turid; Yonescu, Raluca; Wangsa, Danny; Dorritie, Kathleen; Barenboim, Linda; Hu, Yue; Ried, Thomas

2011-01-01

Human carcinomas are defined by recurrent chromosomal aneuploidies, which result in tissue-specific distribution of genomic imbalances. In order to develop models for these genome mutations and determine their role in tumorigenesis, we generated 45 spontaneously transformed murine cell lines from normal epithelial cells derived from bladder, cervix, colon, kidney, lung, and mammary gland. Phenotypic changes, chromosomal aberrations, centrosome number, and telomerase activity were assayed in control uncultured cells and in three subsequent stages of transformation. Supernumerary centrosomes, bi-nucleate cells, and tetraploidy were observed as early as 48 hr after explantation. In addition, telomerase activity increased throughout progression. Live-cell imaging revealed that failure of cytokinesis, not cell fusion, promoted genome duplication. Spectral karyotyping demonstrated that aneuploidy preceded immortalization, consisting predominantly of whole chromosome losses (4, 9, 12, 13, 16, and Y) and gains (1, 10, 15, and 19). After transformation, focal amplifications of the oncogenes Myc and Mdm2 were frequently detected. Fifty percent of the transformed lines resulted in tumors upon injection into immuno-compromised mice. The phenotypic and genomic alterations observed in spontaneously transformed murine epithelial cells recapitulated the aberration pattern observed during human carcinogenesis. The dominant aberration of these cell lines was the presence of specific chromosomal aneuploidies. We propose that our newly derived cancer models will be useful tools to dissect the sequential steps of genome mutations during malignant transformation, and also to identify cancer-specific genes, signaling pathways, and the role of chromosomal instability in this process. PMID:22161874

Transformation of lymphocytes by Herpesvirus papio.

PubMed

Falk, L A; Henle, G; Henle, W; Deinhardt, F; Schudel, A

1977-08-15

Cotton-topped (CT) or white-lipped (WL) marmoset lymphocytes were transformed in vitro with herpesvirus papio (HVP) into permanently growing lymphoblastoid cell lines (LCL). Five of 9 HVP-transformed CT cell lines contained cells with antigens reacting with antibodies to Epstein-Barr virus (EBV) capsid antigen (VCA) and/or to EBV-induced early antigens (EA). None of 12 WL LCL revealed such antigen-producing cells. Cells from both groups of cultures failed to react with antibodies to the EBV-specified nuclear antigen (EBNA). Exposure of baboon circulating lymphocytes to X-irradiated HVP or EBV-carring cells, or to suspensions of EBV resulted in establishment of LCL which all contained VCA and/or EA-positive, but no EBNA-positive cells. Nuclear antigens were undetectable also with anti-VCA-positive sera from baboons, chimpanzees, or other non-human primates. DNA-complementary RNA (cRNA) filter hybridization with EBV cRNA showed that with one exception transformed CT or WL marmoset cells contained at least 1-2 virus genome equivalents per cell, while at least 12-25 virus genome equivalents per cell were detected in transformed baboon cells. These data need confirmation by DNA-DNA reassociation kinetics.

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

Satyapal, Sunita

The 2011 Annual Progress Report summarizes fiscal year 2011 activities and accomplishments by projects funded by the DOE Hydrogen Program. It covers the program areas of hydrogen production and delivery; hydrogen storage; fuel cells; manufacturing; technology validation; safety, codes and standards; education; market transformation; and systems analysis.

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

none,

The 2013 Annual Progress Report summarizes fiscal year 2013 activities and accomplishments by projects funded by the DOE Hydrogen Program. It covers the program areas of hydrogen production and delivery; hydrogen storage; fuel cells; manufacturing; technology validation; safety, codes and standards; market transformation; and systems analysis.

Viability and DNA damage responses of TPPII-deficient Myc- and Ras-transformed fibroblasts

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

Tsurumi, Chizuko; Firat, Elke; Gaedicke, Simone

2009-09-04

Tripeptidyl peptidase II (TPPII) is a giant cytosolic protease. Previous protease inhibitor, overexpression and siRNA studies suggested that TPPII is important for viability and proliferation of tumor cells, and for their ionizing radiation-induced DNA damage response. The possibility that TPPII could be targeted for tumor therapy prompted us to study its role in transformed cells following genetic TPPII deletion. We generated cell lines from primary fibroblasts having conditional (floxed) TPPII alleles, transformed them with both the c-myc and H-ras oncogenes, and deleted TPPII using retroviral self-deleting Cre recombinase. Clonally derived TPPIIflox/flox and TPPII-/- transformed fibroblasts showed no influences of TPPIImore » expression on basal cell survival and proliferation, nor on radiation-induced p53 activation, p21 induction, cell cycle arrest, apoptosis, or clonogenic cell death. Thus, our results do not support a generally important role of TPPII for viability and proliferation of transformed cells or their p53-mediated DNA damage response.« less

Viability and DNA damage responses of TPPII-deficient Myc- and Ras-transformed fibroblasts.

PubMed

Tsurumi, Chizuko; Firat, Elke; Gaedicke, Simone; Huai, Jisen; Mandal, Pankaj Kumar; Niedermann, Gabriele

2009-09-04

Tripeptidyl peptidase II (TPPII) is a giant cytosolic protease. Previous protease inhibitor, overexpression and siRNA studies suggested that TPPII is important for viability and proliferation of tumor cells, and for their ionizing radiation-induced DNA damage response. The possibility that TPPII could be targeted for tumor therapy prompted us to study its role in transformed cells following genetic TPPII deletion. We generated cell lines from primary fibroblasts having conditional (floxed) TPPII alleles, transformed them with both the c-myc and H-ras oncogenes, and deleted TPPII using retroviral self-deleting Cre recombinase. Clonally derived TPPIIflox/flox and TPPII-/- transformed fibroblasts showed no influences of TPPII expression on basal cell survival and proliferation, nor on radiation-induced p53 activation, p21 induction, cell cycle arrest, apoptosis, or clonogenic cell death. Thus, our results do not support a generally important role of TPPII for viability and proliferation of transformed cells or their p53-mediated DNA damage response.

Epidermal cell-shape regulation and subpopulation kinetics during butyrate-induced terminal maturation of normal and SV40-transformed human keratinocytes: epithelial models of differentiation therapy.

PubMed

Staiano-Coico, L; Steinberg, M; Higgins, P J

1990-10-15

Recent data indicate that malignant human epidermal cells may be appropriate targets for sodium butyrate (NaB)-mediated differentiation therapy. The response of pre- and post-crisis populations of SV40-transformed human keratinocytes (SVKs) to this differentiation-inducing agent was assessed, therefore, within the framework of NaB-directed normal human keratinocyte (NHK) maturation. NaB augmented cornified envelope (CE) production in NHK and pre-crisis SVK cultures; the time-course and efficiency of induced maturation were similar in the 2 cell systems. In NHKs, the percentage of amplifying ("B" substate) cells decreased with time in NaB correlating with increases in both "C" stage keratinocytes and CEs. The latter formed over one or 2 layers of nucleated basal-like cells. Inductions were accompanied by immediate cell cycle blocks (in both the G1 and G2/M phases), reorganization within the actin cytoskeleton, and transient early increases in cellular actin content. Increased NHK and pre-crisis SVK cytoskeletal-associated actin reached a maximum approximately 48 hr after NaB addition and preceded development of CEs. The CE precursors, thus, probably reside in the "B" substate. Post-crisis SVKs, in contrast, were refractive to NaB-induced terminal maturation or cell-cycle perturbation, failed to initiate actin filament rearrangements, and retained a basal cell-like phenotype. Stable transformation of human SVKs in post-crisis phase, therefore, appears to be associated with loss of maturation "competence" within the "B" keratinocyte subpopulation.

Exact Solutions of Coupled Multispecies Linear Reaction–Diffusion Equations on a Uniformly Growing Domain

PubMed Central

Simpson, Matthew J.; Sharp, Jesse A.; Morrow, Liam C.; Baker, Ruth E.

2015-01-01

Embryonic development involves diffusion and proliferation of cells, as well as diffusion and reaction of molecules, within growing tissues. Mathematical models of these processes often involve reaction–diffusion equations on growing domains that have been primarily studied using approximate numerical solutions. Recently, we have shown how to obtain an exact solution to a single, uncoupled, linear reaction–diffusion equation on a growing domain, 0 < x < L(t), where L(t) is the domain length. The present work is an extension of our previous study, and we illustrate how to solve a system of coupled reaction–diffusion equations on a growing domain. This system of equations can be used to study the spatial and temporal distributions of different generations of cells within a population that diffuses and proliferates within a growing tissue. The exact solution is obtained by applying an uncoupling transformation, and the uncoupled equations are solved separately before applying the inverse uncoupling transformation to give the coupled solution. We present several example calculations to illustrate different types of behaviour. The first example calculation corresponds to a situation where the initially–confined population diffuses sufficiently slowly that it is unable to reach the moving boundary at x = L(t). In contrast, the second example calculation corresponds to a situation where the initially–confined population is able to overcome the domain growth and reach the moving boundary at x = L(t). In its basic format, the uncoupling transformation at first appears to be restricted to deal only with the case where each generation of cells has a distinct proliferation rate. However, we also demonstrate how the uncoupling transformation can be used when each generation has the same proliferation rate by evaluating the exact solutions as an appropriate limit. PMID:26407013

Exact Solutions of Coupled Multispecies Linear Reaction-Diffusion Equations on a Uniformly Growing Domain.

PubMed

Simpson, Matthew J; Sharp, Jesse A; Morrow, Liam C; Baker, Ruth E

2015-01-01

Embryonic development involves diffusion and proliferation of cells, as well as diffusion and reaction of molecules, within growing tissues. Mathematical models of these processes often involve reaction-diffusion equations on growing domains that have been primarily studied using approximate numerical solutions. Recently, we have shown how to obtain an exact solution to a single, uncoupled, linear reaction-diffusion equation on a growing domain, 0 < x < L(t), where L(t) is the domain length. The present work is an extension of our previous study, and we illustrate how to solve a system of coupled reaction-diffusion equations on a growing domain. This system of equations can be used to study the spatial and temporal distributions of different generations of cells within a population that diffuses and proliferates within a growing tissue. The exact solution is obtained by applying an uncoupling transformation, and the uncoupled equations are solved separately before applying the inverse uncoupling transformation to give the coupled solution. We present several example calculations to illustrate different types of behaviour. The first example calculation corresponds to a situation where the initially-confined population diffuses sufficiently slowly that it is unable to reach the moving boundary at x = L(t). In contrast, the second example calculation corresponds to a situation where the initially-confined population is able to overcome the domain growth and reach the moving boundary at x = L(t). In its basic format, the uncoupling transformation at first appears to be restricted to deal only with the case where each generation of cells has a distinct proliferation rate. However, we also demonstrate how the uncoupling transformation can be used when each generation has the same proliferation rate by evaluating the exact solutions as an appropriate limit.

A near-infrared Fourier transform Raman spectroscopy of epidermal keratinocytes: changes in the protein?DNA structure following malignant transformation

NASA Astrophysics Data System (ADS)

Gao, Xiaoling; Butler, Ian S.; Kremer, Richard

2005-01-01

We report here the use of near-infrared (NIR) Fourier transform (FT) Raman spectroscopy to analyze normal human epidermal keratinocytes prior to and following malignant transformation. Our analysis indicates specific Raman spectral differences between immortalized (HPK1A) and malignant ras transformed (HPK1A- ras) cells. In addition, striking spectral differences are seen in the DNA isolated from these cells and particularly in the 843/810 cm -1 ratio with values of 1.6 ± 0.13 in HPK1A cells and 0.68 ± 0.09 in HPK1A- ras cells (mean ± S.D., n = 12, P < 0.001) indicating specific alterations in the backbone conformation markers following malignant transformation. Subsequently, we analysed the effect of a strong inhibitor of keratinocyte growth, the Vitamin D analog EB1089, on the Raman spectra of intact cells and on the 843/810 cm -1 ratio in the DNA isolated from both cell lines. Specific changes were observed in intact cells in the 1300-750 cm -1 region. Furthermore, the 843/810cm -1 ratio of isolated DNA from HPK1A cells was not affected by EB1089 but significantly increased in DNA isolated from HPK1A-ras cells so much that it became closer to the value observed for HPK1A cells (1.07 ± 0.10). Our data suggest that Raman analysis of DNA and in particular the 843/810cm -1 ratio can provide useful indices of malignant transformation and efficacy of anticancer agents.

Adenovirus Vector Pseudotyping in Fiber-Expressing Cell Lines: Improved Transduction of Epstein-Barr Virus-Transformed B Cells

PubMed Central

Von Seggern, Dan J.; Huang, Shuang; Fleck, Shonna Kaye; Stevenson, Susan C.; Nemerow, Glen R.

2000-01-01

While adenovirus (Ad) gene delivery vectors are useful in many gene therapy applications, their broad tropism means that they cannot be directed to a specific target cell. There are also a number of cell types involved in human disease which are not transducible with standard Ad vectors, such as Epstein-Barr virus (EBV)-transformed B lymphocytes. Adenovirus binds to host cells via the viral fiber protein, and Ad vectors have previously been retargeted by modifying the fiber gene on the viral chromosome. This requires that the modified fiber be able to bind to the cell in which the vector is grown, which prevents truly specific vector targeting. We previously reported a gene delivery system based on a fiber gene-deleted Ad type 5 (Ad5) vector (Ad5.βgal.ΔF) and packaging cells that express the viral fiber protein. Expression of different fibers in packaging cells will allow Ad retargeting without modifying the viral chromosome. Importantly, fiber proteins which can no longer bind to the producer cells can also be used. Using this approach, we generated for the first time pseudotyped Ad5.βgal.ΔF particles containing either the wild-type Ad5 fiber protein or a chimeric fiber with the receptor-binding knob domain of the Ad3 fiber. Particles equipped with the chimeric fiber bound to the Ad3 receptor rather than the coxsackievirus-adenovirus receptor protein used by Ad5. EBV-transformed B lymphocytes were infected efficiently by the Ad3-pseudotyped particles but poorly by virus containing the Ad5 fiber protein. The strategy described here represents a broadly applicable method for targeting gene delivery to specific cell types. PMID:10590124

Cell-Free Synthetic Biology Chassis for Nanocatalytic Photon-to-Hydrogen Conversion

DOE PAGES

Wang, Peng; Chang, Angela Y.; Novosad, Valentyn; ...

2017-06-11

We report on entirely man-made nanobio hybrid fabricated through assembly of cell-free expressed transmembrane proton pump and semiconductor nanoparticles as an efficient nanocatalysis for photocatalytic H 2 evolution. The system produces H 2 at a turnover rate of 239 (μmole protein) -1 h -1 under green and 17742 (μmole protein) -1 h -1 under white light at ambient conditions, in water at neutral pH with methanol as a sacrificial electron donor. Robustness and flexibility of this approach allows for systemic manipulation at nanoparticle-bio interface toward directed evolution of energy transformation materials and artificial systems.

Cell-Free Synthetic Biology Chassis for Nanocatalytic Photon-to-Hydrogen Conversion

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

Wang, Peng; Chang, Angela Y.; Novosad, Valentyn

We report on entirely man-made nanobio hybrid fabricated through assembly of cell-free expressed transmembrane proton pump and semiconductor nanoparticles as an efficient nanocatalysis for photocatalytic H 2 evolution. The system produces H 2 at a turnover rate of 239 (μmole protein) -1 h -1 under green and 17742 (μmole protein) -1 h -1 under white light at ambient conditions, in water at neutral pH with methanol as a sacrificial electron donor. Robustness and flexibility of this approach allows for systemic manipulation at nanoparticle-bio interface toward directed evolution of energy transformation materials and artificial systems.

Epithelial self-defense against cancer.

PubMed

Yamauchi, Hajime; Fujita, Yasuyuki

2012-11-01

It is not clearly understood what happens at the interface between normal and transformed epithelial cells at the first step of carcinogenesis. A recent study reveals that the organized epithelial structure suppresses clonal expansion of transformed cells. Translocation from the epithelium or perturbation of intercellular adhesions may be required for transformed cells to evade the suppressive environments.

Insect cell transformation vectors that support high level expression and promoter assessment in insect cell culture

USDA-ARS?s Scientific Manuscript database

A somatic transformation vector, pDP9, was constructed that provides a simplified means of producing permanently transformed cultured insect cells that support high levels of protein expression of foreign genes. The pDP9 plasmid vector incorporates DNA sequences from the Junonia coenia densovirus th...

Vaccinia virus, herpes simplex virus, and carcinogens induce DNA amplification in a human cell line and support replication of a helpervirus dependent parvovirus

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

Schlehofer, J.R.; Ehrbar, M.; zur Hausen, H.

1986-07-15

The SV40-transformed human kidney cell line, NB-E, amplifies integrated as well as episomal SV40 DNA upon treatment with chemical (DMBA) or physical (uv irradiation) carcinogens (initiators) as well as after infection with herpes simplex virus (HSV) type 1 or with vaccinia virus. In addition it is shown that vaccinia virus induces SV40 DNA amplification also in the SV40-transformed Chinese hamster embryo cell line, CO631. These findings demonstrate that human cells similar to Chinese hamster cells amplify integrated DNA sequences after treatment with carcinogens or infection with specific viruses. Furthermore, a poxvirus--vaccinia virus--similar to herpes group viruses induces DNA amplification. Asmore » reported for other systems, the vaccinia virus-induced DNA amplification in NB-E cells is inhibited by coinfection with adeno-associated virus (AAV) type 5. This is in line with previous studies on inhibition of carcinogen- or HSV-induced DNA amplification in CO631 cells. The experiments also demonstrate that vaccinia virus, in addition to herpes and adenoviruses acts as a helper virus for replication and structural antigen synthesis of AAV-5 in NB-E cells.« less

Ultra-sensitive detection of tumorigenic cellular impurities in human cell-processed therapeutic products by digital analysis of soft agar colony formation.

PubMed

Kusakawa, Shinji; Yasuda, Satoshi; Kuroda, Takuya; Kawamata, Shin; Sato, Yoji

2015-12-08

Contamination with tumorigenic cellular impurities is one of the most pressing concerns for human cell-processed therapeutic products (hCTPs). The soft agar colony formation (SACF) assay, which is a well-known in vitro assay for the detection of malignant transformed cells, is applicable for the quality assessment of hCTPs. Here we established an image-based screening system for the SACF assay using a high-content cell analyzer termed the digital SACF assay. Dual fluorescence staining of formed colonies and the dissolution of soft agar led to accurate detection of transformed cells with the imaging cytometer. Partitioning a cell sample into multiple wells of culture plates enabled digital readout of the presence of colonies and elevated the sensitivity for their detection. In practice, the digital SACF assay detected impurity levels as low as 0.00001% of the hCTPs, i.e. only one HeLa cell contained in 10,000,000 human mesenchymal stem cells, within 30 days. The digital SACF assay saves time, is more sensitive than in vivo tumorigenicity tests, and would be useful for the quality control of hCTPs in the manufacturing process.

Ultra-sensitive detection of tumorigenic cellular impurities in human cell-processed therapeutic products by digital analysis of soft agar colony formation

PubMed Central

Kusakawa, Shinji; Yasuda, Satoshi; Kuroda, Takuya; Kawamata, Shin; Sato, Yoji

2015-01-01

Contamination with tumorigenic cellular impurities is one of the most pressing concerns for human cell-processed therapeutic products (hCTPs). The soft agar colony formation (SACF) assay, which is a well-known in vitro assay for the detection of malignant transformed cells, is applicable for the quality assessment of hCTPs. Here we established an image-based screening system for the SACF assay using a high-content cell analyzer termed the digital SACF assay. Dual fluorescence staining of formed colonies and the dissolution of soft agar led to accurate detection of transformed cells with the imaging cytometer. Partitioning a cell sample into multiple wells of culture plates enabled digital readout of the presence of colonies and elevated the sensitivity for their detection. In practice, the digital SACF assay detected impurity levels as low as 0.00001% of the hCTPs, i.e. only one HeLa cell contained in 10,000,000 human mesenchymal stem cells, within 30 days. The digital SACF assay saves time, is more sensitive than in vivo tumorigenicity tests, and would be useful for the quality control of hCTPs in the manufacturing process. PMID:26644244

Both cell fusion and transdifferentiation account for the transformation of human peripheral blood CD34-positive cells into cardiomyocytes in vivo.

PubMed

Zhang, Sui; Wang, Dachun; Estrov, Zeev; Raj, Sean; Willerson, James T; Yeh, Edward T H

2004-12-21

Adult human peripheral blood CD34-positive (CD34+) cells appear to transform into cardiomyocytes in the injured hearts of severe combined immunodeficient mice. It remains unclear, however, whether the apparent transformation is the result of transdifferentiation of the donor stem cells or of fusion of the donor cell with the cardiomyocyte of the recipients. We performed flow cytometry analyses of cells isolated from the hearts of mice that received human CD34+ cells. Human HLA-ABC antigen and cardiac troponin T or Nkx2.5 were used as markers for cardiomyocytes derived from human CD34+ cells, and HLA-ABC and VE-cadherin were used to identify the transformed endothelial cells. The double-positive cells were collected and interphase fluorescence in situ hybridization was used to detect the expression of human and mouse X chromosomes in these cells. We found that 73.3% of nuclei derived from HLA+ and troponin T+ or Nkx2.5+ cardiomyocytes contain both human and mouse X chromosomes and 23.7% contain only human X chromosome. In contrast, the nuclei of HLA-, troponin T+ cells contain only mouse X chromosomes. Furthermore, 97.3% of endothelial cells derived from CD34+ cells contained human X chromosome only. Thus, both cell fusion and transdifferentiation may account for the transformation of peripheral blood CD34+ cells into cardiomyocytes in vivo.

A comprehensive review of metal-induced cellular transformation studies.

PubMed

Chen, Qiao Yi; Costa, Max

2017-09-15

In vitro transformation assays not only serve practical purposes in screening for potential carcinogenic substances in food, drug, and cosmetic industries, but more importantly, they provide a means of understanding the critical biological processes behind in vivo cancer development. In resemblance to cancer cells in vivo, successfully transformed cells display loss of contact inhibition, gain of anchorage independent growth, resistant to proper cell cycle regulation such as apoptosis, faster proliferation rate, potential for cellular invasion, and ability to form tumors in experimental animals. Cells purposely transformed using metal exposures enable researchers to examine molecular changes, dissect various stages of tumor formation, and ultimately elucidate metal induced cancer mode of action. For practical purposes, this review specifically focuses on studies incorporating As-, Cd-, Cr-, and Ni-induced cell transformation. Through investigating and comparing an extensive list of studies using various methods of metal-induced transformation, this review serves to bridge an information gap and provide a guide for avoiding procedural discrepancies as well as maximizing experimental efficiency. Copyright © 2017 Elsevier Inc. All rights reserved.

v-myb transformation of Xeroderma pigmentosum human fibroblasts: Overexpression of the c-Ha-ras oncogene in the transformed cells

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

Michelin, S.; Varlet, I.; Sarasin, A.

1991-10-01

Human Xeroderma pigmentosum normal' fibroblasts AS16 (XP4 VI) were transformed after transfection with a recombinant v-myb clone. In this clone (pKXA 3457) derived from avian myeloblastosis virus (AMV), the expression of the oncogene sequences is driven by the AMV U-5 LTR promoter. The transformed cells (ASKXA), which have integrated a rearranged v-myb oncogene, grow in agar, are not tumorigenic in nude mice, and express a 45-kDa v-myb protein. The HMW DNA of these cells transform chicken embryo fibroblasts. The c-Ha-ras oncogene is overexpressed in the ASKXA cells but not in the parental normal' AS16 cells and a revertant clone (ASKXAmore » Cl 1.1 G). The results lead to the conclusion that the XP fibroblasts are phenotypically transformed by the presence of the transfected v-myb oncogene, which is able to induce an overexpression of the c-Ha-ras gene.« less

Arsenicals produce stable progressive changes in DNA methylation patterns that are linked to malignant transformation of immortalized urothelial cells

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

Jensen, Taylor J.; Arizona Cancer Center, University of Arizona, Tucson, AZ 85724; Novak, Petr

2009-12-01

Aberrant DNA methylation participates in carcinogenesis and is a molecular hallmark of a tumor cell. Tumor cells generally exhibit a redistribution of DNA methylation resulting in global hypomethylation with regional hypermethylation; however, the speed in which these changes emerge has not been fully elucidated and may depend on the temporal location of the cell in the path from normal, finite lifespan to malignant transformation. We used a model of arsenical-induced malignant transformation of immortalized human urothelial cells and DNA methylation microarrays to examine the extent and temporal nature of changes in DNA methylation that occur during the transition from immortalmore » to malignantly transformed. Our data presented herein suggest that during arsenical-induced malignant transformation, aberrant DNA methylation occurs non-randomly, progresses gradually at hundreds of gene promoters, and alters expression of the associated gene, and these changes are coincident with the acquisition of malignant properties, such as anchorage independent growth and tumor formation in immunocompromised mice. The DNA methylation changes appear stable, since malignantly transformed cells removed from the transforming arsenical exhibited no reversion in DNA methylation levels, associated gene expression, or malignant phenotype. These data suggest that arsenicals act as epimutagens and directly link their ability to induce malignant transformation to their actions on the epigenome.« less

Inhibition of Fas (CD95) expression and Fas-mediated apoptosis by oncogenic Ras.

PubMed

Fenton, R G; Hixon, J A; Wright, P W; Brooks, A D; Sayers, T J

1998-08-01

The ras oncogene plays an important role in the multistep progression to cancer by activation of signal transduction pathways that contribute to aberrant growth regulation. Although many of these effects are cell autonomous, the ras oncogene also regulates the expression of genes that alter host/tumor interactions. We now extend the mechanisms through which ras promotes tumor survival by demonstrating that oncogenic Ras inhibits expression of the fas gene and renders Ras-transformed cells resistant to Fas-induced apoptosis. A panel of Ras-transformed clones exhibited a marked inhibition in fas mRNA and Fas cell surface expression as compared with untransformed parental cell lines. Fas expression was induced by culture in the presence of IFN-gamma + tumor necrosis factor alpha; however, the maximal level attained in Ras transformants was approximately 10-fold below the level of untransformed cells. Whereas untransformed cells were sensitive to apoptotic death induced by cross-linking surface Fas (especially after cytokine treatment), Ras-transformed cells were very resistant to Fas-induced death even under the most stringent assay conditions. To demonstrate that this resistance was mediated by oncogenic Ras and not secondary genetic events, pools of Ras-transformed cells were generated using a highly efficient retroviral transduction technique. Transformed pools were assayed 6 days after infection and demonstrated a marked decrease in fas gene expression and Fas-mediated apoptosis. Oncogenic Ras did not promote general resistance to apoptosis, because ectopic expression of a fas cDNA in Ras-transformed cells restored sensitivity to Fas-induced apoptosis. These data indicate that oncogenic Ras inhibits basal levels of expression of the fas gene, and although cytokine signal transduction pathways are functional in these cells, the level of surface Fas expression remains below the threshold required for induction of apoptosis. These data identify a mechanism by which Ras-transformed cells may escape from host-mediated immune destruction.

The effect of space radiation of the nervous system

NASA Astrophysics Data System (ADS)

Gauger, Grant E.; Tobias, Cornelius A.; Yang, Tracy; Whitney, Monroe

The long-term effects of irradiation by accelerated heavy ions on the structure and function of the nervous system have not been studied extensively. Although the adult brain is relatively resistant to low LET radiation, cellular studies indicate that individual heavy ions can produce serious membrane lesions and multiple chromatin breaks. Capillary hemorrhages may follow high LET particle irradiation of the developing brain as high RBE effects. Evidence has been accumulating that the glial system and blood-brain barrier (BBB) are relatively sensitive to injury by ionizing radiation. While DNA repair is active in neural systems, it may be assumed that a significant portion of this molecular process is misrepair. Since the expression of cell lethality usually requires cell division, and nerve cells have an extremely low rate of division, it is possible that some of the characteristic changes of premature aging may represent a delayed effect of chromatin misrepair in brain. Altered microcirculation, decreased local metabolism, entanglement and reduction in synaptic density, premature loss of neurons, myelin degeneration, and glial proliferation are late signs of such injuries. HZE particles are very efficient in producing carcinogenic cell transformation, reaching a peak for iron particles. The promotion of viral transformation is also efficient up to an energy transfer of approximately 300 keV/micron. The RBE for carcinogenesis in nerve tissues remains unknown. On the basis of available information concerning HZE particle flux in interplanetary space, only general estimates of the magnitude of the effects of long-term spaceflight on some nervous system parameters may be constructed.

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

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

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

1987-01-01

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

Methods for identifying an essential gene in a prokaryotic microorganism

DOEpatents

Shizuya, Hiroaki

2006-01-31

Methods are provided for the rapid identification of essential or conditionally essential DNA segments in any species of haploid cell (one copy chromosome per cell) that is capable of being transformed by artificial means and is capable of undergoing DNA recombination. This system offers an enhanced means of identifying essential function genes in diploid pathogens, such as gram-negative and gram-positive bacteria.

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

Park, Youn-hee; Kim, Donghern; Dai, Jin

Occupational and environmental exposure to arsenic (III) and chromium VI (Cr(VI)) have been confirmed to cause lung cancer. Mechanisms of these metals carcinogenesis are still under investigation. Selection of cell lines to be used is essential for the studies. Human bronchial epithelial BEAS-2B cells are the cells to be utilized by most of scientists. However, due to p53 missense mutation (CCG → TCG) at codon 47 and the codon 72 polymorphism (CGC → CCC) in BEAS-2B cells, its usage has frequently been questioned. The present study has examined activity and expression of 53 and its downstream target protein p21 uponmore » acute or chronic exposure of BEAS-2B cells to arsenic and Cr(VI). The results show that short-term exposure of BEAS-2B cells to arsenic or Cr(VI) was able to activate both p53 and p21. Chronic exposure of BEAS-2B cells to these two metals caused malignant cell transformation and tumorigenesis. In arsenic-transformed BEAS-2B cells reductions in p53 promoter activity, mRNA expression, and phosphorylation of p53 at Ser392 were observed, while the total p53 protein level remained the same compared to those in passage-matched parent ones. p21 promoter activity and expression were decreased in arsenic-transformed cells. Cr(VI)-transformed cells exhibit elevated p53 promoter activity, mRNA expression, and phosphorylation at Ser15, but reduced phosphorylation at Ser392 and total p53 protein level compared to passage-matched parent ones. p21 promoter activity and expression were elevated in Cr(VI)-transformed cells. These results demonstrate that p53 is able to respond to exposure of arsenic or Cr(VI), suggesting that BEAS-2B cells are an appropriate in vitro model to investigate arsenic or Cr(VI) induced lung cancer. - Highlights: • Short-term exposure of BEAS-2B cells to arsenic or Cr(VI) activates p53 and p21. • Chronic exposure of BEAS-2B cells to arsenic or Cr(VI) causes cell transformation and tumorigenesis. • Arsenic-transformed cells exhibit reduced activities of p53 and p21. • Cr(VI)-transformed cells exhibit increased activities of p53 and p21.« less

European Workshop on Bacterial Protein Toxins (4th) Held in Urbino, Italy on July 3-6, 1989

DTIC Science & Technology

1990-02-28

is described in Table Ill. Two Epstein - Barr virus transformed B cell lines which had been pulsed with intact t.t. present the antigen equally well to... Epstein - Barr virus transformed human B lymphocytes (B-LCL) homozygous for HLA-DRI. These cells were chosen because they were hcmozygous, well characterized...using RJ 2.2.5 cells (16). These cells, like the MAJA line, are human B-lymphoblastoid cells transformed by Epstein - Barr virus , except that they do

Recombinant protein production and insect cell culture and process

NASA Technical Reports Server (NTRS)

Spaulding, Glenn (Inventor); Prewett, Tacey (Inventor); Goodwin, Thomas (Inventor); Francis, Karen (Inventor); Andrews, Angela (Inventor); Oconnor, Kim (Inventor)

1993-01-01

A process has been developed for recombinant production of selected polypeptides using transformed insect cells cultured in a horizontally rotating culture vessel modulated to create low shear conditions. A metabolically transformed insect cell line is produced using the culture procedure regardless of genetic transformation. The recombinant polypeptide can be produced by an alternative process using the cultured insect cells as host for a virus encoding the described polypeptide such as baculovirus. The insect cells can also be a host for viral production.

Glass Bead-based Genetic Transformation:An Efficient Method for Transformation of Thraustochytrid Microorganisms.

PubMed

Adachi, Takumi; Sahara, Takehiko; Okuyama, Hidetoshi; Morita, Naoki

2017-07-01

Here, we describe a new method for genetic transformation of thraustochytrids, well-known producers of polyunsaturated fatty acids (PUFAs) like docosahexaenoic acid, by combining mild glass (zirconia) bead treatment and electroporation. Because the cell wall is a barrier against transfer of exogenous DNA into cells, gentle vortexing of cells with glass beads was performed prior to electroporation for partial cell wall disruption. G418-resistant transformants of thraustochytrid cells (Aurantiochytrium limacinum strain SR21 and thraustochytrid strain 12B) were successfully obtained with good reproducibility. The method reported here is simpler than methods using enzymes to generate spheroplasts and may provide advantages for PUFA production by using genetically modified thraustochytrids.

Relationship Among Tau Antigens Isolated from Various Lines of Simian Virus 40-Transformed Cells

PubMed Central

Simmons, Daniel T.; Martin, Malcolm A.; Mora, Peter T.; Chang, Chungming

1980-01-01

In addition to the virus-specified tumor antigens, simian virus 40-transformed cells contain at least one other protein which can be immunoprecipitated with serum from animals bearing simian virus 40-induced tumors. This protein, which is designated Tau antigen, has an apparent molecular weight of 56,000 as determined by electrophoresis on acrylamide gels. The relationship among Tau antigens isolated from different lines of simian virus 40-transformed cells was examined by comparing the methionine-labeled tryptic peptides of these proteins by two-dimensional fingerprinting on thin-layer cellulose plates. In this fashion, we initially determined that the Tau antigens isolated from three different lines of transformed mouse cells were very similar. Second, we found that Tau antigen isolated from a line of rat transformants was closely related, but not identical, to the mouse cell Tau antigens. Approximately 70% of their methionine peptides comigrated in two dimensions. Finally, we showed that Tau antigen isolated from a line of transformed human cells was only partially related to the mouse and rat proteins. About 40% of the methionine peptides of the human protein were also contained in the Tau antigens from the other two species. These results strongly indicate that the Tau antigens isolated from these various simian virus 40-transformed cell lines contain common amino acid sequences. Images PMID:6247503

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

PubMed Central

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

2016-01-01

The increased cancer mortality of diabetes type 2 patients is most likely an evidence of the tight connection between tumor development and energy metabolism. A major focus of today’s research is still the identification of key proteins of both diseases and the development of corresponding inhibitors. In this study we combined the two-stage BALB/c-3T3 cell transformation assay (BALB-CTA) with the IR/IGF-1R inhibitor OSI-906 (linsitinib) and analyzed alterations in protein activity and energy parameters in non-transformed as well as transformed cells. OSI-906 successfully inhibited the phosphorylation of IR/IGF-1R and decreased cell growth in non-transformed cells. In the BALB-CTA, a permanent treatment with OSI-906 reduced cellular transformation dose-dependently, whereas a temporary treatment gave evidence for a preventive effect in the promotion phase. Furthermore, even though several key proteins were affected, it was possible to show that the phosphorylation of GSK3, Erk 1/2 and the S6 protein are not crucial for the cell foci reducing effect of OSI-906. Taken together, the BALB-CTA confirmed results of OSI-906 from animal studies and enhanced the knowledge of its mode of action. Therefore, the BALB-CTA offers the opportunity to analyze alterations in the transformation process more precisely and will be helpful to identify effective cancer treatments. PMID:27849005

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

PubMed

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

2016-11-16

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

Loss of Canonical Smad4 Signaling Promotes KRAS Driven Malignant Transformation of Human Pancreatic Duct Epithelial Cells and Metastasis

PubMed Central

Leung, Lisa; Radulovich, Nikolina; Zhu, Chang-Qi; Wang, Dennis; To, Christine; Ibrahimov, Emin; Tsao, Ming-Sound

2013-01-01

Pancreatic ductal adenocarcinoma (PDAC) is the fourth most common cause of cancer death in North America. Activating KRAS mutations and Smad4 loss occur in approximately 90% and 55% of PDAC, respectively. While their roles in the early stages of PDAC development have been confirmed in genetically modified mouse models, their roles in the multistep malignant transformation of human pancreatic duct cells have not been directly demonstrated. Here, we report that Smad4 represents a barrier in KRAS-mediated malignant transformation of the near normal immortalized human pancreatic duct epithelial (HPDE) cell line model. Marked Smad4 downregulation by shRNA in KRAS G12V expressing HPDE cells failed to cause tumorigenic transformation. However, KRAS-mediated malignant transformation occurred in a new HPDE-TGF-β resistant (TβR) cell line that completely lacks Smad4 protein expression and is resistant to the mito-inhibitory activity of TGF-β. This transformation resulted in tumor formation and development of metastatic phenotype when the cells were implanted orthotopically into the mouse pancreas. Smad4 restoration re-established TGF-β sensitivity, markedly increased tumor latency by promoting apoptosis, and decreased metastatic potential. These results directly establish the critical combination of the KRAS oncogene and complete Smad4 inactivation in the multi-stage malignant transformation and metastatic progression of normal human HPDE cells. PMID:24386371

Stromal cells can contribute oncogenic signals

NASA Technical Reports Server (NTRS)

Tlsty, T. D.

2001-01-01

The majority of studies of neoplastic transformation have focused attention on events that occur within transformed cells. These cell autonomous events result in the disruption of molecular pathways that regulate basic activities of the cells such as proliferation, death, movement and genomic integrity. Other studies have addressed the microenvironment of tumor cells and documented its importance in supporting tumor progression. Recent work has begun to expand on these initial studies of tumor microenvironment and now provide novel insights into the possible initiation and progression of malignant cells. This review will address the transforming effect of stromal cells on epithelial components. Copyright 2001 Academic Press.

Comparative Transcriptome Profiling of an SV40-Transformed Human Fibroblast (MRC5CVI) and Its Untransformed Counterpart (MRC-5) in Response to UVB Irradiation

PubMed Central

Chang, Cheng-Wei; Chen, Chaang-Ray; Huang, Chao-Ying; Shu, Wun-Yi; Chiang, Chi-Shiun; Hong, Ji-Hong; Hsu, Ian C.

2013-01-01

Simian virus 40 (SV40) transforms cells through the suppression of tumor-suppressive responses by large T and small t antigens; studies on the effects of these two oncoproteins have greatly improved our knowledge of tumorigenesis. Large T antigen promotes cellular transformation by binding and inactivating p53 and pRb tumor suppressor proteins. Previous studies have shown that not all of the tumor-suppressive responses were inactivated in SV40-transformed cells; however, the underlying cause is not fully studied. In this study, we investigated the UVB-responsive transcriptome of an SV40-transformed fibroblast (MRC5CVI) and that of its untransformed counterpart (MRC-5). We found that, in response to UVB irradiation, MRC-5 and MRC5CVI commonly up-regulated the expression of oxidative phosphorylation genes. MRC-5 up-regulated the expressions of chromosome condensation, DNA repair, cell cycle arrest, and apoptotic genes, but MRC5CVI did not. Further cell death assays indicated that MRC5CVI was more sensitive than MRC-5 to UVB-induced cell death with increased caspase-3 activation; combining with the transcriptomic results suggested that MRC5CVI may undergo UVB-induced cell death through mechanisms other than transcriptional regulation. Our study provides a further understanding of the effects of SV40 transformation on cellular stress responses, and emphasizes the value of SV40-transformed cells in the researches of sensitizing neoplastic cells to radiations. PMID:24019915

Activation of antioxidant pathways in ras-mediated oncogenic transformation of human surface ovarian epithelial cells revealed by functional proteomics and mass spectrometry.

PubMed

Young, Travis W; Mei, Fang C; Yang, Gong; Thompson-Lanza, Jennifer A; Liu, Jinsong; Cheng, Xiaodong

2004-07-01

Cellular transformation is a complex process involving genetic alterations associated with multiple signaling pathways. Development of a transformation model using defined genetic elements has provided an opportunity to elucidate the role of oncogenes and tumor suppressor genes in the initiation and development of ovarian cancer. To study the cellular and molecular mechanisms of Ras-mediated oncogenic transformation of ovarian epithelial cells, we used a proteomic approach involving two-dimensional electrophoresis and mass spectrometry to profile two ovarian epithelial cell lines, one immortalized with SV40 T/t antigens and the human catalytic subunit of telomerase and the other transformed with an additional oncogenic ras(V12) allele. Of approximately 2200 observed protein spots, we have identified >30 protein targets that showed significant changes between the immortalized and transformed cell lines using peptide mass fingerprinting. Among these identified targets, one most notable group of proteins altered significantly consists of enzymes involved in cellular redox balance. Detailed analysis of these protein targets suggests that activation of Ras-signaling pathways increases the threshold of reactive oxidative species (ROS) tolerance by up-regulating the overall antioxidant capacity of cells, especially in mitochondria. This enhanced antioxidant capacity protects the transformed cells from high levels of ROS associated with the uncontrolled growth potential of tumor cells. It is conceivable that an enhanced antioxidation capability may constitute a common mechanism for tumor cells to evade apoptosis induced by oxidative stresses at high ROS levels.

Pepper, sweet (Capsicum annuum).

PubMed

Heidmann, Iris; Boutilier, Kim

2015-01-01

Capsicum (pepper) species are economically important crops that are recalcitrant to genetic transformation by Agrobacterium (Agrobacterium tumefaciens). A number of protocols for pepper transformation have been described but are not routinely applicable. The main bottleneck in pepper transformation is the low frequency of cells that are both susceptible for Agrobacterium infection and have the ability to regenerate. Here, we describe a protocol for the efficient regeneration of transgenic sweet pepper (C. annuum) through inducible activation of the BABY BOOM (BBM) AP2/ERF transcription factor. Using this approach, we can routinely achieve a transformation efficiency of at least 0.6 %. The main improvements in this protocol are the reproducibility in transforming different genotypes and the ability to produce fertile shoots. An added advantage of this protocol is that BBM activity can be induced subsequently in stable transgenic lines, providing a novel regeneration system for clonal propagation through somatic embryogenesis.

A Modular and Affordable Time-Lapse Imaging and Incubation System Based on 3D-Printed Parts, a Smartphone, and Off-The-Shelf Electronics

PubMed Central

Schwan, Emil; Fatsis-Kavalopoulos, Nikos; Kreuger, Johan

2016-01-01

Time-lapse imaging is a powerful tool for studying cellular dynamics and cell behavior over long periods of time to acquire detailed functional information. However, commercially available time-lapse imaging systems are expensive and this has limited a broader implementation of this technique in low-resource environments. Further, the availability of time-lapse imaging systems often present workflow bottlenecks in well-funded institutions. To address these limitations we have designed a modular and affordable time-lapse imaging and incubation system (ATLIS). The ATLIS enables the transformation of simple inverted microscopes into live cell imaging systems using custom-designed 3D-printed parts, a smartphone, and off-the-shelf electronic components. We demonstrate that the ATLIS provides stable environmental conditions to support normal cell behavior during live imaging experiments in both traditional and evaporation-sensitive microfluidic cell culture systems. Thus, the system presented here has the potential to increase the accessibility of time-lapse microscopy of living cells for the wider research community. PMID:28002463

A Modular and Affordable Time-Lapse Imaging and Incubation System Based on 3D-Printed Parts, a Smartphone, and Off-The-Shelf Electronics.

PubMed

Hernández Vera, Rodrigo; Schwan, Emil; Fatsis-Kavalopoulos, Nikos; Kreuger, Johan

2016-01-01

Time-lapse imaging is a powerful tool for studying cellular dynamics and cell behavior over long periods of time to acquire detailed functional information. However, commercially available time-lapse imaging systems are expensive and this has limited a broader implementation of this technique in low-resource environments. Further, the availability of time-lapse imaging systems often present workflow bottlenecks in well-funded institutions. To address these limitations we have designed a modular and affordable time-lapse imaging and incubation system (ATLIS). The ATLIS enables the transformation of simple inverted microscopes into live cell imaging systems using custom-designed 3D-printed parts, a smartphone, and off-the-shelf electronic components. We demonstrate that the ATLIS provides stable environmental conditions to support normal cell behavior during live imaging experiments in both traditional and evaporation-sensitive microfluidic cell culture systems. Thus, the system presented here has the potential to increase the accessibility of time-lapse microscopy of living cells for the wider research community.

T-DNA transfer and integration in the ectomycorrhizal fungus Suillus bovinus using hygromycin B as a selectable marker.

PubMed

Hanif, Mubashir; Pardo, Alejandro Guillermo; Gorfer, Markus; Raudaskoski, Marjatta

2002-06-01

The T-DNA of Agrobacterium tumefaciens can be transferred to plants, yeasts, fungi and human cells. Using this system, dikaryotic mycelium of the ectomycorrhizal fungus Suillus bovinus was transformed with recombinant hygromycin B phosphotransferase (hph)and enhanced green fluorescent protein (EGFP) genes fused with a heterologous fungal promoter and CaMV35S terminator. Transformation resulted in hygromycin B-resistant clones, which were mitotically stable. Putative transformants were analysed for the presence of hph and EGFP genes by PCR and Southern analysis. The latter analysis proved both multiple- and single-copy integrations of the genes in the S. bovinus genome. A. tumeficiens transformation should make possible the development of tagged mutagenesis and targeted gene disruption technology for S. bovinus.

Recommended GIS Analysis Methods for Global Gridded Population Data

NASA Astrophysics Data System (ADS)

Frye, C. E.; Sorichetta, A.; Rose, A.

2017-12-01

When using geographic information systems (GIS) to analyze gridded, i.e., raster, population data, analysts need a detailed understanding of several factors that affect raster data processing, and thus, the accuracy of the results. Global raster data is most often provided in an unprojected state, usually in the WGS 1984 geographic coordinate system. Most GIS functions and tools evaluate data based on overlay relationships (area) or proximity (distance). Area and distance for global raster data can be either calculated directly using the various earth ellipsoids or after transforming the data to equal-area/equidistant projected coordinate systems to analyze all locations equally. However, unlike when projecting vector data, not all projected coordinate systems can support such analyses equally, and the process of transforming raster data from one coordinate space to another often results unmanaged loss of data through a process called resampling. Resampling determines which values to use in the result dataset given an imperfect locational match in the input dataset(s). Cell size or resolution, registration, resampling method, statistical type, and whether the raster represents continuous or discreet information potentially influence the quality of the result. Gridded population data represent estimates of population in each raster cell, and this presentation will provide guidelines for accurately transforming population rasters for analysis in GIS. Resampling impacts the display of high resolution global gridded population data, and we will discuss how to properly handle pyramid creation using the Aggregate tool with the sum option to create overviews for mosaic datasets.

A 3D tension bioreactor platform to study the interplay between ECM stiffness and tumor phenotype.

PubMed

Cassereau, Luke; Miroshnikova, Yekaterina A; Ou, Guanqing; Lakins, Johnathon; Weaver, Valerie M

2015-01-10

Extracellular matrix (ECM) structure, composition, and stiffness have profound effects on tissue development and pathologies such as cardiovascular disease and cancer. Accordingly, a variety of synthetic hydrogel systems have been designed to study the impact of ECM composition, density, mechanics, and topography on cell and tissue phenotype. However, these synthetic systems fail to accurately recapitulate the biological properties and structure of the native tissue ECM. Natural three dimensional (3D) ECM hydrogels, such as collagen or hyaluronic acid, feature many of the chemical and physical properties of tissue, yet, these systems have limitations including the inability to independently control biophysical properties such as stiffness and pore size. Here, we present a 3D tension bioreactor system that permits precise mechanical tuning of collagen hydrogel stiffness, while maintaining consistent composition and pore size. We achieve this by mechanically loading collagen hydrogels covalently-conjugated to a polydimethylsiloxane (PDMS) membrane to induce hydrogel stiffening. We validated the biological application of this system with oncogenically transformed mammary epithelial cell organoids embedded in a 3D collagen I hydrogel, either uniformly stiffened or calibrated to create a gradient of ECM stiffening, to visually demonstrate the impact of ECM stiffening on transformation and tumor cell invasion. As such, this bioreactor presents the first tunable 3D natural hydrogel system that is capable of independently assessing the role of ECM stiffness on tissue phenotype. Copyright © 2014 Elsevier B.V. All rights reserved.

Development of an optimized tetracycline-inducible expression system to increase the accumulation of interleukin-10 in tobacco BY-2 suspension cells

PubMed Central

2012-01-01

Background Plant cell suspension cultures can be used for the production of valuable pharmaceutical and industrial proteins. When the recombinant protein is secreted into the culture medium, restricting expression to a defined growth phase can improve both the quality and quantity of the recovered product by minimizing proteolytic activity. Temporal restriction is also useful for recombinant proteins whose constitutive expression affects cell growth and viability, such as viral interleukin-10 (vIL-10). Results We have developed a novel, tetracycline-inducible system suitable for tobacco BY-2 suspension cells which increases the yields of vIL-10. The new system is based on a binary vector that is easier to handle than conventional vectors, contains an enhanced inducible promoter and 5′-UTR to improve yields, and incorporates a constitutively-expressed visible marker gene to allow the rapid and straightforward selection of the most promising transformed clones. Stable transformation of BY-2 cells with this vector, without extensive optimization of the induction conditions, led to a 3.5 fold increase in vIL-10 levels compared to constitutive expression in the same host. Conclusions We have developed an effective and straightforward molecular farming platform technology that improves both the quality and the quantity of recombinant proteins produced in plant cells, particularly those whose constitutive expression has a negative impact on plant growth and development. Although we tested the platform using vIL-10 produced in BY-2 cells, it can be applied to other host/product combinations and is also useful for basic research requiring strictly controlled transgene expression. PMID:22784336

[Transforming gene in human esophageal carcinoma tissue].

PubMed

Jiang, W

1988-09-01

The transforming gene in human esophageal carcinoma (HEC) tissues collected from Lin-xian county, a high incidence area of esophageal cancer was studied. Eight primary HEC tissues were used as sources for the preparation of DNA. High molecular weight DNAs were separately added to NIH 3T3 cells by the calcium phosphate coprecipitation method. Of the 8 HEC tissues examined, 3 DNAs showed transforming activity and produced secondary transformants. The use of uncloned NIH 3T3 cells resulted in the appearances of non-transforming. The efficiency of primary transfection foci was low (0.025--0.05 focus per ug of DNA). In the secondary transfection, the efficiency was increased (0.30 focus per ug of DNA). The primary and secondary transformants were capable of forming colonies in soft agar (0.33%) in contrast to the control NIH 3T3 cells, which did not show any anchorage-independent growth. About 1 X 10(6) cells of the cloned secondary transformants were injected subcutaneously into athymic BALB/c nude mice. The mice developed large tumors (approximately 20-30 mm in diameter) within 5--15 days after injection. No tumor developed in mice injected with control NIH 3T3 cells even after 2 months. The transforming DNA had a linkage to the Alu sequence, indicating that a common human DNA fragment is conserved in the tumors. H-ras was found in the transforming DNA using Southern blot assay.

Species-Specific Elements in the Large T-Antigen J Domain Are Required for Cellular Transformation and DNA Replication by Simian Virus 40

PubMed Central

Sullivan, Christopher S.; Tremblay, James D.; Fewell, Sheara W.; Lewis, John A.; Brodsky, Jeffrey L.; Pipas, James M.

2000-01-01

The J domain of simian virus 40 (SV40) large T antigen is required for efficient DNA replication and transformation. Despite previous reports demonstrating the promiscuity of J domains in heterologous systems, results presented here show the requirement for specific J-domain sequences in SV40 large-T-antigen-mediated activities. In particular, chimeric-T-antigen constructs in which the SV40 T-antigen J domain was replaced with that from the yeast Ydj1p or Escherichia coli DnaJ proteins failed to replicate in BSC40 cells and did not transform REF52 cells. However, T antigen containing the JC virus J domain was functional in these assays, although it was less efficient than the wild type. The inability of some large-T-antigen chimeras to promote DNA replication and elicit cellular transformation was not due to a failure to interact with hsc70, since a nonfunctional chimera, containing the DnaJ J domain, bound hsc70. However, this nonfunctional chimeric T antigen was reduced in its ability to stimulate hsc70 ATPase activity and unable to liberate E2F from p130, indicating that transcriptional activation of factors required for cell growth and DNA replication may be compromised. Our data suggest that the T-antigen J domain harbors species-specific elements required for viral activities in vivo. PMID:10891510

Improved ethanol production in the presence of cadmium ions by a Saccharomyces cerevisiae transformed with a novel cadmium-resistance gene DvCRP1.

PubMed

Hu, Jiajun; Xu, Qingyun; Wu, Mengnan; Meng, Xiangzong; Song, Rentao; Gao, Mintian

2016-11-01

The DvCRP1 gene obtained from Dunaliella viridis is a cadmium-resistance gene that induces cadmium accumulation in microbial and plant cells. In the present study, Saccharomyces cerevisiae was used as a model system to investigate the effect of DvCRP1 on both cadmium detoxification and ethanol production. Inhibitory effects of cadmium (50-300 µmol/L) on growth (29-92%), glucose consumption (23-89%), and ethanol production (17-92%) were observed at 24 h by S. cerevisiae. DvCRP1 alleviated the inhibitory effect of cadmium, with increase in the ethanol production. The established mathematical model showed that the initial inoculation concentration, cadmium concentration, and transformation of DvCRP1 were the most important factors for cell growth, glucose consumption, and ethanol production. Cadmium detoxification of yeast was also enhanced by increasing the initial concentration of yeast cells. Transforming with DvCRP1 further enhanced detoxification, especially at high cadmium concentrations. Transforming with DvCRP1 further enhanced detoxification, especially at high cadmium concentrations (200 µmol/L). The present results evidenced the potential of the insertion of the DvCRP1 gene into yeast for use in bio-refineries during fermentation of heavy metals-contaminated substrates. In addition, this is a promising method for phytoremediation of agricultural soils highly contaminated by heavy metals.

Mycoplasmal Infections Prevent Apoptosis and Induce Malignant Transformation of Interleukin-3-Dependent 32D Hematopoietic Cells

PubMed Central

Feng, Shaw-Huey; Tsai, Shien; Rodriguez, Jose; Lo, Shyh-Ching

1999-01-01

32D cells, a murine myeloid cell line, rapidly undergo apoptosis upon withdrawal of interleukin-3 (IL-3) supplement in culture. We found that 32D cells, if infected by several species of human mycoplasmas that rapidly activated NF-κB, would live and continue to grow in IL-3-depleted culture. Mycoplasma-infected cells showed no evidence of autocrine production of IL-3. Pyrrolidine dithiocarbamate (PDTC) blocked activation of NF-κB and led to prominent cell death. Heat-killed mycoplasmas or mycoplasmal membrane preparations alone could support continued growth of 32D cells in culture without IL-3 supplement for a substantial period of time. However, upon removal of heat-inactivated mycoplasmas, 32D cells quickly became apoptotic. In comparison, live Mycoplasma fermentans or M. penetrans infection for 4 to 5 weeks induced malignant transformation of 32D cells. Transformed 32D cells grew autonomously and no longer required support of growth-stimulating factors including IL-3 and mycoplasmas. The transformed 32D cells quickly formed tumors when injected into nude mice. Karyotyping showed that development of chromosomal changes and trisomy 19 was often associated with malignant transformation and tumorigenicity of 32D cells. Mycoplasmal infections apparently affected the fidelity of genomic transmission in cell division as well as checkpoints coordinating the progression of cell cycle events. PMID:10567525

The neurovascular unit, matrix proteases, and innate inflammation.

PubMed

del Zoppo, Gregory J

2010-10-01

In the central nervous system, microvessel-neuron interactions appear highly coordinated. The rapid simultaneous responses of the microvasculature, neurons, and glia to focal ischemia in experimental ischemic stroke suggest that these responses could be viewed in a unitary fashion, rather than as individual components. The "neurovascular unit" consists of microvessels (endothelial cells-basal lamina matrix-astrocyte end-feet [and pericytes]), astrocytes, neurons and their axons, and other supporting cells that are likely to modulate the function of the "unit." Each cell component generates an inflammatory response to ischemia. Matrix metalloproteinase (MMP)-9 was first associated with hemorrhagic transformation following focal ischemia in an experimental model. A series of studies of ischemic stroke patients also suggests a relationship between MMP-9 levels and several consequences of ischemic injury, including hemorrhagic transformation. Recent experimental work suggests specific cell sources for MMP-9 generation and for matrix proteases from four distinct families that could impact neurovascular unit integrity. © 2010 New York Academy of Sciences.

Quantifying Multistate Cytoplasmic Molecular Diffusion in Bacterial Cells via Inverse Transform of Confined Displacement Distribution.

PubMed

Chen, Tai-Yen; Jung, Won; Santiago, Ace George; Yang, Feng; Krzemiński, Łukasz; Chen, Peng

2015-11-12

Single-molecule tracking (SMT) of fluorescently tagged cytoplasmic proteins can provide valuable information on the underlying biological processes in living cells via subsequent analysis of the displacement distributions; however, the confinement effect originated from the small size of a bacterial cell skews the protein's displacement distribution and complicates the quantification of the intrinsic diffusive behaviors. Using the inverse transformation method, we convert the skewed displacement distribution (for both 2D and 3D imaging conditions) back to that in free space for systems containing one or multiple (non)interconverting Brownian diffusion states, from which we can reliably extract the number of diffusion states as well as their intrinsic diffusion coefficients and respective fractional populations. We further demonstrate a successful application to experimental SMT data of a transcription factor in living E. coli cells. This work allows a direct quantitative connection between cytoplasmic SMT data with diffusion theory for analyzing molecular diffusive behavior in live bacteria.

Quantifying Multistate Cytoplasmic Molecular Diffusion in Bacterial Cells via Inverse Transform of Confined Displacement Distribution

PubMed Central

2016-01-01

Single-molecule tracking (SMT) of fluorescently tagged cytoplasmic proteins can provide valuable information on the underlying biological processes in living cells via subsequent analysis of the displacement distributions; however, the confinement effect originated from the small size of a bacterial cell skews the protein’s displacement distribution and complicates the quantification of the intrinsic diffusive behaviors. Using the inverse transformation method, we convert the skewed displacement distribution (for both 2D and 3D imaging conditions) back to that in free space for systems containing one or multiple (non)interconverting Brownian diffusion states, from which we can reliably extract the number of diffusion states as well as their intrinsic diffusion coefficients and respective fractional populations. We further demonstrate a successful application to experimental SMT data of a transcription factor in living E. coli cells. This work allows a direct quantitative connection between cytoplasmic SMT data with diffusion theory for analyzing molecular diffusive behavior in live bacteria. PMID:26491971

Shape Transformations of Epithelial Shells

PubMed Central

Misra, Mahim; Audoly, Basile; Kevrekidis, Ioannis G.; Shvartsman, Stanislav Y.

2016-01-01

Regulated deformations of epithelial sheets are frequently foreshadowed by patterning of their mechanical properties. The connection between patterns of cell properties and the emerging tissue deformations is studied in multiple experimental systems, but the general principles remain poorly understood. For instance, it is in general unclear what determines the direction in which the patterned sheet is going to bend and whether the resulting shape transformation will be discontinuous or smooth. Here these questions are explored computationally, using vertex models of epithelial shells assembled from prismlike cells. In response to rings and patches of apical cell contractility, model epithelia smoothly deform into invaginated or evaginated shapes similar to those observed in embryos and tissue organoids. Most of the observed effects can be captured by a simpler model with polygonal cells, modified to include the effects of the apicobasal polarity and natural curvature of epithelia. Our models can be readily extended to include the effects of multiple constraints and used to describe a wide range of morphogenetic processes. PMID:27074691

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

Carney, H.J.; Hass, B.S.

In order to test the thousands of man-made chemicals in the environment for carcinogenic and genetic hazards, a multitude of short-term screening tests has been developed to complement long-term mammalian bioassays and epidemiological studies. These tests cover a broad spectrum of organisms, and include the use of naked and viral nucleic acids, bacteria, fungi, higher plants, insects in vitro mammalian cell cultures (cell transformation, cell-mediated mutagenesis, DNA repair, and chromosome aberration tests) and live mammals. Assay end points include effects on nucleic acids, DNA repair synthesis, point or gene mutation, structural and numerical chromosome aberrations, cytological alterations, and in vitromore » cell transformation. The present review describes and compares these assays. In addition, it discusses their historical development, the problems and limitations associated with their use, and their implementation in comprehensive testing programs. It is intended to provide overview and specific information to the laboratory that is in the process of establishing genetic toxicological systems. (The literature is reviewed to January 1978.)« less

Pineapple [Ananas comosus (L.) Merr].

PubMed

Yabor, Lourdes; Espinosa, Patricia; Arencibia, Ariel D; Lorenzo, José C

2006-01-01

A procedure for pineapple [Ananas comosus (L.) Merr.] genetic transformation is described, which involves temporary immersion bioreactors (TIB) for selection of transgenic plants. Success in the production of transgenic pineapple plants combines tissue culture factors. Firstly, the use of regenerable pineapple callus as starting material for transformation whose cells shown to be competent for Agrobacterium infection. Secondly, the used of filtered callus, resulting in homogeneously sized clusters, thereby increasing the contact between the cell surfaces and A. tumefaciens and releasing phenolic compounds which induce Agrobacterium virulence. Thirdly, regeneration of primary plants without selection pressure, that allowing a massive production of putative transgenic pineapples. Finally, we support that TIB technology is a powerful system to recover nonchimera transgenic plants by micropropagation with the use of an adequate selection agent.

Adaptive Filtering to Enhance Noise Immunity of Impedance and Admittance Spectroscopy: Comparison with Fourier Transformation

NASA Astrophysics Data System (ADS)

Stupin, Daniil D.; Koniakhin, Sergei V.; Verlov, Nikolay A.; Dubina, Michael V.

2017-05-01

The time-domain technique for impedance spectroscopy consists of computing the excitation voltage and current response Fourier images by fast or discrete Fourier transformation and calculating their relation. Here we propose an alternative method for excitation voltage and current response processing for deriving a system impedance spectrum based on a fast and flexible adaptive filtering method. We show the equivalence between the problem of adaptive filter learning and deriving the system impedance spectrum. To be specific, we express the impedance via the adaptive filter weight coefficients. The noise-canceling property of adaptive filtering is also justified. Using the RLC circuit as a model system, we experimentally show that adaptive filtering yields correct admittance spectra and elements ratings in the high-noise conditions when the Fourier-transform technique fails. Providing the additional sensitivity of impedance spectroscopy, adaptive filtering can be applied to otherwise impossible-to-interpret time-domain impedance data. The advantages of adaptive filtering are justified with practical living-cell impedance measurements.

DNA Uptake by Transformable Bacteria

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

Lacks, Sanford A.

1999-03-31

The various processes of DNA uptake by cells can be categorized as: viral DNA entry, conjugation, or transformation. Within each category, a variety of mechanisms have been found. However, considerable similarities occur among the different mechanisms of conjugation and, especially, transformation. All of these natural mechanisms of DNA transfer are quite elaborate and involve multiple protein components, as the case may be, of the virus, the donor cell, and the recipient cell. The mechanisms of viral infection and conjugation will be discussed mainly with respect to their relevance to transformation.

DNA UPTAKE BY TRANSFORMABLE BACTERIA

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

LACKS,S.A.

1999-09-07

The various processes of DNA uptake by cells can be categorized as: viral DNA entry, conjugation, or transformation. Within each category, a variety of mechanisms have been found. However, considerable similarities occur among the different mechanisms of conjugation and, especially, transformation. All of these natural mechanisms of DNA transfer are quite elaborate and involve multiple protein components, as the case may be, of the virus, the donor cell, and the recipient cell. The mechanisms of viral infection and conjugation will be discussed mainly with respect to their relevance to transformation.

High-speed cell recognition algorithm for ultrafast flow cytometer imaging system.

PubMed

Zhao, Wanyue; Wang, Chao; Chen, Hongwei; Chen, Minghua; Yang, Sigang

2018-04-01

An optical time-stretch flow imaging system enables high-throughput examination of cells/particles with unprecedented high speed and resolution. A significant amount of raw image data is produced. A high-speed cell recognition algorithm is, therefore, highly demanded to analyze large amounts of data efficiently. A high-speed cell recognition algorithm consisting of two-stage cascaded detection and Gaussian mixture model (GMM) classification is proposed. The first stage of detection extracts cell regions. The second stage integrates distance transform and the watershed algorithm to separate clustered cells. Finally, the cells detected are classified by GMM. We compared the performance of our algorithm with support vector machine. Results show that our algorithm increases the running speed by over 150% without sacrificing the recognition accuracy. This algorithm provides a promising solution for high-throughput and automated cell imaging and classification in the ultrafast flow cytometer imaging platform. (2018) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE).

High-speed cell recognition algorithm for ultrafast flow cytometer imaging system

NASA Astrophysics Data System (ADS)

Zhao, Wanyue; Wang, Chao; Chen, Hongwei; Chen, Minghua; Yang, Sigang

2018-04-01

An optical time-stretch flow imaging system enables high-throughput examination of cells/particles with unprecedented high speed and resolution. A significant amount of raw image data is produced. A high-speed cell recognition algorithm is, therefore, highly demanded to analyze large amounts of data efficiently. A high-speed cell recognition algorithm consisting of two-stage cascaded detection and Gaussian mixture model (GMM) classification is proposed. The first stage of detection extracts cell regions. The second stage integrates distance transform and the watershed algorithm to separate clustered cells. Finally, the cells detected are classified by GMM. We compared the performance of our algorithm with support vector machine. Results show that our algorithm increases the running speed by over 150% without sacrificing the recognition accuracy. This algorithm provides a promising solution for high-throughput and automated cell imaging and classification in the ultrafast flow cytometer imaging platform.

New algorithm and system for measuring size distribution of blood cells

NASA Astrophysics Data System (ADS)

Yao, Cuiping; Li, Zheng; Zhang, Zhenxi

2004-06-01

In optical scattering particle sizing, a numerical transform is sought so that a particle size distribution can be determined from angular measurements of near forward scattering, which has been adopted in the measurement of blood cells. In this paper a new method of counting and classification of blood cell, laser light scattering method from stationary suspensions, is presented. The genetic algorithm combined with nonnegative least squared algorithm is employed to inverse the size distribution of blood cells. Numerical tests show that these techniques can be successfully applied to measuring size distribution of blood cell with high stability.

Downregulation of proapoptotic Bim augments IL-2-independent T-cell transformation by human T-cell leukemia virus type-1 Tax.

PubMed

Higuchi, Masaya; Takahashi, Masahiko; Tanaka, Yuetsu; Fujii, Masahiro

2014-12-01

Human T-cell leukemia virus type 1 (HTLV-1), an etiological agent of adult T-cell leukemia, immortalizes and transforms primary human T cells in vitro in both an interleukin (IL)-2-dependent and IL-2-independent manner. Expression of the HTLV-1 oncoprotein Tax transforms the growth of the mouse T-cell line CTLL-2 from being IL-2-dependent to IL-2-independent. Withdrawal of IL-2 from normal activated T cells induces apoptosis, which is mediated through the inducible expression of several proapoptotic proteins, including Bim. In this study, we found that Tax protects IL-2-depleted T cells against Bim-induced apoptosis. Withdrawal of IL-2 from CTLL-2 cells induced a prominent increase in the level of Bim protein in CTLL-2 cells, but not in Tax-transformed CTLL-2 cells. This inhibition of Bim in Tax-transformed CTLL-2 cells was mediated by two mechanisms: downregulation of Bim mRNA and posttranscriptional reduction of Bim protein. Transient expression of Tax in CTLL-2 cells also inhibited IL-2 depletion-induced expression of Bim, however, this decrease in Bim protein expression was not due to downregulation of Bim mRNA, thus indicating that Bim mRNA downregulation in Tax-transformed CTLL-2 occurs only after long-term expression of Tax. Transient expression of Tax in CTLL-2 cells also induced Erk activation, however, this was not involved in the reduction of Bim protein. Knockdown of Bim expression in CTLL-2 cells augmented Tax-induced IL-2-independent transformation. HTLV-1 infection of human T cells also reduced their levels of Bim protein, and restoring Bim expression in HTLV-1-infected cells reduced their proliferation by inducing apoptosis. Taken together, these results indicate that Tax-induced downregulation of Bim in HTLV-1-infected T cells promotes their IL-2-independent growth, thereby supporting the persistence of HTLV-1 infection in vivo. © 2014 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Althaea rosea Cavanil and Plantago major L. suppress neoplastic cell transformation through the inhibition of epidermal growth factor receptor kinase.

PubMed

Choi, Eun-Sun; Cho, Sung-Dae; Shin, Ji-Ae; Kwon, Ki Han; Cho, Nam-Pyo; Shim, Jung-Hyun

2012-10-01

For thousands of years in Asia, Althaea rosea Cavanil (ARC) and Plantago major L. (PML) have been used as powerful non-toxic therapeutic agents that inhibit inflammation. However, the anticancer mechanisms and molecular targets of ARC and PML are poorly understood, particularly in epidermal growth factor (EGF)-induced neoplastic cell transformation. The aim of this study was to evaluate the chemopreventive effects and mechanisms of the methanol extracts from ARC (MARC) and PML (MPML) in EGF-induced neoplastic cell transformation of JB6 P+ mouse epidermal cells using an MTS assay, anchorage-independent cell transformation assay and western blotting. Our results showed that MARC and MPML significantly suppressed neoplastic cell transformation by inhibiting the kinase activity of the EGF receptor (EGFR). The activation of EGFR by EGF was suppressed by MARC and MPML treatment in EGFR(+/+) cells, but not in EGFR(-/-) cells. In addition, MARC and MPML inhibited EGF-induced cell proliferation in EGFR-expressing murine embryonic fibroblasts (EGFR(+/+)). These results strongly indicate that EGFR targeting by MARC and MPML may be a good strategy for chemopreventive or chemotherapeutic applications.

Differential DNA methylation profile of key genes in malignant prostate epithelial cells transformed by inorganic arsenic or cadmium.

PubMed

Pelch, Katherine E; Tokar, Erik J; Merrick, B Alex; Waalkes, Michael P

2015-08-01

Previous work shows altered methylation patterns in inorganic arsenic (iAs)- or cadmium (Cd)-transformed epithelial cells. Here, the methylation status near the transcriptional start site was assessed in the normal human prostate epithelial cell line (RWPE-1) that was malignantly transformed by 10μM Cd for 11weeks (CTPE) or 5μM iAs for 29weeks (CAsE-PE), at which time cells showed multiple markers of acquired cancer phenotype. Next generation sequencing of the transcriptome of CAsE-PE cells identified multiple dysregulated genes. Of the most highly dysregulated genes, five genes that can be relevant to the carcinogenic process (S100P, HYAL1, NTM, NES, ALDH1A1) were chosen for an in-depth analysis of the DNA methylation profile. DNA was isolated, bisulfite converted, and combined bisulfite restriction analysis was used to identify differentially methylated CpG sites, which was confirmed with bisulfite sequencing. Four of the five genes showed differential methylation in transformants relative to control cells that was inversely related to altered gene expression. Increased expression of HYAL1 (>25-fold) and S100P (>40-fold) in transformants was correlated with hypomethylation near the transcriptional start site. Decreased expression of NES (>15-fold) and NTM (>1000-fold) in transformants was correlated with hypermethylation near the transcriptional start site. ALDH1A1 expression was differentially expressed in transformed cells but was not differentially methylated relative to control. In conclusion, altered gene expression observed in Cd and iAs transformed cells may result from altered DNA methylation status. Published by Elsevier Inc.

Role of Oxidative Stress in Transformation Induced by Metal Mixture

PubMed Central

Martín, Silva-Aguilar; Emilio, Rojas; Mahara, Valverde

2011-01-01

Metals are ubiquitous pollutants present as mixtures. In particular, mixture of arsenic-cadmium-lead is among the leading toxic agents detected in the environment. These metals have carcinogenic and cell-transforming potential. In this study, we used a two step cell transformation model, to determine the role of oxidative stress in transformation induced by a mixture of arsenic-cadmium-lead. Oxidative damage and antioxidant response were determined. Metal mixture treatment induces the increase of damage markers and the antioxidant response. Loss of cell viability and increased transforming potential were observed during the promotion phase. This finding correlated significantly with generation of reactive oxygen species. Cotreatment with N-acetyl-cysteine induces effect on the transforming capacity; while a diminution was found in initiation, in promotion phase a total block of the transforming capacity was observed. Our results suggest that oxidative stress generated by metal mixture plays an important role only in promotion phase promoting transforming capacity. PMID:22191014

Down-regulation of Rab5 decreases characteristics associated with maintenance of cell transformation

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

Silva, Patricio; Soto, Nicolás; Díaz, Jorge

2015-08-21

The early endosomal protein Rab5 is highly expressed in tumor samples, although a causal relationship between Rab5 expression and cell transformation has not been established. Here, we report the functional effects of targeting endogenous Rab5 with specific shRNA sequences in different tumor cell lines. Rab5 down-regulation in B16-F10 cells decreased tumor formation by subcutaneous injection into C57/BL6 mice. Accordingly, Rab5 targeting in B16-F10 and A549, but not MDA-MB-231 cells was followed by decreased cell proliferation, increased apoptosis and decreased anchorage-independent growth. These findings suggest that Rab5 expression is required to maintain characteristics associated with cell transformation. - Highlights: • Rab5more » is important to the maintenance of cell transformation characteristics. • Down-regulation of Rab5 decreases cell proliferation and increases apoptosis in different cancer cells. • Rab5 is required for anchorage-independent growth and tumorigenicity in-vivo.« less

Establishment and characterization of Macaca fascicularis lymphoblastoid cell lines.

PubMed

Manning, C H; Heise, E R

1992-01-01

A panel of cynomolgus macaque lymphoblastoid cell lines (LCL) was established by transforming peripheral blood mononuclear cells (PBMC) with Herpesvirus papio (HVP), and selected lines were examined by flow cytometry. Results indicate that HVP-transformed macaque LCL are phenotypically heterogeneous and resemble human Epstein-Barr virus (EBV)-transformed LCL in the abundant expression of major histocompatibility complex (MHC) class I and class II molecules. At least some lines are of B cell origin.

Endometriosis

MedlinePlus

... bleed over the course of each menstrual cycle. Transformation of peritoneal cells. In what's known as the " ... experts propose that hormones or immune factors promote transformation of peritoneal cells — cells that line the inner ...

Smad2-dependent glycosaminoglycan elongation in aortic valve interstitial cells enhances binding of LDL to proteoglycans.

PubMed

Osman, Narin; Grande-Allen, K Jane; Ballinger, Mandy L; Getachew, Robel; Marasco, Silvana; O'Brien, Kevin D; Little, Peter J

2013-01-01

Calcific aortic valve disease is a progressive condition that shares some common pathogenic features with atherosclerosis. Transforming growth factor-β1 is a recognized mediator of atherosclerosis and is expressed in aortic valve lesions. Transforming growth factorβ1 stimulates glycosaminoglycan elongation of proteoglycans that is associated with increased lipid binding. We investigated the presence of transforming growth factor-β1 and downstream signaling intermediates in diseased human aortic valves and the effects of activated transforming growth factor-β1 receptor signaling on aortic valve interstitial cell proteoglycan synthesis and lipid binding as a possible mechanism for the initiation of the early lesion of calcific aortic valve disease. Diseased human aortic valve leaflets demonstrated strong immunohistochemical staining for transforming growth factor-β1 and phosphorylated Smad2/3. In primary porcine aortic valve interstitial cells, Western blots showed that transforming growth factor-β1 stimulated phosphorylation in both the carboxy and linker regions of Smad2/3, which was inhibited by the transforming growth factor-β1 receptor inhibitor SB431542. Gel electrophoresis and size exclusion chromatography demonstrated that SB431542 decreased transforming growth factor-β1-mediated [(35)S]-sulfate incorporation into proteoglycans in a dose-dependent manner. Further, in proteoglycans derived from transforming growth factor-β1-treated valve interstitial cells, gel mobility shift assays demonstrated that inhibition of transforming growth factor-β1 receptor signaling resulted in decreased lipid binding. Classic transforming growth factor-β1 signaling is present in human aortic valves in vivo and contributes to the modification of proteoglycans expressed by valve interstitial cells in vitro. These findings suggest that transforming growth factor-β1 may promote increased low-density lipoprotein binding in the early phases of calcific aortic valve disease. Copyright © 2013 Elsevier Inc. All rights reserved.

Pre-crisis mouse cells show strain-specific covariation in the amount of 54-kilodalton phosphoprotein and in susceptibility to transformation by simian virus 40.

PubMed

Chen, S; Blanck, G; Pollack, R E

1983-09-01

We have used several inbred mouse strains to examine the role of the 54-kilodalton (kDa) cellular phosphoprotein in transformation by the papovavirus simian virus 40. We have measured the endogenous 54-kDa phosphoprotein in cells obtained from these inbred mouse strains. To study the effect of passage, cell cultures were measured for amount of the 54-kDa phosphoprotein at the 2nd and 12th passages. In the absence of any transforming agent, the amount of endogenous 54-kDa phosphoprotein in early pre-crisis mouse cells varied in a strain-specific way. Transformation frequency varied coordinately with endogenous 54-kDa expression. Mouse strains whose cells produced a high level of endogenous 54-kDa phosphoprotein on passage did not further increase its expression after simian virus 40 transformation.

Pre-crisis mouse cells show strain-specific covariation in the amount of 54-kilodalton phosphoprotein and in susceptibility to transformation by simian virus 40.

PubMed Central

Chen, S; Blanck, G; Pollack, R E

1983-01-01

We have used several inbred mouse strains to examine the role of the 54-kilodalton (kDa) cellular phosphoprotein in transformation by the papovavirus simian virus 40. We have measured the endogenous 54-kDa phosphoprotein in cells obtained from these inbred mouse strains. To study the effect of passage, cell cultures were measured for amount of the 54-kDa phosphoprotein at the 2nd and 12th passages. In the absence of any transforming agent, the amount of endogenous 54-kDa phosphoprotein in early pre-crisis mouse cells varied in a strain-specific way. Transformation frequency varied coordinately with endogenous 54-kDa expression. Mouse strains whose cells produced a high level of endogenous 54-kDa phosphoprotein on passage did not further increase its expression after simian virus 40 transformation. Images PMID:6310588

Utility of temporally distinct baculovirus promoters for constitutive and baculovirus-inducible transgene expression in transformed insect cells.

PubMed

Lin, Chi-Hung; Jarvis, Donald L

2013-05-10

Genetically transformed lepidopteran insect cell lines have biotechnological applications as constitutive recombinant protein production platforms and improved hosts for baculovirus-mediated recombinant protein production. Insect cell transformation is often accomplished with a DNA construct(s) encoding a foreign protein(s) under the transcriptional control of a baculovirus immediate early promoter, such as the ie1 promoter. However, the potential utility of increasingly stronger promoters from later baculovirus gene classes, such as delayed early (39K), late (p6.9), and very late (polh), has not been systematically assessed. Hence, we produced DNA constructs encoding secreted alkaline phosphatase (SEAP) under the transcriptional control of each of the four temporally distinct classes of baculovirus promoters, used them to transform insect cells, and compared the levels of SEAP RNA and protein production obtained before and after baculovirus infection. The ie1 construct was the only one that supported SEAP protein production by transformed insect cells prior to baculovirus infection, confirming that only immediate early promoters can be used to isolate transformed insect cells for constitutive recombinant protein production. However, baculovirus infection activated transgene expression by all four classes of baculovirus promoters. After infection, cells transformed with the very late (polh) and late (p6.9) promoter constructs produced the highest levels of SEAP RNA, but only low levels of SEAP protein. Conversely, cells transformed with the immediate early (ie1) and delayed early (39K) promoter constructs produced lower levels of RNA, but equal or higher levels of SEAP protein. Unexpectedly, the 39K promoter construct provided tightly regulated, baculovirus-inducible protein production at higher levels than the later promoter constructs. Thus, this study demonstrated the utility of the 39K promoter for insect cell engineering, particularly when one requires higher levels of effector protein production than obtained with ie1 and/or when constitutive transgene expression adversely impacts host cell fitness and/or genetic stability. Copyright © 2013 Elsevier B.V. All rights reserved.

Arsenic transformation predisposes human skin keratinocytes to UV-induced DNA damage yet enhances their survival apparently by diminishing oxidant response

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

Sun Yang; Kojima, Chikara; Chignell, Colin

2011-09-15

Inorganic arsenic and UV, both human skin carcinogens, may act together as skin co-carcinogens. We find human skin keratinocytes (HaCaT cells) are malignantly transformed by low-level arsenite (100 nM, 30 weeks; termed As-TM cells) and with transformation concurrently undergo full adaptation to arsenic toxicity involving reduced apoptosis and oxidative stress response to high arsenite concentrations. Oxidative DNA damage (ODD) is a possible mechanism in arsenic carcinogenesis and a hallmark of UV-induced skin cancer. In the current work, inorganic arsenite exposure (100 nM) did not induce ODD during the 30 weeks required for malignant transformation. Although acute UV-treatment (UVA, 25 J/cm{supmore » 2}) increased ODD in passage-matched control cells, once transformed by arsenic to As-TM cells, acute UV actually further increased ODD (> 50%). Despite enhanced ODD, As-TM cells were resistant to UV-induced apoptosis. The response of apoptotic factors and oxidative stress genes was strongly mitigated in As-TM cells after UV exposure including increased Bcl2/Bax ratio and reduced Caspase-3, Nrf2, and Keap1 expression. Several Nrf2-related genes (HO-1, GCLs, SOD) showed diminished responses in As-TM cells after UV exposure consistent with reduced oxidant stress response. UV-exposed As-TM cells showed increased expression of cyclin D1 (proliferation gene) and decreased p16 (tumor suppressor). UV exposure enhanced the malignant phenotype of As-TM cells. Thus, the co-carcinogenicity between UV and arsenic in skin cancer might involve adaptation to chronic arsenic exposure generally mitigating the oxidative stress response, allowing apoptotic by-pass after UV and enhanced cell survival even in the face of increased UV-induced oxidative stress and increased ODD. - Highlights: > Arsenic transformation adapted to UV-induced apoptosis. > Arsenic transformation diminished oxidant response. > Arsenic transformation enhanced UV-induced DNA damage.« less

Upregulation of glycolysis and oxidative phosphorylation in benzo[β]pyrene and arsenic-induced rat lung epithelial transformed cells

PubMed Central

Li, Guanwu; Tsao, Sai-Wah; Chiu, Jen-Fu

2016-01-01

Arsenic and benzo[β]pyrene (B[a]P) are common contaminants in developing countries. Many studies have investigated the consequences of arsenic and/or B[a]P-induced cellular transformation, including altered metabolism. In the present study, we show that, in addition to elevated glycolysis, B[a]P/arsenic-induced transformation also stimulates oxidative phosphorylation (OXPHOS). Proteomic data and immunoblot studies demonstrated that enzymatic activities, involved in both glycolysis and OXPHOS, are upregulated in the primary transformed rat lung epithelial cell (TLEC) culture, as well as in subcloned TLEC cell lines (TMCs), indicating that OXPHOS was active and still contributed to energy production. LEC expression, of the glycolytic enzyme phosphoglycerate mutase (PGAM) and the TCA cycle enzyme alpha-ketoglutarate dehydrogenase (OGDH), revealed an alternating cyclic pattern of glycolysis and OXPHOS during cell transformation. We also found that the expression levels of hypoxia-inducible factor-1β were consistent with the pattern of glycolysis during the course of transformation. Low doses of an ATP synthase inhibitor depleted endogenous ATP levels to a greater extent in TLECs, compared to parental LECs, indicating greater sensitivity of B[a]P/arsenic-transformed cells to ATP depletion. However, TLEC cells exhibited better survival under hypoxia, possibly due to further induction of anaerobic glycolysis. Collectively, our data indicate that B[a]P/arsenic-transformed cells can maintain energy production through upregulation of both glycolysis and OXPHOS. Selective inhibition of metabolic pathways may serve as a therapeutic option for cancer therapy. PMID:27276679

The role of miR-200a in mammalian epithelial cell transformation

PubMed Central

Becker, Lindsey E.; Takwi, Apana Agha L.; Lu, Zhongxin; Li, Yong

2015-01-01

Cancer is a multistep disease that begins with malignant cell transformation and frequently culminates in metastasis. MicroRNAs (miRNAs) are small regulatory 21–25 nt RNA molecules and are frequently deregulated in cancer. miR-200a is a member of the miR-200 family, which are known inhibitors of the epithelial-to-mesenchymal transition. As such, the tumor-suppressive role of miR-200a in oncogenesis has been well documented; however, recent studies have found a proliferative role for this miRNA as well as a prometastatic role in the later steps of cancer progression. Little is known about the role of this miRNA in the early stages of cancer, namely, malignant cell transformation. Here, we show that miR-200a alone transforms an immortalized rat epithelial cell line, and miR-200a cooperates with Ras to enhance malignant transformation of an immortalized human epithelial cell line. Furthermore, miR-200a induces cell transformation and tumorigenesis in immunocompromised mice by cooperating with a Ras mutant that activates only the RalGEF effector pathway, but not Ras mutants activating PI3K or Raf effector pathways. This transformative ability is in accordance with miR-200a targeting Fog2 and p53 to activate Akt and directly repress p53 protein levels, respectively. These results demonstrate an oncogenic role for miR-200a and provide a specific cellular context where miR-200a acts as an oncomiR rather than a tumor suppressor by cooperating with an oncogene in malignant cell transformation. PMID:25239643

Malignant Transformation Potentials of Human Umbilical Cord Mesenchymal Stem Cells Both Spontaneously and via 3-Methycholanthrene Induction

PubMed Central

Lai, Xiulan; Liu, Sizheng; Chen, Yezeng; Zheng, Zexin; Xie, Qingdong; Maldonado, Martin; Cai, Zhiwei; Qin, Shan; Ho, Guyu; Ma, Lian

2013-01-01

Human umbilical cord mesenchymal stem cells (HUMSCs) are highly proliferative and can be induced to differentiate into advanced derivatives of all three germ layers. Thus, HUMSCs are considered to be a promising source for cell-targeted therapies and tissue engineering. However there are reports on spontaneous transformation of mesenchymal stem cells (MSCs) derived from human bone marrows. The capacity for HUMSCs to undergo malignant transform spontaneously or via induction by chemical carcinogens is presently unknown. Therefore, we isolated HUMSCs from 10 donors and assessed their transformation potential either spontaneously or by treating them with 3-methycholanthrene (3-MCA), a DNA-damaging carcinogen. The malignant transformation of HUMSCs in vitro was evaluated by morphological changes, proliferation rates, ability to enter cell senescence, the telomerase activity, chromosomal abnormality, and the ability to form tumors in vivo. Our studies showed that HUMSCs from all 10 donors ultimately entered senescence and did not undergo spontaneous malignant transformation. However, HUMSCs from two of the 10 donors treated with 3-MCA displayed an increased proliferation rate, failed to enter senescence, and exhibited an altered cell morphology. When these cells (tHUMSCs) were injected into immunodeficient mice, they gave rise to sarcoma-like or poorly differentiated tumors. Moreover, in contrast to HUMSCs, tHUMSCs showed a positive expression of human telomerase reverse transcriptase (hTERT) and did not exhibit a shortening of the relative telomere length during the long-term culture in vitro. Our studies demonstrate that HUMSCs are not susceptible to spontaneous malignant transformation. However, the malignant transformation could be induced by chemical carcinogen 3-MCA. PMID:24339974

System Design, Implementation, and Evaluation of the Optical Broadband Correlator

DTIC Science & Technology

1994-09-20

shear-mode TeO2 , Model No. N45075-6-20, manufactured by Newport Electro- Optic Systems with a length of 75 pjs, acoustic direction 1110], optical...optical aperture (or useful length) TOA of our cells are shown in Table 3. The Bragg cells are shear-mode TeO2 , Model No. N45075-6-20, manufactured by...focusing or integrating (Fourier transform) lens is a laser diode glass doublet Model 06LAI013/076, from Melles Griot. Its focal length is 145 nun at 830

Pioneering a Biobased UAS

NASA Technical Reports Server (NTRS)

Block, Eli; Byemerwa, Jovita; Dispenza, Ross; Doughty, Benjamin; Gillyard, KaNesha; Godbole, Poorwa; Gonzales-Wright, Jeanette; Hull, Ian; Kannappan, Jotthe; Levine, Alexander;

Pioneering a Biobased UAS

NASA Technical Reports Server (NTRS)

Block, Eli; Byemerwa, Jovita; Dispenza, Ross; Doughty, Benjamin; Gillyard, KaNesha; Godbole, Poorwa; Gonzalez-Wright, Jeanette; Hull, Ian; Kannappan, Jotthe; Levine, Alexander;

Discrete diffusion models to study the effects of Mg2+ concentration on the PhoPQ signal transduction system

PubMed Central

2010-01-01

Background The challenge today is to develop a modeling and simulation paradigm that integrates structural, molecular and genetic data for a quantitative understanding of physiology and behavior of biological processes at multiple scales. This modeling method requires techniques that maintain a reasonable accuracy of the biological process and also reduces the computational overhead. This objective motivates the use of new methods that can transform the problem from energy and affinity based modeling to information theory based modeling. To achieve this, we transform all dynamics within the cell into a random event time, which is specified through an information domain measure like probability distribution. This allows us to use the “in silico” stochastic event based modeling approach to find the molecular dynamics of the system. Results In this paper, we present the discrete event simulation concept using the example of the signal transduction cascade triggered by extra-cellular Mg2+ concentration in the two component PhoPQ regulatory system of Salmonella Typhimurium. We also present a model to compute the information domain measure of the molecular transport process by estimating the statistical parameters of inter-arrival time between molecules/ions coming to a cell receptor as external signal. This model transforms the diffusion process into the information theory measure of stochastic event completion time to get the distribution of the Mg2+ departure events. Using these molecular transport models, we next study the in-silico effects of this external trigger on the PhoPQ system. Conclusions Our results illustrate the accuracy of the proposed diffusion models in explaining the molecular/ionic transport processes inside the cell. Also, the proposed simulation framework can incorporate the stochasticity in cellular environments to a certain degree of accuracy. We expect that this scalable simulation platform will be able to model more complex biological systems with reasonable accuracy to understand their temporal dynamics. PMID:21143785

Discrete diffusion models to study the effects of Mg2+ concentration on the PhoPQ signal transduction system.

PubMed

Ghosh, Preetam; Ghosh, Samik; Basu, Kalyan; Das, Sajal K; Zhang, Chaoyang

2010-12-01

The challenge today is to develop a modeling and simulation paradigm that integrates structural, molecular and genetic data for a quantitative understanding of physiology and behavior of biological processes at multiple scales. This modeling method requires techniques that maintain a reasonable accuracy of the biological process and also reduces the computational overhead. This objective motivates the use of new methods that can transform the problem from energy and affinity based modeling to information theory based modeling. To achieve this, we transform all dynamics within the cell into a random event time, which is specified through an information domain measure like probability distribution. This allows us to use the "in silico" stochastic event based modeling approach to find the molecular dynamics of the system. In this paper, we present the discrete event simulation concept using the example of the signal transduction cascade triggered by extra-cellular Mg2+ concentration in the two component PhoPQ regulatory system of Salmonella Typhimurium. We also present a model to compute the information domain measure of the molecular transport process by estimating the statistical parameters of inter-arrival time between molecules/ions coming to a cell receptor as external signal. This model transforms the diffusion process into the information theory measure of stochastic event completion time to get the distribution of the Mg2+ departure events. Using these molecular transport models, we next study the in-silico effects of this external trigger on the PhoPQ system. Our results illustrate the accuracy of the proposed diffusion models in explaining the molecular/ionic transport processes inside the cell. Also, the proposed simulation framework can incorporate the stochasticity in cellular environments to a certain degree of accuracy. We expect that this scalable simulation platform will be able to model more complex biological systems with reasonable accuracy to understand their temporal dynamics.

p53 functional impairment and high p21waf1/cip1 expression in human T-cell lymphotropic/leukemia virus type I-transformed T cells.

PubMed

Cereseto, A; Diella, F; Mulloy, J C; Cara, A; Michieli, P; Grassmann, R; Franchini, G; Klotman, M E

1996-09-01

Human T-cell lymphotropic/leukemia virus type I (HTLV-I) is associated with T-cell transformation both in vivo and in vitro. Although some of the mechanisms responsible for transformation remain unknown, increasing evidence supports a direct role of viral as well as dysregulated cellular proteins in transformation. We investigated the potential role of the tumor suppressor gene p53 and of the p53-regulated gene, p21waf1/cip1 (wild-type p53 activated fragment 1/cycling dependent kinases [cdks] interacting protein 1), in HTLV-I-infected T cells. We have found that the majority of HTLV-I-infected T cells have the wild-type p53 gene. However, its function in HTLV-I-transformed cells appears to be impaired, as shown by the lack of appropriate p53-mediated responses to ionizing radiation (IR). Interestingly, the expression of the p53 inducible gene, p21waf1/cip1, is elevated at the messenger ribonucleic acid and protein levels in all HTLV-I-infected T-cell lines examined as well as in Taxl-1, a human T-cell line stably expressing Tax. Additionally, Tax induces upregulation of a p21waf1/cip1 promoter-driven luciferase gene in p53 null cells, and increases p21waf1/cip1 expression in Jurkat T cells. These findings suggest that the Tax protein is at least partially responsible for the p53-independent expression of p21waf1/cip1 in HTLV-I-infected cells. Dysregulation of p53 and p21waf1/cip1 proteins regulating cell-cycle progression, may represent an important step in HTLV-I-induced T-cell transformation.

Variation of Keratin 7 Expression and Other Phenotypic Characteristics of Independent Isolates of Cadmium Transformed Human Urothelial Cells (UROtsa)

PubMed Central

Somji, Seema; Zhou, Xu Dong; Mehus, Aaron; Sens, Mary Ann; Garrett, Scott H.; Lutz, Krista L.; Dunlevy, Jane R.; Zheng, Yun; Sens, Donald. A.

2009-01-01

This laboratory has shown that a human urothelial cell line (UROtsa) transformed by cadmium (Cd+2) produced subcutaneous tumor heterotransplants that resemble human transitional cell carcinoma (TCC). In the present study, additional Cd+2 transformed cell lines were isolated to determine if independent exposures of the cell line to Cd+2 would result in malignantly transformed cell lines possessing similar phenotypic properties. Seven independent isolates were isolated and assessed for their doubling times, morphology, ability to heterotransplant subcutaneously and in the peritoneal cavity of nude mice and for the expression keratin 7. The 7 cell lines all displayed an epithelial morphology with no evidence of squamous differentiation. Doubling times were variable among the isolates, being significantly reduced or similar to the parental cells. All 7 isolates were able to form subcutaneous tumor heterotransplants with a TCC morphology and all heterotransplants displayed areas of squamous differentiation of the transitional cells. The degree of squamous differentiation varied among the isolates. In contrast to subcutaneous tumor formation, only 1 isolate of the Cd+2 transformed cells (UTCd#1) was able to effectively colonize multiple sites within the peritoneal cavity. An analysis of keratin 7 expression showed no correlation with squamous differentiation for the subcutaneous heterotransplants generated from the 7 cell lines. Keratin 7 was expressed in 6 of the 7 cell lines and their subcutaneous tumor heterotransplants. Keratin 7 was not expressed in the cell line that was able to form tumors within the peritoneal cavity. These results show that individual isolates of Cd+2 transformed cells have both similarities and differences in their phenotype. PMID:19921857

The transfer and transformation of collective network information in gene-matched networks.

PubMed

Kitsukawa, Takashi; Yagi, Takeshi

2015-10-09

Networks, such as the human society network, social and professional networks, and biological system networks, contain vast amounts of information. Information signals in networks are distributed over nodes and transmitted through intricately wired links, making the transfer and transformation of such information difficult to follow. Here we introduce a novel method for describing network information and its transfer using a model network, the Gene-matched network (GMN), in which nodes (neurons) possess attributes (genes). In the GMN, nodes are connected according to their expression of common genes. Because neurons have multiple genes, the GMN is cluster-rich. We show that, in the GMN, information transfer and transformation were controlled systematically, according to the activity level of the network. Furthermore, information transfer and transformation could be traced numerically with a vector using genes expressed in the activated neurons, the active-gene array, which was used to assess the relative activity among overlapping neuronal groups. Interestingly, this coding style closely resembles the cell-assembly neural coding theory. The method introduced here could be applied to many real-world networks, since many systems, including human society and various biological systems, can be represented as a network of this type.

Reduction of estrogen-induced transformation of mouse mammary epithelial cells by N-acetylcysteine

PubMed Central

Venugopal, Divya; Zahid, Muhammad; Mailander, Paula C; Meza, Jane L.; Rogan, Eleanor G.; Cavalieri, Ercole L.; Chakravarti, Dhrubajyoti

2009-01-01

A growing number of studies indicate that breast cancer initiation is related to abnormal estrogen oxidation to form an excess of estrogen-3,4-quinones, which react with DNA to form depurinating adducts and induce mutations. This mechanism is often called estrogen genotoxicity. 4-catechol estrogens, precursors of the estrogen-3,4-quinones, were previously shown to account for most of the transforming and tumorigenic activity. We examined whether estrogen-induced transformation can be reduced by inhibiting the oxidation of a 4-catechol estrogen to its quinone. We demonstrate that E6 cells (a normal mouse epithelial cell line) can be transformed by a single treatment with a catechol estrogen or its quinone. The transforming activities of 4-hydroxyestradiol and estradiol-3,4-quinone were comparable. N-acetylcysteine, a common antioxidant, inhibited the oxidation of 4-hydroxyestradiol to the quinone and consequent formation of DNA adducts. It also drastically reduced estrogen-induced transformation of E6 cells. These results strongly implicate estrogen genotoxicity in mammary cell transformation. Since N-acetylcysteine is well-tolerated in clinical studies, it may be a promising candidate for breast cancer prevention. PMID:18226522

The contribution of heavy metals in cigarette smoke condensate to malignant transformation of breast epithelial cells and in vivo initiation of neoplasia through induction of a PI3K–AKT–NFκB cascade

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

Mohapatra, Purusottam; Preet, Ranjan; Das, Dipon

Cigarette smoking is a crucial factor in the development and progression of multiple cancers including breast. Here, we report that repeated exposure to a fixed, low dose of cigarette smoke condensate (CSC) prepared from Indian cigarettes is capable of transforming normal breast epithelial cells, MCF-10A, and delineate the biochemical basis for cellular transformation. CSC transformed cells (MCF-10A-Tr) were capable of anchorage-independent growth, and their anchorage dependent growth and colony forming ability were higher compared to the non-transformed MCF-10A cells. Increased expression of biomarkers representative of oncogenic transformation (NRP-1, Nectin-4), and anti-apoptotic markers (PI3K, AKT, NFκB) were also noted in themore » MCF-10A-Tr cells. Short tandem repeat (STR) profiling of MCF-10A and MCF-10A-Tr cells revealed that transformed cells acquired allelic variation during transformation, and had become genetically distinct. MCF-10A-Tr cells formed solid tumors when implanted into the mammary fat pads of Balb/c mice. Data revealed that CSC contained approximately 1.011 μg Cd per cigarette equivalent, and Cd (0.0003 μg Cd/1 × 10{sup 7} cells) was also detected in the lysates from MCF-10A cells treated with 25 μg/mL CSC. In similar manner to CSC, CdCl{sub 2} treatment in MCF-10A cells caused anchorage independent colony growth, higher expression of oncogenic proteins and increased PI3K–AKT–NFκB protein expression. An increase in the expression of PI3K–AKT–NFκB was also noted in the mice xenografts. Interestingly, it was noted that CSC and CdCl{sub 2} treatment in MCF-10A cells increased ROS. Collectively, results suggest that heavy metals present in cigarettes of Indian origin may substantially contribute to tumorigenesis by inducing intercellular ROS accumulation and increased expression of PI3K, AKT and NFκB proteins. - Highlights: • Repeated exposure of CSC causes malignant transformation in MCF-10A. • MCF-10A-Tr cells showed a distinct STR profile and tumor inducing characteristics. • Increased expression of PI3K, AKT, and NFκB protein in MCF-10A-Tr and solid tumor. • Increased ROS and PI3K-AKT-NFκB proteins in smoke carcinogen exposed MCF-10A cells. • Cadmium may be a strong contributor to the transformation of MCF-10A cells.« less

Mis-specified cells die by an active gene-directed process, and inhibition of this death results in cell fate transformation in Drosophila

PubMed Central

Werz, Christian; Lee, Tom V.; Lee, Peter L.; Lackey, Melinda; Bolduc, Clare; Stein, David S.; Bergmann, Andreas

2009-01-01

Summary Incorrectly specified or mis-specified cells often undergo cell death or are transformed to adopt a different cell fate during development. The underlying cause for this distinction is largely unknown. In many developmental mutants in Drosophila, large numbers of mis-specified cells die synchronously, providing a convenient model for analysis of this phenomenon. The maternal mutant bicoid is particularly useful model with which to address this issue because its mutant phenotype is a combination of both transformation of tissue (acron to telson) and cell death in the presumptive head and thorax regions. We show that a subset of these mis-specified cells die through an active gene-directed process involving transcriptional upregulation of the cell death inducer hid. Upregulation of hid also occurs in oskar mutants and other segmentation mutants. In hid bicoid double mutants, mis-specified cells in the presumptive head and thorax survive and continue to develop, but they are transformed to adopt a different cell fate. We provide evidence that the terminal torso signaling pathway protects the mis-specified telson tissue in bicoid mutants from hid-induced cell death, whereas mis-specified cells in the head and thorax die, presumably because equivalent survival signals are lacking. These data support a model whereby mis-specification can be tolerated if a survival pathway is provided, resulting in cellular transformation. PMID:16280349

Mechanisms of cell transformation in the embryonic heart.

PubMed

Huang, J X; Potts, J D; Vincent, E B; Weeks, D L; Runyan, R B

1995-03-27

The process of cell transformation in the heart is a complex one. By use of the invasion bioassay, we have been able to identify several critical components of the cell transformation process in the heart. TGF beta 3 can be visualized as a switch in the environment that contributes to the initial process of cell transformation. Our data show that it is a critical switch in the transformation process. Even so, it is apparently only one of the factors involved. Others may include other TGF beta family members, the ES antigens described by Markwald and co-workers and additional unknown substances. Observing the sensitivity of the process to pertussis toxin, there is likely to be a G-protein-linked receptor involved, yet we have not identified a known ligand for this type of receptor. Clearly, there are several different signal transduction processes involved. The existence of multiple pathways is consistent with the idea that the target endothelial cells receive a variety of environmental imputs, the sum of which will produce cell transformation at the correct time and place. Adjacent endothelial cells of the ventricle that do not undergo cell transformation are apparently refractory to one or more of the stimuli. Figure 4 depicts a summary diagram of this invasion process with localization of most of the molecules mentioned in this narrative. As hypothesized here, elements of the transformation process may recapitulate aspects of gastrulation. Since some conservation of mechanism is expected in cells, it is not surprising that cells undergoing phenotypic change might reutilize mechanisms used previously to produce mesenchyme from the blastodisk. Though we have preliminary data to suggest this point, confirmation of the hypothesis by perturbation of genes such as brachyury, msx-1, etc. will be required to establish this point. The advantage of this hypothesis is that it provides, from the work of others in the area of gastrulation, a ready source of molecules and mechanisms that can be tested in the transforming heart. Whereas, perturbation of such mechanisms at gastrulation may be lethal to the embryo, such molecules and mechanisms may be responsible for the high incidence of birth defects in the heart.

DNA Tumor Viruses and Cell Metabolism

PubMed Central

Mushtaq, Muhammad; Darekar, Suhas

2016-01-01

Viruses play an important role in cancerogenesis. It is estimated that approximately 20% of all cancers are linked to infectious agents. The viral genes modulate the physiological machinery of infected cells that lead to cell transformation and development of cancer. One of the important adoptive responses by the cancer cells is their metabolic change to cope up with continuous requirement of cell survival and proliferation. In this review we will focus on how DNA viruses alter the glucose metabolism of transformed cells. Tumor DNA viruses enhance “aerobic” glycolysis upon virus-induced cell transformation, supporting rapid cell proliferation and showing the Warburg effect. Moreover, viral proteins enhance glucose uptake and controls tumor microenvironment, promoting metastasizing of the tumor cells. PMID:27034740

Human Induced Pluripotent Stem Cell-Derived Cardiac Progenitor Cells in Phenotypic Screening: A Transforming Growth Factor-β Type 1 Receptor Kinase Inhibitor Induces Efficient Cardiac Differentiation.

PubMed

Drowley, Lauren; Koonce, Chad; Peel, Samantha; Jonebring, Anna; Plowright, Alleyn T; Kattman, Steven J; Andersson, Henrik; Anson, Blake; Swanson, Bradley J; Wang, Qing-Dong; Brolen, Gabriella

2016-02-01

Several progenitor cell populations have been reported to exist in hearts that play a role in cardiac turnover and/or repair. Despite the presence of cardiac stem and progenitor cells within the myocardium, functional repair of the heart after injury is inadequate. Identification of the signaling pathways involved in the expansion and differentiation of cardiac progenitor cells (CPCs) will broaden insight into the fundamental mechanisms playing a role in cardiac homeostasis and disease and might provide strategies for in vivo regenerative therapies. To understand and exploit cardiac ontogeny for drug discovery efforts, we developed an in vitro human induced pluripotent stem cell-derived CPC model system using a highly enriched population of KDR(pos)/CKIT(neg)/NKX2.5(pos) CPCs. Using this model system, these CPCs were capable of generating highly enriched cultures of cardiomyocytes under directed differentiation conditions. In order to facilitate the identification of pathways and targets involved in proliferation and differentiation of resident CPCs, we developed phenotypic screening assays. Screening paradigms for therapeutic applications require a robust, scalable, and consistent methodology. In the present study, we have demonstrated the suitability of these cells for medium to high-throughput screens to assess both proliferation and multilineage differentiation. Using this CPC model system and a small directed compound set, we identified activin-like kinase 5 (transforming growth factor-β type 1 receptor kinase) inhibitors as novel and potent inducers of human CPC differentiation to cardiomyocytes. Significance: Cardiac disease is a leading cause of morbidity and mortality, with no treatment available that can result in functional repair. This study demonstrates how differentiation of induced pluripotent stem cells can be used to identify and isolate cell populations of interest that can translate to the adult human heart. Two separate examples of phenotypic screens are discussed, demonstrating the value of this biologically relevant and reproducible technology. In addition, this assay system was able to identify novel and potent inducers of differentiation and proliferation of induced pluripotent stem cell-derived cardiac progenitor cells. ©AlphaMed Press.

T-DNA transfer and T-DNA integration efficiencies upon Arabidopsis thaliana root explant cocultivation and floral dip transformation.

PubMed

Ghedira, Rim; De Buck, Sylvie; Van Ex, Frédéric; Angenon, Geert; Depicker, Ann

2013-12-01

T-DNA transfer and integration frequencies during Agrobacterium-mediated root explant cocultivation and floral dip transformations of Arabidopsis thaliana were analyzed with and without selection for transformation-competent cells. Based on the presence or absence of CRE recombinase activity without or with the CRE T-DNA being integrated, transient expression versus stable transformation was differentiated. During root explant cocultivation, continuous light enhanced the number of plant cells competent for interaction with Agrobacterium and thus the number of transient gene expression events. However, in transformation competent plant cells, continuous light did not further enhance cotransfer or cointegration frequencies. Upon selection for root transformants expressing a first T-DNA, 43-69 % of these transformants showed cotransfer of another non-selected T-DNA in two different light regimes. However, integration of the non-selected cotransferred T-DNA occurred only in 19-46 % of these transformants, indicating that T-DNA integration in regenerating root cells limits the transformation frequencies. After floral dip transformation, transient T-DNA expression without integration could not be detected, while stable T-DNA transformation occurred in 0.5-1.3 % of the T1 seedlings. Upon selection for floral dip transformants with a first T-DNA, 8-34 % of the transformants showed cotransfer of the other non-selected T-DNA and in 93-100 % of them, the T-DNA was also integrated. Therefore, a productive interaction between the agrobacteria and the female gametophyte, rather than the T-DNA integration process, restricts the floral dip transformation frequencies.

Karyotyping of Transformed Human Epithelial Cells from Exposures of Heavy Ions

NASA Technical Reports Server (NTRS)

Yeshitla, Samrawit

2013-01-01

It is most likely that the untreated transformed single clone (clone #2) cell undergoes unequal segregation of chromosome in two daughter cell that result in 94 chromosome during mitosis, particularly in anaphase stage. Chromosome aberration observed. I. Breakage of part of chromosome 7. II. One additional number of chromosome 8 instead of the total chromosome can only be explained by early abnormal cell division. III. Complete lost of chromosome and translocation and fusion of chromosome 3 and X-chromosome. IV. Our result for translocation and fusion of chromosome 3 and X- Chromosome is conformed by mBAND pattern. There is no different between the transformed parental cell and the single cloned transformed cell. Both harbor the chromosome 5 and 16 translocation and both harbor has the trisomy chromosome 20. Transformed cells may have the number of chromosomes greater or less than 46. Doubling of chromosome numbers is a signature of tumor. Chromosomal aberration was observed on HBEC-3kt non-irradiated-soft agar (Clone #2) sample, and indication of chromosome instability in the tumor development process.

Activated platelet-derived growth factor autocrine pathway drives the transformed phenotype of a human glioblastoma cell line.

PubMed

Vassbotn, F S; Ostman, A; Langeland, N; Holmsen, H; Westermark, B; Heldin, C H; Nistér, M

1994-02-01

Human glioblastoma cells (A172) were found to concomitantly express PDGF-BB and PDGF beta-receptors. The receptors were constitutively autophosphorylated in the absence of exogenous ligand, suggesting the presence of an autocrine PDGF pathway. Neutralizing PDGF antibodies as well as suramin inhibited the autonomous PDGF receptor tyrosine kinase activity and resulted in up-regulation of receptor protein. The interruption of the autocrine loop by the PDGF antibodies reversed the transformed phenotype of the glioblastoma cell, as determined by (1) diminished DNA synthesis, (2) inhibition of tumor colony growth, and (3) reversion of the transformed morphology of the tumor cells. The PDGF antibodies showed no effect on the DNA synthesis of another glioblastoma cells line (U-343MGa 31L) or on Ki-ras-transformed fibroblasts. The present study demonstrates an endogenously activated PDGF pathway in a spontaneous human glioblastoma cell line. Furthermore, we provide evidence that the autocrine PDGF pathway drives the transformed phenotype of the tumor cells, a process that can be blocked by extracellular antagonists.

Gas Measurement Using Static Fourier Transform Infrared Spectrometers.

PubMed

Köhler, Michael H; Schardt, Michael; Rauscher, Markus S; Koch, Alexander W

2017-11-13

Online monitoring of gases in industrial processes is an ambitious task due to adverse conditions such as mechanical vibrations and temperature fluctuations. Whereas conventional Fourier transform infrared (FTIR) spectrometers use rather complex optical and mechanical designs to ensure stable operation, static FTIR spectrometers do not require moving parts and thus offer inherent stability at comparatively low costs. Therefore, we present a novel, compact gas measurement system using a static single-mirror Fourier transform spectrometer (sSMFTS). The system works in the mid-infrared range from 650 cm - 1 to 1250 cm - 1 and can be operated with a customized White cell, yielding optical path lengths of up to 120 cm for highly sensitive quantification of gas concentrations. To validate the system, we measure different concentrations of 1,1,1,2-Tetrafluoroethane (R134a) and perform a PLS regression analysis of the acquired infrared spectra. Thereby, the measured absorption spectra show good agreement with reference data. Since the system additionally permits measurement rates of up to 200 Hz and high signal-to-noise ratios, an application in process analysis appears promising.

Gas Measurement Using Static Fourier Transform Infrared Spectrometers

PubMed Central

Schardt, Michael; Rauscher, Markus S.; Koch, Alexander W.

2017-01-01

Online monitoring of gases in industrial processes is an ambitious task due to adverse conditions such as mechanical vibrations and temperature fluctuations. Whereas conventional Fourier transform infrared (FTIR) spectrometers use rather complex optical and mechanical designs to ensure stable operation, static FTIR spectrometers do not require moving parts and thus offer inherent stability at comparatively low costs. Therefore, we present a novel, compact gas measurement system using a static single-mirror Fourier transform spectrometer (sSMFTS). The system works in the mid-infrared range from 650 cm−1 to 1250 cm−1 and can be operated with a customized White cell, yielding optical path lengths of up to 120 cm for highly sensitive quantification of gas concentrations. To validate the system, we measure different concentrations of 1,1,1,2-Tetrafluoroethane (R134a) and perform a PLS regression analysis of the acquired infrared spectra. Thereby, the measured absorption spectra show good agreement with reference data. Since the system additionally permits measurement rates of up to 200 Hz and high signal-to-noise ratios, an application in process analysis appears promising. PMID:29137193

Agrobacterium-mediated transformation of the haploid liverwort Marchantia polymorpha L., an emerging model for plant biology.

PubMed

Ishizaki, Kimitsune; Chiyoda, Shota; Yamato, Katsuyuki T; Kohchi, Takayuki

2008-07-01

Agrobacterium-mediated transformation has not been practical in pteridophytes, bryophytes and algae to date, although it is commonly used in model plants including Arabidopsis and rice. Here we present a rapid Agrobacterium-mediated transformation system for the haploid liverwort Marchantia polymorpha L. using immature thalli developed from spores. Hundreds of hygromycin-resistant plants per sporangium were obtained by co-cultivation of immature thalli with Agrobacterium carrying the binary vector that contains a reporter, the beta-glucuronidase (GUS) gene with an intron, and a selection marker, the hygromycin phosphotransferase (hpt) gene. In this system, individual gemmae, which arise asexually from single initial cells, were analyzed as isogenic transformants. GUS activity staining showed that all hygromycin-resistant plants examined expressed the GUS transgene in planta. DNA analyses verified random integration of 1-5 copies of the intact T-DNA between the right and the left borders into the M. polymorpha genome. The efficient and rapid Agrobacterium-mediated transformation of M. polymorpha should provide molecular techniques to facilitate comparative genomics, taking advantage of this unique model plant that retains many features of the common ancestor of land plants.

Federal Task Force on the Boston Central Artery Tunnel Project. Review of Project Oversight and Costs

DOT National Transportation Integrated Search

2012-01-01

Connected vehicles have the potential to transform the way Americans travel through the creation of a safe, interoperable wireless communications networka system that includes cars, buses, trucks, trains, traffic signals, cell phones, and other de...

Electric-field-induced phase transformation at a lead-free morphotropic phase boundary: Case study in a 93%(Bi0.5Na0.5)TiO3-7% BaTiO3 piezoelectric ceramic

NASA Astrophysics Data System (ADS)

Daniels, John E.; Jo, Wook; Rödel, Jürgen; Jones, Jacob L.

2009-07-01

The electric-field-induced strain in 93%(Bi0.5Na0.5)TiO3-7%BaTiO3 polycrystalline ceramic is shown to be the result of an electric-field-induced phase transformation from a pseudocubic to tetragonal symmetry. High-energy x-ray diffraction is used to illustrate the microstructural nature of the transformation. A combination of induced unit cell volumetric changes, domain texture, and anisotropic lattice strains are responsible for the observed macroscopic strain. This strain mechanism is not analogous to the high electric-field-induced strains observed in lead-based morphotropic phase boundary systems. Thus, systems which appear cubic under zero field should not be excluded from the search for lead-free piezoelectric compositions.

Dominant-negative mutants of platelet-derived growth factor revert the transformed phenotype of human astrocytoma cells.

PubMed Central

Shamah, S M; Stiles, C D; Guha, A

1993-01-01

Malignant astrocytoma is the most common primary human brain tumor. Most astrocytomas express a combination of platelet-derived growth factor (PDGF) and PDGF receptor which could close an autocrine loop. It is not known whether these autocrine loops contribute to the transformed phenotype of astrocytoma cells or are incidental to that phenotype. Here we show that dominant-negative mutants of the PDGF ligand break the autocrine loop and revert the phenotype of BALB/c 3T3 cells transformed by the PDGF-A or PDGF-B (c-sis) gene. Then, we show that these mutants are selective in that they do not alter the phenotype of 3T3 cells transformed by an activated Ha-ras or v-src gene or by simian virus 40. Finally, we show that these mutants revert the transformed phenotype of two independent human astrocytoma cell lines. They have no effect on the growth of human medulloblastoma, bladder carcinoma, or colon carcinoma cell lines. These observations are consistent with the view that PDGF autocrine loops contribute to the transformed phenotype of at least some human astrocytomas. Images PMID:8246942

The contribution of heavy metals in cigarette smoke condensate to malignant transformation of breast epithelial cells and in vivo initiation of neoplasia through induction of a PI3K-AKT-NFκB cascade.

PubMed

Mohapatra, Purusottam; Preet, Ranjan; Das, Dipon; Satapathy, Shakti Ranjan; Siddharth, Sumit; Choudhuri, Tathagata; Wyatt, Michael D; Kundu, Chanakya Nath

2014-01-01

Cigarette smoking is a crucial factor in the development and progression of multiple cancers including breast. Here, we report that repeated exposure to a fixed, low dose of cigarette smoke condensate (CSC) prepared from Indian cigarettes is capable of transforming normal breast epithelial cells, MCF-10A, and delineate the biochemical basis for cellular transformation. CSC transformed cells (MCF-10A-Tr) were capable of anchorage-independent growth, and their anchorage dependent growth and colony forming ability were higher compared to the non-transformed MCF-10A cells. Increased expression of biomarkers representative of oncogenic transformation (NRP-1, Nectin-4), and anti-apoptotic markers (PI3K, AKT, NFκB) were also noted in the MCF-10A-Tr cells. Short tandem repeat (STR) profiling of MCF-10A and MCF-10A-Tr cells revealed that transformed cells acquired allelic variation during transformation, and had become genetically distinct. MCF-10A-Tr cells formed solid tumors when implanted into the mammary fat pads of Balb/c mice. Data revealed that CSC contained approximately 1.011μg Cd per cigarette equivalent, and Cd (0.0003μg Cd/1×10(7) cells) was also detected in the lysates from MCF-10A cells treated with 25μg/mL CSC. In similar manner to CSC, CdCl2 treatment in MCF-10A cells caused anchorage independent colony growth, higher expression of oncogenic proteins and increased PI3K-AKT-NFκB protein expression. An increase in the expression of PI3K-AKT-NFκB was also noted in the mice xenografts. Interestingly, it was noted that CSC and CdCl2 treatment in MCF-10A cells increased ROS. Collectively, results suggest that heavy metals present in cigarettes of Indian origin may substantially contribute to tumorigenesis by inducing intercellular ROS accumulation and increased expression of PI3K, AKT and NFκB proteins. © 2013.

Nuclear events of apoptosis in vitro in cell-free mitotic extracts: a model system for analysis of the active phase of apoptosis

PubMed Central

1993-01-01

We have developed a cell-free system that induces the morphological transformations characteristic of apoptosis in isolated nuclei. The system uses extracts prepared from mitotic chicken hepatoma cells following a sequential S phase/M phase synchronization. When nuclei are added to these extracts, the chromatin becomes highly condensed into spherical domains that ultimately extrude through the nuclear envelope, forming apoptotic bodies. The process is highly synchronous, and the structural changes are completed within 60 min. Coincident with these morphological changes, the nuclear DNA is cleaved into a nucleosomal ladder. Both processes are inhibited by Zn2+, an inhibitor of apoptosis in intact cells. Nuclear lamina disassembly accompanies these structural changes in added nuclei, and we show that lamina disassembly is a characteristic feature of apoptosis in intact cells of mouse, human and chicken. This system may provide a powerful means of dissecting the biochemical mechanisms underlying the final stages of apoptosis. PMID:8408207

Endosymbiosis and the design of eukaryotic electron transport.

PubMed

Berry, Stephan

2003-09-30

The bioenergetic organelles of eukaryotic cells, mitochondria and chloroplasts, are derived from endosymbiotic bacteria. Their electron transport chains (ETCs) resemble those of free-living bacteria, but were tailored for energy transformation within the host cell. Parallel evolutionary processes in mitochondria and chloroplasts include reductive as well as expansive events: On one hand, bacterial complexes were lost in eukaryotes with a concomitant loss of metabolic flexibility. On the other hand, new subunits have been added to the remaining bacterial complexes, new complexes have been introduced, and elaborate folding patterns of the thylakoid and mitochondrial inner membranes have emerged. Some bacterial pathways were reinvented independently by eukaryotes, such as parallel routes for quinol oxidation or the use of various anaerobic electron acceptors. Multicellular organization and ontogenetic cycles in eukaryotes gave rise to further modifications of the bioenergetic organelles. Besides mitochondria and chloroplasts, eukaryotes have ETCs in other membranes, such as the plasma membrane (PM) redox system, or the cytochrome P450 (CYP) system. These systems have fewer complexes and simpler branching patterns than those in energy-transforming organelles, and they are often adapted to non-bioenergetic functions such as detoxification or cellular defense.

Breast fibroblasts in both cancer and normal tissues induce phenotypic transformation of breast cancer stem cells: a preliminary study

PubMed Central

Xi, Chunfang; Liu, Mingwei; Sun, Haichen; Liu, Shuang; Song, Lei

2018-01-01

Background Breast cancer stem cells (BCSCs) are associated with the invasion of breast cancer. In recent years, studies have demonstrated different phenotypes among BCSCs. Furthermore, BCSCs of diverse phenotypes are present at different tumour sites and different histological stages. Fibroblasts are involved in the phenotypic transformation of BCSCs. Cancer-associated fibroblasts (CAFs) participate in the induction of epithelial–mesenchymal transition, thereby promoting the acquisition of stem cell characteristics, but little is known about the role of normal fibroblasts (NFs) in the phenotypic transformation of BCSCs or about the effect of CAFs and NFs on BCSC phenotypes. Methods A total of six pairs of primary CAFs and NFs were isolated from surgical samples of breast cancer patients and subjected to morphological, immunohistochemical, cell invasion and proteomics analyses. After establishing a cell culture system with conditioned medium from CAFs and NFs, we used the mammosphere formation assay to explore the effect of CAFs and NFs on the self-renewal ability of BCSCs. The effect of CAFs and NFs on the phenotypic differentiation of BCSCs was further analysed by flow cytometry and immunofluorescence. Results The isolated CAFs and NFs did not show significant differences in cell morphology or alpha-smooth muscle actin (α-SMA) expression, but cell invasion and proteomics analyses demonstrated heterogeneity among these fibroblasts. Both CAFs and NFs could promote the generation of BCSCs, but CAFs displayed a greater ability than NFs in promoting mammosphere formation. Conditioned medium from CAFs increased the proportion of aldehyde dehydrogenase-1 positive (ALDH1+) BCSCs, but conditioned medium from NFs was more likely to promote the generation of CD44+CD24− BCSCs from MCF-7 cells. Discussion This study validated the heterogeneity among CAFs and NFs and expanded on the conclusion that fibroblasts promote the generation of cancer stem cells. Our results particularly emphasized the effect of NFs on the phenotypic transformation of BCSCs. In addition, this study further highlighted the roles of CAFs and NFs in the induction of different phenotypes in BCSCs. PMID:29780673

Breast fibroblasts in both cancer and normal tissues induce phenotypic transformation of breast cancer stem cells: a preliminary study.

PubMed

Wang, Bixiao; Xi, Chunfang; Liu, Mingwei; Sun, Haichen; Liu, Shuang; Song, Lei; Kang, Hua

2018-01-01

Breast cancer stem cells (BCSCs) are associated with the invasion of breast cancer. In recent years, studies have demonstrated different phenotypes among BCSCs. Furthermore, BCSCs of diverse phenotypes are present at different tumour sites and different histological stages. Fibroblasts are involved in the phenotypic transformation of BCSCs. Cancer-associated fibroblasts (CAFs) participate in the induction of epithelial-mesenchymal transition, thereby promoting the acquisition of stem cell characteristics, but little is known about the role of normal fibroblasts (NFs) in the phenotypic transformation of BCSCs or about the effect of CAFs and NFs on BCSC phenotypes. A total of six pairs of primary CAFs and NFs were isolated from surgical samples of breast cancer patients and subjected to morphological, immunohistochemical, cell invasion and proteomics analyses. After establishing a cell culture system with conditioned medium from CAFs and NFs, we used the mammosphere formation assay to explore the effect of CAFs and NFs on the self-renewal ability of BCSCs. The effect of CAFs and NFs on the phenotypic differentiation of BCSCs was further analysed by flow cytometry and immunofluorescence. The isolated CAFs and NFs did not show significant differences in cell morphology or alpha-smooth muscle actin (α-SMA) expression, but cell invasion and proteomics analyses demonstrated heterogeneity among these fibroblasts. Both CAFs and NFs could promote the generation of BCSCs, but CAFs displayed a greater ability than NFs in promoting mammosphere formation. Conditioned medium from CAFs increased the proportion of aldehyde dehydrogenase-1 positive (ALDH1 + ) BCSCs, but conditioned medium from NFs was more likely to promote the generation of CD44 + CD24 - BCSCs from MCF-7 cells. This study validated the heterogeneity among CAFs and NFs and expanded on the conclusion that fibroblasts promote the generation of cancer stem cells. Our results particularly emphasized the effect of NFs on the phenotypic transformation of BCSCs. In addition, this study further highlighted the roles of CAFs and NFs in the induction of different phenotypes in BCSCs.

Biolistic transformation of cotton embryogenic cell suspension cultures

USDA-ARS?s Scientific Manuscript database

Genetic transformation of cotton is highly dependent on the ability to regenerate fertile plants from transgenic cells through somatic embryogenesis. Induction of embryogenic cell cultures is genotype-dependant. However, once embryogenic cell cultures are available, they can be effectively used fo...

Friend leukemia virus transformed cells exposed to microgravity in the presence of DMSO (7-IML-1)

NASA Technical Reports Server (NTRS)

Cogoli, Augusto

1992-01-01

The purpose of this experiment is to study the adaptation of living cells to microgravity. The in vitro transformation of Friend cells by Dimethylsufoxide (DMSO) is a good model for the study of cell differentiation and protein biosynthesis. Cultures of cells will be prepared shortly before launch. Once in space, transformation will be induced by injection of DMSO. One set of cultures will be chemically fixed with glutaraldehyde for electron microscope investigations; another set will be preserved for determining the amount of hemogloben produced and the extent of cell proliferation.

Caveolin-1 down-regulation is required for Wnt5a-Frizzled 2 signalling in Ha-RasV12 -induced cell transformation.

PubMed

Lin, Hsiu-Kuan; Lin, Hsi-Hui; Chiou, Yu-Wei; Wu, Ching-Lung; Chiu, Wen-Tai; Tang, Ming-Jer

2018-05-01

Caveolin-1 (Cav1) is down-regulated during MK4 (MDCK cells harbouring inducible Ha-Ras V12 gene) transformation by Ha-Ras V12 . Cav1 overexpression abrogates the Ha-Ras V12 -driven transformation of MK4 cells; however, the targeted down-regulation of Cav1 is not sufficient to mimic this transformation. Cav1-silenced cells, including MK4/shCav1 cells and MDCK/shCav1 cells, showed an increased cell area and discontinuous junction-related proteins staining. Cellular and mechanical transformations were completed when MDCK/shCav1 cells were treated with medium conditioned by MK4 cells treated with IPTG (MK4+I-CM) but not with medium conditioned by MK4 cells. Nanoparticle tracking analysis showed that Ha-Ras V12 -inducing MK4 cells increased exosome-like microvesicles release compared with their normal counterparts. The cellular and mechanical transformation activities of MK4+I-CM were abolished after heat treatment and exosome depletion and were copied by exosomes derived from MK4+I-CM (MK4+I-EXs). Wnt5a, a downstream product of Ha-Ras V12 , was markedly secreted by MK4+I-CM and MK4+I-EXs. Suppression of Wnt5a expression and secretion using the porcupine inhibitor C59 or Wnt5a siRNA inhibited the Ha-Ras V12 - and MK4+I-CM-induced transformation of MK4 cells and MDCK/shCav1 cells, respectively. Cav1 down-regulation, either by Ha-Ras V12 or targeted shRNA, increased frizzled-2 (Fzd2) protein levels without affecting its mRNA levels, suggesting a novel role of Cav1 in negatively regulating Fzd2 expression. Additionally, silencing Cav1 facilitated the internalization of MK4+I-EXs in MDCK cells. These data suggest that Cav1-dependent repression of Fzd2 and exosome uptake is potentially relevant to its antitransformation activity, which hinders the activation of Ha-Ras V12 -Wnt5a-Stat3 pathway. Altogether, these results suggest that both decreasing Cav1 and increasing exosomal Wnt5a must be implemented during Ha-Ras V12 -driven cell transformation. © 2018 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Cellular and molecular alterations in human epithelial cells transformed by high let radiation

NASA Astrophysics Data System (ADS)

Hei, T. K.; Piao, C. Q.; Sutter, T.; Willey, J. C.; Suzuki, K.

An understanding of the radiobiological effects of high LET radiation is essential for human risk estimation and radiation protection. In the present study, we show that a single, 30 cGy dose of 150 keV/mum ^4He ions can malignantly transform human papillomavirus immortalized human bronchial epithelial [BEP2D] cells. Transformed cells produce progressively growing tumors in nude mice. The transformation frequency by the single dose of alpha particles is estimated to be approximately 4 x 10^-7. Based on the average cross-sectional area of BEP2D cells, it can be calculated that a mean traversal of 1.4 particles per cell is sufficient to induce tumorigenic conversion of these cells 3 to 4 months post-irradiation. Tumorigenic BEP2D cells overexpress mutated p53 tumor suppressor oncoproteins in addition to the cell cycle control gene cyclin D1 and D2. This model provides an opportunity to study the cellular and molecular changes at the various stages in radiation carcinogenesis involving human cells.

Reciprocal influence of B cells and tumor macro and microenvironments in the ApcMin/+ model of colorectal cancer.

PubMed

Mion, Francesca; Vetrano, Stefania; Tonon, Silvia; Valeri, Viviana; Piontini, Andrea; Burocchi, Alessia; Petti, Luciana; Frossi, Barbara; Gulino, Alessandro; Tripodo, Claudio; Colombo, Mario P; Pucillo, Carlo E

2017-01-01

One of the most fascinating aspects of the immune system is its dynamism, meant as the ability to change and readapt according to the organism needs. Following an insult, we assist to the spontaneous organization of different immune cells which cooperate, locally and at distance, to build up an appropriate response. Throughout tumor progression, adaptations within the systemic tumor environment, or macroenvironment, result in the promotion of tumor growth, tumor invasion and metastasis to distal organs, but also to dramatic changes in the activity and composition of the immune system. In this work, we show the changes of the B-cell arm of the immune system following tumor progression in the Apc Min/+ model of colorectal cancer. Tumor macroenvironment leads to an increased proportion of total and IL-10-competent B cells in draining LNs while activates a differentiation route that leads to the expansion of IgA + lymphocytes in the spleen and peritoneum. Importantly, serum IgA levels were significantly higher in Apc Min/+ than Wt mice. The peculiar involvement of IgA response in the adenomatous transformation had correlates in the gut-mucosal compartment where IgA-positive elements increased from normal mucosa to areas of low grade dysplasia while decreasing upon overt carcinomatous transformation. Altogether, our findings provide a snapshot of the tumor education of B lymphocytes in the Apc Min/+ model of colorectal cancer. Understanding how tumor macroenvironment affects the differentiation, function and distribution of B lymphocytes is pivotal to the generation of specific therapies, targeted to switching B cells to an anti-, rather than pro-, tumoral phenotype.

A rapid, highly efficient and economical method of Agrobacterium-mediated in planta transient transformation in living onion epidermis.

PubMed

Xu, Kedong; Huang, Xiaohui; Wu, Manman; Wang, Yan; Chang, Yunxia; Liu, Kun; Zhang, Ju; Zhang, Yi; Zhang, Fuli; Yi, Liming; Li, Tingting; Wang, Ruiyue; Tan, Guangxuan; Li, Chengwei

2014-01-01

Transient transformation is simpler, more efficient and economical in analyzing protein subcellular localization than stable transformation. Fluorescent fusion proteins were often used in transient transformation to follow the in vivo behavior of proteins. Onion epidermis, which has large, living and transparent cells in a monolayer, is suitable to visualize fluorescent fusion proteins. The often used transient transformation methods included particle bombardment, protoplast transfection and Agrobacterium-mediated transformation. Particle bombardment in onion epidermis was successfully established, however, it was expensive, biolistic equipment dependent and with low transformation efficiency. We developed a highly efficient in planta transient transformation method in onion epidermis by using a special agroinfiltration method, which could be fulfilled within 5 days from the pretreatment of onion bulb to the best time-point for analyzing gene expression. The transformation conditions were optimized to achieve 43.87% transformation efficiency in living onion epidermis. The developed method has advantages in cost, time-consuming, equipment dependency and transformation efficiency in contrast with those methods of particle bombardment in onion epidermal cells, protoplast transfection and Agrobacterium-mediated transient transformation in leaf epidermal cells of other plants. It will facilitate the analysis of protein subcellular localization on a large scale.

The farnesyltransferase inhibitor, LB42708, inhibits growth and induces apoptosis irreversibly in H-ras and K-ras-transformed rat intestinal epithelial cells

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

Kim, Han-Soo; Kim, Ju Won; Gang, Jingu

2006-09-15

LB42708 (LB7) and LB42908 (LB9) are pyrrole-based orally active farnesyltransferase inhibitors (FTIs) that have similar structures. The in vitro potencies of these compounds against FTase and GGTase I are remarkably similar, and yet they display different activity in apoptosis induction and morphological reversion of ras-transformed rat intestinal epithelial (RIE) cells. Both FTIs induced cell death despite K-ras prenylation, implying the participation of Ras-independent mechanism(s). Growth inhibition by these two FTIs was accompanied by G1 and G2/M cell cycle arrests in H-ras and K-ras-transformed RIE cells, respectively. We identified three key markers, p21{sup CIP1/WAF1}, RhoB and EGFR, that can explain themore » differences in the molecular mechanism of action between two FTIs. Only LB7 induced the upregulation of p21{sup CIP1/WAF1} and RhoB above the basal level that led to the cell cycle arrest and to distinct morphological alterations of ras-transformed RIE cells. Both FTIs successfully inhibited the ERK and activated JNK in RIE/K-ras cells. While the addition of conditioned medium from RIE/K-ras reversed the growth inhibition of ras-transformed RIE cells by LB9, it failed to overcome the growth inhibitory effect of LB7 in both H-ras- and K-ras-transformed RIE cells. We found that LB7, but not LB9, decreased the expression of EGFRs that confers the cellular unresponsiveness to EGFR ligands. These results suggest that LB7 causes the induction of p21{sup CIP1/WAF1} and RhoB and downregulation of EGFR that may serve as critical steps in the mechanism by which FTIs trigger irreversible inhibitions on the cell growth and apoptosis in ras-transformed cells.« less

Investigations of the functional states of dendritic cells under different conditioned microenvironments by Fourier transformed infrared spectroscopy.

PubMed

Dong, Rong; Long, Jinhua; Xu, Xiaoli; Zhang, Chunlin; Wen, Zongyao; Li, Long; Yao, Weijuan; Zeng, Zhu

2014-01-10

Dendritic cells are potent and specialized antigen presenting cells, which play a crucial role in initiating and amplifying both the innate and adaptive immune responses. The dendritic cell-based vaccination against cancer has been clinically achieved promising successes. But there are still many challenges in its clinical application, especially for how to identify the functional states. The CD14+ monocytes were isolated from human peripheral blood after plastic adherence and purified to approximately 98% with cocktail immunomagnetic beads. The immature dendritic cells and mature dendritic cells were induced by traditional protocols. The resulting dendritic cells were cocultured with normal cells and cancer cells. The functional state of dendritic cells including immature dendritic cells (imDCs) and mature dendritic cells (mDCs) under different conditioned microenvironments were investigated by Fourier transformed infrared spectroscopy (FTIR) and molecular biological methods. The results of Fourier transformed infrared spectroscopy showed that the gene transcription activity and energy states of dendritic cells were specifically suppressed by tumor cells (P < 0.05 or 0.01). The expression levels of NF-kappa B (NF-κB) in dendritic cells were also specifically inhibited by tumor-derived factors (P < 0.05 or 0.01). Moreover, the ratios of absorption intensities of Fourier transformed infrared spectroscopy at given wave numbers were closely correlated with the expression levels of NF-κB (R2:0.69 and R2:0.81, respectively). Our results confirmed that the ratios of absorption intensities of Fourier transformed infrared spectroscopy at given wave numbers were positively correlated with the expression levels of NF-κB, suggesting that Fourier transformed infrared spectroscopy technology could be clinically applied to identify the functional states of dendritic cell when performing dendritic cell-based vaccination. It's significant for the simplification and standardization of dendritic cell-based vaccination clinical preparation protocols.

Radiological health risks for exploratory class missions in space

NASA Technical Reports Server (NTRS)

Nachtwey, D. Stuart; Yang, Tracy Chui-Hsu

1991-01-01

The radiation risks to crewmembers on missions to the moon and Mars are studied. A graph is presented of the cross section as a function of linear energy transfer (LET) for cell inactivation and neoplastic cell transformation. Alternatives to conventional approaches to radiation protection using dose and Q are presented with attention given to a hybrid of the conventional system for particles with LET less than 100 keV/micron.

A novel dominant selectable system for the selection of transgenic plants under in vitro and greenhouse conditions based on phosphite metabolism.

PubMed

López-Arredondo, Damar L; Herrera-Estrella, Luis

2013-05-01

Antibiotic and herbicide resistance genes are currently the most frequently used selectable marker genes for plant research and crop development. However, the use of antibiotics and herbicides must be carefully controlled because the degree of susceptibility to these compounds varies widely among plant species and because they can also affect plant regeneration. Therefore, new selectable marker systems that are effective for a broad range of plant species are still needed. Here, we report a simple and inexpensive system based on providing transgenic plant cells the capacity to convert a nonmetabolizable compound (phosphite, Phi) into an essential nutrient for cell growth (phosphate) trough the expression of a bacterial gene encoding a phosphite oxidoreductase (PTXD). This system is effective for the selection of Arabidopsis transgenic plants by germinating T0 seeds directly on media supplemented with Phi and to select transgenic tobacco shoots from cocultivated leaf disc explants using nutrient media supplemented with Phi as both a source of phosphorus and selective agent. Because the ptxD/Phi system also allows the establishment of large-scale screening systems under greenhouse conditions completely eliminating false transformation events, it should facilitate the development of novel plant transformation methods. © 2013 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.

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

Subramanian, T.; Zhao, Ling-jun; Chinnadurai, G., E-mail: chinnag@slu.edu

Adenovirus E1A induces cell proliferation, oncogenic transformation and promotes viral replication through interaction with p300/CBP, TRRAP/p400 multi-protein complex and the retinoblastoma (pRb) family proteins through distinct domains in the E1A N-terminal region. The C-terminal region of E1A suppresses E1A/Ras co-transformation and interacts with FOXK1/K2, DYRK1A/1B/HAN11 and CtBP1/2 (CtBP) protein complexes. To specifically dissect the role of CtBP interaction with E1A, we engineered a mutation (DL→AS) within the CtBP-binding motif, PLDLS, and investigated the effect of the mutation on immortalization and Ras cooperative transformation of primary cells and viral replication. Our results suggest that CtBP–E1A interaction suppresses immortalization and Ras co-operativemore » transformation of primary rodent epithelial cells without significantly influencing the tumorigenic activities of transformed cells in immunodeficient and immunocompetent animals. During productive infection, CtBP–E1A interaction enhances viral replication in human cells. Between the two CtBP family proteins, CtBP2 appears to restrict viral replication more than CtBP1 in human cells. - Highlights: • Adenovirus E1A C-terminal region suppresses E1A/Ras co-transformation. • This E1A region binds with FOXK, DYRK1/HAN11 and CtBP cellular protein complexes. • We found that E1A–CtBP interaction suppresses immortalization and transformation. • The interaction enhances viral replication in human cells.« less

Transformation of fruit trees. Useful breeding tool or continued future prospect?

PubMed

Petri, César; Burgos, Lorenzo

2005-02-01

Regeneration and transformation systems using mature plant material of woody fruit species have to be achieved as a necessary requirement for the introduction of useful genes into specific cultivars and the rapid evaluation of resulting horticultural traits. Although the commercial production of transgenic annual crops is a reality, commercial genetically-engineered fruit trees are still far from common. In most woody fruit species, transformation and regeneration of commercial cultivars are not routine, generally being limited to a few genotypes or to seedlings. The future of genetic transformation as a tool for the breeding of fruit trees requires the development of genotype-independent procedures, based on the transformation of meristematic cells with high regeneration potential and/or the use of regeneration-promoting genes. The public concern with the introduction of antibiotic resistance into food and the restrictions due to new European laws that do not allow deliberate release of plants transformed with antibiotic-resistance genes highlight the development of methods that avoid the use of antibiotic-dependent selection or allow elimination of marker genesfrom the transformed plant as a research priority in coming years.

Transforming Boolean models to continuous models: methodology and application to T-cell receptor signaling

PubMed Central

Wittmann, Dominik M; Krumsiek, Jan; Saez-Rodriguez, Julio; Lauffenburger, Douglas A; Klamt, Steffen; Theis, Fabian J

2009-01-01

Background The understanding of regulatory and signaling networks has long been a core objective in Systems Biology. Knowledge about these networks is mainly of qualitative nature, which allows the construction of Boolean models, where the state of a component is either 'off' or 'on'. While often able to capture the essential behavior of a network, these models can never reproduce detailed time courses of concentration levels. Nowadays however, experiments yield more and more quantitative data. An obvious question therefore is how qualitative models can be used to explain and predict the outcome of these experiments. Results In this contribution we present a canonical way of transforming Boolean into continuous models, where the use of multivariate polynomial interpolation allows transformation of logic operations into a system of ordinary differential equations (ODE). The method is standardized and can readily be applied to large networks. Other, more limited approaches to this task are briefly reviewed and compared. Moreover, we discuss and generalize existing theoretical results on the relation between Boolean and continuous models. As a test case a logical model is transformed into an extensive continuous ODE model describing the activation of T-cells. We discuss how parameters for this model can be determined such that quantitative experimental results are explained and predicted, including time-courses for multiple ligand concentrations and binding affinities of different ligands. This shows that from the continuous model we may obtain biological insights not evident from the discrete one. Conclusion The presented approach will facilitate the interaction between modeling and experiments. Moreover, it provides a straightforward way to apply quantitative analysis methods to qualitatively described systems. PMID:19785753

Upregulation of SQSTM1/p62 contributes to nickel-induced malignant transformation of human bronchial epithelial cells.

PubMed

Huang, Haishan; Zhu, Junlan; Li, Yang; Zhang, Liping; Gu, Jiayan; Xie, Qipeng; Jin, Honglei; Che, Xun; Li, Jingxia; Huang, Chao; Chen, Lung-Chi; Lyu, Jianxin; Gao, Jimin; Huang, Chuanshu

2016-10-02

Chronic lung inflammation is accepted as being associated with the development of lung cancer caused by nickel exposure. Therefore, identifying the molecular mechanisms that lead to a nickel-induced sustained inflammatory microenvironment that causes transformation of human bronchial epithelial cells is of high significance. In the current studies, we identified SQSTM1/p62 as a novel nickel-upregulated protein that is important for nickel-induced inflammatory TNF expression, subsequently resulting in transformation of human bronchial epithelial cells. We found that nickel exposure induced SQSTM1 protein upregulation in human lung epithelial cells in vitro and in mouse lung tissues in vivo. The SQSTM1 upregulation was also observed in human lung squamous cell carcinoma. Further studies revealed that the knockdown of SQSTM1 expression dramatically inhibited transformation of human lung epithelial cells upon chronic nickel exposure, whereas ectopic expression of SQSTM1 promoted such transformation. Mechanistic studies showed that the SQSTM1 upregulation by nickel was the compromised result of upregulating SQSTM1 mRNA transcription and promoting SQSTM1 protein degradation. We demonstrated that nickel-initiated SQSTM1 protein degradation is mediated by macroautophagy/autophagy via an MTOR-ULK1-BECN1 axis, whereas RELA is important for SQSTM1 transcriptional upregulation following nickel exposure. Furthermore, SQSTM1 upregulation exhibited its promotion of nickel-induced cell transformation through exerting an impetus for nickel-induced inflammatory TNF mRNA stability. Consistently, the MTOR-ULK1-BECN1 autophagic cascade acted as an inhibitory effect on nickel-induced TNF expression and cell transformation. Collectively, our results demonstrate a novel SQSTM1 regulatory network that promotes a nickel-induced tumorigenic effect in human bronchial epithelial cells, which is negatively controlled by an autophagic cascade following nickel exposure.

Upregulation of SQSTM1/p62 contributes to nickel-induced malignant transformation of human bronchial epithelial cells

PubMed Central

Huang, Haishan; Zhu, Junlan; Li, Yang; Zhang, Liping; Gu, Jiayan; Xie, Qipeng; Jin, Honglei; Che, Xun; Li, Jingxia; Huang, Chao; Chen, Lung-Chi; Lyu, Jianxin; Gao, Jimin; Huang, Chuanshu

2016-01-01

ABSTRACT Chronic lung inflammation is accepted as being associated with the development of lung cancer caused by nickel exposure. Therefore, identifying the molecular mechanisms that lead to a nickel-induced sustained inflammatory microenvironment that causes transformation of human bronchial epithelial cells is of high significance. In the current studies, we identified SQSTM1/p62 as a novel nickel-upregulated protein that is important for nickel-induced inflammatory TNF expression, subsequently resulting in transformation of human bronchial epithelial cells. We found that nickel exposure induced SQSTM1 protein upregulation in human lung epithelial cells in vitro and in mouse lung tissues in vivo. The SQSTM1 upregulation was also observed in human lung squamous cell carcinoma. Further studies revealed that the knockdown of SQSTM1 expression dramatically inhibited transformation of human lung epithelial cells upon chronic nickel exposure, whereas ectopic expression of SQSTM1 promoted such transformation. Mechanistic studies showed that the SQSTM1 upregulation by nickel was the compromised result of upregulating SQSTM1 mRNA transcription and promoting SQSTM1 protein degradation. We demonstrated that nickel-initiated SQSTM1 protein degradation is mediated by macroautophagy/autophagy via an MTOR-ULK1-BECN1 axis, whereas RELA is important for SQSTM1 transcriptional upregulation following nickel exposure. Furthermore, SQSTM1 upregulation exhibited its promotion of nickel-induced cell transformation through exerting an impetus for nickel-induced inflammatory TNF mRNA stability. Consistently, the MTOR-ULK1-BECN1 autophagic cascade acted as an inhibitory effect on nickel-induced TNF expression and cell transformation. Collectively, our results demonstrate a novel SQSTM1 regulatory network that promotes a nickel-induced tumorigenic effect in human bronchial epithelial cells, which is negatively controlled by an autophagic cascade following nickel exposure. PMID:27467530

Rat embryo cells immortalized with transfected oncogenes are transformed by gamma irradiation.

PubMed

Endlich, B; Salavati, R; Sullivan, T; Ling, C C

1992-12-01

Cesium-137 gamma rays were used to transform rat embryo cells (REC) which were first transfected with activated c-myc or c-Ha-ras oncogenes to produce immortal cell lines (REC:myc and REC:ras). When exposed to 6 Gy of 137Cs gamma rays, some cells became morphologically transformed with focus formation frequencies of approximately 3 x 10(-4) for REC:myc and approximately 1 x 10(-4) for REC:ras, respectively. Cells isolated from foci of gamma-ray-transformed REC:myc (REC:myc:gamma) formed anchorage-independent colonies and were tumorigenic in nude mice, but foci from gamma-ray-transformed REC:ras (REC:ras:gamma) did not exhibit either of these criteria of transformation. Similar to the results with gamma irradiation, we observed a sequence-dependent phenomenon when myc and ras were transfected into REC, one at a time. REC immortalized by ras transfection were not converted to a tumorigenic phenotype by secondary transfection with myc, but REC transfected with myc were very susceptible to transformation by subsequent ras transfection. This suggests that myc-immortalized cells are more permissive to transformation via secondary treatments. In sequentially transfected REC, myc expression was high whether it was transfected first or second, whereas ras expression was highest when the ras gene was transfected secondarily into myc-containing REC. Molecular analysis of REC:ras:gamma transformants showed no alterations in structure of the transfected ras or of the endogenous ras, myc, p53, or fos genes. The expression of ras and p53 was increased in some isolates of REC:ras:gamma, but myc and fos expression were not affected. Similarly, REC:myc:gamma transformants did not demonstrate rearrangement or amplification of the transfected or the endogenous myc genes, or of the potentially cooperating Ha-, Ki-, or N-ras genes. Northern hybridization analysis revealed increased expression of N-ras in two isolates, REC:myc:gamma 33 and gamma 41, but no alterations in the expression of myc, raf, Ha-ras, or Ki-ras genes in any REC:myc transformant. DNA from several transformed REC:myc:gamma cell lines induced focus formation in recipient C3H 10T1/2 and NIH 3T3 cells. The NIH 3T3 foci tested positive when hybridized to a probe for rat repetitive DNA. A detailed analysis of the NIH 3T3 transformants generated from REC:myc:gamma 33 and gamma 41 DNA failed to detect Ha-ras, Ki-ras, raf, neu, trk, abl, fms, or src oncogenes of rat origin.(ABSTRACT TRUNCATED AT 400 WORDS)

MCF-10A-NeoST: A New Cell System for Studying Cell-ECM and Cell-Cell Interactions in Breast Cancer

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

Zantek, Nicole Dodge; Walker-Daniels, Jennifer; Stewart, Jane

2001-08-22

There is a continuing need for genetically matched cell systems to model cellular behaviors that are frequently observed in aggressive breast cancers. We report here the isolation and initial characterization of a spontaneously arising variant of MCF-10A cells, NeoST, which provides a new model to study cell adhesion and signal transduction in breast cancer. NeoST cells recapitulate important biological and biochemical features of metastatic breast cancer, including anchorage-independent growth, invasiveness in threedimensional reconstituted membranes, loss of E-cadherin expression, and increased tyrosine kinase activity. A comprehensive analysis of tyrosine kinase expression revealed overexpression or functional activation of the Axl, FAK, andmore » EphA2 tyrosine kinases in transformed MCF-10A cells. MCF-10A and these new derivatives provide a genetically matched model to study defects in cell adhesion and signaling that are relevant to cellular behaviors that often typify aggressive breast cancer cells.« less

Continuous activation of Nrf2 and its target antioxidant enzymes leads to arsenite-induced malignant transformation of human bronchial epithelial cells

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

Yang, Xu; Wang, Dapeng; Department of Toxicology, School of Public Health, Guizhou Medical University, Guiyang, Guizhou

Long-term exposure to arsenite leads to human lung cancer, but the underlying mechanisms of carcinogenesis remain obscure. The transcription factor of nuclear factor-erythroid-2 p45-related factor (Nrf2)-mediated antioxidant response represents a critical cellular defense mechanism and protection against various diseases. Paradoxically, emerging data suggest that the constitutive activation of Nrf2 is associated with cancer development, progression and chemotherapy resistance. However, the role of Nrf2 in the occurrence of cancer induced by long-term arsenite exposure remains to be fully understood. By establishing transformed human bronchial epithelial (HBE) cells via chronic low-dose arsenite treatment, we showed that, in acquiring this malignant phenotype, continuousmore » low level of ROS and sustained enhancement of Nrf2 and its target antioxidant enzyme levels were observed in the later-stage of arsenite-induced cell transformation. The downregulation of Keap1 level may be responsible for the over-activation of Nrf2 and its target enzymes. To validate these observations, Nrf2 was knocked down in arsenite-transformed HBE cells by SiRNA transfection, and the levels of Nrf2 and its target antioxidant enzymes, ROS, cell proliferation, migration, and colony formation were determined following these treatments. Results showed that blocked Nrf2 expression significantly reduced Nrf2 and its target antioxidant enzyme levels, restored ROS levels, and eventually suppressed cell proliferation, migration, and colony formation of the transformed cells. In summary, the results of the study strongly suggested that the continuous activation of Nrf2 and its target antioxidant enzymes led to the over-depletion of intracellular ROS levels, which contributed to arsenite-induced HBE cell transformation. - Highlights: • Low level, long term arsenite exposure induces malignant transformation in vitro. • Long term arsenite exposure reduces ROS and MDA levels. • Long term arsenite exposure enhances Nrf2-mediated antioxidant levels. • Knockdown of Nrf2 reduces malignant degree of arsenite-transformed cells.« less

Moloney leukemia virus immortalizes B lymphocytes in vitro.

PubMed Central

Runnels, J; Serunian, L; Thursby, M; Rosenberg, N

1991-01-01

An in vitro culture system in which Moloney murine leukemia virus induces immortalization of mature B lymphocytes has been developed. The cell lines derived in this way are nontumorigenic, and virus production is not required to sustain them. This system provides a new in vitro model with which to study the stepwise process of transformation by retroviruses lacking oncogenes. Images PMID:1895405

Multistep carcinogenesis of normal human fibroblasts. Human fibroblasts immortalized by repeated treatment with Co-60 gamma rays were transformed into tumorigenic cells with Ha-ras oncogenes.

PubMed

Namba, M; Nishitani, K; Fukushima, F; Kimoto, T

1988-01-01

Two normal mortal human fibroblast cell strains were transformed into immortal cell lines, SUSM-1 and KMST-6, by treatment with 4-nitroquinoline 1-oxide (4NQO) and Co-60 gamma rays, respectively. These immortalized cell lines showed morphological changes of cells and remarkable chromosome aberrations, but neither of them grew in soft agar or formed tumors in nude mice. The immortal cell line, KMST-6, was then converted into neoplastic cells by treatment with Harvey murine sarcoma virus (Ha-MSV) or the c-Ha-ras oncogene derived from a human lung carcinoma. These neoplastically transformed cells acquired anchorage-independent growth potential and developed tumors when transplanted into nude mice. All the tumors grew progressively without regression until the animals died of tumors. In addition, the tumors were transplantable into other nude mice. Normal human fibroblasts, on the other hand, were not transformed into either immortal or tumorigenic cells by treatment with Ha-MSV or c-Ha-ras alone. Our present data indicate that (1) the chemical carcinogen, 4NQO, or gamma rays worked as an initiator of carcinogenesis in normal human cells, giving rise to immortality, and (2) the ras gene played a role in the progression of the immortally transformed cells to more malignant cells showing anchorage-independent growth and tumorigenicity. In other words, the immortalization process of human cells seems to be a pivotal or rate-limiting step in the carcinogenesis of human cells.

Influence of processing parameters on microstructure and biocompatibility of surface laser sintered hydroxyapatite-SiO2 composites.

PubMed

Kivitz, E; Görke, R; Schilling, A F; Zhang, J; Heinrich, J G

2013-05-01

Silica-doped hydroxyapatite (HA) is a promising material concerning biocompatibility to natural bone, bioactivity and osteoconductive characteristics. HA exhibits phase transformations during sintering which are attendant to the change in volume and thermal strain. To avoid cracks during sintering, the exact knowledge of the phase transition temperatures is necessary. The sintering behavior of HA can be improved by adding amorphous silica with a low coefficient of thermal expansion. Therefore, the phase transformations in the system HA-SiO2 were analyzed by using differential scanning calorimetry followed by quantitative phase analysis by X-ray diffraction with the Riedveld method. The maximum sintering temperature without reversible phase transformation was defined as 1265°C. In laser surface sintered (LSS) samples, amorphous SiO2 , HA, and Si-α-TCP (or α-TCP) were detected. By comparison, only crystalline phases, such as cristobalite, HA, β-TCP, and Si-α-TCP (or α-TCP), were determined after furnace sintering. Scanning electron microscopy micrographs of furnace sintered and LSS samples show the differences in the resulting microstructures. Biocompatibility was determined by measuring cell activity of osteoblasts cultivated on four laser-sintered materials in the HA-SiO2 system in comparison to normal cell culture plastic. Cell proliferation was similar on all surfaces. The level of the cell activity on day 8 varied depending on the composition of the material and increased linearly as the amorphous SiO2 content rose. Taken together a laser-based method to develop novel biocompatible HA-SiO2 ceramics with adjustable properties and possible applications as orthopedic bioceramics are discussed. Copyright © 2012 Wiley Periodicals, Inc.

Accelerated Stem Growth Rates and Improved Fiber Properties of Loblolly Pine: Functional Analysis Of CyclinD from Pinus taeda

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

Dr. John Cairney, School of Biology and Institute of Paper Science and Technology @ Georgia Tech, Georgia Institute of Technology; Dr. Gary Peter, University of Florida; Dr. Ulrika Egertsdotter, Dept. of Forestry, Virgina Tech

A sustained supply of low-cost, high quality raw materials is essential for the future success of the U.S. forest products industry. To maximize stem (trunk) growth, a fundamental understanding of the molecular mechanisms that regulate cell divisions within the cambial meristem is essential. We hypothesize that auxin levels within the cambial meristem regulate cyclin gene expression and this in turn controls cell cycle progression as occurs in all eukaryotic cells. Work with model plant species has shown that ectopic overexpression of cyclins promotes cell division thereby increasing root growth > five times. We intended to test whether ectopic overexpression ofmore » cambial cyclins in the cambial zone of loblolly pine also promotes cell division rates that enhance stem growth rates. Results generated in model annual angiosperm systems cannot be reliably extrapolated to perennial gymnosperms, thus while the generation and development of transgenic pine is time consuming, this is the necessary approach for meaningful data. We succeeded in isolating a cyclin D gene and Clustal analysis to the Arabidopsis cyclin D gene family indicates that it is more closely related to cyclin D2 than D1 or D3 Using this gene as a probe we observed a small stimulation of cyclin D expression in somatic embryo culture upon addition of auxin. We hypothesized that trees with more cells in the vascular cambial and expansion zones will have higher cyclin mRNA levels. We demonstrated that in trees under compressive stress where the rates of cambial divisions are increased on the underside of the stem relative to the top or opposite side, there was a 20 fold increase in the level of PtcyclinD1 mRNA on the compressed side of the stem relative to the opposite. This suggests that higher secondary growth rates correlate with PtcyclinD1 expression. We showed that larger diameter trees show more growth during each year and that the increased growth in loblolly pine trees correlates with more cell divisions in the cambial meristem as expected. We isolated a promoter from a cambial specific gene and commenced development of transformation protocols for loblolly pine. Since our results show that cyclin D expression correlates with increased growth we continued with experiments to demonstrate the effect of cyclin overexpression upon tree growth. Vectors which constitutively express the cyclin D cDNA were constructed and transformed into a transgenic pine system through the collaboration with Forest Research, New Zealand. The transformation system for Pinus radiata is well established and we hoped to gain phenotypic information in a closely related pine, rather than await development of a robust loblolly pine transformation method. Transformation experiments were conducted by a biolistic method developed at Forest Research, NZ. A total of 78 transgenic embryogenic lines were generated and bulked up with a good representation of transgenic lines per construct. Transformed calli were originally identified by resistance to the antibiotic Geneticin contained in the medium. The transgenic nature of the selected lines was subsequently confirmed using histochemical GUS staining. To date, 10 out of 13 selected transgenic lines have produced embryos and we are currently harvesting the first transgenic plantlets. At present time 22 of those plantlets have been moved to GMO facilities. We will soon develop a strategy for assessing potential phenotypic differences between the transclones and non-transformed controls. Transgenic plants are being grown to a stage (approx. 1 year) when meaningful phenotypic evaluation can be conducted. The recent availability of 10,000 element loblolly pine cDNA microarray will permit the evaluation of cyclinD overexpression upon gene expression in transgenic Pinus.« less

Neoplastic transformation induced by carbon ions.

PubMed

Bettega, Daniela; Calzolari, Paola; Hessel, Petra; Stucchi, Claudio G; Weyrather, Wilma K

2009-03-01

The objective of this experiment was to compare the oncogenic potential of carbon ion beams and conventional photon beams for use in radiotherapy. The HeLa X human skin fibroblast cell line CGL1 was irradiated with carbon ions of three different energies (270, 100, and 11.4 MeV/u). Inactivation and transformation data were compared with those for 15 MeV photons. Inactivation and transformation frequencies for the 270 MeV/u carbon ions were similar to those for 15-MeV photons. The maximal relative biologic effectiveness (RBE(alpha)) values for 100MeV/u and 11.4 MeV/u carbon ions, respectively, were as follows: inactivation, 1.6 +/- 0.2 and 6.7 +/- 0.7; and transformation per surviving cell, 2.5 +/- 0.6 and 12 +/- 3. The curve for dose-transformation per cell at risk exhibited a maximum that was shifted toward lower doses at lower energies. Transformation induction per cell at risk for carbon ions in the entrance channel was comparable to that for photons, whereas for the lower energies, 100 MeV/u and 11 MeV/u, which are representative of the energies delivered to the tumor margins and volume, respectively, the probability of transformation in a single cell was greater than it was for photons. In addition, at isoeffective doses with respect to cell killing, the 11.4-MeV/u beam was more oncogenic than were photons.

Mannan oligosaccharide requires functional ETC and TLR for biological radiation protection to normal cells.

PubMed

Sanguri, Sweta; Gupta, Damodar

2018-06-27

Low LET Ionizing radiation is known to alter intracellular redox balance by inducing free radical generation, which may cause oxidative modification of various cellular biomolecules. The extent of biomolecule-modifications/ damages and changes in vital processes (viz. cellular homeostasis, inter-/intra-cellular signaling, mitochondrial physiology/dynamics antioxidant defence systems) are crucial which in turn determine fate of cells. In the present study, we expended TLR expressing (normal/ transformed) and TLR null cells; and we have shown that mannan pretreatment in TLR expressing normal cells offers survival advantage against lethal doses of ionizing radiation. On the contrary, mannan pretreatment does not offer any protection against radiation to TLR null cells, NKE ρ° cells and transformed cells. In normal cells, abrupt decrease in mitochondrial membrane potential and endogenous ROS levels occurs following treatment with mannan. We intend to irradiate mannan-pretreated cells at a specific stage of perturbed mitochondrial functioning and ROS levels to comprehend if mannan pretreatment offers any survival advantage against radiation exposure to cells. Interestingly, pre-irradiation treatment of cells with mannan activates NFκB, p38 and JNK, alters mitochondrial physiology, increases expression of Cu/ZnSOD and MnSOD, minimizes oxidation of mitochondrial phospholipids and offers survival advantage in comparison to irradiated group, in TLR expressing normal cells. The study demonstrates that TLR and mitochondrial ETC functions are inevitable in radio-protective efficacy exhibited by mannan.

Generation and function of immunosuppressive human and murine CD8+ T cells by transforming growth factor-β and retinoic acid

PubMed Central

Fleissner, Diana; Frede, Annika; Knott, Markus; Knuschke, Torben; Geffers, Robert; Hansen, Wiebke; Dobos, Gustav; Langhorst, Jost; Buer, Jan; Westendorf, Astrid M

2011-01-01

The intestinal immune system is constantly challenged by foreign antigens and commensal bacteria. Therefore, proper control of the intestinal microenvironment is required. One important arm of this regulatory network consists of regulatory T cells. In contrast to CD4+ Foxp3+ regulatory T cells, which have been well characterized, immunomodulatory CD8+ T cells that express Foxp3 are less well defined in terms of their generation and function. Failures of these regulatory mechanisms contribute to the development of inflammatory bowel disease. In this study we demonstrate that the frequency of CD8+ Foxp3+ T cells is reduced in the peripheral blood of patients with ulcerative colitis. As these cells might play a currently underestimated role in the maintenance of intestinal homeostasis, we have investigated human and murine CD8+ Foxp3+ T cells generated by stimulating naive CD8+ T cells in the presence of transforming growth factor-β and retinoic acid, mediators that are abundantly produced in the intestinal mucosa. These CD8+ Foxp3+ fully competent regulatory T cells show strong expression of regulatory molecules CD25, Gpr83 and CTLA-4 and exhibit cell–cell contact-dependent immunosuppressive activity in vitro. Our study illustrates a previously unappreciated critical role of CD8+ Foxp3+ T cells in controlling potentially dangerous T cells and in the maintenance of intestinal homeostasis. PMID:21711349

Rapid DNA transformation in Salmonella Typhimurium by the hydrogel exposure method.

PubMed

Elabed, Hamouda; Hamza, Rim; Bakhrouf, Amina; Gaddour, Kamel

2016-07-01

Even with advances in molecular cloning and DNA transformation, new or alternative methods that permit DNA penetration in Salmonella enterica subspecies enterica serovar Typhimurium are required in order to use this pathogen in biotechnological or medical applications. In this work, an adapted protocol of bacterial transformation with plasmid DNA based on the "Yoshida effect" was applied and optimized on Salmonella enterica serovar Typhimurium LT2 reference strain. The plasmid transference based on the use of sepiolite as acicular materials to promote cell piercing via friction forces produced by spreading on the surface of a hydrogel. The transforming mixture containing sepiolite nanofibers, bacterial cells to be transformed and plasmid DNA were plated directly on selective medium containing 2% agar. In order to improve the procedure, three variables were tested and the transformation of Salmonella cells was accomplished using plasmids pUC19 and pBR322. Using the optimized protocol on Salmonella LT2 strain, the efficiency was about 10(5) transformed cells per 10(9) subjected to transformation with 0.2μg plasmid DNA. In summary, the procedure is fast, offers opportune efficiency and promises to become one of the widely used transformation methods in laboratories. Copyright © 2016 Elsevier B.V. All rights reserved.

Area and power efficient DCT architecture for image compression

NASA Astrophysics Data System (ADS)

Dhandapani, Vaithiyanathan; Ramachandran, Seshasayanan

2014-12-01

The discrete cosine transform (DCT) is one of the major components in image and video compression systems. The final output of these systems is interpreted by the human visual system (HVS), which is not perfect. The limited perception of human visualization allows the algorithm to be numerically approximate rather than exact. In this paper, we propose a new matrix for discrete cosine transform. The proposed 8 × 8 transformation matrix contains only zeros and ones which requires only adders, thus avoiding the need for multiplication and shift operations. The new class of transform requires only 12 additions, which highly reduces the computational complexity and achieves a performance in image compression that is comparable to that of the existing approximated DCT. Another important aspect of the proposed transform is that it provides an efficient area and power optimization while implementing in hardware. To ensure the versatility of the proposal and to further evaluate the performance and correctness of the structure in terms of speed, area, and power consumption, the model is implemented on Xilinx Virtex 7 field programmable gate array (FPGA) device and synthesized with Cadence® RTL Compiler® using UMC 90 nm standard cell library. The analysis obtained from the implementation indicates that the proposed structure is superior to the existing approximation techniques with a 30% reduction in power and 12% reduction in area.

Nontransgenic Genome Modification in Plant Cells1[W][OA

PubMed Central

Marton, Ira; Zuker, Amir; Shklarman, Elena; Zeevi, Vardit; Tovkach, Andrey; Roffe, Suzy; Ovadis, Marianna; Tzfira, Tzvi; Vainstein, Alexander

2010-01-01

Zinc finger nucleases (ZFNs) are a powerful tool for genome editing in eukaryotic cells. ZFNs have been used for targeted mutagenesis in model and crop species. In animal and human cells, transient ZFN expression is often achieved by direct gene transfer into the target cells. Stable transformation, however, is the preferred method for gene expression in plant species, and ZFN-expressing transgenic plants have been used for recovery of mutants that are likely to be classified as transgenic due to the use of direct gene-transfer methods into the target cells. Here we present an alternative, nontransgenic approach for ZFN delivery and production of mutant plants using a novel Tobacco rattle virus (TRV)-based expression system for indirect transient delivery of ZFNs into a variety of tissues and cells of intact plants. TRV systemically infected its hosts and virus ZFN-mediated targeted mutagenesis could be clearly observed in newly developed infected tissues as measured by activation of a mutated reporter transgene in tobacco (Nicotiana tabacum) and petunia (Petunia hybrida) plants. The ability of TRV to move to developing buds and regenerating tissues enabled recovery of mutated tobacco and petunia plants. Sequence analysis and transmission of the mutations to the next generation confirmed the stability of the ZFN-induced genetic changes. Because TRV is an RNA virus that can infect a wide range of plant species, it provides a viable alternative to the production of ZFN-mediated mutants while avoiding the use of direct plant-transformation methods. PMID:20876340

A Discrete Global Grid System Programming Language Using MapReduce

NASA Astrophysics Data System (ADS)

Peterson, P.; Shatz, I.

2016-12-01

A discrete global grid system (DGGS) is a powerful mechanism for storing and integrating geospatial information. As a "pixelization" of the Earth, many image processing techniques lend themselves to the transformation of data values referenced to the DGGS cells. It has been shown that image algebra, as an example, and advanced algebra, like Fast Fourier Transformation, can be used on the DGGS tiling structure for geoprocessing and spatial analysis. MapReduce has been shown to provide advantages for processing and generating large data sets within distributed and parallel computing. The DGGS structure is ideally suited for big distributed Earth data. We proposed that basic expressions could be created to form the atoms of a generalized DGGS language using the MapReduce programming model. We created three very efficient expressions: Selectors (aka filter) - A selection function that generate a set of cells, cell collections, or geometries; Calculators (aka map) - A computational function (including quantization of raw measurements and data sources) that generate values in a DGGS cell; and Aggregators (aka reduce) - A function that generate spatial statistics from cell values within a cell. We found that these three basic MapReduce operations along with a forth function, the Iterator, for horizontal and vertical traversing of any DGGS structure, provided simple building block resulting in very efficient operations and processes that could be used with any DGGS. We provide examples and a demonstration of their effectiveness using the ISEA3H DGGS on the PYXIS Studio.

CRISPR-Cas9 vectors for genome editing and host engineering in the baculovirus-insect cell system.

PubMed

Mabashi-Asazuma, Hideaki; Jarvis, Donald L

2017-08-22

The baculovirus-insect cell system (BICS) has been widely used to produce many different recombinant proteins for basic research and is being used to produce several biologics approved for use in human or veterinary medicine. Early BICS were technically complex and constrained by the relatively primordial nature of insect cell protein glycosylation pathways. Since then, recombination has been used to modify baculovirus vectors-which has simplified the system-and transform insect cells, which has enhanced its protein glycosylation capabilities. Now, CRISPR-Cas9 tools for site-specific genome editing are needed to facilitate further improvements in the BICS. Thus, in this study, we used various insect U6 promoters to construct CRISPR-Cas9 vectors and assessed their utility for site-specific genome editing in two insect cell lines commonly used as hosts in the BICS. We demonstrate the use of CRISPR-Cas9 to edit an endogenous insect cell gene and alter protein glycosylation in the BICS.

Validation of an entirely in vitro approach for rapid prototyping of DNA regulatory elements for synthetic biology

PubMed Central

Chappell, James; Jensen, Kirsten; Freemont, Paul S.

2013-01-01

A bottleneck in our capacity to rationally and predictably engineer biological systems is the limited number of well-characterized genetic elements from which to build. Current characterization methods are tied to measurements in living systems, the transformation and culturing of which are inherently time-consuming. To address this, we have validated a completely in vitro approach for the characterization of DNA regulatory elements using Escherichia coli extract cell-free systems. Importantly, we demonstrate that characterization in cell-free systems correlates and is reflective of performance in vivo for the most frequently used DNA regulatory elements. Moreover, we devise a rapid and completely in vitro method to generate DNA templates for cell-free systems, bypassing the need for DNA template generation and amplification from living cells. This in vitro approach is significantly quicker than current characterization methods and is amenable to high-throughput techniques, providing a valuable tool for rapidly prototyping libraries of DNA regulatory elements for synthetic biology. PMID:23371936

CONTINUOUS FORMALDEHYDE MEASUREMENT SYSTEM BASED ON MODIFIED FOURIER TRANSFORM INFRARED SPECTROSCOPY

EPA Science Inventory

EPA is developing advanced open-path and cell-based optical techniques for time-resolved measurement of priority hazardous air pollutants such as formaldehyde (HCHO). Due to its high National Air Toxics Assessment risk factor, there is increasing interest in continuous measuremen...

Development of a Multifaceted Ovarian Cancer Therapeutic and Imaging Agent

DTIC Science & Technology

2008-04-01

generate recombinant disintegrins, we have successfully adapted a commercially-available E. coli expression system consisting of the Origami B (DE3...thioredoxin fusion partner from the expressed VN. Briefly for recombinant VN production, multiple colonies of transformed Origami B cells were used to

Static harmonization of dynamically harmonized Fourier transform ion cyclotron resonance cell.

PubMed

Zhdanova, Ekaterina; Kostyukevich, Yury; Nikolaev, Eugene

2017-08-01

Static harmonization in the Fourier transform ion cyclotron resonance cell improves the resolving power of the cell and prevents dephasing of the ion cloud in the case of any trajectory of the charged particle, not necessarily axisymmetric cyclotron (as opposed to dynamic harmonization). We reveal that the Fourier transform ion cyclotron resonance cell with dynamic harmonization (paracell) is proved to be statically harmonized. The volume of the statically harmonized potential distribution increases with an increase in the number of trap segments.

Transformation of NIH3T3 Cells with Synthetic c‐Ha‐ras Genes

PubMed Central

Kamiya, Hiroyuki; Miura, Kazunobu; Ohtomo, Noriko; Koda, Toshiaki; Kakinuma, Mitsuaki; Nishimura, Susumu

1989-01-01

Synthetic human c‐Ha‐ras genes in which amino acid codons were altered to those which are frequently used in highly expressed Escherichia coli genes were ligated to the 3′‐end of Rous sarcoma virus long terminal repeat. When NIH3T3 cells were transfected with the plasmids having those genes with valine at codon 12, leucine at codon 61 or arginine at codon 61, transformants were efficiently produced. These results indicated that the synthetic c‐Ha‐ras genes are expressed in a mammalian system even though their codon usage is altered to correspond with that of E. colt. This expression vector system should he useful for studies on the structure‐function relationships of c‐Ha‐ras, since the synthetic gene can be easily modified to have multiple base alterations, and can also be used simultaneously for the production of large amounts of p21 in E. coli for biochemical and biophysical studies. PMID:2542206

Absence of a dose-fractionation effect on neoplastic transformation induced by fission-spectrum neutrons in C3H 10T1/2 cells

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

Saran, A.; Pazzaglia, S.; Coppola, M.

1991-06-01

We have investigated the effect of fission-spectrum neutron dose fractionation on neoplastic transformation of exponentially growing C3H 10T1/2 cells. Total doses of 10.8, 27, 54, and 108 cGy were given in single doses or in five equal fractions delivered at 24-h intervals in the biological channel of the RSV-TAPIRO reactor at CRE-Casaccia. Both cell inactivation and neoplastic transformation were more effectively induced by fission neutrons than by 250-kVp X rays. No significant effect on cell survival or neoplastic transformation was observed with split doses compared to single doses of fission-spectrum neutrons. Neutron RBE values relative to X rays determined frommore » data for survival and neoplastic transformation were comparable.« less

Cloning of transgenic tobacco BY-2 cells; an efficient method to analyse and reduce high natural heterogeneity of transgene expression.

PubMed

Nocarova, Eva; Fischer, Lukas

2009-04-22

Phenotypic characterization of transgenic cell lines, frequently used in plant biology studies, is complicated because transgene expression in individual cells is often heterogeneous and unstable. To identify the sources and to reduce this heterogeneity, we transformed tobacco (Nicotiana tabacum L.) BY-2 cells with a gene encoding green fluorescent protein (GFP) using Agrobacterium tumefaciens, and then introduced a simple cloning procedure to generate cell lines derived from the individual transformed cells. Expression of the transgene was monitored by analysing GFP fluorescence in the cloned lines and also in lines obtained directly after transformation. The majority ( approximately 90%) of suspension culture lines derived from calli that were obtained directly from transformation consisted of cells with various levels of GFP fluorescence. In contrast, nearly 50% of lines generated by cloning cells from the primary heterogeneous suspensions consisted of cells with homogenous GFP fluorescence. The rest of the lines exhibited "permanent heterogeneity" that could not be resolved by cloning. The extent of fluorescence heterogeneity often varied, even among genetically identical clones derived from the primary transformed lines. In contrast, the offspring of subsequent cloning of the cloned lines was uniform, showing GFP fluorescence intensity and heterogeneity that corresponded to the original clone. The results demonstrate that, besides genetic heterogeneity detected in some lines, the primary lines often contained a mixture of epigenetically different cells that could be separated by cloning. This indicates that a single integration event frequently results in various heritable expression patterns, which are probably accidental and become stabilized in the offspring of the primary transformed cells early after the integration event. Because heterogeneity in transgene expression has proven to be a serious problem, it is highly advisable to use transgenes tagged with a visual marker for BY-2 transformation. The cloning procedure can be used not only for efficient reduction of expression heterogeneity of such transgenes, but also as a useful tool for studies of transgene expression and other purposes.

Effect of a labile methyl donor on the transformation of 5-demethyltangeretin and the related implication on bioactivity.

PubMed

Ting, Yuwen; Li, Colin C; Pan, Min-Hsiung; Ho, Chi-Tang; Huang, Qingrong

2013-08-28

Polymethoxyflavones (PMFs) belong to a subgroup of flavonoids that particularly exist in the peels of citrus fruits. Despite their many health-beneficial biofunctionalities, the lipophilic nature of PMFs limits their water solubility and oral bioavailability. To investigate the effect of the delivery system on the improvement of PMF bioavailibility, a lecithin-based emulsion was formulated for the delivery of two PMF compounds, tangeretin and 5-demethyltangeretin. While the emulsion system improved the digestion kinetics and the total solubilized PMF concentrations in in vitro lipolysis studies, the concentration of 5-demethyltangeretin decreased due to chemical transformation to its permethoxylated counterpart, tangeretin. The emulsifier lecithin used in this emulsion formulation contained a choline headgroup as a labile methyl group donor. The presence of a methyl donor potentially caused the transformation of 5-demethyltangeretin and reduced its anti-cancer-cell-proliferation activities. Moreover, this is the first report in the literature of the transformation from 5-demethyltangeretin to tangeretin in a lecithin-based emulsion during lipolysis, and the mechanism underlying this phenomenon has also been proposed for the first time.

Protoplast isolation, transient transformation of leaf mesophyll protoplasts and improved Agrobacterium-mediated leaf disc infiltration of Phaseolus vulgaris: tools for rapid gene expression analysis.

PubMed

Nanjareddy, Kalpana; Arthikala, Manoj-Kumar; Blanco, Lourdes; Arellano, Elizabeth S; Lara, Miguel

2016-06-24

Phaseolus vulgaris is one of the most extensively studied model legumes in the world. The P. vulgaris genome sequence is available; therefore, the need for an efficient and rapid transformation system is more imperative than ever. The functional characterization of P. vulgaris genes is impeded chiefly due to the non-amenable nature of Phaseolus sp. to stable genetic transformation. Transient transformation systems are convenient and versatile alternatives for rapid gene functional characterization studies. Hence, the present work focuses on standardizing methodologies for protoplast isolation from multiple tissues and transient transformation protocols for rapid gene expression analysis in the recalcitrant grain legume P. vulgaris. Herein, we provide methodologies for the high-throughput isolation of leaf mesophyll-, flower petal-, hypocotyl-, root- and nodule-derived protoplasts from P. vulgaris. The highly efficient polyethylene glycol-mannitol magnesium (PEG-MMG)-mediated transformation of leaf mesophyll protoplasts was optimized using a GUS reporter gene. We used the P. vulgaris SNF1-related protein kinase 1 (PvSnRK1) gene as proof of concept to demonstrate rapid gene functional analysis. An RT-qPCR analysis of protoplasts that had been transformed with PvSnRK1-RNAi and PvSnRK1-OE vectors showed the significant downregulation and ectopic constitutive expression (overexpression), respectively, of the PvSnRK1 transcript. We also demonstrated an improved transient transformation approach, sonication-assisted Agrobacterium-mediated transformation (SAAT), for the leaf disc infiltration of P. vulgaris. Interestingly, this method resulted in a 90 % transformation efficiency and transformed 60-85 % of the cells in a given area of the leaf surface. The constitutive expression of YFP further confirmed the amenability of the system to gene functional characterization studies. We present simple and efficient methodologies for protoplast isolation from multiple P. vulgaris tissues. We also provide a high-efficiency and amenable method for leaf mesophyll transformation for rapid gene functional characterization studies. Furthermore, a modified SAAT leaf disc infiltration approach aids in validating genes and their functions. Together, these methods help to rapidly unravel novel gene functions and are promising tools for P. vulgaris research.

Fourier analysis of the cell shape of paired human urothelial cell lines of the same origin but of different grades of transformation.

PubMed

Ostrowski, K; Dziedzic-Goclawska, A; Strojny, P; Grzesik, W; Kieler, J; Christensen, B; Mareel, M

1986-01-01

The rationale of the present investigation is the observations made by many authors of changes in the molecular structure of the cell surface during the multistep process of malignant transformation. These changes may influence cell-matrix and cell-cell interactions and thereby cause changes in cell adhesiveness and cell shape. The aim of the present work was to investigate whether the development of various grades of transformation in vivo and in vitro of human urothelial cells is accompanied by significant changes in cell shape as measured by Fourier analysis. The following transformation grades (TGr) have been defined (Christensen et al. 1984; Kieler 1984): TGr I = nonmalignant, mortal cell lines that grow independently of fibroblasts and have a prolonged life span. TGr II = nonmalignant cell lines with an infinite life span. TGr III = malignant and immortal cell lines that grow invasively in co-cultures with embryonic chick heart fragments and possess tumorigenic properties after s.c. injection into nude mice. Comparisons of 4 pairs of cell lines were performed; each pair was of the same origin. Two pairs--each including a TGr I cell line (Hu 961b and Hu 1703S) compared to a TGr III cell line (Hu 961a or Hu 1703He)--were derived from two transitional cell carcinomas (TCC) containing a heterogeneous cell population. Two additional cell lines classified as TGr II (HCV-29 and Hu 609) were compared to two TGr III sublines (HCV-29T and Hu 609T, respectively) which arose by "spontaneous" transformation during propagation in vitro of the respective maternal TGr II-cell lines.(ABSTRACT TRUNCATED AT 250 WORDS)

JunD/AP-1 Antagonizes the Induction of DAPK1 To Promote the Survival of v-Src-Transformed Cells.

PubMed

Maślikowski, Bart M; Wang, Lizhen; Wu, Ying; Fielding, Ben; Bédard, Pierre-André

2017-01-01

The increase in AP-1 activity is a hallmark of cell transformation by tyrosine kinases. Previously, we reported that blocking AP-1 using the c-Jun dominant negative mutant TAM67 induced senescence, adipogenesis, or apoptosis in v-Src-transformed chicken embryo fibroblasts (CEFs) whereas inhibition of JunD by short hairpin RNA (shRNA) specifically induced apoptosis. To investigate the role of AP-1 in Src-mediated transformation, we undertook a gene profiling study to characterize the transcriptomes of v-Src-transformed CEFs expressing either TAM67 or the JunD shRNA. Our study revealed a cluster of 18 probe sets upregulated exclusively in response to AP-1/JunD impairment and v-Src transformation. Four of these probe sets correspond to genes involved in the interferon pathway. One gene in particular, death-associated protein kinase 1 (DAPK1), is a C/EBPβ-regulated mediator of apoptosis in gamma interferon (IFN-γ)-induced cell death. Here, we show that inhibition of DAPK1 abrogates cell death in v-Src-transformed cells expressing the JunD shRNA. Chromatin immunoprecipitation data indicated that C/EBPβ was recruited to the DAPK1 promoter while the expression of a dominant negative mutant of C/EBPβ abrogated the induction of DAPK1 in response to the inhibition of AP-1. In contrast, as determined by chromatin immunoprecipitation (ChIP) assays, JunD was not detected on the DAPK1 promoter under any conditions, suggesting that JunD promotes survival by indirectly antagonizing the expression of DAPK1 in v-Src transformed cells. Transformation by the v-Src oncoprotein causes extensive changes in gene expression in primary cells such as chicken embryo fibroblasts. These changes, determining the properties of transformed cells, are controlled in part at the transcriptional level. Much attention has been devoted to transcription factors such as AP-1 and NF-κB and the control of genes associated with a more aggressive phenotype. In this report, we describe a novel mechanism of action determined by the JunD component of AP-1, a factor enhancing cell survival in v-Src-transformed cells. We show that the loss of JunD results in the aberrant activation of a genetic program leading to cell death. This program requires the activation of the tumor suppressor death-associated protein kinase 1 (DAPK1). Since DAPK1 is phosphorylated and inhibited by v-Src, these results highlight the importance of this kinase and the multiple mechanisms controlled by v-Src to antagonize the tumor suppressor function of DAPK1. Copyright © 2016 American Society for Microbiology.

Establishment of an efficient genetic transformation method in Dunaliella tertiolecta mediated by Agrobacterium tumefaciens.

PubMed

Norzagaray-Valenzuela, Claudia D; Germán-Báez, Lourdes J; Valdez-Flores, Marco A; Hernández-Verdugo, Sergio; Shelton, Luke M; Valdez-Ortiz, Angel

2018-05-16

Microalgae are photosynthetic microorganisms widely used for the production of highly valued compounds, and recently they have been shown to be promising as a system for the heterologous expression of proteins. Several transformation methods have been successfully developed, from which the Agrobacterium tumefaciens-mediated method remains the most promising. However, microalgae transformation efficiency by A. tumefaciens is shown to vary depending on several transformation conditions. The present study aimed to establish an efficient genetic transformation system in the green microalgae Dunaliella tertiolecta using the A. tumefaciens method. The parameters assessed were the infection medium, the concentration of the A. tumefaciens and co-culture time. As a preliminary screening, the expression of the gusA gene and the viability of transformed cells were evaluated and used to calculate a novel parameter called Transformation Efficiency Index (TEI). The statistical analysis of TEI values showed five treatments with the highest gusA gene expression. To ensure stable transformation, transformed colonies were cultured on selective medium using hygromycin B and the DNA of resistant colonies were extracted after five subcultures and molecularly analyzed by PCR. Results revealed that treatments which use solid infection medium, A. tumefaciens OD 600  = 0.5 and co-culture times of 72 h exhibited the highest percentage of stable gusA expression. Overall, this study established an efficient, optimized A. tumefaciens-mediated genetic transformation of D. tertiolecta, which represents a relatively easy procedure with no expensive equipment required. This simple and efficient protocol opens the possibility for further genetic manipulation of this commercially-important microalgae for biotechnological applications. Copyright © 2018 Elsevier B.V. All rights reserved.

Histological transformation after acquired resistance to epidermal growth factor tyrosine kinase inhibitors.

PubMed

Shao, Yi; Zhong, Dian-Sheng

2018-04-01

Non-small-cell lung cancer patients with sensitive epidermal growth factor receptor mutations generally respond well to tyrosine kinase inhibitors (TKIs). However, acquired resistance will eventually develop place after 8-16 months. Several mechanisms contribute to the resistance including T790M mutation, c-Met amplification, epithelial mesenchymal transformation and PIK3CA mutation; however, histological transformation is a rare mechanism. The patterns and mechanisms underlying histological transformation need to be explored. We searched PubMed, EMBASE and search engines Google Scholar, Medical Matrix for literature related to histological transformation. Case reports, cases series, and clinical and basic medical research articles were reviewed. Sixty-one articles were included in this review. Cases of transformation to small-cell lung cancer, squamous cell carcinoma, large-cell neuroendocrine carcinoma and sarcoma after TKI resistance have all been reported. As the clinical course differed dramatically between cases, a new treatment scheme needs to be recruited. The mechanisms underlying histological transformation have not been fully elucidated and probably relate to cancer stem cells, driver genetic alterations under selective pressure or the heterogeneity of the tumor. When TKI resistance develops, we recommend that patients undergo a second biopsy to determine the reason, guide the next treatment and predict the prognosis.

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

Bradbury, Andrew M.

The invention relates to a novel phagemid display system for packaging phagemid DNA into phagemid particles which completely avoids the use of helper phage. The system of the invention incorporates the use of bacterial packaging cell lines which have been transformed with helper plasmids containing all required phage proteins but not the packaging signals. The absence of packaging signals in these helper plasmids prevents their DNA from being packaged in the bacterial cell, which provides a number of significant advantages over the use of both standard and modified helper phage. Packaged phagemids expressing a protein or peptide of interest, inmore » fusion with a phage coat protein such as g3p, are generated simply by transfecting phagemid into the packaging cell line.« less

In vitro CpG methylation increases the transformation efficiency of Borrelia burgdorferi strains harboring the endogenous linear plasmid lp56.

PubMed

Chen, Qiang; Fischer, Joshua R; Benoit, Vivian M; Dufour, Nicholas P; Youderian, Philip; Leong, John M

2008-12-01

Borrelia burgdorferi is the causative agent of Lyme disease, the most common vector-borne illness in the Northern hemisphere. Low-passage-number infectious strains of B. burgdorferi exhibit extremely low transformation efficiencies-so low, in fact, as to hinder the genetic study of putative virulence factors. Two putative restriction-modification (R-M) systems, BBE02 contained on linear plasmid 25 (lp25) and BBQ67 contained on lp56, have been postulated to contribute to this poor transformability. Restriction barriers posed by other bacteria have been overcome by the in vitro methylation of DNA prior to transformation. To test whether a methylation-sensitive restriction system contributes to poor B. burgdorferi transformability, shuttle plasmids were treated with the CpG methylase M.SssI prior to the electroporation of a variety of strains harboring different putative R-M systems. We found that for B. burgdorferi strains that harbor lp56, in vitro methylation increased transformation by at least 1 order of magnitude. These results suggest that in vitro CpG methylation protects exogenous DNA from degradation by an lp56-contained R-M system, presumably BBQ67. The utility of in vitro methylation for the genetic manipulation of B. burgdorferi was exemplified by the ease of plasmid complementation of a B. burgdorferi B31 A3 BBK32 kanamycin-resistant (B31 A3 BBK32::Kan(r)) mutant, deficient in the expression of the fibronectin- and glycosaminoglycan (GAG)-binding adhesin BBK32. Consistent with the observation that several surface proteins may promote GAG binding, the B. burgdorferi B31 A3 BBK32::Kan(r) mutant demonstrated no defect in the ability to bind purified GAGs or GAGs expressed on the surfaces of cultured cells.

Effect of cell-surface hydrophobicity on bacterial conversion of water-immiscible chemicals in two-liquid-phase culture systems.

PubMed

Hamada, Takahiro; Maeda, Yusuke; Matsuda, Hiroyuki; Sameshima, Yuka; Honda, Kohsuke; Omasa, Takeshi; Kato, Junichi; Ohtake, Hisao

2009-08-01

The effect of bacterial cell-surface hydrophobicity on the bioconversion of water-immiscible chemicals in an aqueous-organic (A/O) two-liquid-phase culture system was investigated. Escherichia coli JM109 and Rhodococcus opacus B-4 were used as hydrophilic and hydrophobic whole-cell catalysts, respectively. Hydroxylation reactions of monoaromatics, including toluene (log P(ow)=2.9), ethylbenzene (3.1), n-propylbenzene (3.4), and sec-butylbenzene (3.7), were employed as model conversions. When the todC1C2BA genes encoding Pseudomonas putida toluene dioxygenase were expressed in E. coli JM109, the yield of hydroxylated monoaromatics decreased with increasing substrate hydrophobicity. By contrast, R. opacus transformants, which expressed the todC1C2BA genes, showed high performance in the hydroxylation of monoaromatics, irrespective of substrate hydrophobicity. When the R. opacus transformants were examined for their ability to hydroxylate monoaromatics in an aqueous single-liquid-phase culture, the reaction velocity was markedly lower than that observed in the A/O two-liquid-phase culture. These results suggested that R. opacus B-4 accessed the hydrophobic substrates in the oil phase, thus making it more effective for the bioconversion reactions.

Caffeine toxicity is inversely related to DNA repair in simian virus 40-transformed xeroderma pigmentosum cells irradiated with ultraviolet light

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

Cleaver, J.E.

1989-01-01

Human cells transformed by simian virus 40 (SV40) are more sensitive to killing by ultraviolet light when grown in caffeine after irradiation. The degree of sensitization at 2 mM caffeine (expressed as the ratio of the 37% survival dose for control cells divided by the 37% survival dose for cells grown in caffeine, i.e., the dose modification factor) was approximately 1.9 in transformed normal cells and 3.8-5.8 in excision-defective xeroderma pigmentosum (XP) groups A, C, and D cells. A large dose modification factor of 12 was observed in a transformed XP variant cell line. Chinese hamster ovary cells were notmore » significantly different from transformed normal human cells, with a maximum dose modification factor of 1.5. Two radioresistant XP revertants that do not excise cyclobutane dimers gave different responses; one resembled its group A parent in being sensitized by caffeine, and one did not. These results can be interpreted on the basis of a single hypothesis that cells are killed as a result of attempts to replicate damaged DNA. Increased replication rates caused by transformation, increased numbers of replication forks in DNA caused by caffeine, and increased numbers of damaged sites ahead of replication forks in excision-defective cells are all processes that will consequently increase killing according to this hypothesis. A corollary is that the XP variant may be highly sensitized to caffeine because of excision defects at the DNA replication forks, an idea that may be important in designing cloning strategies for the XP variant gene.« less

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

Pelch, Katherine E.; Tokar, Erik J.; Merrick, B. Alex

Previous work shows altered methylation patterns in inorganic arsenic (iAs)- or cadmium (Cd)-transformed epithelial cells. Here, the methylation status near the transcriptional start site was assessed in the normal human prostate epithelial cell line (RWPE-1) that was malignantly transformed by 10 μM Cd for 11 weeks (CTPE) or 5 μM iAs for 29 weeks (CAsE-PE), at which time cells showed multiple markers of acquired cancer phenotype. Next generation sequencing of the transcriptome of CAsE-PE cells identified multiple dysregulated genes. Of the most highly dysregulated genes, five genes that can be relevant to the carcinogenic process (S100P, HYAL1, NTM, NES, ALDH1A1)more » were chosen for an in-depth analysis of the DNA methylation profile. DNA was isolated, bisulfite converted, and combined bisulfite restriction analysis was used to identify differentially methylated CpG sites, which was confirmed with bisulfite sequencing. Four of the five genes showed differential methylation in transformants relative to control cells that was inversely related to altered gene expression. Increased expression of HYAL1 (> 25-fold) and S100P (> 40-fold) in transformants was correlated with hypomethylation near the transcriptional start site. Decreased expression of NES (> 15-fold) and NTM (> 1000-fold) in transformants was correlated with hypermethylation near the transcriptional start site. ALDH1A1 expression was differentially expressed in transformed cells but was not differentially methylated relative to control. In conclusion, altered gene expression observed in Cd and iAs transformed cells may result from altered DNA methylation status. - Highlights: • Cd and iAs are known human carcinogens, yet neither appears directly mutagenic. • Prior data suggest epigenetic modification plays a role in Cd or iAs induced cancer. • Altered methylation of four misregulated genes was found in Cd or iAs transformants. • The resulting altered gene expression may be relevant to cellular transformation.« less

Neoplastic transformation of hamster embryo cells by heavy ions

NASA Astrophysics Data System (ADS)

Han, Z.; Suzuki, H.; Suzuki, F.; Suzuki, M.; Furusawa, Y.; Kato, T.; Ikenaga, M.

1998-11-01

We have studied the induction of morphological transformation of Syrian hamster embryo cells by low doses of heavy ions with different linear energy transfer (LET), ranging from 13 to 400 keV/μm. Exponentially growing cells were irradiated with 12C or 28Si ion beams generated by the Heavy Ion Medical Accelerator in Chiba (HIMAC), inoculated to culture dishes, and transformed colonies were identified when the cells were densely stacked and showed a crisscross pattern. Over the LET range examined, the frequency of transformation induced by the heavy ions increased sharply at very low doses no greater than 5 cGy. The relative biological effectiveness (RBE) of the heavy ions relative to 250 kVp X-rays showed an initial increase with LET, reaching a maximum value of about 7 at 100 keV/μm, and then decreased with the further increase in LET. Thus, we confirmed that high LET heavy ions are significantly more effective than X-rays for the induction of in vitro cell transformation.

Neoplastic transformation of hamster embyro cells by heavy ions.

PubMed

Han, Z; Suzuki, H; Suzuki, F; Suzuki, M; Furusawa, Y; Kato, T; Ikenaga, M

1998-01-01

We have studied the induction of morphological transformation of Syrian hamster embryo cells by low doses of heavy ions with different linear energy transfer (LET), ranging from 13 to 400 keV/micrometer. Exponentially growing cells were irradiated with 12C or 28Si ion beams generated by the Heavy Ion Medical Accelerator in Chiba (HIMAC), inoculated to culture dishes, and transformed colonies were identified when the cells were densely stacked and showed a crisscross pattern. Over the LET range examined, the frequency of transformation induced by the heavy ions increased sharply at very low doses no greater than 5 cGy. The relative biological effectiveness (RBE) of the heavy ions relative to 250 kVp X-rays showed an initial increase with LET, reaching a maximum value of about 7 at 100 keV/micrometer, and then decreased with the further increase in LET. Thus, we confirmed that high LET heavy ions are significantly more effective than X-rays for the induction of in vitro cell transformation.

Transformation of follicular lymphoma - Why does it happen and can it be prevented?

PubMed

Link, Brian K

2018-03-01

Follicular lymphoma is a clinical disease with a multitude of presentations and behaviors. Although infrequent, transformation of follicular lymphoma to a more aggressive behaving subtype - prototypically diffuse large B-cell lymphoma - confers a substantially adverse prognosis. There is no consensus for optimal management after transformation is recognized. Historically considered a distinct clinical event, this review highlights the multiple subclinical transformational events that either variably or cumulatively result in clinical recognition of transformed follicular lymphoma. Known and suspected events include genetic and epigenetic perturbations, metabolomic changes, and alterations in the microenvironment. This diverse spectrum of pathways leads to heterogeneous clinical presentations and outcomes of transformed follicular lymphoma. Current options for prevention of transformation are limited to known strategies of managing follicular lymphoma before the transformation is recognized. Although most retrospectively analyzed studies suggest an association of lower transformation rates with early systemic therapy, specific components of therapy such as anti-CD20 antibodies, anthracyclines, or purine analogues are less strongly associated with "preventative' value. Thus, the goal of preventing transformation is of limited value among all factors that go into decisions on early management of follicular lymphoma. Future opportunities to prevent clinical evidence of transformation will benefit from early detection of markers of subclinical transformation and development of therapies to specifically target the biology implied by those markers. Copyright © 2017. Published by Elsevier Ltd.

Modeling Neuropsychiatric and Neurodegenerative Diseases With Induced Pluripotent Stem Cells.

PubMed

LaMarca, Elizabeth A; Powell, Samuel K; Akbarian, Schahram; Brennand, Kristen J

2018-01-01

Human-induced pluripotent stem cells (hiPSCs) have revolutionized our ability to model neuropsychiatric and neurodegenerative diseases, and recent progress in the field is paving the way for improved therapeutics. In this review, we discuss major advances in generating hiPSC-derived neural cells and cutting-edge techniques that are transforming hiPSC technology, such as three-dimensional "mini-brains" and clustered, regularly interspersed short palindromic repeats (CRISPR)-Cas systems. We examine specific examples of how hiPSC-derived neural cells are being used to uncover the pathophysiology of schizophrenia and Parkinson's disease, and consider the future of this groundbreaking research.

Cytoskeletal dynamics in interphase, mitosis and cytokinesis analysed through Agrobacterium-mediated transient transformation of tobacco BY-2 cells.

PubMed

Buschmann, H; Green, P; Sambade, A; Doonan, J H; Lloyd, C W

2011-04-01

Transient transformation with Agrobacterium is a widespread tool allowing rapid expression analyses in plants. However, the available methods generate expression in interphase and do not allow the routine analysis of dividing cells. Here, we present a transient transformation method (termed 'TAMBY2') to enable cell biological studies in interphase and cell division. Agrobacterium-mediated transient gene expression in tobacco BY-2 was analysed by Western blotting and quantitative fluorescence microscopy. Time-lapse microscopy of cytoskeletal markers was employed to monitor cell division. Double-labelling in interphase and mitosis enabled localization studies. We found that the transient transformation efficiency was highest when BY-2/Agrobacterium co-cultivation was performed on solid medium. Transformants produced in this way divided at high frequency. We demonstrated the utility of the method by defining the behaviour of a previously uncharacterized microtubule motor, KinG, throughout the cell cycle. Our analyses demonstrated that TAMBY2 provides a flexible tool for the transient transformation of BY-2 with Agrobacterium. Fluorescence double-labelling showed that KinG localizes to microtubules and to F-actin. In interphase, KinG accumulates on microtubule lagging ends, suggesting a minus-end-directed function in vivo. Time-lapse studies of cell division showed that GFP-KinG strongly labels preprophase band and phragmoplast, but not the metaphase spindle. © 2010 The Authors. New Phytologist © 2010 New Phytologist Trust.

[Mechanism study on difference of biotransformation between Mycobacterium fortuitum MF2 and MF96].

PubMed

Ling, Liang-Fei; Ge, Mei; Fu, Lei; Huang, Wei-Yi; Chen, Dai-Jie

2005-08-01

Biotransformation difference between parent strain (MF2) and mutant strain (MF96) of Mycobacterium fortuitum was observed. Biotransformation with resting cells showed that the major products of biotransformation by both parent and mutant strains are delta4-androstenedione(4AD) and testosterone(TS). Experiments with cell-free extract system showed that the proportion of 4AD/TS obtained from parent and mutant strains was almost same when enough NAD+ and NADH were supplied in this system. It was suggested that the difference of the ratio of products transformed by both strains in resting cell system may result from their different ratio of NAD+/NADH. This speculation was verified to be true by determination of the amount of NAD+ and NADH presented in both strains.

Parameters affecting frequency of CRISPR/Cas9 mediated targeted mutagenesis in rice.

PubMed

Mikami, Masafumi; Toki, Seiichi; Endo, Masaki

2015-10-01

Frequency of CRISPR/Cas9-mediated targeted mutagenesis varies depending on Cas9 expression level and culture period of rice callus. Recent reports have demonstrated that the CRISPR/Cas9 system can function as a sequence-specific nuclease in various plant species. Induction of mutation in proliferating tissue during embryogenesis or in germline cells is a practical means of generating heritable mutations. In the case of plant species in which cultured cells are used for transformation, non-chimeric plants can be obtained when regeneration occurs from mutated cells. Since plantlets are regenerated from both mutated and non-mutated cells in a random manner, any increment in the proportion of mutated cells in Cas9- and guide RNA (gRNA)-expressing cells will help increase the number of plants containing heritable mutations. In this study, we examined factors affecting mutation frequency in rice calli. Following sequential transformation of rice calli with Cas9- and gRNA- expression constructs, the mutation frequency in independent Cas9 transgenic lines was analyzed. A positive correlation between Cas9 expression level and mutation frequency was found. This positive relationship was observed regardless of whether the transgene or an endogenous gene was used as the target for CRISPR/Cas9-mediated mutagenesis. Furthermore, we found that extending the culture period increased the proportion of mutated cells as well as the variety of mutations obtained. Because mutated and non-mutated cells might proliferate equally, these results suggest that a prolonged tissue culture period increases the chance of inducing de novo mutations in non-mutated cells. This fundamental knowledge will help improve systems for obtaining non-chimeric regenerated plants in many plant species.

Host nuclear proteins expressed in simian virus 40-transformed and -infected cells.

PubMed Central

Melero, J A; Tur, S; Carroll, R B

1980-01-01

Two new families of host proteins (Mr, 48,000 and 55,000), in additional to the viral large (T) and small tumor antigens, are precipitable, with anti-T antiserum, from cells transformed or infected by the DNA tumor virus simian virus 40 (SV40). Rabbit anti-mouse 48,000 protein antiserum reacts specifically with SV40-infected or -transformed mouse cells to give nuclear staining indistinguishable from T-antigen staining but does not react with SV40-transformed human cells which nevertheless have structurally analogous 48,000 proteins, nor does it give nuclear fluorescence with untransformed mouse cells. Comparison of the partial proteolytic digests of the 48,000 proteins from cultured cells of various mammalian species shows that they are structurally related but not related to the 55,000 or large T-antigen proteins. The 55,000 proteins from the various mammalian species were also structurally related. Images PMID:6244576

In vitro reestablishment of cell-cell contacts in adult rat cardiomyocytes. Functional role of transmembrane components in the formation of new intercalated disk-like cell contacts.

PubMed

Eppenberger, H M; Zuppinger, C

1999-01-01

Primary adult rat cardiomyocytes (ARC)in culture are shown to be a model system for cardiac cell hypertrophy in vitro. ARC undergo a process of morphological transformation and grow only by increase in cell size, however, without loss of the cardiac phenotype. The isolated cells spread and establish new cell-cell contacts, eventually forming a two-dimensional heart tissue-like synchronously beating cell sheet. The reformation of specific cell contacts (intercalated disks) is shown also between ventricular and atrial cardiomyocytes by using antibodies against the gap junction protein connexin-43 and after microinjection into ARC of N-cadherin cDNA fused to reporter green fluorescent protein (GFP) cDNA. The expressed fusion protein allowed the study of live cell cultures and of the dynamics of the adherens junction protein N-cadherin during the formation of new cell-cell contacts. The possible use of the formed ARC cell-sheet cells under microgravity conditions as a test system for the reformation of the cytoskeleton of heart muscle cells is proposed.

Primary EBV infection of human umbilical cord lymphocytes and EBV genome-negative lymphoblastoid cell lines (BJAB and Ramos).

PubMed

Takimoto, T; Sato, H; Ogura, H

1986-01-01

The appearance of Epstein-Barr virus (EBV)-associated nuclear antigen (EBNA) and induction of EBV-induced early antigen (EA) in human umbilical cord blood lymphocytes (HUCLs) and two EBV genome-negative Burkitt's lymphoma (BL) lines (BJAB and Ramos) were studied by infection with EBVs prepared from three different cell lines: marmoset cell line (B95-8) derived from infections mononucleosis, BL-derived cell line (P3HR-1) and human epithelial hybrid cell line (NPC-KT) derived from nasopharyngeal carcinoma. B95-8 virus can transform HUCLs but cannot superinfect Raji cells. P3HR-1 virus can transform HUCLs cells but cannot transform HUCLs. NPC-KT virus can transform HUCLs and can superinfect Raji cells. We have examined the time sequence of EBNA appearance and EA induction in HUCLs, BJAB cells and Ramos cells, in order to determine if three different strains of EBV differ in their abilities to infect their cells. We found that all three strains of EBV can induce EBNA in HUCLs, BJAB cells and Ramos cells. On the other hand, we found that P3HR-1 virus and NPC-KT virus can induce EA in BJAB cells and Ramos cells, but B95-8 virus cannot induce EA in their cells.

Methods for genetic transformation of filamentous fungi.

PubMed

Li, Dandan; Tang, Yu; Lin, Jun; Cai, Weiwen

2017-10-03

Filamentous fungi have been of great interest because of their excellent ability as cell factories to manufacture useful products for human beings. The development of genetic transformation techniques is a precondition that enables scientists to target and modify genes efficiently and may reveal the function of target genes. The method to deliver foreign nucleic acid into cells is the sticking point for fungal genome modification. Up to date, there are some general methods of genetic transformation for fungi, including protoplast-mediated transformation, Agrobacterium-mediated transformation, electroporation, biolistic method and shock-wave-mediated transformation. This article reviews basic protocols and principles of these transformation methods, as well as their advantages and disadvantages.

Identification of Novel Prognostic Genetic Markers in Prostate Cancer

DTIC Science & Technology

2000-02-01

alterations in two normal- and three malignant-derived prostate epithelial cell lines immortalized with the E6 and E7 transforming genes of human papilloma virus (HPV...malignant-derived prostate epithelial cell lines immortalized with the E6 and E7 transforming genes of human papilloma virus (HPV) 16. These studies...transforming genes of human papilloma virus (HPV) 16 (13). The cell lines demonstrated several numerical and structural chromosomal alterations

Water Carcinogenicity and Prevalence of HPV Infection in Esophageal Cancer Patients in Huaihe River Basin, China

PubMed Central

Li, Jintao; Khodahemmati, Sara; Wang, Minglian; Wang, Yangjunqi; Zhao, Lijiao; Jia, Runqing; Chen, Su

2018-01-01

Objective The incidence of the upper gastrointestinal tumor has increased rapidly during recent decades. The relationship between local water pollution and the tumor is still not much clear, so this study was conducted to further investigate the local water pollution and its influence on the malignant cell transformation. Prevalence of human papillomavirus (HPV) in local esophageal cancer (EC) patients was also analyzed in Shenqiu County for the first time. Methods Two-step cell transformation was used to study different sources of water in the malignant cell transformation, and the existence of 3-methylcholanthrene (3-MC) in water was analyzed from the river and shallow and deep wells. HPV DNA in tissue samples of EC patients was detected by polymerase chain reaction (PCR) and HPV diagnostic kit. Results The river water has higher cytotoxicity than the shallow well water and induced significant cell malignant transformation, while deep well water has not shown the malignant cell transformation. In Huaihe River water, the 3-MC concentration was found higher than shallow and deep wells. An HPV infection rate was found high in patients with esophageal cancer. Conclusion Long-term consumption of polluted water can induce malignant cell transformation, and the presence of HPV may be an important cause of cancer. PMID:29853858

Pycnogenol reduces talc-induced neoplastic transformation in human ovarian cell cultures.

PubMed

Buz'Zard, Amber R; Lau, Benjamin H S

2007-06-01

Talc and poor diet have been suggested to increase the risk of developing ovarian cancer; which can be reduced by a diet rich in fruit and vegetables. Talc is ubiquitous despite concern about its safety, role as a possible carcinogen and known ability to cause irritation and inflammation. It was recently shown that Pycnogenol (Pyc; a proprietary mixture of water-soluble bioflavonoids extracted from French maritime pine bark) was selectively toxic to established malignant ovarian germ cells. This study investigated talc-induced carcinogenesis and Pyc-induced chemoprevention. Normal human epithelial and granulosa ovarian cell lines and polymorphonuclear neutrophils (PMN) were treated with talc, or pretreated with Pyc then talc. Cell viability, reactive oxygen species (ROS) generation and neoplastic transformation by soft agar assay were measured. Talc increased proliferation, induced neoplastic transformation and increased ROS generation time-dependently in the ovarian cells and dose-dependently in the PMN. Pretreatment with Pyc inhibited the talc-induced increase in proliferation, decreased the number of transformed colonies and decreased the ROS generation in the ovarian cells. The data suggest that talc may contribute to ovarian neoplastic transformation and Pyc reduced the talc-induced transformation. Taken together, Pyc may prove to be a potent chemopreventative agent against ovarian carcinogenesis. (c) 2007 John Wiley & Sons, Ltd.

Mammalian synthetic biology for studying the cell

PubMed Central

Mathur, Melina; Xiang, Joy S.

2017-01-01

Synthetic biology is advancing the design of genetic devices that enable the study of cellular and molecular biology in mammalian cells. These genetic devices use diverse regulatory mechanisms to both examine cellular processes and achieve precise and dynamic control of cellular phenotype. Synthetic biology tools provide novel functionality to complement the examination of natural cell systems, including engineered molecules with specific activities and model systems that mimic complex regulatory processes. Continued development of quantitative standards and computational tools will expand capacities to probe cellular mechanisms with genetic devices to achieve a more comprehensive understanding of the cell. In this study, we review synthetic biology tools that are being applied to effectively investigate diverse cellular processes, regulatory networks, and multicellular interactions. We also discuss current challenges and future developments in the field that may transform the types of investigation possible in cell biology. PMID:27932576

Improved electroporation procedure for genetic transformation of Dekkera/Brettanomyces bruxellensis.

PubMed

Miklenić, Marina; Žunar, Bojan; Štafa, Anamarija; Svetec, Ivan-Krešimir

2015-12-01

Yeast Dekkera/Brettanomyces bruxellensis is one of the most common contaminants in wine industry, but also one of the most promising candidates for large-scale bioethanol production. Brettanomyces bruxellensis not only produces and tolerates high ethanol concentrations, but can also ferment cellobiose and adapt to lignocellulose hydrolasate. Furthermore, genome sequences of several B. bruxellensis strains are available, and efforts have been made to develop tools for genetic transformation of this yeast. Previously, we reported a successful transformation using lithium acetate/PEG method and electroporation, however, with very low transformation efficiency (10-20 transformants μg(-1)). Here we describe an optimization of electroporation procedure which resulted in a significant increase of transformation efficiency (2.8 × 10(3) transformants μg(-1)). Several key transformation parameters were optimized including cell growth phase, density of cells in the transformation sample and electroporation settings. We determined that treating the cells with both lithium acetate (100 mM) and dithiothreitol (35 mM) synergistically improves transformation efficiency. Using the described procedure around 500 transformants can be obtained per transformation sample with 180 ng of non-homologous linear transforming fragment. Additionally, several transformants were obtained with less than 1 ng of DNA demonstrating that this procedure is adequate even when very limited amount of DNA is available. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Evaluating the biological risk of functionalized multiwalled carbon nanotubes and functionalized oxygen-doped multiwalled carbon nanotubes as possible toxic, carcinogenic, and embryotoxic agents

PubMed Central

Lara-Martínez, Luis A; Massó, Felipe; Palacios González, Eduardo; García-Peláez, Isabel; Contreras–Ramos, Alejandra; Valverde, Mahara; Rojas, Emilio; Cervantes-Sodi, Felipe; Hernández-Gutiérrez, Salomón

2017-01-01

Carbon nanotubes (CNTs) have been a focus of attention due to their possible applications in medicine, by serving as scaffolds for cell growth and proliferation and improving mesenchymal cell transplantation and engraftment. The emphasis on the benefits of CNTs has been offset by the ample debate on the safety of nanotechnologies. In this study, we determine whether functionalized multiwalled CNTs (fMWCNTs) and functionalized oxygen-doped multiwalled CNTs (fCOxs) have toxic effects on rat mesenchymal stem cells (MSCs) in vitro by analyzing morphology and cell proliferation and, using in vivo models, whether they are able to transform MSCs in cancer cells or induce embryotoxicity. Our results demonstrate that there are statistically significant differences in cell proliferation and the cell cycle of MSCs in culture. We identified dramatic changes in cells that were treated with fMWCNTs. Our evaluation of the transformation to cancer cells and cytotoxicity process showed little effect. However, we found a severe embryotoxicity in chicken embryos that were treated with fMWCNTs, while fCOxs seem to exert cardioembryotoxicity and a discrete teratogenicity. Furthermore, it seems that the time of contact plays an important role during cell transformation and embryotoxicity. A single contact with fMWCNTs is not sufficient to transform cells in a short time; an exposure of fMWCNTs for 2 weeks led to cell transformation risk and cardioembryotoxicity effects. PMID:29089764

Evaluating the biological risk of functionalized multiwalled carbon nanotubes and functionalized oxygen-doped multiwalled carbon nanotubes as possible toxic, carcinogenic, and embryotoxic agents.

PubMed

Lara-Martínez, Luis A; Massó, Felipe; Palacios González, Eduardo; García-Peláez, Isabel; Contreras-Ramos, Alejandra; Valverde, Mahara; Rojas, Emilio; Cervantes-Sodi, Felipe; Hernández-Gutiérrez, Salomón

2017-01-01

Carbon nanotubes (CNTs) have been a focus of attention due to their possible applications in medicine, by serving as scaffolds for cell growth and proliferation and improving mesenchymal cell transplantation and engraftment. The emphasis on the benefits of CNTs has been offset by the ample debate on the safety of nanotechnologies. In this study, we determine whether functionalized multiwalled CNTs (fMWCNTs) and functionalized oxygen-doped multiwalled CNTs (fCOxs) have toxic effects on rat mesenchymal stem cells (MSCs) in vitro by analyzing morphology and cell proliferation and, using in vivo models, whether they are able to transform MSCs in cancer cells or induce embryotoxicity. Our results demonstrate that there are statistically significant differences in cell proliferation and the cell cycle of MSCs in culture. We identified dramatic changes in cells that were treated with fMWCNTs. Our evaluation of the transformation to cancer cells and cytotoxicity process showed little effect. However, we found a severe embryotoxicity in chicken embryos that were treated with fMWCNTs, while fCOxs seem to exert cardioembryotoxicity and a discrete teratogenicity. Furthermore, it seems that the time of contact plays an important role during cell transformation and embryotoxicity. A single contact with fMWCNTs is not sufficient to transform cells in a short time; an exposure of fMWCNTs for 2 weeks led to cell transformation risk and cardioembryotoxicity effects.

Immortalization of cat iris sphincter smooth muscle cells by SV40 virus: growth, morphological, biochemical and pharmacological characteristics.

PubMed

Ocklind, A; Yousufzai, S Y; Ghosh, S; Coca-Prados, M; St Jernschantz, J; Abdel-Latif, A A

1995-11-01

The purpose of this study was to establish immortalized cell cultures of cat iris sphincter smooth muscle cells for a model investigating ocular receptors and their signal transduction pathways. Cultured cat iris sphincter muscle cells were immortalized by viral transformation with SV40 virus and the morphological and immunocytochemical properties of the normal and immortalized cells were investigated. The transformed cell clone, SV-CISM-2, was further characterized biochemically and pharmacologically. The normal muscle cells showed characteristics of smooth muscle cells, as judged by their growth and the presence of smooth muscle alpha-actin and desmin. After seven passages the normal cells ceased to proliferate. In contrast, the immortalized cells retained their proliferative ability for more than 220 population doublings over 55 passages. The transformation phenotype in these cells was confirmed by their expression of the large T-antigen, the incorporation of viral DNA into cellular DNA, growth in agarose and in low-serum medium, and complete loss of contact inhibition. The immortalized cells expressed smooth muscle alpha-actin, desmin and MLC protein. Biochemical and pharmacological studies on the SV-CISM cells revealed the presence of several functional receptors including muscarinic cholinergic, beta-adrenergic, peptidergic (substance P and endothelin). Platelet-activating factor, and prostaglandin (PG). Muscarinic stimulation of these cells resulted in: (a) a dose-dependent increase in the release of arachidonic acid (AA) and (PGs) and enhancement in the production of inositol trisphosphate (IP3); and (b) a substantial increase in MLC phosphorylation (118%), an indicator of smooth muscle contractility. The stimulatory effects of carbachol on these responses were completely blocked by atropine, a muscarinic receptor antagonist. This study constitutes the first successful immortalization of iris sphincter smooth muscle cells. The SV-CISM-2 cells can serve as an important model system for investigations on the biochemical and pharmacological properties of receptors and their signal transduction pathways in smooth muscle. The advantage of these cells over normal iris sphincter cells is that they can be propagated over many generations without alterations in their morphological, biochemical and physiological characteristics.

A Rapid, Highly Efficient and Economical Method of Agrobacterium-Mediated In planta Transient Transformation in Living Onion Epidermis

PubMed Central

Xu, Kedong; Huang, Xiaohui; Wu, Manman; Wang, Yan; Chang, Yunxia; Liu, Kun; Zhang, Ju; Zhang, Yi; Zhang, Fuli; Yi, Liming; Li, Tingting; Wang, Ruiyue; Tan, Guangxuan; Li, Chengwei

2014-01-01

Transient transformation is simpler, more efficient and economical in analyzing protein subcellular localization than stable transformation. Fluorescent fusion proteins were often used in transient transformation to follow the in vivo behavior of proteins. Onion epidermis, which has large, living and transparent cells in a monolayer, is suitable to visualize fluorescent fusion proteins. The often used transient transformation methods included particle bombardment, protoplast transfection and Agrobacterium-mediated transformation. Particle bombardment in onion epidermis was successfully established, however, it was expensive, biolistic equipment dependent and with low transformation efficiency. We developed a highly efficient in planta transient transformation method in onion epidermis by using a special agroinfiltration method, which could be fulfilled within 5 days from the pretreatment of onion bulb to the best time-point for analyzing gene expression. The transformation conditions were optimized to achieve 43.87% transformation efficiency in living onion epidermis. The developed method has advantages in cost, time-consuming, equipment dependency and transformation efficiency in contrast with those methods of particle bombardment in onion epidermal cells, protoplast transfection and Agrobacterium-mediated transient transformation in leaf epidermal cells of other plants. It will facilitate the analysis of protein subcellular localization on a large scale. PMID:24416168

Quantitative two-dimensional gel electrophoresis analysis of human fibroblasts transformed by ras oncogenes.

PubMed

Miller, M J; Maher, V M; McCormick, J J

1992-11-01

Quantitative two-dimensional gel electrophoresis was used to compare the cellular protein patterns of a normal foreskin-derived human fibroblasts cell line (LG1) and three immortal derivatives of LG1. One derivative, designated MSU-1.1 VO, was selected for its ability to grow in the absence of serum and is non-tumorigenic in athymic mice. The other two strains were selected for focus-formation following transfection with either Ha-ras or N-ras oncogenes and form high grade malignant tumors. Correspondence and cluster analysis provided a nonbiased estimate of the relative similarity of the different two-dimensional patterns. These techniques separated the gel patterns into three distinct classes: LG1, MSU-1.1 VO, and the ras transformed cell strains. The MSU-1.1 VO cells were more closely related to the parental LG1 than to the ras-transformed cells. The differences between the three classes were primarily quantitative in nature: 16% of the spots demonstrated statistically significant changes (P < 0.01, T test, mean ratio of intensity > 2) in the rate of incorporation of radioactive amino acids. The patterns from the two ras-transformed cell strains were similar, and variations in the expression of proteins that occurred between the separate experiments obscured consistent differences between the Ha-ras and N-ras transformed cells. However, while only 9 out of 758 spots were classified as different (1%), correspondence analysis could consistently separate the two ras transformants. One of these spots was five times more intense in the Ha-ras transformed cells than the N-ras.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell-free synthetic biology for environmental sensing and remediation.

PubMed

Karig, David K

2017-06-01

The fields of biosensing and bioremediation leverage the phenomenal array of sensing and metabolic capabilities offered by natural microbes. Synthetic biology provides tools for transforming these fields through complex integration of natural and novel biological components to achieve sophisticated sensing, regulation, and metabolic function. However, the majority of synthetic biology efforts are conducted in living cells, and concerns over releasing genetically modified organisms constitute a key barrier to environmental applications. Cell-free protein expression systems offer a path towards leveraging synthetic biology, while preventing the spread of engineered organisms in nature. Recent efforts in the areas of cell-free approaches for sensing, regulation, and metabolic pathway implementation, as well as for preserving and deploying cell-free expression components, embody key steps towards realizing the potential of cell-free systems for environmental sensing and remediation. Copyright © 2017 The Author. Published by Elsevier Ltd.. All rights reserved.

Approaching Resistance to Targeted Inhibition of PI3K in Breast Cancer

DTIC Science & Technology

2011-10-01

promise, there are concerns that drug resistance may emerge within the cancerous cells, thus limiting clinical efficacy. Using genetically defined human...mechanism of such resistance. Using genetically defined human mammary epithelial cells (HMECs), a model system which has previously been used for PI3K...pathway driven transformation due to its dependence on oncogenic PI3K signaling, we screened for emergence of BEZ235-resistance and identified genetic

New restriction enzymes discovered from Escherichia coli clinical strains using a plasmid transformation method

PubMed Central

Kasarjian, Julie K. A.; Iida, Masatake; Ryu, Junichi

2003-01-01

The presence of restriction enzymes in bacterial cells has been predicted by either classical phage restriction-modification (R-M) tests, direct in vitro enzyme assays or more recently from bacterial genome sequence analysis. We have applied phage R-M test principles to the transformation of plasmid DNA and established a plasmid R-M test. To validate this test, six plasmids that contain BamHI fragments of phage lambda DNA were constructed and transformed into Escherichia coli strains containing known R-M systems including: type I (EcoBI, EcoAI, Eco124I), type II (HindIII) and type III (EcoP1I). Plasmid DNA with a single recognition site showed a reduction of relative efficiency of transformation (EOT = 10–1–10–2). When multiple recognition sites were present, greater reductions in EOT values were observed. Once established in the cell, the plasmids were subjected to modification (EOT = 1.0). We applied this test to screen E.coli clinical strains and detected the presence of restriction enzymes in 93% (14/15) of cells. Using additional subclones and the computer program, RM Search, we identified four new restriction enzymes, Eco377I, Eco585I, Eco646I and Eco777I, along with their recognition sequences, GGA(8N)ATGC, GCC(6N)TGCG, CCA(7N)CTTC, and GGA(6N)TATC, respectively. Eco1158I, an isoschizomer of EcoBI, was also found in this study. PMID:12595571

Cajal-body formation correlates with differential coilin phosphorylation in primary and transformed cell lines.

PubMed

Hearst, Scoty M; Gilder, Andrew S; Negi, Sandeep S; Davis, Misty D; George, Eric M; Whittom, Angela A; Toyota, Cory G; Husedzinovic, Alma; Gruss, Oliver J; Hebert, Michael D

2009-06-01

Cajal bodies (CBs) are nuclear structures that are thought to have diverse functions, including small nuclear ribonucleoprotein (snRNP) biogenesis. The phosphorylation status of coilin, the CB marker protein, might impact CB formation. We hypothesize that primary cells, which lack CBs, contain different phosphoisoforms of coilin compared with that found in transformed cells, which have CBs. Localization, self-association and fluorescence recovery after photobleaching (FRAP) studies on coilin phosphomutants all suggest this modification impacts the function of coilin and may thus contribute towards CB formation. Two-dimensional gel electrophoresis demonstrates that coilin is hyperphosphorylated in primary cells compared with transformed cells. mRNA levels of the nuclear phosphatase PPM1G are significantly reduced in primary cells and expression of PPM1G in primary cells induces CBs. Additionally, PPM1G can dephosphorylate coilin in vitro. Surprisingly, however, expression of green fluorescent protein alone is sufficient to form CBs in primary cells. Taken together, our data support a model whereby coilin is the target of an uncharacterized signal transduction cascade that responds to the increased transcription and snRNP demands found in transformed cells.

RUNX1T1 Amplification Induces Small Cell Cancer

DTIC Science & Technology

and it is believed that the second, more differentiated component has transformed into a small cell cancer. Similarly, extra-pulmonary small cell...tumors have primary tumors that arise outside the lung, such as in the prostate or GI tract, and transform into a small cell cancer. So in reality the

Human Herpesvirus-8-Transformed Endothelial Cells Have Functionally Activated Vascular Endothelial Growth Factor/Vascular Endothelial Growth Factor Receptor

PubMed Central

Masood, Rizwan; Cesarman, Ethel; Smith, D. Lynne; Gill, Parkash S.; Flore, Ornella

2002-01-01

Kaposi’s sarcoma is a vascular tumor commonly associated with human immunodeficiency virus (HIV)-1 and human herpesvirus (HHV-8) also known as Kaposi’s sarcoma-associated herpesvirus. The principal features of this tumor are abnormal proliferation of vascular structures lined with spindle-shaped endothelial cells. HHV-8 may transform a subpopulation of endothelial cells in vitro via viral and cellular gene expression. We hypothesized that among the cellular genes, vascular endothelial growth factors (VEGFs) and their cognate receptors may be involved in viral-mediated transformation. We have shown that HHV-8-transformed endothelial cells (EC-HHV-8) express higher levels of VEGF, VEGF-C, VEGF-D, and PlGF in addition to VEGF receptors-1, -2, and -3. Furthermore, antibodies to VEGF receptor-2 inhibited cell proliferation and viability. Similarly, inhibition of VEGF gene expression with antisense oligonucleotides inhibited EC-HHV-8 cell proliferation/viability. The growth and viability of primary endothelial cells and a fibroblast cell line however were unaffected by either the VEGF receptor-2 antibody or the VEGF antisense oligodeoxynucleotides. VEGF and VEGF receptors are thus induced in EC-HHV-8 and participate in the transformation. Inhibitors of VEGF may thus modulate the disease process during development and progression. PMID:11786394