scholarly journals Association of mitotane with chylomicrons and serum lipoproteins: practical implications for treatment of adrenocortical carcinoma

2016 ◽  
Vol 174 (3) ◽  
pp. 343-353 ◽  
Author(s):  
Matthias Kroiss ◽  
Dietmar Plonné ◽  
Sabine Kendl ◽  
Diana Schirmer ◽  
Cristina L Ronchi ◽  
...  
Keyword(s):  
Er Stress ◽  
Adrenocortical Carcinoma ◽  
Marker Gene ◽  
Index Patient ◽  
Cholesterol Content ◽  
Serum Lipoprotein ◽  
Marker Genes ◽  
Stress Marker ◽  
High Concentrations

ObjectiveOral mitotane (o,p′-DDD) is a cornerstone of medical treatment for adrenocortical carcinoma (ACC). AimSerum mitotane concentrations >14 mg/l are targeted for improved efficacy but not achieved in about half of patients. Here we aimed at a better understanding of intestinal absorption and lipoprotein association of mitotane and metabolites o,p′-dichlorodiphenylacetic acid (o,p′-DDA) and o,p′-dichlorodiphenyldichloroethane (o,p′-DDE).DesignLipoproteins were isolated by ultracentrifugation from the chyle of a 29-year-old patient and serum from additional 14 ACC patients treated with mitotane. HPLC was applied for quantification of mitotane and metabolites. We assessed NCI–H295 cell viability, cortisol production, and expression of endoplasmic reticulum (ER) stress marker genes to study the functional consequences of mitotane binding to lipoproteins.ResultsChyle of the index patient contained 197 mg/ml mitotane, 53 mg/ml o,p′-DDA, and 51 mg/l o,p′-DDE. Of the total mitotane in serum, lipoprotein fractions contained 21.7±21.4% (VLDL), 1.9±0.8% (IDL), 8.9±5.5% (LDL1), 18.9±9.6% (LDL2), 10.1±4.0% (LDL3), and 26.3±13.0% (HDL2). Only 12.3±5.5% were in the lipoprotein-depleted fraction.DiscussionMitotane content of lipoproteins directly correlated with their triglyceride and cholesterol content. O,p′-DDE was similarly distributed, but 87.9±4.2% of o,p′-DDA found in the HDL2 and lipoprotein-depleted fractions. Binding of mitotane to human lipoproteins blunted its anti-proliferative and anti-hormonal effects on NCI–H295 cells and reduced ER stress marker gene expression.ConclusionMitotane absorption involves chylomicron binding. High concentrations of o,p′-DDA and o,p′-DDE in chyle suggest intestinal mitotane metabolism. In serum, the majority of mitotane is bound to lipoproteins. In vitro, lipoprotein binding inhibits activity of mitotane suggesting that lipoprotein-free mitotane is the therapeutically active fraction.

scholarly journals Insertion of EGFP into the replicase gene of Semliki Forest virus results in a novel, genetically stable marker virus

2007 ◽  
Vol 88 (4) ◽  
pp. 1225-1230 ◽  
Author(s):  
Nele Tamberg ◽  
Valeria Lulla ◽  
Rennos Fragkoudis ◽  
Aleksei Lulla ◽  
John K. Fazakerley ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Semliki Forest Virus ◽  
Cultured Cells ◽  
Marker Gene ◽  
Virus Production ◽  
Marker Genes ◽  
Replicase Gene ◽  
Marker Virus

Alphavirus-based vector and replicon systems have been extensively used experimentally and are likely to be used in human and animal medicine. Whilst marker genes can be inserted easily under the control of a duplicated subgenomic promoter, these constructs are often genetically unstable. Here, a novel alphavirus construct is described in which an enhanced green fluorescent protein (EGFP) marker gene is inserted into the virus replicase open reading frame between nsP3 and nsP4, flanked by nsP2 protease-recognition sites. This construct has correct processing of the replicase polyprotein, produces viable virus and expresses detectable EGFP fluorescence upon infection of cultured cells and cells of the mouse brain. In comparison to parental virus, the marker virus has an approximately 1 h delay in virus RNA and infectious virus production. Passage of the marker virus in vitro and in vivo demonstrates good genetic stability. Insertion of different markers into this novel construct has potential for various applications.

scholarly journals Pilot investigation on the dose-dependent impact of irradiation on primary human alveolar osteoblasts in vitro

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Anna-Klara Amler ◽  
Domenic Schlauch ◽  
Selin Tüzüner ◽  
Alexander Thomas ◽  
Norbert Neckel ◽  
...  
Keyword(s):  
Bone Cells ◽  
Marker Gene ◽  
Marker Genes ◽  
Dependent Manner ◽  
Current Standard ◽  
Functional Changes ◽  
Standard Procedures ◽  
The Impact ◽  
Dose Dependent

AbstractRadiotherapy of head and neck squamous cell carcinoma can lead to long-term complications like osteoradionecrosis, resulting in severe impairment of the jawbone. Current standard procedures require a 6-month wait after irradiation before dental reconstruction can begin. A comprehensive characterization of the irradiation-induced molecular and functional changes in bone cells could allow the development of novel strategies for an earlier successful dental reconstruction in patients treated by radiotherapy. The impact of ionizing radiation on the bone-forming alveolar osteoblasts remains however elusive, as previous studies have relied on animal-based models and fetal or animal-derived cell lines. This study presents the first in vitro data obtained from primary human alveolar osteoblasts. Primary human alveolar osteoblasts were isolated from healthy donors and expanded. After X-ray irradiation with 2, 6 and 10 Gy, cells were cultivated under osteogenic conditions and analyzed regarding their proliferation, mineralization, and expression of marker genes and proteins. Proliferation of osteoblasts decreased in a dose-dependent manner. While cells recovered from irradiation with 2 Gy, application of 6 and 10 Gy doses not only led to a permanent impairment of proliferation, but also resulted in altered cell morphology and a disturbed structure of the extracellular matrix as demonstrated by immunostaining of collagen I and fibronectin. Following irradiation with any of the examined doses, a decrease of marker gene expression levels was observed for most of the investigated genes, revealing interindividual differences. Primary human alveolar osteoblasts presented a considerably changed phenotype after irradiation, depending on the dose administered. Mechanisms for these findings need to be further investigated. This could facilitate improved patient care by re-evaluating current standard procedures and investigating faster and safer reconstruction concepts, thus improving quality of life and social integrity.

scholarly journals Directing Stem Cell Commitment by Amorphous Calcium Phosphate Nanoparticles Incorporated in PLGA: Relevance of the Free Calcium Ion Concentration

2020 ◽  
Vol 21 (7) ◽  
pp. 2627
Author(s):  
Olivier Gröninger ◽  
Samuel Hess ◽  
Dirk Mohn ◽  
Elia Schneider ◽  
Wendelin Stark ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Calcium Phosphate ◽  
Amorphous Calcium Phosphate ◽  
Marker Gene ◽  
Ion Concentration ◽  
Marker Genes ◽  
Calcium Phosphate Nanoparticles ◽  
Cell Commitment

The microenvironment of mesenchymal stem cells (MSCs) is responsible for the modulation in MSC commitment. Nanocomposites with an inorganic and an organic component have been investigated, and osteogenesis of MSCs has been attributed to inorganic phases such as calcium phosphate under several conditions. Here, electrospun meshes and two-dimensional films of poly(lactic-co-glycolic acid) (PLGA) or nanocomposites of PLGA and amorphous calcium phosphate nanoparticles (PLGA/aCaP) seeded with human adipose-derived stem cells (ASCs) were analyzed for the expression of selected marker genes. In a two-week in vitro experiment, osteogenic commitment was not found to be favored on PLGA/aCaP compared to pure PLGA. Analysis of the medium revealed a significant reduction of the Ca2+ concentration when incubated with PLGA/aCaP, caused by chemical precipitation of hydroxyapatite (HAp) on aCaP seeds of PLGA/aCaP. Upon offering a constant Ca2+ concentration, however, the previously observed anti-osteogenic effect was reversed: alkaline phosphatase, an early osteogenic marker gene, was upregulated on PLGA/aCaP compared to pristine PLGA. Hence, in addition to the cell–material interaction, the material–medium interaction was also important for the stem cell commitment here, affecting the cell–medium interaction. Complex in vitro models should therefore consider all factors, as coupled impacts might emerge.

scholarly journals A plant regeneration platform to apply new breeding techniques for improving disease resistance in grapevine rootstocks and cultivars

2019 ◽  
Vol 12 ◽  
pp. 01019 ◽  
Author(s):  
S. Sabbadini ◽  
L. Capriotti ◽  
C. Limera ◽  
O. Navacchi ◽  
G. Tempesta ◽  
...  
Keyword(s):  
Marker Gene ◽  
Marker Genes ◽  
Wine Grape ◽  
Thompson Seedless ◽  
Multiple Virus ◽  
Almost All ◽  
New Breeding Techniques ◽  
Breeding Techniques ◽  
Selection Of

Worldwide grapevine cultivation is based on the use of elite cultivars, in many cases strictly linked to local important wine brands. Most of Vitis viniferacultivars have high susceptibility to fungal and viral diseases therefore, new breeding techniques (e.g. Cisgenesis, RNAi and gene editing) offer the possibility to introduce new clones of the main cultivars with increased diseases resistance, in order to reduce environmental impact and improve quality in the intensive wine grape industry. This study is finalized to develop efficient in vitro regeneration and transformation protocols to extend the application of these technologies in wine grape cultivars and rootstocks. With this aim, in vitro regeneration protocols based on the production of meristematic bulks (Mezzetti et al., 2002) were optimized for different grapevine cultivars (Glera, Vermentino, Sangiovese, Thompson Seedless) and rootstocks (1103 Paulsen, and 110 Richter). The meristematic bulks were then used as explants for Agrobacteriummediated genetic transformation protocols, by comparing the use of NPTII and e-GFP as marker genes. Results confirmed the efficiency of meristematic bulks as the regenerating tissue to produce new modified plants in almost all the above genotypes. The highest regeneration efficiency in some genotypes allowed the selection of stable modified lines/calli with only the use of e-GFP marker gene. This protocol can be applied in the use of MYB marker gene for the production of cisgenic lines. Genotypes having the highest regeneration and transformation efficiency were also used for transformation experiments using a hairpin gene construct designed to silence the RNA-dependent RNA polymerase (RpRd) of the GFLV and GLRaV3, which would induce multiple virus resistances, and the Dicer-like protein 1 (Bc-DCL1) and Bc-DCL2 to control B. cinerea infection.

scholarly journals StarD5: an ER stress protein regulates plasma membrane and intracellular cholesterol homeostasis

2019 ◽  
Vol 60 (6) ◽  
pp. 1087-1098 ◽  
Author(s):  
Daniel Rodriguez-Agudo ◽  
Leonel Malacrida ◽  
Genta Kakiyama ◽  
Tavis Sparrer ◽  
Carolina Fortes ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Er Stress ◽  
Stress Protein ◽  
Cholesterol Content ◽  
Cholesterol Homeostasis ◽  
Fluorescence Lifetime Imaging ◽  
Lipid Transfer ◽  
Altered Expression ◽  
Intracellular Cholesterol

How plasma membrane (PM) cholesterol is controlled is poorly understood. Ablation of the gene encoding the ER stress steroidogenic acute regulatory-related lipid transfer domain (StarD)5 leads to a decrease in PM cholesterol content, a decrease in cholesterol efflux, and an increase in intracellular neutral lipid accumulation in macrophages, the major cell type that expresses StarD5. ER stress increases StarD5 expression in mouse hepatocytes, which results in an increase in accessible PM cholesterol in WT but not in StarD5−/− hepatocytes. StarD5−/− mice store higher levels of cholesterol and triglycerides, which leads to altered expression of cholesterol-regulated genes. In vitro, a recombinant GST-StarD5 protein transfers cholesterol between synthetic liposomes. StarD5 overexpression leads to a marked increase in PM cholesterol. Phasor analysis of 6-dodecanoyl-2-dimethylaminonaphthalene fluorescence lifetime imaging microscopy data revealed an increase in PM fluidity in StarD5−/− macrophages. Taken together, these studies show that StarD5 is a stress-responsive protein that regulates PM cholesterol and intracellular cholesterol homeostasis.

scholarly journals Selenate Prevents Adipogenesis through Induction of Selenoprotein S and Attenuation of Endoplasmic Reticulum Stress

Molecules ◽  
2018 ◽  
Vol 23 (11) ◽  
pp. 2882 ◽  
Author(s):  
Choon Kim ◽  
Kee-Hong Kim
Keyword(s):  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Early Stage ◽  
Marker Genes ◽  
Peroxisome Proliferator ◽  
Peroxisome Proliferator Activated Receptor ◽  
Homologous Protein ◽  
Selenoprotein S ◽  
The Individual

The conversion of preadipocytes to adipocytes (adipogenesis) is a potential target to treat or prevent obesity. Selenate, an inorganic form of selenium, elicits diverse health benefits, mainly through its incorporation into selenoproteins. The individual roles of selenium and certain selenoproteins have been reported. However, the effects of selenate treatment on selenoproteins in adipocytes are unclear. In this study, the effects of selenate pretreatment on selenoprotein and endoplasmic reticulum (ER) stress during adipogenesis were examined in vitro. The selenate pretreatment dose-dependently suppressed the adipogenesis of 3T3-L1 preadipocytes. The selenate pretreatment at 50 μM for 24 h almost completely suppressed adipogenesis without cytotoxic effects. The expression of the adipogenic genes peroxisome proliferator-activated receptor gamma, CCAAT-enhancer binding protein alpha, and leptin was suppressed by selenate. This pretreatment also upregulated selenoprotein S (SEPS1), an ER resident selenoprotein that reduces ER stress, and prevented dexamethasone-induced SEPS1 degradation during the early stage of adipogenesis. The selenate-inhibited adipogenesis was associated with an attenuation of ER stress. The expression of the ER stress marker genes was upregulated during the early stage of differentiation, whereas the selenate pretreatment suppressed the mRNA expression of the XBP1 and C/EBP homologous protein. The collective data suggest a preventive role of selenate and SEPS1 in adipogenesis, and support a novel dietary approach to prevent obesity.

scholarly journals Development of marker-free transgenic pigeon pea (Cajanus cajan) expressing a pod borer insecticidal protein

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Snehasish Sarkar ◽  
Souri Roy ◽  
Sudip K. Ghosh
Keyword(s):  
Insect Pests ◽  
Marker Gene ◽  
Pigeon Pea ◽  
Small Subunit ◽  
Grain Legume ◽  
Marker Genes ◽  
35S Promoter ◽  
Selectable Marker Genes ◽  
Insect Resistant

AbstractPigeon pea, a grain legume of the semiarid tropics, is a rich source of high-quality protein. The productivity of this pulse is seriously affected by lepidopteron insect pests. To generate a sustainable insect-resistant plant, synthetically prepared bioactive key constituents of a crystal protein (Syn Cry1Ab) of Bacillus thuringiensis were expressed in pigeon pea under the guidance of a tissue-specific promoter of the RuBP carboxylase/oxygenase small subunit (rbcS) gene. Regenerated transgenic plants with the cry1Ab expression cassette (cry1Ab-lox-bar-lox) showed the optimum insect motility rate (90%) in an in vitro insect bioassay with second instar larvae, signifying the insecticidal potency of Syn Cry1Ab. In parallel, another plant line was also generated with a chimaeric vector harbouring a cre recombinase gene under the control of the CaMV 2 × 35S promoter. Crossing between T1 plants with a single insertion of cry1Ab-lox-bar-lox T-DNA and T1 plants with moderate expression of a cre gene with a linked hygromycin resistance (hptII) gene was performed to exclude the bialaphos resistance (bar) marker gene. Excision of the bar gene was achieved in T1F1 hybrids, with up to 35.71% recombination frequency. Insect-resistant pigeon pea plants devoid of selectable marker genes (syn Cry1Ab- bar and cre-hptII) were established in a consecutive generation (T1F2) through genetic segregation.

scholarly journals Microtubule Acetylation Controls MDA-MB-231 Breast Cancer Cell Invasion through the Modulation of Endoplasmic Reticulum Stress

2021 ◽  
Vol 22 (11) ◽  
pp. 6018
Author(s):  
Panseon Ko ◽  
Jee-Hye Choi ◽  
Seongeun Song ◽  
Seula Keum ◽  
Jangho Jeong ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Cancer Cells ◽  
Cancer Progression ◽  
Marker Genes ◽  
Stress Marker ◽  
Stress Markers ◽  
Cancer Types ◽  
Cellular Stresses

During aggressive cancer progression, cancer cells adapt to unique microenvironments by withstanding various cellular stresses, including endoplasmic reticulum (ER) stress. However, the mechanism whereby cancer cells overcome the ER stress to survive remains to be elucidated. Herein, we demonstrated that microtubule acetylation in cancer cells grown on a stiff matrix promotes cancer progression by preventing excessive ER stress. Downregulation of microtubule acetylation using shRNA or CRSIPR/Cas9 techniques targeting ATAT1, which encodes α-tubulin N-acetyltransferase (αTAT1), resulted in the upregulation of ER stress markers, changes in ER morphology, and enhanced tunicamycin-induced UPR signaling in cancer cells. A set of genes involved in cancer progression, especially focal adhesion genes, were downregulated in both ATAT1-knockout and tunicamycin-treated cells, whereas ATAT1 overexpression restored the gene expression inhibited by tunicamycin. Finally, the expression of ATAT1 and ER stress marker genes were negatively correlated in various breast cancer types. Taken together, our results suggest that disruption of microtubule acetylation is a potent therapeutic tool for preventing breast cancer progression through the upregulation of ER stress. Moreover, ATAT1 and ER stress marker genes may be useful diagnostic markers in various breast cancer types.

203 ESTABLISHMENT OF AN IN VITRO SCREENING ASSAY FOR DEVELOPMENTAL TOXICITY USING MOUSE EMBRYOID BODIES

2014 ◽  
Vol 26 (1) ◽  
pp. 216
Author(s):  
Y. Choi ◽  
E. B. Jeung
Keyword(s):  
Developmental Toxicity ◽  
Marker Gene ◽  
Embryoid Bodies ◽  
Toxic Chemicals ◽  
Penicillin G ◽  
Marker Genes ◽  
High Dose ◽  
Germ Layers ◽  
Hes Cells

Toxicity is generally referred to as the degree to which a substance can damage an organism. According to numerous reports, determination of toxicity through an in vitro system is an economical and ethical method, compared with an in vivo system using animals. The hES cells can differentiate into three germ layers and the unique feature of hES cells from the three germ layers via the EB formation adds good insight in understanding human developmental biology in vitro. Evaluation of developmental toxicity of a compound on early embryo states can therefore be based on its effect on EB formation. In this study, we used mouse embryonic stem cells (mESC) to investigate the effects of several developmental toxic chemicals on the formation of EBs. We used the EB hanging drop method and tested five toxic chemicals; cytosine arabinoside, dexamethasone, hydroxyurea, indomethacin, 5-fluorouracil, and two negative controls; ascorbic acid and penicillin G. We demonstrated a significant reduction of EB size after treatment with a high dose of each chemical. We evaluated cell toxicity by performing measurements of cell viability after treatment with each chemical. Expression of apoptosis-related genes (p53, Sirt1, p21, Puma, Noxa, Mdm2) and pluripotency marker genes (Oct4, Nanog, Sox2, ZFP206) and differentiation marker genes, such as endoderm (HNF4, AFP), mesoderm (T-brachy), and ectoderm (Pax6) were determined by quantitative real-time PCR. The chemicals induced abnormal differentiation and apoptosis. We confirmed apoptotic positive cells by TUNEL assay. In addition, we also demonstrate the reduction of the size of EBs through expression of apoptosis-related marker genes (p53, Caspase-3, PARP) and necrosis-related marker gene (HMGB1). The results obtained demonstrate that EBs can be used as an in vitro model for testing developmental toxicity.

Ceiling culture-derived proliferative adipocytes retain high adipogenic potential suitable for use as a vehicle for gene transduction therapy

2011 ◽  
Vol 301 (1) ◽  
pp. C181-C185 ◽  
Author(s):  
Sakiyo Asada ◽  
Masayuki Kuroda ◽  
Yasuyuki Aoyagi ◽  
Yoshitaka Fukaya ◽  
Shigeaki Tanaka ◽  
...  
Keyword(s):  
Regenerative Medicine ◽  
Adipogenic Differentiation ◽  
Marker Gene ◽  
Stromal Vascular Fraction ◽  
Marker Genes ◽  
Medicine Research ◽  
Ceiling Culture ◽  
The Difference ◽  
Adipogenic Marker

Adipose tissue is expected to provide a source of proliferative cells for regenerative medicine and cell-transplantation therapies using gene transfer manipulation. We have recently identified ceiling culture-derived proliferative adipocytes (ccdPAs) from the mature adipocyte fraction as cells suitable as a therapeutic gene vehicle because of their stable proliferative capacity. In this study, we examined the capability of adipogenic differentiation of the ccdPAs compared with stromal vascular fraction (SVF)-derived progenitor cells (adipose-derived stem cells, ASCs) with regard to their multipotential ability to be converted to another lineage and therefore their potential to be used for regenerative medicine research. After in vitro passaging, the surface antigen profile and the basal levels of adipogenic marker genes of the ccdPAs were not obviously different from those of the ASCs. However, the ccdPAs showed increased lipid-droplet accumulation accompanied with higher adipogenic marker gene expression after stimulation of differentiation compared with the ASCs. The higher adipogenic potential of the ccdPAs than the ASCs from the SVF was maintained for 42 days in culture. Furthermore, the difference in the adipogenic response was enhanced after partial stimulation without indomethacin. These results indicate that the ccdPAs retain a high adipogenic potential even after in vitro passaging, thus suggesting the commitment of ccdPAs to stable mature adipocytes after autotransplantation, indicating that they may have potential for use in regenerative and gene-manipulated medicine.

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