scholarly journals Nonmetastatic Colon Cancer Model C26 Upregulates Glycolysis in Osteocytes in Vitro and Bone in Vivo

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
M. Roarke Tollar ◽  
Matthew Prideaux ◽  
Fabrizio Pin ◽  
Lynda F. Bonewald
Keyword(s):  
Gene Expression ◽  
Culture Media ◽  
Bone Mineralization ◽  
Metabolic Effects ◽  
Whole Body ◽  
Rt Pcr ◽  
Systemic Complications ◽  
Whole Body Metabolism

Background: Developing effective treatments for musculoskeletal complications in cancer patients requires understanding metabolic effects of cancer on bone, and particularly osteocytes, the most abundant bone cell and key regulator of bone remodeling. However, little is known regarding how cancer impacts normal osteocyte energy metabolic pathways, such as glycolysis. Given that changes in metabolism are important regulators of cellular function, it is essential to determine how osteocyte metabolism is disrupted by cancer and how this may impact skeletal and whole-body health. Methods: Mice inoculated with saline (N=5) or C26 cells (N=6) were sacrificed after 2 weeks. Bones were harvested for metabolic profiling by GC-MS, gene expression by RT-PCR and bone morphology by µCT. Differentiated IDG-SW3 osteocyte-like cells were cocultured with C26 cells for 12-24hrs and metabolites and gene expression analyzed by GC-MS and RT-PCR. Results: Trabecular bone mass was significantly decreased in the C26 mice. GC-MS analysis revealed decreased glucose in C26 mice tibiae, but no change in lactate. The bone resorption promoting gene Rankl was upregulated, whereas the inhibitor Opg was unchanged. Bone mineralization regulators Mepe and Phex were decreased. In vitro metabolic studies revealed increased glucose and lactate in IDG-SW3 cell lysate; culture media glucose levels were decreased whereas lactate was increased in the co-cultures with C26 cells. RT-PCR demonstrated increases in the glycolysis promoter Hif1α in addition to glycolysis pathway genes including Glut1, Hk2, Slc16a3 and Pdk1. Rankl was also increased in the IDG-SW3 cells co-cultured with the C26 cells whereas Opg, Phex, and Mepe were downregulated. Conclusion: Glycolysis is upregulated in mouse bone and in vitro IDG-SW3 cells exposed to cancer. Our study provides novel understanding for how cancer affects bone metabolism. Integrating these results with whole body metabolism will aid in the development of novel therapeutic strategies to target musculoskeletal and systemic complications of cancer.

scholarly journals Enhanced glycogen metabolism in adipose tissue decreases triglyceride mobilization

2010 ◽  
Vol 299 (1) ◽  
pp. E117-E125 ◽  
Author(s):  
Kathleen R. Markan ◽  
Michael J. Jurczak ◽  
Margaret B. Allison ◽  
Honggang Ye ◽  
Maria M. Sutanto ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Lipid Metabolism ◽  
Lactate Production ◽  
Transgenic Animals ◽  
Whole Body ◽  
Nonesterified Fatty Acids ◽  
Transgenic Mouse Line ◽  
Whole Body Metabolism

Adipose tissue is a primary site for lipid storage containing trace amounts of glycogen. However, refeeding after a prolonged partial fast produces a marked transient spike in adipose glycogen, which dissipates in coordination with the initiation of lipid resynthesis. To further study the potential interplay between glycogen and lipid metabolism in adipose tissue, the aP2-PTG transgenic mouse line was utilized since it contains a 100- to 400-fold elevation of adipocyte glycogen levels that are mobilized upon fasting. To determine the fate of the released glucose 1-phosphate, a series of metabolic measurements were made. Basal and isoproterenol-stimulated lactate production in vitro was significantly increased in adipose tissue from transgenic animals. In parallel, basal and isoproterenol-induced release of nonesterified fatty acids (NEFAs) was significantly reduced in transgenic adipose tissue vs. control. Interestingly, glycerol release was unchanged between the genotypes, suggesting that enhanced triglyceride resynthesis was occurring in the transgenic tissue. Qualitatively similar results for NEFA and glycerol levels between wild-type and transgenic animals were obtained in vivo during fasting. Additionally, the physiological upregulation of the phospho enolpyruvate carboxykinase cytosolic isoform (PEPCK-C) expression in adipose upon fasting was significantly blunted in transgenic mice. No changes in whole body metabolism were detected through indirect calorimetry. Yet weight loss following a weight gain/loss protocol was significantly impeded in the transgenic animals, indicating a further impairment in triglyceride mobilization. Cumulatively, these results support the notion that the adipocyte possesses a set point for glycogen, which is altered in response to nutritional cues, enabling the coordination of adipose glycogen turnover with lipid metabolism.

MLL-AF9 and MLL-ENL Induce Deregulation of Similar Downstream Candidate Genes: An Analysis across Experimental Protocols and Laboratories.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3372-3372
Author(s):  
Ashish R. Kumar ◽  
Robert K. Slany ◽  
Jay L. Hess ◽  
John H. Kersey
Keyword(s):  
Gene Expression ◽  
Fusion Protein ◽  
Fusion Gene ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Expression Systems ◽  
Rt Pcr ◽  
Conditional Expression

Expression profiling has become an important tool for understanding gene deregulation in MLL-fusion leukemias. However, the results of gene profiling experiments are difficult to interpret when applied to leukemia cells because (i) leukemias arise in cells that differ greatly in their gene expression profiles, and (ii) leukemias most often require secondary genetic events in addition to the MLL fusion gene. Two principal model systems have been used to understand the direct effects of MLL-fusion genes. Knock-in models have the advantage of the fusion gene being under control of the physiologic promoter. On the other hand, conditional expression systems offer the ability to conduct short term experiments, permitting the analysis of direct effects on downstream genes. In the present combined-analysis, we used the Affymetrix U74Av2 oligonucleotide microarray to evaluate the effects of the MLL-fusion gene in vivo and in vitro respectively using two closely related MLL fusion genes - MLL-AF9 for knock-in and MLL-ENL for conditional expression. In the MLL-AF9 study, we compared gene expression profiles of bone marrow cells from MLL-AF9 knock-in mice (C57Bl/6, MLL-AF9+/−) to those of age-matched wild type mice (Kumar et. al. 2004, Blood). We used a t-test (p<0.05) to selected genes that showed significant changes in expression levels. In the MLL-ENL study, we transformed murine primary hematopoietic cells with a conditional MLL-ENL vector (MLL-ENL fused to the modified ligand-binding domain of the estrogen receptor) such that the fusion protein was active only in the presence of tamoxifen. We then studied the downstream effects of the fusion protein by comparing gene expression profiles of the cells in the presence and absence of tamoxifen. We used a pair-wise comparison analysis to select genes that showed a change in expression level of 1.5 fold or greater in at least two of three experiments (Zeisig et. al. 2004, Mol. Cell Biol.). Those genes that were up-regulated in both datasets were then compiled together. This list included Hoxa7, Hoxa9 and Meis1. The results for these 3 genes were confirmed by quantitative RT-PCR in both the MLL-AF9-knock-in and the MLL-ENL-conditional-expression systems. The remaining candidate genes in the common up-regulated gene set (not yet tested by quantitative RT-PCR) include protein kinases (Bmx, Mapk3, Prkcabp, Acvrl1, Cask), RAS-associated proteins (Rab7, Rab3b), signal transduction proteins (Notch1, Eat2, Shd, Fpr1), cell membrane proteins (Igsf4), chaperones (Hsp70.2), transcription factors (Isgf3g), proteins with unknown functions (Olfm1, Flot1), and hypothetical proteins. The results of the combined analysis demonstrate that these over-expressions are (i) a direct and sustained effect of the MLL-fusion protein, (ii) are independent of secondary events that might be involved in leukemogensis, and (iii) are independent of the two partner genes that participate in these fusions. The over-expression of a few genes in both the -in vitro and in vivo experimental systems makes these molecules very interesting for further studies, to understand the biology of MLL-fusion leukemias and for development of new therapeutic strategies.

The Outgrowth of CLL Nurselike Cells in Vitro, and Its Relation to Clinical and Hematological Parameters.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2360-2360
Author(s):  
Agata A Filip ◽  
Dorota Koczkodaj ◽  
Tomasz Kubiatowski ◽  
Ewa Wasik-Szczepanek ◽  
Anna Dmoszynska
Keyword(s):  
Gene Expression ◽  
Serum Level ◽  
Ex Vivo ◽  
Hematological Parameters ◽  
Tp53 Gene ◽  
Leukemic Cells ◽  
Rt Pcr ◽  
Gene Status

Abstract Abstract 2360 Poster Board II-337 Introduction: Despite their longevity in vivo, CLL lymphocytes die rapidly when put to in vitro cultures, what proves that the resistance to apoptosis is not an intrinsic feature of leukemic cells, but depends on environmental signals. Recently it was shown that mononuclear cells from peripheral blood of CLL patients differentiate in vitro into large, adherent cells that grow in close contact with CLL lymphocytes. They were termed “nurselike cells” (NLCs), because they support leukemic lymphocyte survival in culture. The presence of the cells morphologically and phenotypically similar to NLCs was demonstrated in peripheral lymphatic organs of CLL patients. It may suggest their role in CLL lymphocytes protection in vivo and, as a consequence, point the new target in CLL treatment. Patients and Methods: The study included the group of 65 previously untreated CLL patients, 24 women and 41 men, aged from 36 to 86 yrs. 12 patients (18%) were diagnosed with stage 0 according to Rai, 15 patients (23%) with stage I, 30 patients (46%) with stage II, 5 patients (8%) with stage III and 3 patients (5%) with stage IV. Peripheral blood lymphocytes ex vivo were examined for CD14, CD38, BCL2 and ZAP70 expression by flow cytometry and for BCL2, SURVIVIN and ZAP70 gene expression by RT-PCR. TP53 gene status was assessed by FISH. Lymphocytes of 20 patients were assayed for apoptosis-related gene expression by means of cDNA macroarrays (Clontech). To generate NLCs, PB leukemic cells were cultured in vitro for 14 days on standard medium (RPMI 1640 with L-glutamine, 15% FCS, antibiotics/antimycotics; cell density 3 × 106/ml) and the outgrowth and number of NLCs was assessed in relation to clinical and hematological parameters. NLCs were identified morphologically and by CD31/VIMENTIN protein expression. Results: In 58 cases (89%) the outgrowth of NLCs was observed, while their number differed in cultures of the cells of different patients: in 49 cultures (84.5%) there were over 20 NLCs/mm2 (up to 52 NLCs/mm2), and in 9 cases (15.5%) less than 20 NLCs/mm2. Positive correlation was shown between NLC number and B2M serum level (p=0.044) and absolute monocyte count (p=0.019). Significantly higher NLC number was observed in case of patients with higher CD14+ cell number (p<0.0001) and higher SURVIVIN gene expression assessed by RT-PCR (p<0.0001) and macroarrays (p=0.013). We found no statistically significant relation of NLCs number and: the Rai stage of the disease, WBC, lymphocyte count, LDH serum level, BCL2, CD38 and ZAP70 expression and TP53 gene status. During the follow-up period of 6 years we observed the tendency for longer overall survival in patients that produce less than 20 NLCs/mm2 (fig. 1), but it was not statistically significant. Conclusions: The number of NLC cells obtained in vitro from PBL of CLL patients correlates with B2M serum level and SURVIVIN gene expression in CLL cells ex vivo. High B2M level is a marker of poor prognosis. SURVIVIN represents a family of IAP (Inhibitor of APoptosis) proteins. While rare in PBL of CLL patients, its expression is typical for proliferating leukemic cells pool in pseudofollicle microenvironment. SURVIVIN inhibits apoptosis by blocking caspase-3 and -7. Considering the protective role of NLC cells towards CLL lymphocytes in vitro, these results altogether with observed tendency to shorter survival of patients generating high NLCs number may prove the presence of supportive mechanisms exerted by NLCs in vivo. Disclosures: No relevant conflicts of interest to declare.

179 EFFECT OF SERUM DURING CULTURE ON DAY 14 ELONGATED BOVINE EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 191 ◽  
Author(s):  
J. Angulo ◽  
G. T. Gentry ◽  
R. A. Godke ◽  
K. R. Bondioli
Keyword(s):  
Gene Expression ◽  
Messenger Rna ◽  
Culture Media ◽  
Calf Serum ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Expression Levels ◽  
The Mean

It has been reported that the addition of serum to embryo culture media alters gene expression and triggers the development of large offspring syndrome. The objectives of this study were to determine gene expression levels in embryos cultured in the absence or presence of 5% calf serum and in vivo-derived (IVD) embryos and to determine the effects of serum on the length of elongated embryos. Abattoir-derived oocytes were obtained from a commercial provider and fertilized at 24 h of maturation with semen from a bull previously used for IVF. At 18 h post-insemination (hpi), embryos were denuded and groups of 15 presumptive zygotes were cultured in 30-μL drops of modified SOF medium with amino acids and 6 mg mL–1 of BSA (mSOFaa). At 72 hpi, cleavage rate was assessed and embryos were randomly allocated into 2 treatments: mSOFaa without and with 5% calf serum. Embryos were then cultured to 168 hpi and blastocyst rates were assessed and recorded. Blastocysts (n = 5 to 10) from each treatment were transferred into synchronized recipients, and Day 14 embryos were recovered 7 days post-transfer. Embryos were photographed, measured, and immediately stored at –80°C in a minimal volume of PBS + 0.1% polyvinyl alcohol. Messenger RNA was isolated using a Dynabeads mRNA Direct Kit™ (Invitrogen, Carlsbad, CA), and reverse transcription was performed using an iScript™ cDNA Synthesis Kit (Bio-Rad Laboratories, Inc., CA). Quantitative PCR was performed to determine the transcript abundance for COX6A, IFNT1a, PLAC8, IGF2R, and GAPDH for each sample. The GAPDH was used as a reference gene, and gene expression was calculated as a ratio of expression levels between each gene of interest and GAPDH. Expression levels for each gene were determined from standard curves generated by serial dilutions of PCR amplicons starting with 0.4 pg/reaction. Blastocyst development rates were higher in embryos cultured with serum compared with the nonserum treatment (14.9 and 7.4% respectively; chi-square, P < 0.001). Lengths of elongated embryos from the serum (3395.3 ± 414.7 μm) and nonserum (2784 ± 741.8 μm) culture treatments differed from the IVD (6297.7 ± 677.2 μm) treatment (mean ± SE; ANOVA, P < 0.0052). There were no differences in the mean expression levels for COX6A, IFNT1a, PLAC8, and IGF2R across treatment groups, but in the serum treatment, 3 out 11 overexpressed IFNT1a, 4 out of 11 overexpressed IGF2R, and 2 out of 11 overexpressed PLAC8, defined as being 2 standard deviations above the mean of the IVD treatment for each respective gene. In the in vitro-produced nonserum and IVD treatments, overexpression by this definition was not observed. Although mean expression levels were not affected by culture with serum under these conditions, very high expression of IFNT1a, IGF2R, and PLAC8 was observed in some embryos cultured with serum, but not in embryos cultured without serum or IVD embryos.

scholarly journals Thyrotropin Activates Guanosine 5′-Diphosphate/Guanosine 5′-Triphosphate Exchange on the Rate-Limiting Endocytic Catalyst, Rab5a, in Human Thyrocytes in Vivo and in Vitro

2007 ◽  
Vol 92 (7) ◽  
pp. 2803-2810 ◽  
Author(s):  
Marie-France van den Hove ◽  
Karine Croizet-Berger ◽  
Donatienne Tyteca ◽  
Charlotte Selvais ◽  
Philippe de Diesbach ◽  
...  
Keyword(s):  
Hormone Secretion ◽  
Thyroid Tissue ◽  
Whole Body ◽  
Nucleotide Exchange ◽  
Membrane Recruitment ◽  
Exchange Factor ◽  
Whole Body Metabolism ◽  
Rate Limiting

Abstract Context: We have previously reported that the TSH receptor/cAMP cascade enhances the coordinate expression of the rate-limiting endocytic catalysts, Rab5a and Rab7, which respectively promote thyroglobulin (Tg) internalization and transfer to lysosomes, thereby accelerating thyroid hormone secretion. Objective: We address whether TSH further controls Rab5a activity by promoting its GTP-bound state. Design: We compared Rab5a activation in seven pairs of hyperactive and corresponding quiescent thyroid tissues; TSH effect was reproduced on polarized cultures of normal human thyrocytes. Patients: We studied seven euthyroid patients bearing hyperactive autonomous adenomas; normal thyroid tissue for culture. Main Outcome Measurements: Rab5a GDP/GTP exchange factor activity [Rab5a-guanine nucleotide exchange factor (GEF)], expression of Rabex-5 (a Rab5a-GEF), and function of thyrocytes in vitro were the main outcome measures. Results: In autonomous adenomas, constitutive activation increased both total activity and sedimentability (membrane recruitment) of Rab5a-GEF, compared with perinodular tissues. Increased Rab5a-GEF activity correlated with increased expression of Rabex-5 and Rab5a, as well as with Tg store depletion. In polarized human thyrocyte monolayers, TSH did not affect total Rab5a-GEF activity after 2 h but promoted its membrane recruitment; after 4 d, TSH increased both Rab5a-GEF activity and Rabex-5 expression and recruitment onto membranes where Rabex-5 coimmunoprecipitated with Rabaptin-5 and Rab5a. Sedimentable Rab5a-GEF perfectly correlated with apical endocytosis and lysosomal transfer of 125I-Tg, and with basolateral secretion of 125I-derived hormones. Conclusion: This study provides the first clinical and experimental evidence that regulation of the activity of a rate-limiting endocytic catalyst finely tunes a tightly controlled cellular function that ultimately governs whole body metabolism.

scholarly journals The Gonococcal Fur-Regulated tbpA and tbpB Genes Are Expressed during Natural Mucosal Gonococcal Infection

2005 ◽  
Vol 73 (7) ◽  
pp. 4281-4287 ◽  
Author(s):  
Sarika Agarwal ◽  
Carol A. King ◽  
Ellen K. Klein ◽  
David E. Soper ◽  
Peter A. Rice ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Profiles ◽  
Iron Concentration ◽  
Pcr Analysis ◽  
Positive Trend ◽  
Gonococcal Infection ◽  
Rt Pcr ◽  
Male Subjects

ABSTRACT Iron is limiting in the human host, and bacterial pathogens respond to this environment by regulating gene expression through the ferric uptake regulator protein (Fur). In vitro studies have demonstrated that Neisseria gonorrhoeae controls the expression of several critical genes through an iron- and Fur-mediated mechanism. While most in vitro experiments are designed to determine the response of N. gonorrhoeae to an exogenous iron concentration of zero, these organisms are unlikely to be exposed to such severe limitations of iron in vivo. To determine if N. gonorrhoeae expresses iron- and Fur-regulated genes in vivo during uncomplicated gonococcal infection, we examined gene expression profiles of specimens obtained from male subjects with urethral infections. RNA was isolated from urethral swab specimens and used as a template to amplify, by reverse transcriptase PCR (RT-PCR), gonococcal genes known to be regulated by iron and Fur (tbpA, tbpB, and fur). The constitutively expressed gonococcal rmp gene was used as a positive control. RT-PCR analysis indicated that gonorrhea-positive specimens where rmp expression was seen were also 93% (51/55) fbpA positive, 87% (48/55) tbpA positive, and 86% (14 of 16 tested) tbpB positive. In addition, we detected a fur transcript in 79% (37 of 47 tested) of positive specimens. We also measured increases in levels of immunoglobulin G antibody against TbpA (91%) and TbpB (73%) antigens in sera from infected male subjects compared to those in uninfected controls. A positive trend between tbpA gene expression and TbpA antibody levels in sera indicated a relationship between levels of gene expression and immune response in male subjects infected with gonorrhea for the first time. These results indicate that gonococcal iron- and Fur-regulated tbpA and tbpB genes are expressed in gonococcal infection and that male subjects with mucosal gonococcal infections exhibit antibodies to these proteins.

scholarly journals Blue light-induced gene expression alterations in cultured neurons are the result of phototoxic interactions with neuronal culture media

2019 ◽  
Author(s):  
Corey G. Duke ◽  
Katherine E. Savell ◽  
Robert A. Phillips ◽  
Jeremy J. Day
Keyword(s):  
Gene Expression ◽  
Blue Light ◽  
Cortical Neurons ◽  
Clock Genes ◽  
Culture Media ◽  
Light Exposure ◽  
Neuronal Culture ◽  
Gene Expression Alterations

Blue waveform light is used as an optical actuator in numerous optogenetic technologies employed in neuronal systems. However, the potential side effects of blue waveform light in neurons has not been thoroughly explored, and recent reports suggest that neuronal exposure to blue light can induce transcriptional alterations in vitro and in vivo. Here, we examined the effects of blue waveform light in cultured primary rat cortical neurons. Exposure to blue light (470nm) resulted in upregulation of several immediate early genes (IEGs) traditionally used as markers of neuronal activity, including Fos and Fosb, but did not alter the expression of circadian clock genes Bmal1, Cry1, Cry2, Clock, or Per2. IEG expression was increased following 4 hours of 5% duty cycle light exposure, and IEG induction was not dependent on light pulse width. Elevated levels of blue light exposure induced a loss of cell viability in vitro, suggestive of overt phototoxicity. Changes in gene expression induced by blue waveform light were prevented when neurons were cultured in a photoinert media supplemented with a photostable neuronal supplement instead of commonly utilized neuronal culture media and supplements. Together, these findings suggest that light-induced gene expression alterations observed in vitro stem from a phototoxic interaction between commonly used media and neurons, and offer a solution to prevent this toxicity when using photoactivatable technology in vitro.

391 A COMPARATIVE GENE EXPRESSION ANALYSIS BETWEEN SHEEP EARLY EMBRYONIC DEVELOPMENT AND PUTATIVE EMBRYONIC STEM CELLS

2010 ◽  
Vol 22 (1) ◽  
pp. 352
Author(s):  
B. M. Murray ◽  
S. Schmoelzl ◽  
N. M. Andronicos ◽  
J. R. Hill ◽  
P. J. Verma ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Embryonic Stem Cells ◽  
In Vitro Culture ◽  
Culture Media ◽  
Embryonic Stem ◽  
Oct4 Expression ◽  
Pluripotency Markers

The optimization of culture media to support the isolation of embryonic stem cells relies on methods to monitor whether the pluripotent state of the cultured cells has been maintained. We developed a panel of gene expression assays that allowed us to correlate molecular measures of pluripotency or lineage differentiation with a developmental time course. By conducting quantitative PCR analysis of sheep embryos over Day 6.5 to 24 and sheep inner cell mass (ICM) cells cultured over 25 days, we tested whether culture media designed to inhibit differentiation are able to maintain sheep ICM cells in a pluripotent state. Briefly, embryos were collected from Merino ewes (n = 50, 3 years) at Day 6.5, 12, 16, 20, and 24 post-AI. Embryos were collected from the dissected uterine tracts of slaughtered ewes, excluding Day 6.5 blastocysts, which were surgically recovered from superovulated ewes. For the in vitro culture, Day 6.5 ICM cells were isolated by immunosurgery and cultured on mitomycin-C-treated mouse embryonic fibroblasts in an inhibitor-based medium (3i, based on Ying Q-L et al. 2008 Nature 453, 519-523). Real-time PCR assays for pluripotency (OCT4, SOX2, NANOG) and differentiation (ectodermal: FGF5, PAX6; endodermal: GATA4, GATA6, Somatostatin; mesodermal: BMP4, Connexin40) of sheep candidate genes were conducted on cDNA prepared from these samples and normalized against the reference genes RPL19 and RPS26. In in vivo embryos, pluripotency markers OCT4, SOX2, and NANOG all decreased between Day 6.5 and Day 20, although OCT4 expression spiked around Day 16. More interestingly, pluripotency expression decreased during in vitro culture, with NANOG expression completely lost by passage 2 at Day 11 and OCT4 expression at an equivalent Day 24 embryo basal level by Day 14. The endodermal markers GATA6 and GATA4 decreased between Day 6.5 and Day 12, respectively, although in vitro GATA4 was only expressed once at Day 7. In vivo FGF5 and both PAX6 and Somatostatin displayed a delayed onset, increasing expression from Day 16 and 20, respectively, whereas the ectodermal markers were already expressed by Day 7 in vitro. Both mesodermal markers Connexin40 and BMP4 presented minor fold changes in both data sets. In conclusion, this study has verified the primer sets and described a sheep in vivo embryo gene expression profile comprising both pluripotent and differentiation candidates. Furthermore, the decrease of pluripotency markers together with the appearance of differentiation markers during in vitro culture of ICM cells suggest that culturing ICM cells in 3i media is not sufficient to maintain a sheep-specific pluripotent population of cells. Therefore, future studies will be aimed at manipulating the current in vitro system to focus on maintaining pluripotent genes such as NANOG and OCT4 in culture.

scholarly journals Tetracycline-Regulatable System To Tightly Control Gene Expression in the Pathogenic Fungus Candida albicans

2000 ◽  
Vol 68 (12) ◽  
pp. 6712-6719 ◽  
Author(s):  
Hironobu Nakayama ◽  
Toshiyuki Mio ◽  
Shigehisa Nagahashi ◽  
Michiko Kokado ◽  
Mikio Arisawa ◽  
...  
Keyword(s):  
Gene Expression ◽  
Candida Albicans ◽  
Culture Media ◽  
Pathogenic Fungus ◽  
Elongation Factor ◽  
Growth Defect ◽  
Control Gene ◽  
Control Gene Expression

ABSTRACT Conventional tools for elucidating gene function are relatively scarce in Candida albicans, the most prevalent human fungal pathogen. To this end, we developed a convenient system to control gene expression in C. albicans by the tetracycline-regulatable (TR) promoters. When the sea pansy Renilla reniformisluciferase gene (RLUC1) was placed under the control of this system, doxycycline (DOX) inhibited the luciferase activity almost completely. In the absence of DOX, the RLUC1 gene was induced to express luciferase at a level 400- to 1,000-fold higher than that in the presence of DOX. The same results were obtained in hypha-forming cells. The replacement ofN-myristoyltransferase or translation elongation factor 3 promoters with TR promoters conferred a DOX-dependent growth defect in culture media. Furthermore, all the mice infected with these mutants, which are still virulent, survived following DOX administration. Consistently, we observed that the number of these mutant cells recovered from the mouse kidneys was significantly reduced following DOX administration. Thus, this system is useful for investigating gene functions, since this system is able to function in both in vitro and in vivo settings.

scholarly journals Matrix metalloproteinase 11 protects from diabesity and promotes metabolic switch

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Nassim Dali-Youcef ◽  
Karim Hnia ◽  
Sébastien Blaise ◽  
Nadia Messaddeq ◽  
Stéphane Blanc ◽  
...  
Keyword(s):  
Cancer Progression ◽  
Energy Homeostasis ◽  
Aerobic Glycolysis ◽  
Whole Body ◽  
Metabolic Switch ◽  
Metabolic Signalling ◽  
Cancer Cell Survival ◽  
Whole Body Metabolism

Abstract MMP11 overexpression is a bad prognostic factor in various human carcinomas. Interestingly, this proteinase is not expressed in malignant cells themselves but is secreted by adjacent non-malignant mesenchymal/stromal cells, such as cancer associated fibroblasts (CAFs) and adipocytes (CAAs), which favors cancer cell survival and progression. As MMP11 negatively regulates adipogenesis in vitro, we hypothesized that it may play a role in whole body metabolism and energy homeostasis. We used an in vivo gain- (Mmp11-Tg mice) and loss- (Mmp11−/− mice) of-function approach to address the systemic function of MMP11. Strikingly, MMP11 overexpression protects against type 2 diabetes while Mmp11−/− mice exhibit hallmarks of metabolic syndrome. Moreover, Mmp11-Tg mice were protected from diet-induced obesity and display mitochondrial dysfunction, due to oxidative stress, and metabolic switch from oxidative phosphorylation to aerobic glycolysis. This Warburg-like effect observed in adipose tissues might provide a rationale for the deleterious impact of CAA-secreted MMP11, favouring tumor progression. MMP11 overexpression also leads to increased circulating IGF1 levels and the activation of the IGF1/AKT/FOXO1 cascade, an important metabolic signalling pathway. Our data reveal a major role for MMP11 in controlling energy metabolism, and provide new clues for understanding the relationship between metabolism, cancer progression and patient outcome.

Sign in / Sign up

Export Citation Format

Share Document