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Sample records for adipocyte marker genes

  1. Endocrine modulators of mouse subcutaneous adipose tissue beige adipocyte markers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The stromal vascular fraction (SVF) of subcutaneous adipose tissue contains precursors that can give rise to beige adipocytes. Beige adipocytes are characterized by the expression of specific markers, but it is not clear which markers best evaluate beige adipocyte differentiation. Both regulators of...

  2. Phytanic acid, a novel activator of uncoupling protein-1 gene transcription and brown adipocyte differentiation.

    PubMed Central

    Schlüter, Agatha; Barberá, Maria José; Iglesias, Roser; Giralt, Marta; Villarroya, Francesc

    2002-01-01

    Phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) is a phytol-derived branched-chain fatty acid present in dietary products. Phytanic acid increased uncoupling protein-1 (UCP1) mRNA expression in brown adipocytes differentiated in culture. Phytanic acid induced the expression of the UCP1 gene promoter, which was enhanced by co-transfection with a retinoid X receptor (RXR) expression vector but not with other expression vectors driving peroxisome proliferator-activated receptor (PPAR)alpha, PPARgamma or a form of RXR devoid of ligand-dependent sensitivity. The effect of phytanic acid on the UCP1 gene required the 5' enhancer region of the gene and the effects of phytanic acid were mediated in an additive manner by three binding sites for RXR. Moreover, phytanic acid activates brown adipocyte differentiation: long-term exposure of brown preadipocytes to phytanic acid promoted the acquisition of the brown adipocyte morphology and caused a co-ordinate induction of the mRNAs for gene markers of brown adipocyte differentiation, such as UCP1, adipocyte lipid-binding protein aP2, lipoprotein lipase, the glucose transporter GLUT4 or subunit II of cytochrome c oxidase. In conclusion, phytanic acid is a natural product of phytol metabolism that activates brown adipocyte thermogenic function. It constitutes a potential nutritional signal linking dietary status to adaptive thermogenesis. PMID:11829740

  3. Atypical antipsychotics induce both proinflammatory and adipogenic gene expression in human adipocytes in vitro

    SciTech Connect

    Sárvári, Anitta K.; Veréb, Zoltán; Uray, Iván P.; Fésüs, László; Balajthy, Zoltán

    2014-08-08

    Highlights: • Antipsychotics modulate the expression of adipogenic genes in human adipocytes. • Secretion of proinflammatory cytokine IL8 and MCP-1 is induced by antipsychotics. • Adipocyte-dependent inflammatory abnormality could develop during chronic treatment. • Infiltrated macrophages would further enhance proinflammatory cytokine production. - Abstract: Schizophrenia requires lifelong treatment, potentially causing systemic changes in metabolic homeostasis. In the clinical setting, antipsychotic treatment may differentially lead to weight gain among individual patients, although the molecular determinants of such adverse effects are currently unknown. In this study, we investigated changes in the expression levels of critical regulatory genes of adipogenesis, lipid metabolism and proinflammatory genes during the differentiation of primary human adipose-derived stem cells (ADSCs). These cells were isolated from patients with body mass indices <25 and treated with the second-generation antipsychotics olanzapine, ziprasidone, clozapine, quetiapine, aripiprazole and risperidone and the first-generation antipsychotic haloperidol. We found that antipsychotics exhibited a marked effect on key genes involved in the regulation of cell cycle, signal transduction, transcription factors, nuclear receptors, differentiation markers and metabolic enzymes. In particular, we observed an induction of the transcription factor NF-KB1 and NF-KB1 target genes in adipocytes in response to these drugs, including the proinflammatory cytokines TNF-α, IL-1β, IL-8 and MCP-1. In addition, enhanced secretion of both IL8 and MCP-1 was observed in the supernatant of these cell cultures. In addition to their remarkable stimulatory effects on proinflammatory gene transcription, three of the most frequently prescribed antipsychotic drugs, clozapine, quetiapine and aripiprazole, also induced the expression of essential adipocyte differentiation genes and the adipocyte hormones leptin

  4. Evaluation of markers of beige adipocytes in white adipose tissue of the mouse

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: There is a growing interest in exploiting the induction of beige or “brite” (brown in white) adipocytes (beigeing) to combat obesity and its comorbidities. However, there is some uncertainty regarding the best markers to evaluate the occurrence or magnitude of beigeing in white adipose t...

  5. PPARy and GLUT-4 expression as developmental regulators/markers for preadipocyte differentiation into an adipocyte

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In this document, we have integrated knowledge about two major cellular markers found in cells of the adipocyte lineage. The first factor is PPARy, which has been identified as an important adipogenic regulator. PPARy plays an important role in converting adipofibroblasts, fibroblasts or preadipocyt...

  6. An siRNA-based method for efficient silencing of gene expression in mature brown adipocytes.

    PubMed

    Isidor, Marie S; Winther, Sally; Basse, Astrid L; Petersen, M Christine H; Cannon, Barbara; Nedergaard, Jan; Hansen, Jacob B

    2016-01-01

    Brown adipose tissue is a promising therapeutic target for opposing obesity, glucose intolerance and insulin resistance. The ability to modulate gene expression in mature brown adipocytes is important to understand brown adipocyte function and delineate novel regulatory mechanisms of non-shivering thermogenesis. The aim of this study was to optimize a lipofection-based small interfering RNA (siRNA) transfection protocol for efficient silencing of gene expression in mature brown adipocytes. We determined that a critical parameter was to deliver the siRNA to mature adipocytes by reverse transfection, i.e. transfection of non-adherent cells. Using this protocol, we effectively knocked down both high- and low-abundance transcripts in a model of mature brown adipocytes (WT-1) as well as in primary mature mouse brown adipocytes. A functional consequence of the knockdown was confirmed by an attenuated increase in uncoupled respiration (thermogenesis) in response to β-adrenergic stimulation of mature WT-1 brown adipocytes transfected with uncoupling protein 1 siRNA. Efficient gene silencing was also obtained in various mouse and human white adipocyte models (3T3-L1, primary mouse white adipocytes, hMADS) with the ability to undergo "browning." In summary, we report an easy and versatile reverse siRNA transfection protocol to achieve specific silencing of gene expression in various models of mature brown and browning-competent white adipocytes, including primary cells. PMID:27386153

  7. ASC-1, PAT2, and P2RX5 are cell surface markers for white, beige, and brown adipocytes.

    PubMed

    Ussar, Siegfried; Lee, Kevin Y; Dankel, Simon N; Boucher, Jeremie; Haering, Max-Felix; Kleinridders, Andre; Thomou, Thomas; Xue, Ruidan; Macotela, Yazmin; Cypess, Aaron M; Tseng, Yu-Hua; Mellgren, Gunnar; Kahn, C Ronald

    2014-07-30

    White, beige, and brown adipocytes are developmentally and functionally distinct but often occur mixed together within individual depots. To target white, beige, and brown adipocytes for diagnostic or therapeutic purposes, a better understanding of the cell surface properties of these cell types is essential. Using a combination of in silico, in vitro, and in vivo methods, we have identified three new cell surface markers of adipose cell types. The amino acid transporter ASC-1 is a white adipocyte-specific cell surface protein, with little or no expression in brown adipocytes, whereas the amino acid transporter PAT2 and the purinergic receptor P2RX5 are cell surface markers expressed in classical brown and beige adipocytes in mice. These markers also selectively mark brown/beige and white adipocytes in human tissue. Thus, ASC-1, PAT2, and P2RX5 are membrane surface proteins that may serve as tools to identify and target white and brown/beige adipocytes for therapeutic purposes.

  8. Asc-1, PAT2 and P2RX5 are novel cell surface markers for white, beige and brown adipocytes

    PubMed Central

    Ussar, Siegfried; Lee, Kevin Y.; Dankel, Simon N.; Boucher, Jeremie; Haering, Max-Felix; Kleinridders, Andre; Thomou, Thomas; Xue, Ruidan; Macotela, Yazmin; Cypess, Aaron M.; Tseng, Yu-Hua; Mellgren, Gunnar; Kahn, C. Ronald

    2015-01-01

    White, beige and brown adipocytes are developmentally and functionally distinct but often occur mixed together within individual depots. To target white, beige and brown adipocytes for diagnostic or therapeutic purposes, a better understanding of the cell surface properties of these cell types is essential. Using a combination of in silico, in vitro and in vivo methods, we have identified three new cell surface markers of adipose cell types. The amino acid transporter Asc-1 is a white adipocyte-specific cell surface protein, with little or no expression in brown adipocytes, whereas the amino acid transporter PAT2 and the purinergic receptor P2RX5 are cell surface markers expressed in classical brown and beige adipocytes in mice. These markers also selectively mark brown/beige and white adipocytes in human tissue. Thus, Asc-1, PAT2 and P2RX5 are membrane surface proteins that may serve as tools to identify and target white and brown/beige adipocytes for therapeutic purposes. PMID:25080478

  9. Alpha-tocopheryl-phosphate regulation of gene expression in pre-adipocytes and adipocytes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A correct function of adipocytes in connection with cellular fatty acid loading and release is a vital aspect of energy homeostasis; dysregulation of these reactions can result in obesity and type 2 diabetes mellitus. In addition, adipocytes have been proposed to play a major role in preventing lipo...

  10. Human coronary artery perivascular adipocytes overexpress genes responsible for regulating vascular morphology, inflammation, and hemostasis

    PubMed Central

    Aronow, Bruce J.; Tong, Wilson S.; Manka, David; Tang, Yaoliang; Bogdanov, Vladimir Y.; Unruh, Dusten; Blomkalns, Andra L.; Piegore, Mark G.; Weintraub, Daniel S.; Rudich, Steven M.; Kuhel, David G.; Hui, David Y.; Weintraub, Neal L.

    2013-01-01

    Inflammatory cross talk between perivascular adipose tissue and the blood vessel wall has been proposed to contribute to the pathogenesis of atherosclerosis. We previously reported that human perivascular (PV) adipocytes exhibit a proinflammatory phenotype and less adipogenic differentiation than do subcutaneous (SQ) adipocytes. To gain a global view of the genomic basis of biologic differences between PV and SQ adipocytes, we performed genome-wide expression analyses to identify differentially expressed genes between adipocytes derived from human SQ vs. PV adipose tissues. Although >90% of well-expressed genes were similarly regulated, we identified a signature of 307 differentially expressed genes that were highly enriched for functions associated with the regulation of angiogenesis, vascular morphology, inflammation, and blood clotting. Of the 156 PV upregulated genes, 59 associate with angiogenesis, vascular biology, or inflammation, noteworthy of which include TNFRSF11B (osteoprotegerin), PLAT, TGFB1, THBS2, HIF1A, GATA6, and SERPINE1. Of 166 PV downregulated genes, 21 associated with vascular biology and inflammation, including ANGPT1, ANGPTL1, and VEGFC. Consistent with the emergent hypothesis that PV adipocytes differentially regulate angiogenesis and inflammation, cell culture-derived adipocyte-conditioned media from PV adipocytes strongly enhanced endothelial cell tubulogenesis and monocyte migration compared with media from SQ adipocytes. These findings demonstrate that PV adipocytes have the potential to significantly modulate vascular inflammatory crosstalk in the setting of atherosclerosis by their ability to signal to both endothelial and inflammatory cells. PMID:23737535

  11. Impact of elvitegravir on human adipocytes: Alterations in differentiation, gene expression and release of adipokines and cytokines.

    PubMed

    Moure, Ricardo; Domingo, Pere; Gallego-Escuredo, José M; Villarroya, Joan; Gutierrez, Maria Del Mar; Mateo, Maria G; Domingo, Joan C; Giralt, Marta; Villarroya, Francesc

    2016-08-01

    Elvitegravir is a recently developed integrase inhibitor used for antiretroviral treatment of HIV infection. Secondary effects, including disturbances in lipid metabolism and, ultimately, in adipose tissue distribution and function, are common concerns associated with antiretroviral treatments. Here, we provide the first study of the effects of elvitegravir (in comparison with efavirenz, a non-nucleoside analog inhibitor of reverse transcriptase; and raltegravir, another integrase inhibitor) on human adipocyte differentiation, gene expression and secretion of adipokines and cytokines. Elvitegravir impaired adipogenesis and adipocyte metabolism in human SGBS adipocytes in a concentration-dependent manner (delaying acquisition of adipocyte morphology and reducing the expression of adipogenesis marker genes such as PPARγ, glucose transporter GLUT4, lipoprotein lipase, and the adipokines adiponectin and leptin). Compared with efavirenz, the effects of elvitegravir were similar but tended to occur at higher concentrations than those elicited by efavirenz, or were somewhat less intense than those caused by efavirenz at similar concentration. Elvitegravir tended to cause a more moderate induction of pro-inflammatory cytokines than efavirenz. Efavirenz induced a marked concentration-dependent increase in interleukin-8 expression and release whereas elvitregravir had little effect. Raltegravir had totally neutral actions of adipogenesis, adipocyte metabolism-related gene expression and release of adipokines and cytokines. In conclusion, elvitegravir alters adipocyte differentiation and function and promotes induction of pro-inflammatory cytokines similarly to efavirenz, but several effects were less intense. Further assessment of lipid metabolism and adipose tissue function in patients administered elvitegravir-based regimes is advisable considering that totally neutral effects of elvitegravir on lipid homeostasis cannot be anticipated from the current study in

  12. PPAR{alpha} does not suppress muscle-associated gene expression in brown adipocytes but does influence expression of factors that fingerprint the brown adipocyte

    SciTech Connect

    Walden, Tomas B.; Petrovic, Natasa; Nedergaard, Jan

    2010-06-25

    Brown adipocytes and myocytes develop from a common adipomyocyte precursor. PPAR{alpha} is a nuclear receptor important for lipid and glucose metabolism. It has been suggested that in brown adipose tissue, PPAR{alpha} represses the expression of muscle-associated genes, in this way potentially acting to determine cell fate in brown adipocytes. To further understand the possible role of PPAR{alpha} in these processes, we measured expression of muscle-associated genes in brown adipose tissue and brown adipocytes from PPAR{alpha}-ablated mice, including structural genes (Mylpf, Tpm2, Myl3 and MyHC), regulatory genes (myogenin, Myf5 and MyoD) and a myomir (miR-206). However, in our hands, the expression of these genes was not influenced by the presence or absence of PPAR{alpha}, nor by the PPAR{alpha} activator Wy-14,643. Similarly, the expression of genes common for mature brown adipocyte and myocytes (Tbx15, Meox2) were not affected. However, the brown adipocyte-specific regulatory genes Zic1, Lhx8 and Prdm16 were affected by PPAR{alpha}. Thus, it would not seem that PPAR{alpha} represses muscle-associated genes, but PPAR{alpha} may still play a role in the regulation of the bifurcation of the adipomyocyte precursor into a brown adipocyte or myocyte phenotype.

  13. Oligopeptide complex for targeted non-viral gene delivery to adipocytes

    NASA Astrophysics Data System (ADS)

    Won, Young-Wook; Adhikary, Partho Protim; Lim, Kwang Suk; Kim, Hyung Jin; Kim, Jang Kyoung; Kim, Yong-Hee

    2014-12-01

    Commercial anti-obesity drugs acting in the gastrointestinal tract or the central nervous system have been shown to have limited efficacy and severe side effects. Anti-obesity drug development is thus focusing on targeting adipocytes that store excess fat. Here, we show that an adipocyte-targeting fusion-oligopeptide gene carrier consisting of an adipocyte-targeting sequence and 9-arginine (ATS-9R) selectively transfects mature adipocytes by binding to prohibitin. Injection of ATS-9R into obese mice confirmed specific binding of ATS-9R to fat vasculature, internalization and gene expression in adipocytes. We also constructed a short-hairpin RNA (shRNA) for silencing fatty-acid-binding protein 4 (shFABP4), a key lipid chaperone in fatty-acid uptake and lipid storage in adipocytes. Treatment of obese mice with ATS-9R/shFABP4 led to metabolic recovery and body-weight reduction (>20%). The ATS-9R/shFABP4 oligopeptide complex could prove to be a safe therapeutic approach to regress and treat obesity as well as obesity-induced metabolic syndromes.

  14. The brown and brite adipocyte marker Cox7a1 is not required for non-shivering thermogenesis in mice

    PubMed Central

    Maurer, Stefanie F.; Fromme, Tobias; Grossman, Lawrence I.; Hüttemann, Maik; Klingenspor, Martin

    2015-01-01

    The cytochrome c oxidase subunit isoform Cox7a1 is highly abundant in skeletal muscle and heart and influences enzyme activity in these tissues characterised by high oxidative capacity. We identified Cox7a1, well-known as brown adipocyte marker gene, as a cold-responsive protein of brown adipose tissue. We hypothesised a mechanistic relationship between cytochrome c oxidase activity and Cox7a1 protein levels affecting the oxidative capacity of brown adipose tissue and thus non-shivering thermogenesis. We subjected wildtype and Cox7a1 knockout mice to different temperature regimens and tested characteristics of brown adipose tissue activation. Cytochrome c oxidase activity, uncoupling protein 1 expression and maximal norepinephrine-induced heat production were gradually increased during cold-acclimation, but unaffected by Cox7a1 knockout. Moreover, the abundance of uncoupling protein 1 competent brite cells in white adipose tissue was not influenced by presence or absence of Cox7a1. Skin temperature in the interscapular region of neonates was lower in uncoupling protein 1 knockout pups employed as a positive control, but not in Cox7a1 knockout pups. Body mass gain and glucose tolerance did not differ between wildtype and Cox7a1 knockout mice fed with high fat or control diet. We conclude that brown adipose tissue function in mice does not require the presence of Cox7a1. PMID:26635001

  15. Apolipoprotein E promotes lipid accumulation and differentiation in human adipocytes

    SciTech Connect

    Lasrich, Dorothee; Bartelt, Alexander; Grewal, Thomas; Heeren, Joerg

    2015-09-10

    Several studies in mice indicate a role for apolipoprotein E (APOE) in lipid accumulation and adipogenic differentiation in adipose tissue. However, little is yet known if APOE functions in a similar manner in human adipocytes. This prompted us to compare lipid loading and expression of adipocyte differentiation markers in APOE-deficient and control adipocytes using the differentiated human mesenchymal stem cell line hMSC-Tert as well as primary human and mouse adipocytes as model systems. Differentiated hMSC-Tert were stably transduced with or without siRNA targeting APOE while murine adipocytes were isolated from wild type and Apoe knockout mice. Human APOE knockdown hMSC-Tert adipocytes accumulated markedly less triglycerides compared to control cells. This correlated with strongly decreased gene expression levels of adipocyte markers such as adiponectin (ADIPOQ) and fatty acid binding protein 4 (FABP4) as well as the key transcription factor driving adipocyte differentiation, peroxisome proliferator activator receptor gamma (PPARG), in particular the PPARG2 isoform. Similarly, differentiation of murine Apoe-deficient adipocytes was characterized by reduced gene expression of Adipoq, Fabp4 and Pparg. Interestingly, incubation of APOE-deficient hMSC-Tert adipocytes with conditioned media from APOE3-overexpressing adipocytes or APOE-containing Very Low Density Lipoprotein (VLDL) partially restored triglyceride accumulation, but were unable to induce adipocyte differentiation, as judged by expression of adipocyte markers. Taken together, depletion of endogenous APOE in human adipocytes severely impairs lipid accumulation, which is associated with an inability to initiate differentiation. - Highlights: • Immortalized human mesenchymal stem cells were used to study adipocyte development. • Knockdown of endogenous APOE lead to impaired lipid accumulation and adipogenesis. • APOE supplementation partially restored lipid accumulation but not differentiation.

  16. Gene expression profiling in multipotent DFAT cells derived from mature adipocytes

    SciTech Connect

    Ono, Hiromasa; Oki, Yoshinao; Bono, Hidemasa; Kano, Koichiro

    2011-04-15

    Highlights: {yields} Adipocyte dedifferentiation is evident in a significant decrease in typical genes. {yields} Cell proliferation is strongly related to adipocyte dedifferentiation. {yields} Dedifferentiated adipocytes express several lineage-specific genes. {yields} Comparative analyses using publicly available datasets boost the interpretation. -- Abstract: Cellular dedifferentiation signifies the withdrawal of cells from a specific differentiated state to a stem cell-like undifferentiated state. However, the mechanism of dedifferentiation remains obscure. Here we performed comparative transcriptome analyses during dedifferentiation in mature adipocytes (MAs) to identify the transcriptional signatures of multipotent dedifferentiated fat (DFAT) cells derived from MAs. Using microarray systems, we explored similarly expressed as well as significantly differentially expressed genes in MAs during dedifferentiation. This analysis revealed significant changes in gene expression during this process, including a significant reduction in expression of genes for lipid metabolism concomitantly with a significant increase in expression of genes for cell movement, cell migration, tissue developmental processes, cell growth, cell proliferation, cell morphogenesis, altered cell shape, and cell differentiation. Our observations indicate that the transcriptional signatures of DFAT cells derived from MAs are summarized in terms of a significant decrease in functional phenotype-related genes and a parallel increase in cell proliferation, altered cell morphology, and regulation of the differentiation of related genes. A better understanding of the mechanisms involved in dedifferentiation may enable scientists to control and possibly alter the plasticity of the differentiated state, which may lead to benefits not only in stem cell research but also in regenerative medicine.

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

    SciTech Connect

    Yuan, Guoyue; Jia, Jue; Di, Liangliang; Zhou, Libin; Dong, Sijing; Ye, Jingjing; Wang, Dong; Yang, Ling; Wang, Jifang; Li, Lianxi; Yang, Ying; Mao, Chaoming; Chen, Mingdao

    2012-08-03

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

  18. IGF-I is a mitogen involved in differentiation-related gene expression in fetal rat brown adipocytes

    PubMed Central

    1993-01-01

    Fetal rat brown adipocytes at time zero of culture constitute a population of cells of broad spectrum, as estimated by cell size, endogenous fluorescence and lipid content, and show an intrinsic mitogenic competence. They express constitutively early growth-related genes such as c-myc, c-fos, and beta-actin, tissue specific-genes such as the uncoupling protein (UCP) and the lipogenic marker malic enzyme (ME). Fetal brown adipocytes bear a high expression of insulin-like growth factor receptor (IGF-IR), and show a high affinity IGF-I specific-binding to its receptor, and a high number of binding sites per cell. After cell quiescence, insulin-like growth factor I (IGF-I) was as potent as 10% FCS in inducing DNA synthesis, cell number increase, and the entry of cells into the cell-cycle. In addition, IGF- I or 10% FCS for 48 h increased the percentage of [3H]thymidine-labeled nuclei as compared to quiescent cells. Single cell autoradiographic microphotographs show typical multilocular fat droplets brown adipocytes, resulting positive to [3H]thymidine-labeled nuclei in response to IGF-I. IGF-I increased mRNA expression of the early- response genes c-fos (30 min), c-myc (2 and 24 h), and H-ras (4 and 24 h). 10% FCS also increased c-fos and c-myc, but failed to increase H- ras as an early event. IGF-I or 10% FCS, however, similarly increased the mRNA late expression of c-myc, H-ras, c-raf, beta-actin, and glucose 6-phosphate dehydrogenase (G6PD) at 72 h, as compared to quiescent cells. IGF-I or FCS maintained at 24 h or increased at 48 and 72 h UCP mRNA expression. The results demonstrate that IGF-I is a mitogen for fetal rat brown adipocytes, capable of inducing the expression of early and late growth-regulated genes, and of increasing the lipogenic marker ME and the tissue-specific gene UCP, suggesting the involvement of IGF-I in the differentiation as well as in the proliferation processes. PMID:8253851

  19. Human adipocytes are highly sensitive to intermittent hypoxia induced NF-kappaB activity and subsequent inflammatory gene expression

    SciTech Connect

    Taylor, Cormac T.; Kent, Brian D.; Crinion, Sophie J.; McNicholas, Walter T.; Ryan, Silke

    2014-05-16

    Highlights: • Intermittent hypoxia (IH) leads to NF-κB activation in human primary adipocytes. • Adipocytes bear higher pro-inflammatory potential than other human primary cells. • IH leads to upregulation of multiple pro-inflammatory genes in human adipocytes. - Abstract: Introduction: Intermittent hypoxia (IH)-induced activation of pro-inflammatory pathways is a major contributing factor to the cardiovascular pathophysiology associated with obstructive sleep apnea (OSA). Obesity is commonly associated with OSA although it remains unknown whether adipose tissue is a major source of inflammatory mediators in response to IH. The aim of this study was to test the hypothesis that IH leads to augmented inflammatory responses in human adipocytes when compared to cells of non-adipocyte lineages. Methods and results: Human primary subcutaneous and visceral adipocytes, human primary microvascular pulmonary endothelial cells (HUMEC-L) and human primary small airway epithelial cells (SAEC) were exposed to 0, 6 or 12 cycles of IH or stimulated with tumor necrosis factor (TNF)-α. IH led to a robust increase in NF-κB DNA-binding activity in adipocytes compared with normoxic controls regardless of whether the source of adipocytes was visceral or subcutaneous. Notably, the NF-κB response of adipocytes to both IH and TNF-α was significantly greater than that in HUMEC-L and SAEC. Western blotting confirmed enhanced nuclear translocation of p65 in adipocytes in response to IH, accompanied by phosphorylation of I-κB. Parallel to p65 activation, we observed a significant increase in secretion of the adipokines interleukin (IL)-8, IL-6 and TNF-α with IH in adipocytes accompanied by significant upregulation of mRNA expression. PCR-array suggested profound influence of IH on pro-inflammatory gene expression in adipocytes. Conclusion: Human adipocytes demonstrate strong sensitivity to inflammatory gene expression in response to acute IH and hence, adipose tissue may be a key

  20. Experimental Model to Study the Role of Retinoblastoma Gene Product (pRb) for Determination of Adipocyte Differentiation.

    PubMed

    Popov, B V; Shilo, P S; Zhidkova, O V; Zaichik, A M; Petrov, N S

    2015-06-01

    Using stable constitutive expression of retinoblastoma gene product (pRb) in polypotent mesenchymal 10T1/2 cells we obtained stable cell lines hyperexpressing functionally active or inactive mutant pRb. The cells producing active exogenous pRb demonstrated high sensitivity to adipocyte differentiation inductors, whereas production of inactive form of the exogenous protein suppressed adipocyte differentiation. The obtained lines can serve as the experimental model for studying the role of pRb in determination of adipocyte differentiation.

  1. Estrogen-related receptor {alpha} modulates the expression of adipogenesis-related genes during adipocyte differentiation

    SciTech Connect

    Ijichi, Nobuhiro; Ikeda, Kazuhiro; Horie-Inoue, Kuniko; Yagi, Ken; Okazaki, Yasushi; Inoue, Satoshi . E-mail: INOUE-GER@h.u-tokyo.ac.jp

    2007-07-06

    Estrogen-related receptor {alpha} (ERR{alpha}) is an orphan nuclear receptor that regulates cellular energy metabolism by modulating gene expression involved in fatty acid oxidation and mitochondrial biogenesis in brown adipose tissue. However, the physiological role of ERR{alpha} in adipogenesis and white adipose tissue development has not been well studied. Here, we show that ERR{alpha} and ERR{alpha}-related transcriptional coactivators, peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) coactivator-1{alpha} (PGC-1{alpha}) and PGC-1{beta}, can be up-regulated in 3T3-L1 preadipocytes at mRNA levels under the adipogenic differentiation condition including the inducer of cAMP, glucocorticoid, and insulin. Gene knockdown by ERR{alpha}-specific siRNA results in mRNA down-regulation of fatty acid binding protein 4, PPAR{gamma}, and PGC-1{alpha} in 3T3-L1 cells in the adipogenesis medium. ERR{alpha} and PGC-1{beta} mRNA expression can be also up-regulated in another preadipocyte lineage DFAT-D1 cells and a pluripotent mesenchymal cell line C3H10T1/2 under the differentiation condition. Furthermore, stable expression of ERR{alpha} in 3T3-L1 cells up-regulates adipogenic marker genes and promotes triglyceride accumulation during 3T3-L1 differentiation. These results suggest that ERR{alpha} may play a critical role in adipocyte differentiation by modulating the expression of various adipogenesis-related genes.

  2. JAZF1 can regulate the expression of lipid metabolic genes and inhibit lipid accumulation in adipocytes

    SciTech Connect

    Ming, Guang-feng; Xiao, Di; Gong, Wei-jing; Liu, Hui-xia; Liu, Jun; Zhou, Hong-hao; Liu, Zhao-qian

    2014-03-14

    Highlights: • JAZF1 was significantly upregulated during the differentiation of 3T3-L1 preadipocytes. • JAZF1 overexpression inhibited lipid accumulation in differentiated mature 3T3-L1 adipocytes. • JAZF1 overexpression inhibited the expression of SREBP1, ACC, and FAS. • JAZF1 overexpression upregulated the expression of HSL and ATGL. • SREBP1 and JAZF1 could regulate each other in adipocytes. - Abstract: JAZF1 is a newly identified gene with unknown functions. A recent genome-wide association study showed that JAZF1 is associated with type 2 diabetes and is highly expressed in liver and adipose tissue. Studies have demonstrated that JAZF1 is the co-repressor for nuclear orphan receptor TAK1, whereas most nuclear orphan receptor family members are involved in the regulation of lipid metabolism. Therefore, JAZF1 could be closely related to glycolipid metabolism. In this study, JAZF1 was significantly upregulated during the induced differentiation process of 3T3-L1 preadipocytes. The overexpression of JAZF1 inhibited lipid accumulation in differentiated mature 3T3-L1 adipocytes and significantly inhibited the expression of SREBPl, ACC, and FAS, which were important in lipid synthesis, while upregulating the expression of key enzyme hormone-sensitive lipase in lipoclasis. Moreover, SREBPl exhibited an inhibitory function on the expression of JAZF1. SREBP1 reversed the inhibitory action on lipid accumulation of JAZF1. SREBP1 and JAZF1 were observed to regulate each other in adipocytes. Therefore, JAZF1 could regulate the expression of particular genes related to lipid metabolism and inhibit lipid accumulation in adipocytes. This result suggests that JAZF1 may be a potential target for the treatment of diseases, such as obesity and lipid metabolism disorders.

  3. Inflammatory markers and adipokines alter adipocyte-derived ASP production through direct and indirect immune interaction.

    PubMed

    Lu, H; Gauvreau, D; Tom, F-Q; Lapointe, M; Luo, X P; Cianflone, K

    2013-04-01

    Obesity and related metabolic diseases are associated with chronic low-grade inflammation, characterized by increased pro-inflammatory proteins. Several studies have demonstrated increases in acylation stimulating protein (ASP) and its precursor protein C3 in obesity, diabetes and dyslipidemia. To evaluate the effects of acute inflammatory factors and adipokines on ASP production and potential mechanisms of action, 3T3-L1 adipocytes were treated for 24 h with adipokines, cytokines, macrophage-conditioned media and direct co-culture with J774 macrophages. ASP and C3 in the media were evaluated in relation to changes in adipocyte lipid metabolism (cellular triglyceride stores). Leptin, adiponectin, IL-10, LPS and TNF-α increased ASP production (151%, 153%, 190%, 318%, 134%, P<0.05, respectively,). C5a and RANTES (Regulated and normal T cell expressed and secreted) decreased ASP production ( - 34%, - 47%, P<0.05), which was also associated with a decrease in the precursor protein C3 ( - 39% to - 51%, P<0.01), while keratinocyte chemoattractant (KC; murine IL-8 ortholog) had no effect on ASP and C3 secretion. By contrast, apelin, omentin and visfatin also decreased ASP ( - 27%, - 49%, - 22%, P<0.05), but without changes in precursor protein C3 secretion. Macrophage-conditioned media alone had little effect on C3 or ASP, while co-culture of adipocytes with macrophages markedly increased ASP and C3 production (272%, 167%, P<0.05). These in vitro results suggest various metabolic hormones and inflammatory factors can affect ASP production through increased precursor C3 production and/or by changing the rate of C3 conversion to ASP. As an adipokine, ASP could constitute a new link between adipocytes and macrophages.

  4. Quercetin Impacts Expression of Metabolism- and Obesity-Associated Genes in SGBS Adipocytes.

    PubMed

    Leiherer, Andreas; Stoemmer, Kathrin; Muendlein, Axel; Saely, Christoph H; Kinz, Elena; Brandtner, Eva M; Fraunberger, Peter; Drexel, Heinz

    2016-05-12

    Obesity is characterized by the rapid expansion of visceral adipose tissue, resulting in a hypoxic environment in adipose tissue which leads to a profound change of gene expression in adipocytes. As a consequence, there is a dysregulation of metabolism and adipokine secretion in adipose tissue leading to the development of systemic inflammation and finally resulting in the onset of metabolic diseases. The flavonoid quercetin as well as other secondary plant metabolites also referred to as phytochemicals have anti-oxidant, anti-inflammatory, and anti-diabetic effects known to be protective in view of obesity-related-diseases. Nevertheless, its underlying molecular mechanism is still obscure and thus the focus of this study was to explore the influence of quercetin on human SGBS (Simpson Golabi Behmel Syndrome) adipocytes' gene expression. We revealed for the first time that quercetin significantly changed expression of adipokine (Angptl4, adipsin, irisin and PAI-1) and glycolysis-involved (ENO2, PFKP and PFKFB4) genes, and that this effect not only antagonized but in part even overcompensated the effect mediated by hypoxia in adipocytes. Thus, these results are explained by the recently proposed hypothesis that the protective effect of quercetin is not solely due to its free radical-scavenging activity but also to a direct effect on mitochondrial processes, and they demonstrate that quercetin might have the potential to counteract the development of obesity-associated complications.

  5. Gene-manipulated Adipocytes for the Treatment of Various Intractable Diseases.

    PubMed

    Kuroda, Masayuki; Bujo, Hideaki; Aso, Masayuki; Saito, Yasushi; Yokote, Koutaro

    2016-01-01

    Although protein replacement is an effective treatment for serum protein deficiencies such as diabetes and hemophilia, recombinant protein products are not available for all rare inherited diseases due to the instability of the recombinant proteins and/or to cost. Gene therapy is the most attractive option for treating patients with such rare diseases. To develop an effective ex vivo gene therapy-based protein replacement treatment requires recipient cells that differ from those used in standard gene therapy, which is performed to correct the function of the recipient cells. Adipose tissue is an expected source of proliferative cells for cell-based therapies, including regenerative medicine and gene transfer applications. Based on recent advances in cell biology and extensive clinical experience in transplantation therapy for adipose tissue, we focused on the mature adipocyte fraction, which is the floating fraction after collagenase digestion and centrifugation of adipose tissue. Proliferative adipocytes were propagated from the floating fraction by the ceiling culture technique. These cells are designated as ceiling culture-derived proliferative adipocytes (ccdPAs). We first focused on lecithin:cholesterol acyltransferase (LCAT) deficiency, an inherited metabolic disorder caused by lcat gene mutation, and ccdPAs as a therapeutic gene vehicle for LCAT replacement therapy. In our recent in vitro and animal model studies, we developed an adipose cell manipulation procedure using advanced gene transduction methods and transplantation scaffolds. We herein introduce the progress made in novel adipose tissue-based therapeutic strategies for the treatment of protein deficiencies and describe their future applications for other intractable diseases. PMID:27150923

  6. The Fto Gene Regulates the Proliferation and Differentiation of Pre-Adipocytes in Vitro

    PubMed Central

    Jiao, Yang; Zhang, Jingying; Lu, Lunjie; Xu, Jiaying; Qin, Liqiang

    2016-01-01

    The highly regulated differentiation and proliferation of pre-adipocytes play a key role in the initiation of obesity. Fat mass and obesity associated (FTO) is a novel gene strongly associated with the risk of obesity. A deficiency of FTO may cause growth retardation in addition to fat mass and adipocyte size reduction in vivo. To investigate the potential role of Fto gene on the proliferation and differentiation of pre-adipocytes, we generated Fto-knockdown and overexpressed 3T3-L1 cells. Using numerous proliferation assays our results suggest that Fto knockdown leads to suppression of proliferation, lower mitochondrial membrane potential, less cellular ATP, and decreased and smaller intracellular lipid droplets compared with controls (p < 0.05). Western blot analysis demonstrated that Fto knockdown can significantly suppress peroxisome proliferator-activated receptor gamma (PPARγ) and glucose transporter type 4 (GLUT4) expression and inhibit Akt phosphorylation. By contrast, overexpression of Fto had the opposing effect on proliferation, mitochondrial membrane potential, ATP generation, in vitro differentiation, Akt phosphorylation, and PPARγ and GLUT4 expression. Moreover, we demonstrated that Wortmannin, a phosphoinositide 3-kinase (PI3K) inhibitor, could inhibit phospho-Akt in Fto overexpressed 3T3-L1 cells. Taken together, the results suggest that Fto regulates the proliferation and differentiation of 3T3-L1 cells via multiple mechanisms, including PPARγ and PI3K/Akt signaling. PMID:26907332

  7. Secretome-derived isotope tags (SDIT) reveal adipocyte-derived apolipoprotein C-I as a predictive marker for cardiovascular disease.

    PubMed

    Li, Rong-Xia; Ding, Yu-Bo; Zhao, Shi-Lin; Xiao, Yuan-Yuan; Li, Qing-run; Xia, Fang-Ying; Sun, Liang; Lin, Xu; Wu, Jia-Rui; Liao, Kan; Zeng, Rong

    2012-05-01

    We developed a quantitative strategy, named secretome-derived isotopic tag (SDIT), to concurrently identify and quantify the adipocyte-secreted plasma proteins from normal and high-fat-diet (HFD) induced obese mice, based on the application of isotope-labeled secreted proteins from cultured mouse adipocytes as internal standards. We detected 197 proteins with significant changes between normal and obese mice plasma. Importantly, a novel adipocyte-secreted plasma protein, apolipoprotein C-I (apoC-I), significantly increased in the obese mice plasma. The expression and secretion of adipocyte apoC-I was detected in differentiated 3T3-L1 and primary rat adipocytes. Our in vitro experiments proved that functional Golgi apparatus was required for apoC-I secretion. Additionally, obese mice had increased apoC-I production in adipose tissue. Population survey of 367 participants showed that the plasma level of apoC-I was significantly increased in obese individuals compared with healthy individuals. After multiple adjustments for age and sex, the odds ratios for risk factors of cardiovascular disease including high LDL cholesterol, hypercholesterolemia, and hypertriglyceridemia, respectively, were used to compare the highest with the lowest apoC-I quartile. Taken together, our studies provide a novel strategy to concurrently identify and quantify tissue-specific secreted proteins. This strategy can be used to identify the largest global characterization of adipocyte-derived plasma proteome and provides a potential disease-related biomarker for clinical diagnoses. By selectively analyzing adipocyte-secreted proteins in plasma from obese vs lean murine and/or human subjects, we discovered that apoC-I is an adipocyte-secreted plasma protein and a predictive marker for cardiovascular disease.

  8. N-Acetylcysteine Reduces Markers of Differentiation in 3T3-L1 Adipocytes

    PubMed Central

    Calzadilla, Pablo; Sapochnik, Daiana; Cosentino, Soledad; Diz, Virginia; Dicelio, Lelia; Calvo, Juan Carlos; Guerra, Liliana N.

    2011-01-01

    Oxidative stress plays a critical role in the pathogenesis of diabetes, hypertension and atherosclerosis. Some authors reported that fat accumulation correlates to systemic oxidative stress in humans and mice, but the relationship of lipid production and oxidative metabolism is still unclear. In our laboratory we used 3T3-L1 preadipocytes, which are able to differentiate into mature adipocytes and accumulate lipids, as obesity model. We showed that intracellular reactive oxygen species (ROS) and antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities increased in parallel with fat accumulation. Meanwhile N-acetylcysteine (NAC), a well known antioxidant and Glutathione (GSH) precursor, inhibited ROS levels as well as fat accumulation in a concentration-dependent manner. NAC also inhibited both adipogenic transcription factors CCAAT/enhancer binding protein beta (C/EBP β) and peroxisomal proliferator activated receptor gamma (PPAR γ) expression; we suggested that intracellular GSH content could be responsible for these effects. PMID:22072928

  9. Quercetin Impacts Expression of Metabolism- and Obesity-Associated Genes in SGBS Adipocytes

    PubMed Central

    Leiherer, Andreas; Stoemmer, Kathrin; Muendlein, Axel; Saely, Christoph H.; Kinz, Elena; Brandtner, Eva M.; Fraunberger, Peter; Drexel, Heinz

    2016-01-01

    Obesity is characterized by the rapid expansion of visceral adipose tissue, resulting in a hypoxic environment in adipose tissue which leads to a profound change of gene expression in adipocytes. As a consequence, there is a dysregulation of metabolism and adipokine secretion in adipose tissue leading to the development of systemic inflammation and finally resulting in the onset of metabolic diseases. The flavonoid quercetin as well as other secondary plant metabolites also referred to as phytochemicals have anti-oxidant, anti-inflammatory, and anti-diabetic effects known to be protective in view of obesity-related-diseases. Nevertheless, its underlying molecular mechanism is still obscure and thus the focus of this study was to explore the influence of quercetin on human SGBS (Simpson Golabi Behmel Syndrome) adipocytes’ gene expression. We revealed for the first time that quercetin significantly changed expression of adipokine (Angptl4, adipsin, irisin and PAI-1) and glycolysis-involved (ENO2, PFKP and PFKFB4) genes, and that this effect not only antagonized but in part even overcompensated the effect mediated by hypoxia in adipocytes. Thus, these results are explained by the recently proposed hypothesis that the protective effect of quercetin is not solely due to its free radical-scavenging activity but also to a direct effect on mitochondrial processes, and they demonstrate that quercetin might have the potential to counteract the development of obesity-associated complications. PMID:27187453

  10. y-Synuclein is an Adipocyte-Neuron Gene Coordinately-Expressed with Leptin & Increased in Obesity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Objective: Recently, we characterized tumor suppressor candidate 5 (Tusc5) as an adipocyte-neuron peroxisome proliferator activated receptor-y (PPARy) target gene (1). Our objective herein was to identify additional candidate genes that play shared roles in neuron-fat physiology. Research Methods an...

  11. Adipocyte gene expression is altered in formerly obese mice and as a function of diet composition.

    PubMed

    Miller, Ryan S; Becker, Kevin G; Prabhu, Vinayakumar; Cooke, David W

    2008-06-01

    In the development of obesity, the source of excess energy may influence appetite and metabolism. To determine the effects of differences in diet composition in obesity, mice were fed either a high-carbohydrate diet (HC; 10% fat energy) or a high-fat energy-restricted diet (HFR; 60% fat energy) over 18 wk in weight-matched groups of mice. To identify obesity-associated genes with persistently altered expression following weight reduction, mice were fed either a standard low-fat diet (LF; 10% fat energy), an unrestricted high-fat diet (HF; 60% fat energy), or a HF diet followed by weight reduction (WR). Mice fed a HF diet had significantly greater gonadal fat mass and higher whole blood glucose concentrations than mice fed an HC diet. Of the mice fed a high-fat diet, total body weight and serum insulin concentrations were greater in HF than in HFR. Microarray analysis revealed that HF vs. HC feeding resulted in global differences in adipocyte gene expression patterns. Although we identified genes whose expression was altered in both moderately and severely obese mice, there were also a large number of genes with altered expression only in severe obesity. Formerly obese, WR mice did not differ significantly from lean controls in total body weight or physiological measures. However, microarray analysis revealed distinctly different patterns of adipocyte gene expression. Furthermore, there were 398 genes with altered expression in HF mice that persisted in WR mice. Genes with persistently altered expression following obesity may play a role in rebound weight gain following weight reduction.

  12. Cholesteryl ester transfer protein gene expression during differentiation of human preadipocytes to adipocytes in primary culture.

    PubMed

    Gauthier, B; Robb, M; McPherson, R

    1999-02-01

    The expression pattern of the CETP gene in relationship to that of LPL, adipsin, PPARgamma, C/EBPalpha, ADD1/SREBPI and actin was examined by RT-PCR during differentiation of human fibroblastic preadipocytes to adipocytes in primary culture. Preadipocytes were isolated from subcutaneous fat obtained from healthy female subjects undergoing mammary reduction procedures, and induced to differentiate in culture. Morphologically, adipogenesis was confirmed by the accumulation of lipid droplets in cells. We show that the gene encoding CETP is expressed in preadipocytes and is present throughout differentiation as compared to LPL and adipsin which were detected in the majority of samples by day 2 or 3 of adipogenesis. The transcription factors, PPARgamma, ADD1/SREBP1 and C/EBPalpha were expressed by day 2, concomitant with the appearance of LPL and adipsin but subsequent to the appearance of CETP. CETP mRNA was not detectable in human skin fibroblasts. These studies demonstrate that CETP. expression is induced at an early stage of commitment to the adipocyte lineage and may be activated by transcription factor(s), which are not members of the PPAR, ADD1/SREBP1 or C/EBP families. PMID:10030381

  13. Regulation of UCP gene expression in brown adipocytes differentiated in primary culture. Effects of a new beta-adrenoceptor agonist.

    PubMed

    Champigny, O; Holloway, B R; Ricquier, D

    1992-07-01

    Primary cultures of precursor cells from mouse and rat brown adipose tissue (BAT) were used to study the effect of a new beta-agonist (ICI D7114) on the uncoupling protein (UCP) gene expression. ICI 215001 (the active metabolite of D7114) increased the expression of UCP and its mRNA in brown adipocytes differentiating in vitro in a dose-dependent manner. This stimulating effect was not inhibited by propranolol, a non-specific beta-antagonist, but was partially reduced by bupranolol, a beta 3-antagonist. No expression of UCP mRNA was ever induced by ICI 215001 in white adipocytes differentiated in vitro. It was concluded that the drug could affect the brown adipose cells through a beta 3-pathway. It could clearly modulate the expression of UCP in brown adipocytes differentiated in vitro, but was not able by itself to turn on the gene. PMID:1355051

  14. Nuclear factor of activated T cell (NFAT) transcription proteins regulate genes involved in adipocyte metabolism and lipolysis

    SciTech Connect

    Holowachuk, Eugene W. . E-mail: geneh@telenet.net

    2007-09-21

    NFAT involvement in adipocyte physiological processes was examined by treatment with CsA and/or GSK3{beta} inhibitors (Li{sup +} or TZDZ-8), which prevent or increase NFAT nuclear translocation, respectively. CsA treatment reduced basal and TNF{alpha}-induced rates of lipolysis by 50%. Adipocytes preincubated with Li{sup +} or TZDZ-8 prior to CsA and/or TNF{alpha}, exhibited enhanced basal rates of lipolysis and complete inhibition of CsA-mediated decreased rates of lipolysis. CsA treatment dramatically reduced the mRNA levels of adipocyte-specific genes (aP2, HSL, PPAR{gamma}, ACS and Adn), compared with control or TNF{alpha}-treatment, whereas Li{sup +} pretreatment blocked the inhibitory effects of CsA, and mRNA levels of aP2, HSL, PPAR{gamma}, and ACS were found at or above control levels. NFAT nuclear localization, assessed by EMSA, confirmed that CsA or Li{sup +} treatments inhibited or increased NFAT nuclear translocation, respectively. These results show that NFAT proteins in mature adipocytes participate in the transcriptional control of genes involved in adipocyte metabolism and lipolysis.

  15. Regulation of GLUT4 gene expression by SREBP-1c in adipocytes

    PubMed Central

    Im, Seung-Soon; Kwon, Sool-Ki; Kang, Seung-Youn; Kim, Tae-Hyun; Kim, Ha-Il; Hur, Man-Wook; Kim, Kyung-Sup; Ahn, Yong-Ho

    2006-01-01

    Expression of the GLUT4 (glucose transporter type 4 isoform) gene in adipocytes is subject to hormonal or metabolic control. In the present study, we have characterized an adipose tissue transcription factor that is influenced by fasting/refeeding regimens and insulin. Northern blotting showed that refeeding increased GLUT4 mRNA levels for 24 h in adipose tissue. Consistent with an increased GLUT4 gene expression, the mRNA levels of SREBP (sterol-regulatory-element-binding protein)-1c in adipose tissue were also increased by refeeding. In streptozotocin-induced diabetic rats, insulin treatment increased the mRNA levels of GLUT4 in adipose tissue. Serial deletion, luciferase reporter assays and electrophoretic mobility-shift assay studies indicated that the putative sterol response element is located in the region between bases −109 and −100 of the human GLUT4 promoter. Transduction of the SREBP-1c dominant negative form to differentiated 3T3-L1 adipocytes caused a reduction in the mRNA levels of GLUT4, suggesting that SREBP-1c mediates the transcription of GLUT4. In vivo chromatin immunoprecipitation revealed that refeeding increased the binding of SREBP-1 to the putative sterol-response element in the GLUT4. Furthermore, treating streptozotocin-induced diabetic rats with insulin restored SREBP-1 binding. In addition, we have identified an Sp1 binding site adjacent to the functional sterol-response element in the GLUT4 promoter. The Sp1 site appears to play an additive role in SREBP-1c mediated GLUT4 gene upregulation. These results suggest that upregulation of GLUT4 gene transcription might be directly mediated by SREBP-1c in adipose tissue. PMID:16787385

  16. Superparamagnetic iron oxide nanoparticles alter expression of obesity and T2D-associated risk genes in human adipocytes

    PubMed Central

    Sharifi, S.; Daghighi, S.; Motazacker, M. M.; Badlou, B.; Sanjabi, B.; Akbarkhanzadeh, A.; Rowshani, A. T.; Laurent, S.; Peppelenbosch, M. P.; Rezaee, F.

    2013-01-01

    Adipocytes hypertrophy is the main cause of obesity and its affliction such as type 2 diabetes (T2D). Since superparamagnetic iron oxide nanoparticles (SPIONs) are used for a wide range of biomedical/medical applications, we aimed to study the effect of SPIONs on 22 and 29 risk genes (Based on gene wide association studies) for obesity and T2D in human adipocytes. The mRNA expression of lipid and glucose metabolism genes was changed upon the treatment of human primary adipocytes with SPIONs. mRNA of GULP1, SLC30A8, NEGR1, SEC16B, MTCH2, MAF, MC4R, and TMEM195 were severely induced, whereas INSIG2, NAMPT, MTMR9, PFKP, KCTD15, LPL and GNPDA2 were down-regulated upon SPIONs stimulation. Since SEC16B gene assist the phagocytosis of apoptotic cells and this gene were highly expressed upon SPIONs treatment in adipocytes, it is logic to assume that SPIONs may play a crucial role in this direction, which requires more consideration in the future. PMID:23838847

  17. Superparamagnetic iron oxide nanoparticles alter expression of obesity and T2D-associated risk genes in human adipocytes

    NASA Astrophysics Data System (ADS)

    Sharifi, S.; Daghighi, S.; Motazacker, M. M.; Badlou, B.; Sanjabi, B.; Akbarkhanzadeh, A.; Rowshani, A. T.; Laurent, S.; Peppelenbosch, M. P.; Rezaee, F.

    2013-07-01

    Adipocytes hypertrophy is the main cause of obesity and its affliction such as type 2 diabetes (T2D). Since superparamagnetic iron oxide nanoparticles (SPIONs) are used for a wide range of biomedical/medical applications, we aimed to study the effect of SPIONs on 22 and 29 risk genes (Based on gene wide association studies) for obesity and T2D in human adipocytes. The mRNA expression of lipid and glucose metabolism genes was changed upon the treatment of human primary adipocytes with SPIONs. mRNA of GULP1, SLC30A8, NEGR1, SEC16B, MTCH2, MAF, MC4R, and TMEM195 were severely induced, whereas INSIG2, NAMPT, MTMR9, PFKP, KCTD15, LPL and GNPDA2 were down-regulated upon SPIONs stimulation. Since SEC16B gene assist the phagocytosis of apoptotic cells and this gene were highly expressed upon SPIONs treatment in adipocytes, it is logic to assume that SPIONs may play a crucial role in this direction, which requires more consideration in the future.

  18. 3T3-L1 adipocytes display phenotypic characteristics of multiple adipocyte lineages

    PubMed Central

    Morrison, Shona; McGee, Sean L

    2015-01-01

    Differentiated 3T3-L1 adipocytes are a widely used in vitro model of white adipocytes. In addition to classical white and brown adipocytes that are derived from different cell lineages, beige adipocytes have also been identified, which have characteristics of both white and brown adipocytes. Here we show that 3T3-L1 adipocytes display features of multiple adipocytes lineages. While the gene expression profile and basal bioenergetics of 3T3-L1 adipocytes was typical of white adipocytes, they responded acutely to catecholamines by increasing oxygen consumption in an UCP1-dependent manner, and by increasing the expression of genes enriched in brown but not beige adipocytes. Chronic exposure to catecholamines exacerbated this phenotype. However, a beige adipocyte differentiation procedure did not induce a beige adipocyte phenotype in 3T3-L1 fibroblasts. These multiple lineage features should be considered when interpreting data from experiments utilizing 3T3-L1 adipocytes. PMID:26451286

  19. Effects of dopamine on leptin release and leptin gene (OB) expression in adipocytes from obese and hypertensive patients

    PubMed Central

    Alvarez-Aguilar, Cleto; Alvarez-Paredes, Alfonso Rafael; Lindholm, Bengt; Stenvinkel, Peter; García-López, Elvia; Mejía-Rodríguez, Oliva; López-Meza, Joel Edmundo; Amato, Dante; Paniagua, Ramon

    2013-01-01

    Background A reduction of dopaminergic (DAergic) activity with increased prolactin levels has been found in obese and hypertensive patients, suggesting its involvement as a pathophysiological mechanism promoting hypertension. Similarly, leptin action increasing sympathetic activity has been proposed to be involved in mechanisms of hypertension. The aim of this study was to analyze the effects of DA, norepinephrine (NE), and prolactin on leptin release and leptin gene (OB) expression in adipocytes from obese and hypertensive patients. Methods Leptin release and OB gene expression were analyzed in cultured adipocytes from 16 obese and hypertensive patients treated with DA (0.001, 0.01, 0.1, and 1.0 μmol/L), NE (1.0 μmol/L), insulin (0.1 μmol/L), and prolactin (1.0 μmol/L), and from five nonobese and normotensive controls treated with DA (1 μmol/L), NE (1 μmol/L), insulin (0.1 μmol/L), and prolactin (1.0 μmol/L). Results A dose-related reduction of leptin release and OB gene messenger ribonucleic acid expression under different doses of DA was observed in adipocytes from obese hypertensive patients. Whereas prolactin treatment elicited a significant increase of both leptin release and OB gene expression, NE reduced these parameters. Although similar effects of DA and NE were observed in adipocytes from controls, baseline values in controls were reduced to 20% of the value in adipocytes from obese hypertensive patients. Conclusion These results suggest that DAergic deficiency contributes to metabolic disorders linked to hyperleptinemia in obese and hypertensive patients. PMID:24348062

  20. SNP marker discovery in koala TLR genes.

    PubMed

    Cui, Jian; Frankham, Greta J; Johnson, Rebecca N; Polkinghorne, Adam; Timms, Peter; O'Meally, Denis; Cheng, Yuanyuan; Belov, Katherine

    2015-01-01

    Toll-like receptors (TLRs) play a crucial role in the early defence against invading pathogens, yet our understanding of TLRs in marsupial immunity is limited. Here, we describe the characterisation of nine TLRs from a koala immune tissue transcriptome and one TLR from a draft sequence of the koala genome and the subsequent development of an assay to study genetic diversity in these genes. We surveyed genetic diversity in 20 koalas from New South Wales, Australia and showed that one gene, TLR10 is monomorphic, while the other nine TLR genes have between two and 12 alleles. 40 SNPs (16 non-synonymous) were identified across the ten TLR genes. These markers provide a springboard to future studies on innate immunity in the koala, a species under threat from two major infectious diseases.

  1. SNP marker discovery in koala TLR genes.

    PubMed

    Cui, Jian; Frankham, Greta J; Johnson, Rebecca N; Polkinghorne, Adam; Timms, Peter; O'Meally, Denis; Cheng, Yuanyuan; Belov, Katherine

    2015-01-01

    Toll-like receptors (TLRs) play a crucial role in the early defence against invading pathogens, yet our understanding of TLRs in marsupial immunity is limited. Here, we describe the characterisation of nine TLRs from a koala immune tissue transcriptome and one TLR from a draft sequence of the koala genome and the subsequent development of an assay to study genetic diversity in these genes. We surveyed genetic diversity in 20 koalas from New South Wales, Australia and showed that one gene, TLR10 is monomorphic, while the other nine TLR genes have between two and 12 alleles. 40 SNPs (16 non-synonymous) were identified across the ten TLR genes. These markers provide a springboard to future studies on innate immunity in the koala, a species under threat from two major infectious diseases. PMID:25799012

  2. CGMD: An integrated database of cancer genes and markers

    PubMed Central

    Pradeepkiran, Jangampalli Adi; Sainath, Sri Bhashyam; Kramthi Kumar, Konidala; Balasubramanyam, Lokanada; Vidya Prabhakar, Kodali; Bhaskar, Matcha

    2015-01-01

    Integrating cancer genes and markers with experimental evidence might provide valuable information for the further investigation of crosstalk between tumor genes and markers in cancer biology. To achieve this objective, we developed a database known as the Cancer Gene Marker Database (CGMD), which integrates data on tumor genes and markers based on experimental evidence. The major goal of CGMD is to provide the following: 1) current systematic treatment approaches and recent advances in different cancer treatments; 2) the aggregation of different genes and markers by their molecular characteristics and pathway associations; and 3) free access to the data compiled by CGMD at http://cgmd.in/. The database consists of 309 genes and 206 markers, as well as a list of 40 different human cancers, with detailed descriptions of all characterized markers. CGMD provides complete cancer annotations and molecular descriptions of cancer genes and markers such as CpG islands, promoters, exons, PDB structures, active sites and domains. PMID:26160459

  3. DNA Methylation Suppresses Leptin Gene in 3T3-L1 Adipocytes

    PubMed Central

    Kuroda, Masashi; Tominaga, Ayako; Nakagawa, Kasumi; Nishiguchi, Misa; Sebe, Mayu; Miyatake, Yumiko; Kitamura, Tadahiro; Tsutsumi, Rie; Harada, Nagakatsu; Nakaya, Yutaka; Sakaue, Hiroshi

    2016-01-01

    Leptin is a key regulator of energy intake and expenditure. This peptide hormone is expressed in mouse white adipose tissue, but hardly expressed in 3T3-L1 adipocytes. Using bisulfite sequencing, we found that CpG islands in the leptin promoter are highly methylated in 3T3-L1cells. 5-azacytidine, an inhibitor of DNA methyltransferase, markedly increased leptin expression as pre-adipocytes matured into adipocytes. Remarkably, leptin expression was stimulated by insulin in adipocytes derived from precursor cells exposed to 5-azacytidine, but suppressed by thiazolidinedione and dexamethasone. In contrast, adipocytes derived from untreated precursor cells were unresponsive to both 5-azacytidine and hormonal stimuli, although lipid accumulation was sufficient to boost leptin expression in the absence of demethylation. Taken together, the results suggest that leptin expression in 3T3-L1 cells requires DNA demethylation prior to adipogenesis, transcriptional activation during adipogenesis, and lipid accumulation after adipogenesis. PMID:27494408

  4. Human imprinted genes as oncodevelopmental markers.

    PubMed

    Biran, H; Ariel, I; de Groot, N; Shani, A; Hochberg, A

    1994-01-01

    Imprinted genes mediate unique maternal or paternal genetic roles and their function is essential in prenatal development. The reciprocally imprinted human insulin-like growth factor 2 (IGF2) and H19 genes are expressed during embryonal life, also in the placenta, and are downregulated postnatally. They reexpress in pediatric tumors (e.g. Wilms' tumor) and in inborn developmental syndromes predisposing to such tumors (e.g., Beckwith-Wiedemann syndrome). H19 (RNA transcripts) and IGF2 are manifested in Wilms' tumor, rhabdomyosarcoma, immature ovarian teratoma and gestational trophoblastic diseases. We have found that in the placenta and in urothelial carcinoma, H19 expression reflects the degree of invasiveness. These genes, displaying a tissue-specific oncofetal pattern of expression, are, therefore, tumor markers.

  5. Markers and mapping revisited: finding your gene.

    PubMed

    Jones, Neil; Ougham, Helen; Thomas, Howard; Pasakinskiene, Izolda

    2009-01-01

    This paper is an update of our earlier review (Jones et al., 1997, Markers and mapping: we are all geneticists now. New Phytologist 137: 165-177), which dealt with the genetics of mapping, in terms of recombination as the basis of the procedure, and covered some of the first generation of markers, including restriction fragment length polymorphisms (RFLPs), random amplified polymorphic DNA (RAPDs), simple sequence repeats (SSRs) and quantitative trait loci (QTLs). In the intervening decade there have been numerous developments in marker science with many new systems becoming available, which are herein described: cleavage amplification polymorphism (CAP), sequence-specific amplification polymorphism (S-SAP), inter-simple sequence repeat (ISSR), sequence tagged site (STS), sequence characterized amplification region (SCAR), selective amplification of microsatellite polymorphic loci (SAMPL), single nucleotide polymorphism (SNP), expressed sequence tag (EST), sequence-related amplified polymorphism (SRAP), target region amplification polymorphism (TRAP), microarrays, diversity arrays technology (DArT), single-strand conformation polymorphism (SSCP), denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE) and methylation-sensitive PCR. In addition there has been an explosion of knowledge and databases in the area of genomics and bioinformatics. The number of flowering plant ESTs is c. 19 million and counting, with all the opportunity that this provides for gene-hunting, while the survey of bioinformatics and computer resources points to a rapid growth point for future activities in unravelling and applying the burst of new information on plant genomes. A case study is presented on tracking down a specific gene (stay-green (SGR), a post-transcriptional senescence regulator) using the full suite of mapping tools and comparative mapping resources. We end with a brief speculation on how genome analysis may progress into the future of

  6. NADPH Oxidase-derived Reactive Oxygen Species Increases Expression of Monocyte Chemotactic Factor Genes in Cultured Adipocytes*

    PubMed Central

    Han, Chang Yeop; Umemoto, Tomio; Omer, Mohamed; Den Hartigh, Laura J.; Chiba, Tsuyoshi; LeBoeuf, Renee; Buller, Carolyn L.; Sweet, Ian R.; Pennathur, Subramaniam; Abel, E. Dale; Chait, Alan

    2012-01-01

    Excess glucose and free fatty acids delivered to adipose tissue causes local inflammation, which contributes to insulin resistance. Glucose and palmitate generate reactive oxygen species (ROS) in adipocytes, leading to monocyte chemotactic factor gene expression. Docosahexaenoate (DHA) has the opposite effect. In this study, we evaluated the potential sources of ROS in the presence of excess nutrients. Differentiated 3T3-L1 adipocytes were exposed to palmitate and DHA (250 μm) in either 5 or 25 mm glucose to evaluate the relative roles of mitochondrial electron transport and NADPH oxidases (NOX) as sources of ROS. Excess glucose and palmitate did not increase mitochondrial oxidative phosphorylation. However, glucose exposure increased glycolysis. Of the NOX family members, only NOX4 was expressed in adipocytes. Moreover, its activity was increased by excess glucose and palmitate and decreased by DHA. Silencing NOX4 inhibited palmitate- and glucose-stimulated ROS generation and monocyte chemotactic factor gene expression. NADPH, a substrate for NOX, and pentose phosphate pathway activity increased with glucose but not palmitate and decreased with DHA exposure. Inhibition of the pentose phosphate pathway by glucose-6-phosphate dehydrogenase inhibitors and siRNA suppressed ROS generation and monocyte chemotactic factor gene expression induced by both glucose and palmitate. Finally, both high glucose and palmitate induced NOX4 translocation into lipid rafts, effects that were blocked by DHA. Excess glucose and palmitate generate ROS via NOX4 rather than by mitochondrial oxidation in cultured adipocytes. NOX4 is regulated by both NADPH generated in the PPP and translocation of NOX4 into lipid rafts, leading to expression of monocyte chemotactic factors. PMID:22287546

  7. MicroRNAs are required for the feature maintenance and differentiation of brown adipocytes.

    PubMed

    Kim, Hye-Jin; Cho, Hyunjii; Alexander, Ryan; Patterson, Heide Christine; Gu, Minxia; Lo, Kinyui Alice; Xu, Dan; Goh, Vera J; Nguyen, Long N; Chai, Xiaoran; Huang, Cher X; Kovalik, Jean-Paul; Ghosh, Sujoy; Trajkovski, Mirko; Silver, David L; Lodish, Harvey; Sun, Lei

    2014-12-01

    Brown adipose tissue (BAT) is specialized to burn lipids for heat generation as a natural defense against cold and obesity. Previous studies established microRNAs (miRNAs) as essential regulators of brown adipocyte differentiation, but whether miRNAs are required for the feature maintenance of mature brown adipocytes remains unknown. To address this question, we ablated Dgcr8, a key regulator of the miRNA biogenesis pathway, in mature brown as well as in white adipocytes. Adipose tissue-specific Dgcr8 knockout mice displayed enlarged but pale interscapular brown fat with decreased expression of genes characteristic of brown fat and were intolerant to cold exposure. Primary brown adipocyte cultures in vitro confirmed that miRNAs are required for marker gene expression in mature brown adipocytes. We also demonstrated that miRNAs are essential for the browning of subcutaneous white adipocytes in vitro and in vivo. Using this animal model, we performed miRNA expression profiling analysis and identified a set of BAT-specific miRNAs that are upregulated during brown adipocyte differentiation and enriched in brown fat compared with other organs. We identified miR-182 and miR-203 as new regulators of brown adipocyte development. Taken together, our study demonstrates an essential role of miRNAs in the maintenance as well as in the differentiation of brown adipocytes.

  8. MicroRNAs Are Required for the Feature Maintenance and Differentiation of Brown Adipocytes

    PubMed Central

    Kim, Hye-Jin; Cho, Hyunjii; Alexander, Ryan; Patterson, Heide Christine; Gu, Minxia; Lo, Kinyui Alice; Xu, Dan; Goh, Vera J.; Nguyen, Long N.; Chai, Xiaoran; Huang, Cher X.; Kovalik, Jean-Paul; Ghosh, Sujoy; Trajkovski, Mirko; Silver, David L.; Lodish, Harvey

    2014-01-01

    Brown adipose tissue (BAT) is specialized to burn lipids for heat generation as a natural defense against cold and obesity. Previous studies established microRNAs (miRNAs) as essential regulators of brown adipocyte differentiation, but whether miRNAs are required for the feature maintenance of mature brown adipocytes remains unknown. To address this question, we ablated Dgcr8, a key regulator of the miRNA biogenesis pathway, in mature brown as well as in white adipocytes. Adipose tissue–specific Dgcr8 knockout mice displayed enlarged but pale interscapular brown fat with decreased expression of genes characteristic of brown fat and were intolerant to cold exposure. Primary brown adipocyte cultures in vitro confirmed that miRNAs are required for marker gene expression in mature brown adipocytes. We also demonstrated that miRNAs are essential for the browning of subcutaneous white adipocytes in vitro and in vivo. Using this animal model, we performed miRNA expression profiling analysis and identified a set of BAT-specific miRNAs that are upregulated during brown adipocyte differentiation and enriched in brown fat compared with other organs. We identified miR-182 and miR-203 as new regulators of brown adipocyte development. Taken together, our study demonstrates an essential role of miRNAs in the maintenance as well as in the differentiation of brown adipocytes. PMID:25008181

  9. Downregulation of Runx2 by 1,25-Dihydroxyvitamin D3 Induces the Transdifferentiation of Osteoblasts to Adipocytes

    PubMed Central

    Kim, Jung Ha; Seong, Semun; Kim, Kabsun; Kim, Inyoung; Jeong, Byung-Chul; Kim, Nacksung

    2016-01-01

    1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) indirectly stimulates bone formation, but little is known about its direct effect on bone formation. In this study, we observed that 1,25(OH)2D3 enhances adipocyte differentiation, but inhibits osteoblast differentiation during osteogenesis. The positive role of 1,25(OH)2D3 in adipocyte differentiation was confirmed when murine osteoblasts were cultured in adipogenic medium. Additionally, 1,25(OH)2D3 enhanced the expression of adipocyte marker genes, but inhibited the expression of osteoblast marker genes in osteoblasts. The inhibition of osteoblast differentiation and promotion of adipocyte differentiation mediated by 1,25(OH)2D3 were compensated by Runx2 overexpression. Our results suggest that 1,25(OH)2D3 induces the transdifferentiation of osteoblasts to adipocytes via Runx2 downregulation in osteoblasts. PMID:27213351

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

    PubMed

    Parray, Hilal Ahmad; Yun, Jong Won

    2016-05-01

    Recruitment of the brown-like phenotype in white adipocytes (browning) and activation of existing brown adipocytes are currently being investigated as a means to combat obesity. Thus, a wide variety of dietary agents that contribute to browning of white adipocytes have been identified. The present study was designed to investigate the effects of cannabidiol (CBD), a major nonpsychotropic phytocannabinoid of Cannabis sativa, on induction of browning in 3T3-L1 adipocytes. CBD enhanced expression of a core set of brown fat-specific marker genes (Ucp1, Cited1, Tmem26, Prdm16, Cidea, Tbx1, Fgf21, and Pgc-1α) and proteins (UCP1, PRDM16, and PGC-1α). Increased expression of UCP1 and other brown fat-specific markers contributed to the browning of 3T3-L1 adipocytes possibly via activation of PPARγ and PI3K. In addition, CBD increased protein expression levels of CPT1, ACSL, SIRT1, and PLIN while down-regulating JNK2, SREBP1, and LPL. These data suggest possible roles for CBD in browning of white adipocytes, augmentation of lipolysis, thermogenesis, and reduction of lipogenesis. In conclusion, the current data suggest that CBD plays dual modulatory roles in the form of inducing the brown-like phenotype as well as promoting lipid metabolism. Thus, CBD may be explored as a potentially promising therapeutic agent for the prevention of obesity. PMID:27067870

  11. Effects of MicroRNA-23a on Differentiation and Gene Expression Profiles in 3T3-L1 Adipocytes

    PubMed Central

    Huang, Yong; Huang, Jinxiu; Qi, Renli; Wang, Qi; Wu, Yongjiang; Wang, Jing

    2016-01-01

    MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate growth, development, and programmed death of cells. A newly-published study has shown that miRNA-23a could regulate 3T3-L1 adipocyte differentiation. Here, we identified miRNA-23a as a negative regulator of 3T3-L1 adipocyte differentiation again. Over-expression of miRNA-23a inhibited differentiation and decreased lipogenesis as well as down-regulated mRNA and protein expression of both peroxisome proliferator-activated receptor (PPAR) γ and fatty acid binding protein (FABP) 4, whereas knock down of miRNA-23a showed the opposite effects on differentiation as well as increasing the number of apoptotic cells. Additionally, digital gene expression profiling sequencing (DGE-Seq) was used to assay changes in gene expression profiles following alterations in the level of miR-23a. In total, over-expression or knock down of miRNA-23a significantly changed the expression of 313 and 425 genes, respectively. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses indicated that these genes were mainly involved in the stress response, immune system, metabolism, cell cycle, among other pathways. Additionally, the signal transducer and activator of transcription 1 (Stat1) was shown to be a target of miRNA-23a by computational and dual-luciferase reporter assays that indicated Janus Kinase (Jak)-Stat signal pathway was implicated in regulating adipogenesis mediated by miRNA-23a in adipocytes. PMID:27783036

  12. Effects of vitamin a status on expression of ucp1 and brown/beige adipocyte-related genes in white adipose tissues of beef cattle.

    PubMed

    Kanamori, Yohei; Yamada, Tomoya; Asano, Hiroki; Kida, Ryosuke; Qiao, Yuhang; Abd Eldaim, Mabrouk A; Tomonaga, Shozo; Matsui, Tohru; Funaba, Masayuki

    2014-09-01

    We previously reported the presence of brown/beige adipocytes in the white fat depots of mature cattle. The present study examined the effects of dietary vitamin A on the expression of brown/beige adipocyte-related genes in the white fat depots of fattening cattle. No significant differences were observed in the expression of Ucp1 between vitamin A-deficient cattle and control cattle. However, the expression of the other brown/beige adipocyte-related genes was slightly higher in the mesenteric fat depots of vitamin A-deficient cattle. The present results suggest that a vitamin A deficiency does not markedly affect the expression of Ucp1 in white fat depots, but imply that it may stimulate the emergence of beige adipocytes in the mesenteric fat depots of fattening cattle.

  13. Activation of peroxisome proliferator-activated receptor-{alpha} enhances fatty acid oxidation in human adipocytes

    SciTech Connect

    Lee, Joo-Young; Hashizaki, Hikari; Goto, Tsuyoshi; Sakamoto, Tomoya; Takahashi, Nobuyuki; Kawada, Teruo

    2011-04-22

    Highlights: {yields} PPAR{alpha} activation increased mRNA expression levels of adipocyte differentiation marker genes and GPDH activity in human adipocytes. {yields} PPAR{alpha} activation also increased insulin-dependent glucose uptake in human adipocytes. {yields} PPAR{alpha} activation did not affect lipid accumulation in human adipocytes. {yields} PPAR{alpha} activation increased fatty acid oxidation through induction of fatty acid oxidation-related genes in human adipocytes. -- Abstract: Peroxisome proliferator-activated receptor-{alpha} (PPAR{alpha}) is a key regulator for maintaining whole-body energy balance. However, the physiological functions of PPAR{alpha} in adipocytes have been unclarified. We examined the functions of PPAR{alpha} using human multipotent adipose tissue-derived stem cells as a human adipocyte model. Activation of PPAR{alpha} by GW7647, a potent PPAR{alpha} agonist, increased the mRNA expression levels of adipocyte differentiation marker genes such as PPAR{gamma}, adipocyte-specific fatty acid-binding protein, and lipoprotein lipase and increased both GPDH activity and insulin-dependent glucose uptake level. The findings indicate that PPAR{alpha} activation stimulates adipocyte differentiation. However, lipid accumulation was not changed, which is usually observed when PPAR{gamma} is activated. On the other hand, PPAR{alpha} activation by GW7647 treatment induced the mRNA expression of fatty acid oxidation-related genes such as CPT-1B and AOX in a PPAR{alpha}-dependent manner. Moreover, PPAR{alpha} activation increased the production of CO{sub 2} and acid soluble metabolites, which are products of fatty acid oxidation, and increased oxygen consumption rate in human adipocytes. The data indicate that activation of PPAR{alpha} stimulates both adipocyte differentiation and fatty acid oxidation in human adipocytes, suggesting that PPAR{alpha} agonists could improve insulin resistance without lipid accumulation in adipocytes. The expected

  14. Sp1 mediates repression of the resistin gene by PPAR{gamma} agonists in 3T3-L1 adipocytes

    SciTech Connect

    Chung, S.S.; Choi, H.H.; Cho, Y.M.; Lee, H.K.; Park, K.S. . E-mail: kspark@snu.ac.kr

    2006-09-15

    Resistin is an adipokine related to obesity and insulin resistance. Expression of the resistin gene is repressed by the treatment of peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) agonists, thiazolidinediones (TZDs). In this study, we investigated the mechanism by which TZDs inhibit the resistin gene expression. Resistin gene expression was decreased by TZD in fully differentiated 3T3-L1 adipocytes, which was abolished after treatment of cycloheximide (a protein synthesis inhibitor). TZD could not repress the expression of the resistin gene in the presence of mithramycin A (an Sp1 binding inhibitor). Sp1 binding site of the resistin promoter (-122/-114 bp) was necessary for the repression. Further investigation of the effect of TZDs on the modification of Sp1 showed that the level of O-glycosylation of Sp1 was decreased in this process. These results suggest that PPAR{gamma} activation represses the expression of the resistin gene by modulating Sp1 activity.

  15. Transcriptional regulatory program in wild-type and retinoblastoma gene-deficient mouse embryonic fibroblasts during adipocyte differentiation

    PubMed Central

    2011-01-01

    Background Although many molecular regulators of adipogenesis have been identified a comprehensive catalogue of components is still missing. Recent studies showed that the retinoblastoma protein (pRb) was expressed in the cell cycle and late cellular differentiation phase during adipogenesis. To investigate this dual role of pRb in the early and late stages of adipogenesis we used microarrays to perform a comprehensive systems-level analysis of the common transcriptional program of the classic 3T3-L1 preadipocyte cell line, wild-type mouse embryonic fibroblasts (MEFs), and retinoblastoma gene-deficient MEFs (Rb-/- MEFs). Findings Comparative analysis of the expression profiles of 3T3-L1 cells and wild-type MEFs revealed genes involved specifically in early regulation of the adipocyte differentiation as well as secreted factors and signaling molecules regulating the later phase of differentiation. In an attempt to identify transcription factors regulating adipogenesis, bioinformatics analysis of the promoters of coordinately and highly expressed genes was performed. We were able to identify a number of high-confidence target genes for follow-up experimental studies. Additionally, combination of experimental data and computational analyses pinpointed a feedback-loop between Pparg and Foxo1. To analyze the effects of the retinoblastoma protein at the transcriptional level we chose a perturbated system (Rb-/- MEFs) for comparison to the transcriptional program of wild-type MEFs. Gene ontology analysis of 64 deregulated genes showed that the Rb-/- MEF model exhibits a brown(-like) adipocyte phenotype. Additionally, the analysis results indicate a different or additional role for pRb family member involvement in the lineage commitment. Conclusion In this study a number of commonly modulated genes during adipogenesis in 3T3-L1 cells and MEFs, potential transcriptional regulation mechanisms, and differentially regulated targets during adipocyte differentiation of Rb

  16. Small Molecule-Induced Complement Factor D (Adipsin) Promotes Lipid Accumulation and Adipocyte Differentiation.

    PubMed

    Song, No-Joon; Kim, Suji; Jang, Byung-Hyun; Chang, Seo-Hyuk; Yun, Ui Jeong; Park, Ki-Moon; Waki, Hironori; Li, Dean Y; Tontonoz, Peter; Park, Kye Won

    2016-01-01

    Adipocytes are differentiated by various transcriptional cascades integrated on the master regulator, Pparγ. To discover new genes involved in adipocyte differentiation, preadipocytes were treated with three newly identified pro-adipogenic small molecules and GW7845 (a Pparγ agonist) for 24 hours and transcriptional profiling was analyzed. Four genes, Peroxisome proliferator-activated receptor γ (Pparγ), human complement factor D homolog (Cfd), Chemokine (C-C motif) ligand 9 (Ccl9), and GIPC PDZ Domain Containing Family Member 2 (Gipc2) were induced by at least two different small molecules but not by GW7845. Cfd and Ccl9 expressions were specific to adipocytes and they were altered in obese mice. Small hairpin RNA (shRNA) mediated knockdown of Cfd in preadipocytes inhibited lipid accumulation and expression of adipocyte markers during adipocyte differentiation. Overexpression of Cfd promoted adipocyte differentiation, increased C3a production, and led to induction of C3a receptor (C3aR) target gene expression. Similarly, treatments with C3a or C3aR agonist (C4494) also promoted adipogenesis. C3aR knockdown suppressed adipogenesis and impaired the pro-adipogenic effects of Cfd, further suggesting the necessity for C3aR signaling in Cfd-mediated pro-adipogenic axis. Together, these data show the action of Cfd in adipogenesis and underscore the application of small molecules to identify genes in adipocytes. PMID:27611793

  17. Small Molecule-Induced Complement Factor D (Adipsin) Promotes Lipid Accumulation and Adipocyte Differentiation

    PubMed Central

    Jang, Byung-Hyun; Chang, Seo-Hyuk; Yun, Ui Jeong; Park, Ki-Moon; Waki, Hironori; Li, Dean Y.; Tontonoz, Peter; Park, Kye Won

    2016-01-01

    Adipocytes are differentiated by various transcriptional cascades integrated on the master regulator, Pparγ. To discover new genes involved in adipocyte differentiation, preadipocytes were treated with three newly identified pro-adipogenic small molecules and GW7845 (a Pparγ agonist) for 24 hours and transcriptional profiling was analyzed. Four genes, Peroxisome proliferator-activated receptor γ (Pparγ), human complement factor D homolog (Cfd), Chemokine (C-C motif) ligand 9 (Ccl9), and GIPC PDZ Domain Containing Family Member 2 (Gipc2) were induced by at least two different small molecules but not by GW7845. Cfd and Ccl9 expressions were specific to adipocytes and they were altered in obese mice. Small hairpin RNA (shRNA) mediated knockdown of Cfd in preadipocytes inhibited lipid accumulation and expression of adipocyte markers during adipocyte differentiation. Overexpression of Cfd promoted adipocyte differentiation, increased C3a production, and led to induction of C3a receptor (C3aR) target gene expression. Similarly, treatments with C3a or C3aR agonist (C4494) also promoted adipogenesis. C3aR knockdown suppressed adipogenesis and impaired the pro-adipogenic effects of Cfd, further suggesting the necessity for C3aR signaling in Cfd-mediated pro-adipogenic axis. Together, these data show the action of Cfd in adipogenesis and underscore the application of small molecules to identify genes in adipocytes. PMID:27611793

  18. Osteocalcin (BGP), gene expression, and protein production by marrow stromal adipocytes.

    PubMed

    Benayahu, D; Shamay, A; Wientroub, S

    1997-02-13

    This study was designed to demonstrate the expression and production of osteocalcin, a bone Gla-protein (BGP), by marrow stromal cells. We were able to accomplish this by using a series of marrow stromal cell lines (MBA cells). A unique expression of the osteocalcin was detected by the adipocyte 14F1.1 cells. This was at the mRNA level by Northern blot and by RT-PCR analysis. The secreted protein was quantitated by radioimmunoassay (RIA), in conditioned medium (CM) harvested from these cultured cells. These findings offer the first evidence that marrow adipocyte 14F1.1 derived cells express mRNA for osteocalcin and produce the protein. PMID:9070297

  19. Excision of plastid marker genes using directly repeated DNA sequences.

    PubMed

    Mudd, Elisabeth A; Madesis, Panagiotis; Avila, Elena Martin; Day, Anil

    2014-01-01

    Excision of marker genes using DNA direct repeats makes use of the predominant homologous recombination pathways present in the plastids of algae and plants. The method is simple, efficient, and widely applicable to plants and microalgae. Marker excision frequency is dependent on the length and number of directly repeated sequences. When two repeats are used a repeat size of greater than 600 bp promotes efficient excision of the marker gene. A wide variety of sequences can be used to make the direct repeats. Only a single round of transformation is required, and there is no requirement to introduce site-specific recombinases by retransformation or sexual crosses. Selection is used to maintain the marker and ensure homoplasmy of transgenic plastid genomes. Release of selection allows the accumulation of marker-free plastid genomes generated by marker excision, which is spontaneous, random, and a unidirectional process. Positive selection is provided by linking marker excision to restoration of the coding region of an herbicide resistance gene from two overlapping but incomplete coding regions. Cytoplasmic sorting allows the segregation of cells with marker-free transgenic plastids. The marker-free shoots resulting from direct repeat-mediated excision of marker genes have been isolated by vegetative propagation of shoots in the T0 generation. Alternatively, accumulation of marker-free plastid genomes during growth, development and flowering of T0 plants allows the collection of seeds that give rise to a high proportion of marker-free T1 seedlings. The simplicity and convenience of direct repeat excision facilitates its widespread use to isolate marker-free crops. PMID:24599849

  20. Evaluation of the synuclein-γ (SNCG) gene as a PPARγ target in murine adipocytes, dorsal root ganglia somatosensory neurons, and human adipose tissue.

    PubMed

    Dunn, Tamara N; Akiyama, Tasuku; Lee, Hyun Woo; Kim, Jae Bum; Knotts, Trina A; Smith, Steven R; Sears, Dorothy D; Carstens, Earl; Adams, Sean H

    2015-01-01

    Recent evidence in adipocytes points to a role for synuclein-γ in metabolism and lipid droplet dynamics, but interestingly this factor is also robustly expressed in peripheral neurons. Specific regulation of the synuclein-γ gene (Sncg) by PPARγ requires further evaluation, especially in peripheral neurons, prompting us to test if Sncg is a bona fide PPARγ target in murine adipocytes and peripheral somatosensory neurons derived from the dorsal root ganglia (DRG). Sncg mRNA was decreased in 3T3-L1 adipocytes (~68%) by rosiglitazone, and this effect was diminished by the PPARγ antagonist T0070907. Chromatin immunoprecipitation experiments confirmed PPARγ protein binding at two promoter sequences of Sncg during 3T3-L1 adipogenesis. Rosiglitazone did not affect Sncg mRNA expression in murine cultured DRG neurons. In subcutaneous human WAT samples from two cohorts treated with pioglitazone (>11 wks), SNCG mRNA expression was reduced, albeit highly variable and most evident in type 2 diabetes. Leptin (Lep) expression, thought to be coordinately-regulated with Sncg based on correlations in human adipose tissue, was also reduced in 3T3-L1 adipocytes by rosiglitazone. However, Lep was unaffected by PPARγ antagonist, and the LXR agonist T0901317 significantly reduced Lep expression (~64%) while not impacting Sncg. The results support the concept that synuclein-γ shares some, but not all, gene regulators with leptin and is a PPARγ target in adipocytes but not DRG neurons. Regulation of synuclein-γ by cues such as PPARγ agonism in adipocytes is logical based on recent evidence for an important role for synuclein-γ in the maintenance and dynamics of adipocyte lipid droplets. PMID:25756178

  1. Factor for adipocyte differentiation 158 gene disruption prevents the body weight gain and insulin resistance induced by a high-fat diet.

    PubMed

    Hayashi, Takahiro; Nozaki, Yuriko; Nishizuka, Makoto; Ikawa, Masahito; Osada, Shigehiro; Imagawa, Masayoshi

    2011-01-01

    To clarify the molecular mechanism of adipocyte differentiation, we previously isolated a novel gene, factor for adipocyte differentiation (fad) 158, whose expression was induced during the earliest stages of adipogenesis, and its product was localized to the endoplasmic reticulum. We found that the knockdown of fad158 expression prevented the differentiation of 3T3-L1 cells into adipocytes. In addition, over-expression of fad158 promoted the differentiation of NIH-3T3 cells, which do not usually differentiate into adipocytes. Although these findings strongly suggest that fad158 has a crucial role in regulating adipocyte differentiation, the physiological role of the gene is still unclear. In this study, we generated mice in which fad158 expression was deleted. The fad158-deficient mice did not show remarkable changes in body weight or the weight of white adipose tissue on a chow diet, but had significantly lower body weights and fat mass than wild-type mice when fed a high-fat diet. Furthermore, although the disruption of fad158 did not influence insulin sensitivity on the chow diet, it improved insulin resistance induced by the high-fat diet. These results indicate that fad158 is a key factor in the development of obesity and insulin resistance caused by a high-fat diet.

  2. [Adipocytic tumors].

    PubMed

    Stock, Nathalie

    2015-01-01

    Adipocytic tumors are the most common mesenchymal neoplasms, liposarcoma accounting for approximately 20% of soft tissue sarcomas. The differential diagnosis between benign and malignant tumors is often problematic and represents a significant proportion of consultation cases. The goal of this article is to review liposarcoma subtypes, the main benign adipocytic neoplasms: lipoblastoma, hibernoma, spindle/pleomorphic cell lipoma, chondroid lipoma, as well as non adipocytic neoplasms with a lipomatous component such as lipomatous solitary fibrous tumor, emphasizing on practical differential diagnosis issues, and immunohistochemical and molecular tools allowing their resolution.

  3. Altered adipocyte structure and function in nutritionally programmed microswine offspring

    PubMed Central

    DuPriest, E. A.; Kupfer, P.; Lin, B.; Sekiguchi, K.; Morgan, T. K.; Saunders, K. E.; Chatkupt, T. T.; Denisenko, O. N.; Purnell, J. Q.; Bagby, S. P.

    2015-01-01

    Adipose tissue (AT) dysfunction links obesity of any cause with cardiometabolic disease, but whether early-life nutritional deficiency can program adipocyte dysfunction independently of obesity is untested. In 3–5-month-old juvenile microswine offspring exposed to isocaloric perinatal maternal protein restriction (MPR) and exhibiting accelerated prepubertal fat accrual without obesity, we assessed markers of acquired obesity: adiponectin and tumor necrosis factor (TNF)-α messenger ribonucleic acid (mRNA) levels and adipocyte size in intra-abdominal (ABD-AT) and subcutaneous (SC-AT) adipose tissues. Plasma cortisol, leptin and insulin levels were measured in fetal, neonatal and juvenile offspring. In juvenile low-protein offspring (LPO), adipocyte size in ABD-AT was reduced 22% (P=0.011 v. controls), whereas adipocyte size in SC-AT was increased in female LPO (P=0.05) and normal in male LPO; yet, adiponectin mRNA in LPO was low in both sexes and in both depots (P<0.001). Plasma leptin (P=0.004) and cortisol (P<0.05) were reduced only in neonatal LPO during MPR. In juveniles, correlations between % body fat and adiponectin mRNA, TNF-α mRNA or plasma leptin were significant in normal-protein offspring (NPO) but absent in LPO. Plasma glucose in juvenile LPO was increased in males but decreased in females (interaction, P=0.023); plasma insulin levels and insulin sensitivity were unaffected. Findings support nutritional programming of adipocyte size and gene expression and subtly altered glucose homeostasis. Reduced adiponectin mRNA and adipokine dysregulation in juvenile LPO following accelerated growth occurred independently of obesity, adipocyte hypertrophy or inflammatory markers; thus, perinatal MPR and/or growth acceleration can alter adipocyte structure and disturb adipokine homeostasis in metabolically adverse patterns predictive of enhanced disease risk. PMID:25102010

  4. HSD1 and AQP7 short-term gene regulation by cortisone in 3T3-L1 adipocytes.

    PubMed

    Quesada-López, Tania; González-Dávalos, Laura; Piña, Enrique; Mora, Ofelia

    2016-01-01

    Adipose Tissue (AT) is a complex organ with a crucial regulatory role in energy metabolism and in the development of obesity and the Metabolic Syndrome (MS). Modified responses and the metabolism of hormones have been observed in visceral adiposity during obesity, specifically as related with cortisone. The objective of this study was to assess, in the 3T3-L1 adipocyte cell line, the short-term effect of cortisone on the expression of 11β-Hydroxysteroid dehydrogenase 1 (Hsd1), which is responsible for activation of cortisone into cortisol, and for Aquaporin 7 (Aqp7), involved in glycerol transport through the cell membrane. Total RNA (tRNA) and complementary DNA (cDNA) were obtained from cell samples treated with cortisone (0.1, 1, and 10 μM) during different times (0, 5, 10, 15, and 20 min, and 48 h) to quantify the expression of the aforementioned genes by real time PCR employing MnSOD and Ppia as housekeeping genes. There was a time-dependent response of Aqp7, a dose-dependent response of Hsd1, and an increase observed in the expression of both genes during min 1 of treatment (5- and 6-fold, respectively), followed by a decrease during the following 5-10 min (P < 0.05). With the 1-μM cortisone treatment, both genes showed cubic tendencies in their expression; the Hsd1 tendency is described by the equation y = 0.18×(3)-1.65×(2)+3.59x+1.31, while the Aqp7 tendency is described by y = 0.33×(3)-2.67×(2)+4.93x+1.84. There are immediate and quantitatively important actions of cortisone on the expression of Aqp7 and Hsd1 in 3T3-L1 adipocytes.

  5. HSD1 and AQP7 short-term gene regulation by cortisone in 3T3-L1 adipocytes.

    PubMed

    Quesada-López, Tania; González-Dávalos, Laura; Piña, Enrique; Mora, Ofelia

    2016-01-01

    Adipose Tissue (AT) is a complex organ with a crucial regulatory role in energy metabolism and in the development of obesity and the Metabolic Syndrome (MS). Modified responses and the metabolism of hormones have been observed in visceral adiposity during obesity, specifically as related with cortisone. The objective of this study was to assess, in the 3T3-L1 adipocyte cell line, the short-term effect of cortisone on the expression of 11β-Hydroxysteroid dehydrogenase 1 (Hsd1), which is responsible for activation of cortisone into cortisol, and for Aquaporin 7 (Aqp7), involved in glycerol transport through the cell membrane. Total RNA (tRNA) and complementary DNA (cDNA) were obtained from cell samples treated with cortisone (0.1, 1, and 10 μM) during different times (0, 5, 10, 15, and 20 min, and 48 h) to quantify the expression of the aforementioned genes by real time PCR employing MnSOD and Ppia as housekeeping genes. There was a time-dependent response of Aqp7, a dose-dependent response of Hsd1, and an increase observed in the expression of both genes during min 1 of treatment (5- and 6-fold, respectively), followed by a decrease during the following 5-10 min (P < 0.05). With the 1-μM cortisone treatment, both genes showed cubic tendencies in their expression; the Hsd1 tendency is described by the equation y = 0.18×(3)-1.65×(2)+3.59x+1.31, while the Aqp7 tendency is described by y = 0.33×(3)-2.67×(2)+4.93x+1.84. There are immediate and quantitatively important actions of cortisone on the expression of Aqp7 and Hsd1 in 3T3-L1 adipocytes. PMID:27617175

  6. Three-dimensional arrangement of genes involved in lipid metabolism in nuclei of porcine adipocytes and fibroblasts in relation to their transcription level.

    PubMed

    Kociucka, B; Cieslak, J; Szczerbal, I

    2012-01-01

    The 3-dimensional arrangement of chromosomes and genes within a nuclear space is considered to represent the level of transcriptional regulation. Understanding how the nuclear architecture of adipocyte cells contributes to gene expression has become the subject of great interest in the context of obesity research. In this study we investigated nuclear positioning of 3 gene loci involved in lipid metabolism in the pig (Sus scrofa, SSC) which is considered as an important animal model for obesity in humans. We found that the position of the SCD gene in the 3-dimensional space of the cell nucleus is not correlated with transcriptional activity. The gene locus as well as chromosome territory SSC14 occupied the same peripheral location in adipocyte and fibroblast cells, in spite of the fact that their transcription level differs significantly between both cell types. For the 2 other investigated genes, i.e. ACACA and SREBF1 and their chromosome territory (SSC12), slightly different nuclear locations were found. They occupied intermediate nuclear positions in fibroblast nuclei, while in adipocytes they were positioned in the nuclear interior. The more internal location of these genes corresponds to increased transcription levels in fat cells. Our results confirm the non-random position of genes and chromosome territories in nuclei of adult porcine cells and indicate that relationship between transcription activity and gene positioning exists only for some but not all genes.

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

    SciTech Connect

    Milton, Flora Aparecida; Cvoro, Aleksandra; Amato, Angelica A.; Sieglaff, Douglas H.; Filgueira, Carly S.; Arumanayagam, Anithachristy Sigamani; Caro Alves de Lima, Maria do; Rocha Pitta, Ivan; Assis Rocha Neves, Francisco de; Webb, Paul

    2015-08-28

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

  8. MicroRNA-192* impairs adipocyte triglyceride storage.

    PubMed

    Mysore, Raghavendra; Zhou, You; Sädevirta, Sanja; Savolainen-Peltonen, Hanna; Nidhina Haridas, P A; Soronen, Jarkko; Leivonen, Marja; Sarin, Antti-Pekka; Fischer-Posovszky, Pamela; Wabitsch, Martin; Yki-Järvinen, Hannele; Olkkonen, Vesa M

    2016-04-01

    We investigated the expression of miR-192* (miR-192-3p) in the visceral adipose tissue (VAT) of obese subjects and its function in cultured human adipocytes. This miRNA is a 3' arm derived from the same pre-miRNA as miR-192 (miR-192-5p) implicated in type 2 diabetes, liver disease and cancers, and is predicted to target key genes in lipid metabolism. In morbidly obese subjects undergoing bariatric surgery preceded by a very low calorie diet, miR-192* in VAT correlated negatively (r=-0.387; p=0.046) with serum triglyceride (TG) and positively with high-density lipoprotein (HDL) concentration (r=0.396; p=0.041). In a less obese patient cohort, the miRNA correlated negatively with the body mass index (r=-0.537; p=0.026). To characterize the function of miR-192*, we overexpressed it in cultured adipocytes and analyzed the expression of adipogenic differentiation markers as well as cellular TG content. Reduced TG and expression of the adipocyte marker proteins aP2 (adipocyte protein 2) and perilipin 1 were observed. The function of miR-192* was further investigated by transcriptomic profiling of adipocytes expressing this miRNA, revealing impacts on key lipogenic genes. A number of the mRNA alterations were validated by qPCR. Western analysis confirmed a marked reduction of the lipogenic enzyme SCD (stearoyl coenzyme A desaturase-1), the fatty aldehyde dehydrogenase ALDH3A2 (aldehyde dehydrogenase 3 family member A2) and the high-density lipoprotein receptor SCARB1 (scavenger receptor B, type I). SCD and ALDH3A2 were demonstrated to be direct targets of miR-192*. To conclude, the present data identify miR-192* as a novel controller of adipocyte differentiation and lipid homeostasis.

  9. Impact of Metabolic Regulators on the Expression of the Obesity Associated Genes FTO and NAMPT in Human Preadipocytes and Adipocytes

    PubMed Central

    Schönberg, Maria; Bernhard, Falk; Büttner, Petra; Landgraf, Kathrin; Kiess, Wieland; Körner, Antje

    2011-01-01

    Background FTO and NAMPT/PBEF/visfatin are thought to play a role in obesity but their transcriptional regulation in adipocytes is not fully understood. In this study, we evaluated the transcriptional regulation of FTO and NAMPT in preadipocytes and adipocytes by metabolic regulators. Methodology and Principal Findings We assessed FTO mRNA expression during human adipocyte differentiation of Simpson-Golabi-Behmel syndrome (SGBS) cells and primary subcutaneous preadipocytes in vitro and evaluated the effect of the metabolic regulators glucose, insulin, dexamethasone, IGF-1 and isoproterenol on FTO and NAMPT mRNA expression in SGBS preadipocytes and adipocytes. FTO mRNA levels were not significantly modulated during adipocyte differentiation. Also, metabolic regulators had no impact on FTO expression in preadipocytes or adipocytes. In SGBS preadipocytes NAMPT expression was more than 3fold induced by dexamethasone and isoproterenol and 1.6fold by dexamethasone in adipocytes. Complete glucose restriction caused an increase in NAMPT mRNA expression by more than 5fold and 1.4fold in SGBS preadipocytes and adipocytes, respectively. Conclusion FTO mRNA expression is not significantly affected by differentiation or metabolic regulators in human adipocytes. The stimulation of NAMPT expression by dexamethasone, isoproterenol and complete glucose restriction may indicate a regulation of NAMPT by metabolic stress, which was more pronounced in preadipocytes compared to mature adipocytes. PMID:21687707

  10. Reverse Differentiation as a Gene Filtering Tool in Genome Expression Profiling of Adipogenesis for Fat Marker Gene Selection and Their Analysis

    PubMed Central

    Ullah, Mujib; Stich, Stefan; Häupl, Thomas; Eucker, Jan; Sittinger, Michael; Ringe, Jochen

    2013-01-01

    Background During mesenchymal stem cell (MSC) conversion into adipocytes, the adipogenic cocktail consisting of insulin, dexamethasone, indomethacin and 3-isobutyl-1-methylxanthine not only induces adipogenic-specific but also genes for non-adipogenic processes. Therefore, not all significantly expressed genes represent adipogenic-specific marker genes. So, our aim was to filter only adipogenic-specific out of all expressed genes. We hypothesize that exclusively adipogenic-specific genes change their expression during adipogenesis, and reverse during dedifferentiation. Thus, MSC were adipogenic differentiated and dedifferentiated. Results Adipogenesis and reverse adipogenesis was verified by Oil Red O staining and expression of PPARG and FABP4. Based on GeneChips, 991 genes were differentially expressed during adipogenesis and grouped in 4 clusters. According to bioinformatic analysis the relevance of genes with adipogenic-linked biological annotations, expression sites, molecular functions, signaling pathways and transcription factor binding sites was high in cluster 1, including all prominent adipogenic genes like ADIPOQ, C/EBPA, LPL, PPARG and FABP4, moderate in clusters 2–3, and negligible in cluster 4. During reversed adipogenesis, only 782 expressed genes (clusters 1–3) were reverted, including 597 genes not reported for adipogenesis before. We identified APCDD1, CHI3L1, RARRES1 and SEMA3G as potential adipogenic-specific genes. Conclusion The model system of adipogenesis linked to reverse adipogenesis allowed the filtration of 782 adipogenic-specific genes out of total 991 significantly expressed genes. Database analysis of adipogenic-specific biological annotations, transcription factors and signaling pathways further validated and valued our concept, because most of the filtered 782 genes showed affiliation to adipogenesis. Based on this approach, the selected and filtered genes would be potentially important for characterization of adipogenesis and

  11. Loss of Oncostatin M Signaling in Adipocytes Induces Insulin Resistance and Adipose Tissue Inflammation in Vivo.

    PubMed

    Elks, Carrie M; Zhao, Peng; Grant, Ryan W; Hang, Hardy; Bailey, Jennifer L; Burk, David H; McNulty, Margaret A; Mynatt, Randall L; Stephens, Jacqueline M

    2016-08-12

    Oncostatin M (OSM) is a multifunctional gp130 cytokine. Although OSM is produced in adipose tissue, it is not produced by adipocytes. OSM expression is significantly induced in adipose tissue from obese mice and humans. The OSM-specific receptor, OSM receptor β (OSMR), is expressed in adipocytes, but its function remains largely unknown. To better understand the effects of OSM in adipose tissue, we knocked down Osmr expression in adipocytes in vitro using siRNA. In vivo, we generated a mouse line lacking Osmr in adiponectin-expressing cells (OSMR(FKO) mice). The effects of OSM on gene expression were also assessed in vitro and in vivo OSM exerts proinflammatory effects on cultured adipocytes that are partially rescued by Osmr knockdown. Osm expression is significantly increased in adipose tissue T cells of high fat-fed mice. In addition, adipocyte Osmr expression is increased following high fat feeding. OSMR(FKO) mice exhibit increased insulin resistance and adipose tissue inflammation and have increased lean mass, femoral length, and bone volume. Also, OSMR(FKO) mice exhibit increased expression of Osm, the T cell markers Cd4 and Cd8, and the macrophage markers F4/80 and Cd11c Interestingly, the same proinflammatory genes induced by OSM in adipocytes are induced in the adipose tissue of the OSMR(FKO) mouse, suggesting that increased expression of proinflammatory genes in adipose tissue arises both from adipocytes and other cell types. These findings suggest that adipocyte OSMR signaling is involved in the regulation of adipose tissue homeostasis and that, in obesity, OSMR ablation may exacerbate insulin resistance by promoting adipose tissue inflammation.

  12. Loss of Oncostatin M Signaling in Adipocytes Induces Insulin Resistance and Adipose Tissue Inflammation in Vivo.

    PubMed

    Elks, Carrie M; Zhao, Peng; Grant, Ryan W; Hang, Hardy; Bailey, Jennifer L; Burk, David H; McNulty, Margaret A; Mynatt, Randall L; Stephens, Jacqueline M

    2016-08-12

    Oncostatin M (OSM) is a multifunctional gp130 cytokine. Although OSM is produced in adipose tissue, it is not produced by adipocytes. OSM expression is significantly induced in adipose tissue from obese mice and humans. The OSM-specific receptor, OSM receptor β (OSMR), is expressed in adipocytes, but its function remains largely unknown. To better understand the effects of OSM in adipose tissue, we knocked down Osmr expression in adipocytes in vitro using siRNA. In vivo, we generated a mouse line lacking Osmr in adiponectin-expressing cells (OSMR(FKO) mice). The effects of OSM on gene expression were also assessed in vitro and in vivo OSM exerts proinflammatory effects on cultured adipocytes that are partially rescued by Osmr knockdown. Osm expression is significantly increased in adipose tissue T cells of high fat-fed mice. In addition, adipocyte Osmr expression is increased following high fat feeding. OSMR(FKO) mice exhibit increased insulin resistance and adipose tissue inflammation and have increased lean mass, femoral length, and bone volume. Also, OSMR(FKO) mice exhibit increased expression of Osm, the T cell markers Cd4 and Cd8, and the macrophage markers F4/80 and Cd11c Interestingly, the same proinflammatory genes induced by OSM in adipocytes are induced in the adipose tissue of the OSMR(FKO) mouse, suggesting that increased expression of proinflammatory genes in adipose tissue arises both from adipocytes and other cell types. These findings suggest that adipocyte OSMR signaling is involved in the regulation of adipose tissue homeostasis and that, in obesity, OSMR ablation may exacerbate insulin resistance by promoting adipose tissue inflammation. PMID:27325693

  13. Tuberous sclerosis complex 1-mechanistic target of rapamycin complex 1 signaling determines brown-to-white adipocyte phenotypic switch.

    PubMed

    Xiang, Xinxin; Lan, He; Tang, Hong; Yuan, Fang; Xu, Yanhui; Zhao, Jing; Li, Yin; Zhang, Weizhen

    2015-02-01

    Interconversion of white and brown adipocytes occurs between anabolic and catabolic states. The molecular mechanism regulating this phenotypic switch remains largely unknown. This study explores the role of tuberous sclerosis complex 1 (TSC1)-mechanistic target of rapamycin (mTOR) signaling in the conversion of brown to white adipose tissue (WAT). A colony of Fabp4-Tsc1(-/-) mice, in which the Tsc1 gene was specifically deleted by the fatty acid binding protein 4 (FABP4)-Cre, was established. Western blotting and immunostaining demonstrated the absence of TSC1 and activation of ribosomal protein S6 kinase 1, the downstream target of mTOR complex 1 (mTORC1) signaling, in the brown adipose tissues (BATs) of Fabp4-Tsc1(-/-) mice. Accumulation of lipid droplets in BAT was significantly increased. Levels of brown adipocyte markers were markedly downregulated, while white adipocyte markers were upregulated. Rapamycin reversed the conversion from BAT to WAT in Fabp4-Tsc1(-/-) mice. Deletion of the Tsc1 gene in cultured brown preadipocytes significantly increased the conversion to white adipocytes. FoxC2 mRNA, the transcriptional factor for brown adipocyte determination, was significantly decreased, while mRNAs for retinoblastoma protein, p107 and RIP140, the transcriptional factors for white adipocyte determination, increased in the BAT of Fabp4-Tsc1(-/-) mice. Our study demonstrates that TSC1-mTORC1 signaling contributes to the brown-to-white adipocyte phenotypic switch. PMID:25213336

  14. Progeny from dedifferentiated adipocytes display protracted adipogenesis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Progeny of adipofibroblast cells, derived from mature bovine adipocytes, were used to determine their ability to redifferentiate into lipid-assimilating adipocytes. Traditional cell biology methods were used, including the expression of adipogenic markers such as PPAR'. When exposed to medium supple...

  15. Adipocyte Accumulation of Long-Chain Fatty Acids in Obesity is Multifactorial, Resulting from Increased Fatty Acid Uptake and Decreased Activity of Genes Involved in Fat Utilization

    PubMed Central

    Walewski, José L.; Ge, Fengxia; Gagner, Michel; Inabnet, William B.; Pomp, Alfons; Branch, Andrea D.

    2010-01-01

    Background The obesity epidemic causes significant morbidity and mortality. Knowledge of cellular function and gene expression in obese adipose tissue will yield insights into obesity pathogenesis and suggest therapeutic targets. The aim of this work is to study the processes determining fat accumulation in adipose tissue from obese patients. Methods Omental fat was collected from two cohorts of obese bariatric surgery patients and sex-matched normal-weight donors. Isolated adipocytes were compared for cell size, volume, and long-chain fatty acid (LCFA) uptake. Omental fat RNAs were screened by 10K microarray (cohort 1: three obese, three normal) or Whole Genome microarray (cohort 2: seven obese, four normal). Statistical differences in gene and pathway expression were identified in cohort 1 using the GeneSifter Software (Geospiza) with key results confirmed in cohort 2 samples by microarray, quantitative real-time polymerase chain reaction, and pathway analysis. Results Obese omental adipocytes had increased surface area, volume, and Vmax for saturable LCFA uptake. Dodecenoyl-coenzyme A delta isomerase, central to LCFA metabolism, was approximately 1.6-fold underexpressed in obese fat in cohorts 1 and 2. Additionally, the Kyoto Encyclopedia of Genes and Genomics pathway analysis identified oxidative phosphorylation and fatty acid metabolism pathways as having coordinate, nonrandom down-regulation of gene expression in both cohorts. Conclusions In obese omental fat, saturable adipocyte LCFA uptake was greater than in controls, and expression of key genes involved in lipolysis, β-oxidation, and metabolism of fatty acids was reduced. Thus, both increased uptake and reduced metabolism of LCFAs contribute to the accumulation of LCFAs in obese adipocytes. PMID:19866242

  16. Exercise Decreases Lipogenic Gene Expression in Adipose Tissue and Alters Adipocyte Cellularity during Weight Regain After Weight Loss

    PubMed Central

    Giles, Erin D.; Steig, Amy J.; Jackman, Matthew R.; Higgins, Janine A.; Johnson, Ginger C.; Lindstrom, Rachel C.; MacLean, Paul S.

    2016-01-01

    Exercise is a potent strategy to facilitate long-term weight maintenance. In addition to increasing energy expenditure and reducing appetite, exercise also favors the oxidation of dietary fat, which likely helps prevent weight re-gain. It is unclear whether this exercise-induced metabolic shift is due to changes in energy balance, or whether exercise imparts additional adaptations in the periphery that limit the storage and favor the oxidation of dietary fat. To answer this question, adipose tissue lipid metabolism and related gene expression were studied in obese rats following weight loss and during the first day of relapse to obesity. Mature, obese rats were weight-reduced for 2 weeks with or without daily treadmill exercise (EX). Rats were weight maintained for 6 weeks, followed by relapse on: (a) ad libitum low fat diet (LFD), (b) ad libitum LFD plus EX, or (c) a provision of LFD to match the positive energy imbalance of exercised, relapsing animals. 24 h retention of dietary- and de novo-derived fat were assessed directly using 14C palmitate/oleate and 3H20, respectively. Exercise decreased the size, but increased the number of adipocytes in both retroperitoneal (RP) and subcutaneous (SC) adipose depots, and prevented the relapse-induced increase in adipocyte size. Further, exercise decreased the expression of genes involved in lipid uptake (CD36 and LPL), de novo lipogenesis (FAS, ACC1), and triacylglycerol synthesis (MGAT and DGAT) in RP adipose during relapse following weight loss. This was consistent with the metabolic data, whereby exercise reduced retention of de novo-derived fat even when controlling for the positive energy imbalance. The decreased trafficking of dietary fat to adipose tissue with exercise was explained by reduced energy intake which attenuated energy imbalance during refeeding. Despite having decreased expression of lipogenic genes, the net retention of de novo-derived lipid was higher in both the RP and SC adipose of exercising

  17. Exercise Decreases Lipogenic Gene Expression in Adipose Tissue and Alters Adipocyte Cellularity during Weight Regain After Weight Loss.

    PubMed

    Giles, Erin D; Steig, Amy J; Jackman, Matthew R; Higgins, Janine A; Johnson, Ginger C; Lindstrom, Rachel C; MacLean, Paul S

    2016-01-01

    Exercise is a potent strategy to facilitate long-term weight maintenance. In addition to increasing energy expenditure and reducing appetite, exercise also favors the oxidation of dietary fat, which likely helps prevent weight re-gain. It is unclear whether this exercise-induced metabolic shift is due to changes in energy balance, or whether exercise imparts additional adaptations in the periphery that limit the storage and favor the oxidation of dietary fat. To answer this question, adipose tissue lipid metabolism and related gene expression were studied in obese rats following weight loss and during the first day of relapse to obesity. Mature, obese rats were weight-reduced for 2 weeks with or without daily treadmill exercise (EX). Rats were weight maintained for 6 weeks, followed by relapse on: (a) ad libitum low fat diet (LFD), (b) ad libitum LFD plus EX, or (c) a provision of LFD to match the positive energy imbalance of exercised, relapsing animals. 24 h retention of dietary- and de novo-derived fat were assessed directly using (14)C palmitate/oleate and (3)H20, respectively. Exercise decreased the size, but increased the number of adipocytes in both retroperitoneal (RP) and subcutaneous (SC) adipose depots, and prevented the relapse-induced increase in adipocyte size. Further, exercise decreased the expression of genes involved in lipid uptake (CD36 and LPL), de novo lipogenesis (FAS, ACC1), and triacylglycerol synthesis (MGAT and DGAT) in RP adipose during relapse following weight loss. This was consistent with the metabolic data, whereby exercise reduced retention of de novo-derived fat even when controlling for the positive energy imbalance. The decreased trafficking of dietary fat to adipose tissue with exercise was explained by reduced energy intake which attenuated energy imbalance during refeeding. Despite having decreased expression of lipogenic genes, the net retention of de novo-derived lipid was higher in both the RP and SC adipose of exercising

  18. Exercise Decreases Lipogenic Gene Expression in Adipose Tissue and Alters Adipocyte Cellularity during Weight Regain After Weight Loss.

    PubMed

    Giles, Erin D; Steig, Amy J; Jackman, Matthew R; Higgins, Janine A; Johnson, Ginger C; Lindstrom, Rachel C; MacLean, Paul S

    2016-01-01

    Exercise is a potent strategy to facilitate long-term weight maintenance. In addition to increasing energy expenditure and reducing appetite, exercise also favors the oxidation of dietary fat, which likely helps prevent weight re-gain. It is unclear whether this exercise-induced metabolic shift is due to changes in energy balance, or whether exercise imparts additional adaptations in the periphery that limit the storage and favor the oxidation of dietary fat. To answer this question, adipose tissue lipid metabolism and related gene expression were studied in obese rats following weight loss and during the first day of relapse to obesity. Mature, obese rats were weight-reduced for 2 weeks with or without daily treadmill exercise (EX). Rats were weight maintained for 6 weeks, followed by relapse on: (a) ad libitum low fat diet (LFD), (b) ad libitum LFD plus EX, or (c) a provision of LFD to match the positive energy imbalance of exercised, relapsing animals. 24 h retention of dietary- and de novo-derived fat were assessed directly using (14)C palmitate/oleate and (3)H20, respectively. Exercise decreased the size, but increased the number of adipocytes in both retroperitoneal (RP) and subcutaneous (SC) adipose depots, and prevented the relapse-induced increase in adipocyte size. Further, exercise decreased the expression of genes involved in lipid uptake (CD36 and LPL), de novo lipogenesis (FAS, ACC1), and triacylglycerol synthesis (MGAT and DGAT) in RP adipose during relapse following weight loss. This was consistent with the metabolic data, whereby exercise reduced retention of de novo-derived fat even when controlling for the positive energy imbalance. The decreased trafficking of dietary fat to adipose tissue with exercise was explained by reduced energy intake which attenuated energy imbalance during refeeding. Despite having decreased expression of lipogenic genes, the net retention of de novo-derived lipid was higher in both the RP and SC adipose of exercising

  19. Intermediate filament genes as differentiation markers in the leech Helobdella

    PubMed Central

    Kuo, Dian-Han

    2011-01-01

    The intermediate filament (IF) cytoskeleton is a general feature of differentiated cells. Its molecular components, IF proteins, constitute a large family including the evolutionarily conserved nuclear lamins and the more diverse collection of cytoplasmic intermediate filament (CIF) proteins. In vertebrates, genes encoding CIFs exhibit cell/tissue type-specific expression profiles and are thus useful as differentiation markers. The expression of invertebrate CIFs, however, is not well documented. Here, we report a whole-genome survey of IF genes and their developmental expression patterns in the leech Helobdella, a lophotrochozoan model for developmental biology research. We found that, as in vertebrates, each of the leech CIF genes is expressed in a specific set of cell/tissue types. This allows us to detect earliest points of differentiation for multiple cell types in leech development and to use CIFs as molecular markers for studying cell fate specification in leech embryos. In addition, to determine the feasibility of using CIFs as universal metazoan differentiation markers, we examined phylogenetic relationships of IF genes from various species. Our results suggest that CIFs, and thus their cell/tissue-specific expression patterns, have expanded several times independently during metazoan evolution. Moreover, comparing the expression patterns of CIF orthologs between two leech species suggests that rapid evolutionary changes in the cell or tissue specificity of CIFs have occurred among leeches. Hence, CIFs are not suitable for identifying cell or tissue homology except among very closely related species, but they are nevertheless useful species-specific differentiation markers. PMID:21938507

  20. QUANTIFICATION OF TRANSGENIC PLANT MARKER GENE PERSISTENCE IN THE FIELD

    EPA Science Inventory

    Methods were developed to monitor persistence of genomic DNA in decaying plants in the field. As a model, we used recombinant neomycin phosphotransferase II (rNPT-II) marker genes present in genetically engineered plants. Polymerase chain reaction (PCR) primers were designed, com...

  1. Effects of leukemia inhibitory factor on 3T3-L1 adipocytes.

    PubMed

    Hogan, Jessica C; Stephens, Jacqueline M

    2005-06-01

    Leukemia inhibitory factor (LIF) is a member of the gp130 cytokine family and signals through the receptor complex of gp130 and the LIF receptor (LIFR) to activate the JAK/STAT signaling cascade. Since LIF activates STATs 1 and 3 in adipocytes, we examined the effects of LIF on 3T3-L1 adipocytes. Our studies clearly demonstrate that LIF treatment had minimal effects on adipocyte differentiation as judged by marker gene expression, but did inhibit triacylglyceride (TAG) accumulation during adipogenesis. Acute treatment with LIF resulted in increased expression of suppressors of cytokine signaling-3 (SOCS3) and CCAAT/enhancer-binding protein-delta (C/EBPdelta) mRNA in 3T3-L1 adipocytes. Moreover, the upregulation of C/EBPdelta correlated with binding to three sites in the C/EBPdelta promoter by LIF-activated protein complexes that contained STAT1 and not STAT3. Chronic treatment with LIF resulted in decreased protein levels of sterol regulatory element binding protein-1 (SREBP1) and fatty acid synthase (FAS), but had no effect on the expression of other adipocyte marker proteins or on TAG levels in mature 3T3-L1 adipocytes. LIF had a small effect on insulin-stimulated glucose uptake in 3T3-L1 adipocytes, but did not cause insulin resistance following chronic treatment. These findings indicate that LIF has similar and distinct effects in comparison with the effects of other gp130 cytokines on cultured fat cells. In summary, our results support a role for LIF in the regulation of proteins involved in lipid synthesis and in the modulation of signal transduction pathways in 3T3-L1 adipocytes.

  2. Stress of endoplasmic reticulum modulates differentiation and lipogenesis of human adipocytes

    SciTech Connect

    Koc, Michal; Mayerová, Veronika; Kračmerová, Jana; Mairal, Aline; Mališová, Lucia; Štich, Vladimír; Langin, Dominique; Rossmeislová, Lenka

    2015-05-08

    Background: Adipocytes are cells specialized for storage of neutral lipids. This storage capacity is dependent on lipogenesis and is diminished in obesity. The reason for the decline in lipogenic activity of adipocytes in obesity remains unknown. Recent data show that lipogenesis in liver is regulated by pathways initiated by endoplasmic reticulum stress (ERS). Thus, we aimed at investigating the effect of ERS on lipogenesis in adipose cells. Methods: Preadipocytes were isolated from subcutaneous abdominal adipose tissue from obese volunteers and in vitro differentiated into adipocytes. ERS was induced pharmacologically by thapsigargin (TG) or tunicamycin (TM). Activation of Unfolded Protein Response pathway (UPR) was monitored on the level of eIF2α phosphorylation and mRNA expression of downstream targets of UPR sensors. Adipogenic and lipogenic capacity was evaluated by Oil Red O staining, measurement of incorporation of radio-labelled glucose or acetic acid into lipids and mRNA analysis of adipogenic/lipogenic markers. Results: Exposition of adipocytes to high doses of TG (100 nM) and TM (1 μg/ml) for 1–24 h enhanced expression of several UPR markers (HSPA5, EDEM1, ATF4, XBP1s) and phosphorylation of eIF2α. This acute ERS substantially inhibited expression of lipogenic genes (DGAT2, FASN, SCD1) and glucose incorporation into lipids. Moreover, chronic exposure of preadipocytes to low dose of TG (2.5 nM) during the early phases of adipogenic conversion of preadipocytes impaired both, lipogenesis and adipogenesis. On the other hand, chronic low ERS had no apparent effect on lipogenesis in mature adipocytes. Conclusions: Acute ERS weakened a capacity of mature adipocytes to store lipids and chronic ERS diminished adipogenic potential of preadipocytes. - Highlights: • High intensity ERS inhibits lipogenic capacity of adipocytes. • ERS impairs adipogenesis when present in early stages of adipogenesis. • Lipogenesis in mature adipocytes is not

  3. Differentiation and characterization of human facial subcutaneous adipocytes.

    PubMed

    Chon, Su-Hyoun; Pappas, Apostolos

    2015-01-01

    Aging is associated with the loss of facial subcutaneous fat and with increased abdominal subcutaneous fat. Site specific differences in adipocyte phenotype and/or gene expression may play a role in these age-related changes. In this study, we isolated and characterized human facial preadipocytes and investigated distinct metabolic properties such as a differentiation pattern in relation to abdominal preadipocytes. Subcutaneous preadipocytes were isolated from human facial and abdominal skin and cultured in the presence of differentiation factors including rosiglitazone, a known peroxisome proliferator-activated receptor gamma (PPAR-γ) agonist, isobutyl-methyl xanthine (IBMX) and insulin. Differentiation was characterized microscopically and by quantitative real-time PCR. Unexpected superior adipogenic capacity of facial preadipocytes was observed; more facial preadipocytes differentiated in response to rosiglitazone than abdominal preadipocytes and facial preadipocytes retained their ability to differentiate through passage 11 compared with passage 5 for abdominal preadipocytes. Experiments confirmed a reduced lipolysis response in facial versus abdominal adipocytes after exposure to isoproterenol, which was consistent with the reduced β2-adrenergic receptor expression by 60% in the facial cells. The expression of other lipid metabolic gene markers was similar in both facial and abdominal adipocytes with the exception of β3-adrenergic receptor which was only found in abdominal adipose tissue. Gene profiling, by microarray analysis, identified that several HOX genes are robustly reduced in facial adipocytes compared to abdominal adipocytes, suggesting different characteristics between the 2 fat depots. These differences may have implications for development of treatments for facial fat loss during aging.

  4. Differentiation and characterization of human facial subcutaneous adipocytes

    PubMed Central

    Chon, Su-Hyoun; Pappas, Apostolos

    2014-01-01

    Aging is associated with the loss of facial subcutaneous fat and with increased abdominal subcutaneous fat. Site specific differences in adipocyte phenotype and/or gene expression may play a role in these age-related changes. In this study, we isolated and characterized human facial preadipocytes and investigated distinct metabolic properties such as a differentiation pattern in relation to abdominal preadipocytes. Subcutaneous preadipocytes were isolated from human facial and abdominal skin and cultured in the presence of differentiation factors including rosiglitazone, a known peroxisome proliferator-activated receptor gamma (PPAR-γ) agonist, isobutyl-methyl xanthine (IBMX) and insulin. Differentiation was characterized microscopically and by quantitative real-time PCR. Unexpected superior adipogenic capacity of facial preadipocytes was observed; more facial preadipocytes differentiated in response to rosiglitazone than abdominal preadipocytes and facial preadipocytes retained their ability to differentiate through passage 11 compared with passage 5 for abdominal preadipocytes. Experiments confirmed a reduced lipolysis response in facial versus abdominal adipocytes after exposure to isoproterenol, which was consistent with the reduced β2-adrenergic receptor expression by 60% in the facial cells. The expression of other lipid metabolic gene markers was similar in both facial and abdominal adipocytes with the exception of β3-adrenergic receptor which was only found in abdominal adipose tissue. Gene profiling, by microarray analysis, identified that several HOX genes are robustly reduced in facial adipocytes compared to abdominal adipocytes, suggesting different characteristics between the 2 fat depots. These differences may have implications for development of treatments for facial fat loss during aging. PMID:26167398

  5. Identification of novel PPAR{gamma} target genes by integrated analysis of ChIP-on-chip and microarray expression data during adipocyte differentiation

    SciTech Connect

    Nakachi, Yutaka; Yagi, Ken; Nikaido, Itoshi; Bono, Hidemasa; Tonouchi, Mio; Schoenbach, Christian; Okazaki, Yasushi

    2008-07-25

    PPAR{gamma} (peroxisome proliferator-activated receptor gamma) acts as a key molecule of adipocyte differentiation, and transactivates multiple target genes involved in lipid metabolic pathways. Identification of PPAR{gamma} target genes will facilitate to predict the extent to which the drugs can affect and also to understand the molecular basis of lipid metabolism. Here, we have identified five target genes regulated directly by PPAR{gamma} during adipocyte differentiation in 3T3-L1 cells using integrated analyses of ChIP-on-chip and expression microarray. We have confirmed the direct PPAR{gamma} regulation of five genes by luciferase reporter assay in NIH-3T3 cells. Of these five genes Hp, Tmem143 and 1100001G20Rik are novel PPAR{gamma} targets. We have also detected PPREs (PPAR response elements) sequences in the promoter region of the five genes computationally. Unexpectedly, most of the PPREs detected proved to be atypical, suggesting the existence of more atypical PPREs than previously thought in the promoter region of PPAR{gamma} regulated genes.

  6. The adipocyte specific transcription factor C/EBPalpha modulates human ob gene expression.

    PubMed Central

    Miller, S G; De Vos, P; Guerre-Millo, M; Wong, K; Hermann, T; Staels, B; Briggs, M R; Auwerx, J

    1996-01-01

    The ob gene product, leptin, apparently exclusively expressed in adipose tissue, is a signaling factor regulating body weight homeostasis and energy balance. ob gene expression is increased in obese rodents and regulated by feeding, insulin, and glucocorticoids, which supports the concept that ob gene expression is under hormonal control, which is expected for a key factor controlling body weight homeostasis and energy balance. In humans, ob mRNA expression is increased in gross obesity; however, the effects of the above factors on human ob expression are unknown. We describe the structure of the human ob gene and initial functional analysis of its promoter. The human ob gene's three exons cover approximately 15 kb of genomic DNA. The entire coding region is contained in exons 2 and 3, which are separated by a 2-kb intron. The first small 30-bp untranslated exon is located >10.5 kb upstream of the initiator ATG codon. Three kilobases of DNA upstream of the transcription start site has been cloned and characterized. Only 217 bp of 5' sequence are required for basal adipose tissue-specific expression of the ob gene as well as enhanced expression by C/EBPalpha. Mutation of the single C/EBPalpha site in this region abolished inducibility of the promoter by C/EBPalpha in cotransfection assays. The gene structure will facilitate our analysis of ob mutations in human obesity, whereas knowledge of sequence elements and factors regulating ob gene expression should be of major importance in the prevention and treatment of obesity. Images Fig. 1 Fig. 2 PMID:8643605

  7. Clustering of multiallele DNA markers near the Huntington's disease gene.

    PubMed

    MacDonald, M E; Cheng, S V; Zimmer, M; Haines, J L; Poustka, A; Allitto, B; Smith, B; Whaley, W L; Romano, D M; Jagadeesh, J

    1989-09-01

    Five highly informative multiallele restriction fragment length polymorphisms (RFLPs) of value for preclinical diagnosis of Huntington's disease (HD) have been genetically characterized. One RFLP was uncovered by expansion of the D4S43 locus while three others are at D4S111 and D4S115, loci defined by NotI-linking clones. The final marker, D4S125, represents a recently discovered VNTR locus. All four loci map closer to the HD gene and to the telomere than D4S10, the original linked marker for HD. In combination with two multiallele RFLPs previously identified for D4S43 and another linked locus, D4S95, these five new multiallele markers will dramatically improve the speed and accuracy of predictive testing in HD, and increase its applicability by maximizing the chances of an informative test for anyone with appropriate family structure.

  8. N-acetylaspartate catabolism determines cytosolic acetyl-CoA levels and histone acetylation in brown adipocytes

    PubMed Central

    Prokesch, A.; Pelzmann, H. J.; Pessentheiner, A. R.; Huber, K.; Madreiter-Sokolowski, C. T.; Drougard, A.; Schittmayer, M.; Kolb, D.; Magnes, C.; Trausinger, G.; Graier, W. F.; Birner-Gruenberger, R.; Pospisilik, J. A.; Bogner-Strauss, J. G.

    2016-01-01

    Histone acetylation depends on the abundance of nucleo-cytoplasmic acetyl-CoA. Here, we present a novel route for cytoplasmic acetyl-CoA production in brown adipocytes. N-acetylaspartate (NAA) is a highly abundant brain metabolite catabolized by aspartoacylase yielding aspartate and acetate. The latter can be further used for acetyl-CoA production. Prior to this work, the presence of NAA has not been described in adipocytes. Here, we show that accumulation of NAA decreases the brown adipocyte phenotype. We increased intracellular NAA concentrations in brown adipocytes via media supplementation or knock-down of aspartoacylase and measured reduced lipolysis, thermogenic gene expression, and oxygen consumption. Combinations of approaches to increase intracellular NAA levels showed additive effects on lipolysis and gene repression, nearly abolishing the expression of Ucp1, Cidea, Prdm16, and Ppara. Transcriptome analyses of aspartoacylase knock-down cells indicate deficiencies in acetyl-CoA and lipid metabolism. Concordantly, cytoplasmic acetyl-CoA levels and global histone H3 acetylation were decreased. Further, activating histone marks (H3K27ac and H3K9ac) in promoters/enhancers of brown marker genes showed reduced acetylation status. Taken together, we present a novel route for cytoplasmic acetyl-CoA production in brown adipocytes. Thereby, we mechanistically connect the NAA pathway to the epigenomic regulation of gene expression, modulating the phenotype of brown adipocytes. PMID:27045997

  9. Integrating objective gene-brain-behavior markers of psychiatric disorders.

    PubMed

    Gordon, Evian; Liddell, Belinda J; Brown, Kerri J; Bryant, Richard; Clark, C Richard; DAS, Pritha; Dobson-Stone, Carol; Falconer, Erin; Felmingham, Kim; Flynn, Gary; Gatt, Justine M; Harris, Anthony; Hermens, Daniel F; Hopkinson, Patrick J; Kemp, Andrew H; Kuan, Stacey A; Lazzaro, Illario; Moyle, Jonson; Paul, Robert H; Rennie, Chris J; Schofield, Peter; Whitford, Thomas; Williams, Leanne M

    2007-03-01

    There is little consensus about which objective markers should be used to assess major psychiatric disorders, and predict/evaluate treatment response for these disorders. Clinical practice relies instead on subjective signs and symptoms, such that there is a "translational gap" between research findings and clinical practice. This gap arises from: a) a lack of integrative theoretical models which provide a basis for understanding links between gene-brain-behavior mechanisms and clinical entities; b) the reliance on studying one measure at a time so that linkages between markers are their specificity are not established; and c) the lack of a definitive understanding of what constitutes normative function. Here, we draw on a standardized methodology for acquiring multiple sources of genomic, brain and behavioral data in the same subjects, to propose candidate markers of selected psychiatric disorders: depression, post-traumatic stress disorder, schizophrenia, attention-deficit/hyperactivity disorder and dementia disorders. This methodology has been used to establish a standardized international database which provides a comprehensive framework and the basis for testing hypotheses derived from an integrative theoretical model of the brain. Using this normative base, we present preliminary findings for a number of disorders in relation to the proposed markers. Establishing these objective markers will be the first step towards determining their sensitivity, specificity and treatment prediction in individual patients.

  10. Characterization of Tusc5, a Unique Adipocyte Gene Co-Expressed in Peripheral Somatosensory Neurons

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Tumor suppressor candidate 5 (Tusc5, GenBank nomenclature) is a cold-repressed gene encoding a member of the CD225 domain-containing family, identified through analysis of transcripts differentially-expressed in brown adipose tissue (BAT) with changes in ambient temperature. Tusc5 mRNA was found to ...

  11. Adipocyte induced arterial calcification is prevented with sodium thiosulfate

    SciTech Connect

    Chen, Neal X.; O’Neill, Kalisha; Akl, Nader Kassis; Moe, Sharon M.

    2014-06-20

    Highlights: • High phosphorus can induce calcification of adipocytes, even when fully differentiated. • Adipocytes can induce vascular calcification in an autocrine manner. • Sodium thiosulfate inhibits adipocyte calcification. - Abstract: Background: Calcification can occur in fat in multiple clinical conditions including in the dermis, breasts and in the abdomen in calciphylaxis. All of these are more common in patients with advanced kidney disease. Clinically, hyperphosphatemia and obesity are risk factors. Thus we tested the hypothesis that adipocytes can calcify in the presence of elevated phosphorus and/or that adipocytes exposed to phosphorus can induce vascular smooth muscle cell (VSMC) calcification. Methods: 3T3-L1 preadipocytes were induced into mature adipocytes and then treated with media containing high phosphorus. Calcification was assessed biochemically and PCR performed to determine the expression of genes for osteoblast and adipocyte differentiation. Adipocytes were also co-cultured with bovine VSMC to determine paracrine effects, and the efficacy of sodium thiosulfate was determined. Results: The results demonstrated that high phosphorus induced the calcification of differentiated adipocytes with increased expression of osteopontin, the osteoblast transcription factor Runx2 and decreased expression of adipocyte transcription factors peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT-enhancer-binding protein α (CEBPα), indicating that high phosphorus led to a phenotypic switch of adipocytes to an osteoblast like phenotype. Sodium thiosulfate, dose dependently decreased adipocyte calcification and inhibited adipocyte induced increase of VSMC calcification. Co-culture studies demonstrated that adipocytes facilitated VSMC calcification partially mediated by changes of secretion of leptin and vascular endothelial growth factor (VEGF) from adipocytes. Conclusion: High phosphorus induced calcification of mature adipocytes, and

  12. Cooperation between HMGA1 and HIF-1 Contributes to Hypoxia-Induced VEGF and Visfatin Gene Expression in 3T3-L1 Adipocytes

    PubMed Central

    Messineo, Sebastiano; Laria, Anna Elisa; Arcidiacono, Biagio; Chiefari, Eusebio; Luque Huertas, Raúl M.; Foti, Daniela P.; Brunetti, Antonio

    2016-01-01

    The architectural transcription factor high-mobility group AT-hook 1 (HMGA1) is a chromatin regulator with implications in several biological processes, including tumorigenesis, inflammation, and metabolism. Previous studies have indicated a role for this factor in promoting the early stages of adipogenesis, while inhibiting adipocyte terminal differentiation, and decreasing fat mass. It has been demonstrated that hypoxia – through the hypoxia-inducible factor 1 (HIF-1) – plays a major role in triggering changes in the adipose tissue of the obese, leading to inhibition of adipocyte differentiation, adipose cell dysfunction, inflammation, insulin resistance, and type 2 diabetes. To examine the possible cooperation between HMGA1 and HIF-1, herein, we investigated the role of HMGA1 in the regulation of Visfatin and VEGF, two genes normally expressed in adipose cells, which are both responsive to hypoxia. We demonstrated that HMGA1 enhanced Visfatin and VEGF gene expression in human embryonic kidney (HEK) 293 cells in hypoxic conditions, whereas HMGA1 knockdown in differentiated 3T3-L1 adipocytes reduced these effects. Reporter gene analysis showed that Visfatin and VEGF transcriptional activity was increased by the addition of either HMGA1 or HIF-1 and even further by the combination of both factors. As demonstrated by chromatin immunoprecipitation in intact cells, HMGA1 directly interacted with the VEGF gene, and this interaction was enhanced in hypoxic conditions. Furthermore, as indicated by co-immunoprecipitation studies, HMGA1 and HIF-1 physically interacted with each other, supporting the notion that this association may corroborate a functional link between these factors. Therefore, our findings provide evidence for molecular cross-talk between HMGA1 and HIF-1, and this may be important for elucidating protein and gene networks relevant to obesity. PMID:27445976

  13. Cooperation between HMGA1 and HIF-1 Contributes to Hypoxia-Induced VEGF and Visfatin Gene Expression in 3T3-L1 Adipocytes.

    PubMed

    Messineo, Sebastiano; Laria, Anna Elisa; Arcidiacono, Biagio; Chiefari, Eusebio; Luque Huertas, Raúl M; Foti, Daniela P; Brunetti, Antonio

    2016-01-01

    The architectural transcription factor high-mobility group AT-hook 1 (HMGA1) is a chromatin regulator with implications in several biological processes, including tumorigenesis, inflammation, and metabolism. Previous studies have indicated a role for this factor in promoting the early stages of adipogenesis, while inhibiting adipocyte terminal differentiation, and decreasing fat mass. It has been demonstrated that hypoxia - through the hypoxia-inducible factor 1 (HIF-1) - plays a major role in triggering changes in the adipose tissue of the obese, leading to inhibition of adipocyte differentiation, adipose cell dysfunction, inflammation, insulin resistance, and type 2 diabetes. To examine the possible cooperation between HMGA1 and HIF-1, herein, we investigated the role of HMGA1 in the regulation of Visfatin and VEGF, two genes normally expressed in adipose cells, which are both responsive to hypoxia. We demonstrated that HMGA1 enhanced Visfatin and VEGF gene expression in human embryonic kidney (HEK) 293 cells in hypoxic conditions, whereas HMGA1 knockdown in differentiated 3T3-L1 adipocytes reduced these effects. Reporter gene analysis showed that Visfatin and VEGF transcriptional activity was increased by the addition of either HMGA1 or HIF-1 and even further by the combination of both factors. As demonstrated by chromatin immunoprecipitation in intact cells, HMGA1 directly interacted with the VEGF gene, and this interaction was enhanced in hypoxic conditions. Furthermore, as indicated by co-immunoprecipitation studies, HMGA1 and HIF-1 physically interacted with each other, supporting the notion that this association may corroborate a functional link between these factors. Therefore, our findings provide evidence for molecular cross-talk between HMGA1 and HIF-1, and this may be important for elucidating protein and gene networks relevant to obesity.

  14. Concomitant beige adipocyte differentiation upon induction of mesenchymal stem cells into brown adipocytes.

    PubMed

    Wang, Yung-Li; Lin, Shih-Pei; Hsieh, Patrick C H; Hung, Shih-Chieh

    2016-09-16

    The accumulation of fat, which results in obesity, is related to many metabolic disorders. Besides white and brown adipose tissue, beige adipose tissue has recently been recognized as a new type of accumulated fat. Mesenchymal stem cells (MSCs) have been shown to differentiate into brown adipocytes. Through analyzing levels of mRNA and protein markers associated with beige adipocyte, we found concomitant beige adipocyte differentiation upon induction of MSCs into brown adipocytes in a defined medium containing triiodothyronine, insulin, dexamethasone, and indomethacin. Moreover, we found that protein kinase A (PKA) modulators regulated MSC differentiation into brown or beige adipocytes. Activation of PKA by isobutylmethylxanthine or forskolin increased brown adipocyte differentiation and reduced beige adipocyte differentiation, while inactivation of PKA by KT-5720 or SC-3010 or the knockdown of PKA downstream cAMP response element-binding protein (CREB) decreased brown adipocyte differentiation and increased beige adipocyte differentiation. We also showed that increased brown adipocyte differentiation was accompanied by an increase in mitochondrial mass. In conclusion, we propose a model of beige/brown co-differentiation in MSCs and develop a method for controlling this differentiation via PKA modulation. PMID:27498007

  15. Concomitant beige adipocyte differentiation upon induction of mesenchymal stem cells into brown adipocytes.

    PubMed

    Wang, Yung-Li; Lin, Shih-Pei; Hsieh, Patrick C H; Hung, Shih-Chieh

    2016-09-16

    The accumulation of fat, which results in obesity, is related to many metabolic disorders. Besides white and brown adipose tissue, beige adipose tissue has recently been recognized as a new type of accumulated fat. Mesenchymal stem cells (MSCs) have been shown to differentiate into brown adipocytes. Through analyzing levels of mRNA and protein markers associated with beige adipocyte, we found concomitant beige adipocyte differentiation upon induction of MSCs into brown adipocytes in a defined medium containing triiodothyronine, insulin, dexamethasone, and indomethacin. Moreover, we found that protein kinase A (PKA) modulators regulated MSC differentiation into brown or beige adipocytes. Activation of PKA by isobutylmethylxanthine or forskolin increased brown adipocyte differentiation and reduced beige adipocyte differentiation, while inactivation of PKA by KT-5720 or SC-3010 or the knockdown of PKA downstream cAMP response element-binding protein (CREB) decreased brown adipocyte differentiation and increased beige adipocyte differentiation. We also showed that increased brown adipocyte differentiation was accompanied by an increase in mitochondrial mass. In conclusion, we propose a model of beige/brown co-differentiation in MSCs and develop a method for controlling this differentiation via PKA modulation.

  16. The effect of plasminogen activator inhibitor-1 -675 4G/5G polymorphism on PAI-1 gene expression and adipocyte differentiation.

    PubMed

    Ozel Demiralp, Duygu; Aktas, Huseyin; Akar, Nejat

    2008-10-01

    Obesity is a complex, multifactorial chronic disease frequently associated with cardiovascular risks, hypertriglyceridemia, low high-density lipoprotein-cholesterol, high blood pressure, and the insulin resistance that appears to be central to the pathogenesis of Type II diabetes. Plasminogen activator inhibitor-1 expression induced in differentiating adipose tissue, but its role in adipogenesis and obesity is poorly understood. Circulating plasminogen activator inhibitor-1 levels are elevated at an early stage of impaired glucose tolerance, resulting in diabetes and metabolic syndrome. Plasminogen activator inhibitor-1 levels are also significantly elevated in the plasma of obese individuals and in adipose tissues of obese mice and humans. Some investigators proposed that the -675 4G/5G polymorphism in plasminogen activator inhibitor-1 promoter caused overexpression of this gene and predisposed carriers to obesity. In this study, we investigated the role of -675 4G/5G polymorphism in plasminogen activator inhibitor-1 promoter in the expression of this gene and the contribution of plasminogen activator inhibitor-1 to adipogenesis. Using a dual-luciferase promoter assay, we determined that the -675 4G/5G polymorphism contributes significantly to overexpression of plasminogen activator inhibitor-1 in the course of adipogenesis. The antidiabetic agents troglitazone and ciglitazone inhibited reporter gene expression driven by wild-type and -675 4G/5G mutant promoter, as well as the expression of endogenous plasminogen activator inhibitor-1, indicating that suppression of plasminogen activator inhibitor-1 expression may contribute to antidiabetic effects of these agents. The results indicate that absence of plasminogen activator inhibitor-1 in adipocytes may protect the cells against insulin resistance by promoting glucose uptake and adipocyte differentiation via a decrease in the peroxisome proliferator activated receptor-gamma expression that modulates the adipocyte

  17. Adipocyte hypoxia promotes epithelial-mesenchymal transition-related gene expression and estrogen receptor-negative phenotype in breast cancer cells

    PubMed Central

    YAO-BORENGASSER, AIWEI; MONZAVI-KARBASSI, BEHJATOLAH; HEDGES, REBECCA A; ROGERS, LORA J; KADLUBAR, SUSAN A; KIEBER-EMMONS, THOMAS

    2015-01-01

    The development of breast cancer is linked to the loss of estrogen receptor (ER) during the course of tumor progression, resulting in loss of responsiveness to hormonal treatment. The mechanisms underlying dynamic ERα gene expression change in breast cancer remain unclear. A range of physiological and biological changes, including increased adipose tissue hypoxia, accompanies obesity. Hypoxia in adipocytes can establish a pro-malignancy environment in breast tissues. Epidemiological studies have linked obesity with basal-like breast cancer risk and poor disease outcome, suggesting that obesity may affect the tumor phenotype by skewing the microenvironment toward support of more aggressive tumor phenotypes. In the present study, human SGBS adipocytes were co-cultured with ER-positive MCF7 cells for 24 h. After co-culture, HIF1α, TGF-β, and lectin-type oxidized LDL receptor 1 (LOX1) mRNA levels in the SGBS cells were increased. Expression levels of the epithelial-mesenchymal transition (EMT)-inducing transcription factors FOXC2 and TWIST1 were increased in the co-cultured MCF7 cells. In addition, the E-cadherin mRNA level was decreased, while the N-cadherin mRNA level was increased in the co-cultured MCF7 cells. ERα mRNA levels were significantly repressed in the co-cultured MCF7 cells. ERα gene expression in the MCF7 cells was decreased due to increased HIF1α in the SGBS cells. These results suggest that adipocytes can modify breast cancer cell ER gene expression through hypoxia and also can promote EMT processes in breast cancer cells, supporting an important role of obesity in aggressive breast cancer development. PMID:25823469

  18. Production of marker-free disease-resistant potato using isopentenyl transferase gene as a positive selection marker.

    PubMed

    Khan, Raham Sher; Ntui, Valentine Otang; Chin, Dong Poh; Nakamura, Ikuo; Mii, Masahiro

    2011-04-01

    The use of antibiotic or herbicide resistant genes as selection markers for production of transgenic plants and their continuous presence in the final transgenics has been a serious problem for their public acceptance and commercialization. MAT (multi-auto-transformation) vector system has been one of the different strategies to excise the selection marker gene and produce marker-free transgenic plants. In the present study, ipt (isopentenyl transferase) gene was used as a selection marker gene. A chitinase gene, ChiC (isolated from Streptomyces griseus strain HUT 6037) was used as a gene of interest. ChiC gene was cloned from the binary vector, pEKH1 to an ipt-type MAT vector, pMAT21 by gateway cloning and transferred to Agrobacterium tumefaciens strain EHA105. The infected tuber discs of potato were cultured on hormone- and antibiotic-free MS medium. Seven of the 35 explants infected with the pMAT21/ChiC produced shoots. The same antibiotic- and hormones-free MS medium was used in subcultures of the shoots (ipt like and normal shoots). Molecular analyses of genomic DNA from transgenic plants confirmed the integration of gene of interest and excision of the selection marker in 3 of the 7 clones. Expression of ChiC gene was confirmed by Northern blot and western blot analyses. Disease-resistant assay of the marker-free transgenic, in vitro and greenhouse-grown plants exhibited enhanced resistance against Alternaria solani (early blight), Botrytis cinerea (gray mold) and Fusarium oxysporum (Fusarium wilt). From these results it could be concluded that ipt gene can be used as a selection marker to produce marker-free disease-resistant transgenic potato plants on PGR- and antibiotic-free MS medium.

  19. Adipocyte expression of PU.1 transcription factor causes insulin resistance through upregulation of inflammatory cytokine gene expression and ROS production

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have reported previously that ETS family transcription factor PU.1 is expressed in mature adipocytes of white adipose tissue. PU.1 expression is increased greatly in mouse models of genetic or diet-induced obesity. Here, we show that PU.1 expression is increased only in visceral but not subcutane...

  20. (-)-Hydroxycitric acid attenuates endoplasmic reticulum stress-mediated alterations in 3T3-L1 adipocytes by protecting mitochondria and downregulating inflammatory markers.

    PubMed

    Nisha, V M; Priyanka, A; Anusree, S S; Raghu, K G

    2014-11-01

    Endoplasmic reticulum (ER) stress is an emerging potential therapeutic target for metabolic syndrome due to its role in synthesis, secretion, and folding of proteins. It leads to an increased production of reactive oxygen species (ROS) which, along with mitochondrial dysfunction and reduced antioxidant defense, causes chronic cell injury. The present investigation aims to observe the alterations in adipocytes due to ER stress and the protective effect of hydroxycitric acid (HCA), a bioactive from Garcinia species, to develop the same as a nutraceutical. ER stress was induced in mature 3T3-L1 adipocytes by treating them with tunicamycin (2μg/ml) for 18 h. Alterations in cell viability, innate antioxidant system (superoxide dismutase, glutathione peroxidase, and glutathione reductase), mitochondria (membrane potential, biogenesis, and transition pore opening), and inflammatory cytokines (tumor necrosis factor, monocyte chemoattractant protein, interferon-γ, interleukin (IL)-10, IL-6, and IL-1β) during ER stress, and co-treatment with HCA were analyzed. Endocrine function of adipocytes was also assessed by measuring adiponectin and leptin secretion levels. HCA protected the cells from ER stress by improving the antioxidant status and mitochondrial functions. The results validate nutraceutical properties of the edible bioactive, commonly used for culinary purpose. A more detailed study on the mechanism of action of HCA is required for developing it as a therapeutic agent for metabolic syndrome. PMID:25175938

  1. Inhibition of hormone-sensitive lipase gene expression by cAMP and phorbol esters in 3T3-F442A and BFC-1 adipocytes.

    PubMed

    Plée-Gautier, E; Grober, J; Duplus, E; Langin, D; Forest, C

    1996-09-15

    Hormone-sensitive lipase (HSL) catalyses the rate-limiting step in adipocyte lipolysis. Short-term hormonal regulation of HSL activity is well characterized, whereas little is known about the control of HSL gene expression. We have measured HSL mRNA content of 3T3-F442A and BFC-1 adipocytes in response to the cAMP analogue 8-(4-chlorophenylthio)-cAMP (8-CPT-cAMP) and to the phorbol ester phorbol 12-myristate 13-acetate (PMA) by Northern blot, using a specific mouse cDNA fragment. Treatment of the cells for 12 or 6 h with, respectively, 0.5 mM 8-CPT-cAMP or 1 microM PMA produced a maximal decrease of about 60% in HSL mRNA. These effects were unaffected by the protein-synthesis inhibitor anisomycin, suggesting that cAMP and PMA actions were direct. The reduction in HSL mRNA was accompanied by a reduction in HSL total activity. The intracellular routes that cAMP and PMA follow for inducing such an effect seemed clearly independent. (i) After desensitization of the protein kinase C regulation pathway by a 24 h treatment of the cells with 1 microM PMA, PMA action was abolished whereas cAMP was still fully active. (ii) Treatment with saturating concentrations of both agents produced an additive effect. (iii) The synthetic glucocorticoid dexamethasone had no proper effect on HSL gene expression but potentiated cAMP action without affecting PMA action. cAMP inhibitory action on HSL is unexpected. Indeed, the second messenger of catecholamines is the main activator of HSL by phosphorylation. We envision that a long-term cAMP treatment of adipocytes induces a counter-regulatory process that reduces HSL content and, ultimately, limits fatty acid depletion from stored triacylglycerols.

  2. The Farnesoid X Receptor Regulates Adipocyte Differentiation and Function by Promoting Peroxisome Proliferator-activated Receptor-γ and Interfering with the Wnt/β-Catenin Pathways*

    PubMed Central

    Abdelkarim, Mouaadh; Caron, Sandrine; Duhem, Christian; Prawitt, Janne; Dumont, Julie; Lucas, Anthony; Bouchaert, Emmanuel; Briand, Olivier; Brozek, John; Kuipers, Folkert; Fievet, Catherine; Cariou, Bertrand; Staels, Bart

    2010-01-01

    The bile acid receptor farnesoid X receptor (FXR) is expressed in adipose tissue, but its function remains poorly defined. Peroxisome proliferator-activated receptor-γ (PPARγ) is a master regulator of adipocyte differentiation and function. The aim of this study was to analyze the role of FXR in adipocyte function and to assess whether it modulates PPARγ action. Therefore, we tested the responsiveness of FXR-deficient mice (FXR−/−) and cells to the PPARγ activator rosiglitazone. Our results show that genetically obese FXR−/−/ob/ob mice displayed a resistance to rosiglitazone treatment. In vitro, rosiglitazone treatment did not induce normal adipocyte differentiation and lipid droplet formation in FXR−/− mouse embryonic fibroblasts (MEFs) and preadipocytes. Moreover, FXR−/− MEFs displayed both an increased lipolysis and a decreased de novo lipogenesis, resulting in reduced intracellular triglyceride content, even upon PPARγ activation. Retroviral-mediated FXR re-expression in FXR−/− MEFs restored the induction of adipogenic marker genes during rosiglitazone-forced adipocyte differentiation. The expression of Wnt/β-catenin pathway and target genes was increased in FXR−/− adipose tissue and MEFs. Moreover, the expression of several endogenous inhibitors of this pathway was decreased early during the adipocyte differentiation of FXR−/− MEFs. These findings demonstrate that FXR regulates adipocyte differentiation and function by regulating two counteracting pathways of adipocyte differentiation, the PPARγ and Wnt/β-catenin pathways. PMID:20851881

  3. Green Tea (-)-Epigallotocatechin-3-Gallate Induces PGC-1α Gene Expression in HepG2 Cells and 3T3-L1 Adipocytes.

    PubMed

    Lee, Mak-Soon; Lee, Seohyun; Doo, Miae; Kim, Yangha

    2016-03-01

    Green tea (Camellia sinensis) is one of the most popular beverages in the world and has been acknowledged for centuries as having significant health benefits. (-)-Epigallocatechin-3-gallate (EGCG) is the most abundant catechin in green tea, and it has been reported to have health benefit effects. Peroxisome proliferator-activated receptor γ coactivator (PGC)-1α is a crucial regulator of mitochondrial biogenesis and hepatic gluconeogenesis. The objective of this study was to investigate whether EGCG from green tea can affect the ability of transcriptional regulation on PGC-1α mRNA expression in HepG2 cells and 3T3-L1 adipocytes. To study the molecular mechanism that allows EGCG to control PGC-1α expression, the promoter activity levels of PGC-1α were examined. The PGC-1α mRNA level was measured using quantitative real-time PCR. The -970/+412 bp of PGC-1α promoter was subcloned into the pGL3-Basic vector that includes luciferase as a reporter gene. EGCG was found to up-regulate the PGC-1α mRNA levels significantly with 10 μmol/L of EGCG in HepG2 cells and differentiated 3T3-L1 adipocytes. PGC-1α promoter activity was also increased by treatment with 10 μmol/L of EGCG in both cells. These results suggest that EGCG may induce PGC-1α gene expression, potentially through promoter activation. PMID:27069908

  4. Green Tea (−)-Epigallotocatechin-3-Gallate Induces PGC-1α Gene Expression in HepG2 Cells and 3T3-L1 Adipocytes

    PubMed Central

    Lee, Mak-Soon; Lee, Seohyun; Doo, Miae; Kim, Yangha

    2016-01-01

    Green tea (Camellia sinensis) is one of the most popular beverages in the world and has been acknowledged for centuries as having significant health benefits. (−)-Epigallocatechin-3-gallate (EGCG) is the most abundant catechin in green tea, and it has been reported to have health benefit effects. Peroxisome proliferator-activated receptor γ coactivator (PGC)-1α is a crucial regulator of mitochondrial biogenesis and hepatic gluconeogenesis. The objective of this study was to investigate whether EGCG from green tea can affect the ability of transcriptional regulation on PGC-1α mRNA expression in HepG2 cells and 3T3-L1 adipocytes. To study the molecular mechanism that allows EGCG to control PGC-1α expression, the promoter activity levels of PGC-1α were examined. The PGC-1α mRNA level was measured using quantitative real-time PCR. The −970/+412 bp of PGC-1α promoter was subcloned into the pGL3-Basic vector that includes luciferase as a reporter gene. EGCG was found to up-regulate the PGC-1α mRNA levels significantly with 10 μmol/L of EGCG in HepG2 cells and differentiated 3T3-L1 adipocytes. PGC-1α promoter activity was also increased by treatment with 10 μmol/L of EGCG in both cells. These results suggest that EGCG may induce PGC-1α gene expression, potentially through promoter activation. PMID:27069908

  5. Adrenergic stimulation of lipoprotein lipase gene expression in rat brown adipocytes differentiated in culture: mediation via beta3- and alpha1-adrenergic receptors.

    PubMed Central

    Kuusela, P; Rehnmark, S; Jacobsson, A; Cannon, B; Nedergaard, J

    1997-01-01

    In order to investigate whether the positive effect of adrenergic stimulation on lipoprotein lipase (LPL) gene expression in brown adipose tissue is a direct effect on the brown adipocytes themselves, the expression of the LPL gene was investigated by measuring LPL mRNA levels in brown adipocytes, isolated as precursors from the brown adipose tissue of rats and grown in culture in a fully defined medium before experimentation. Addition of noradrenaline led to an enhancement of LPL gene expression; the mRNA levels increased as a linear function of time for at least 5 h and were finally approx. 3 times higher than in control cells, an increase commensurate with that seen in vivo in both LPL mRNA levels and LPL activity during physiological stimulation. The increase was dependent on transcription. The effect of noradrenaline showed simple Michaelis-Menten kinetics with an EC50 of approx. 11 nM. beta3-Agonists (BRL-37344 and CGP-12177) could mimic the effect of noradrenaline; the beta1-agonist dobutamine and the beta2-agonist salbutamol could not; the alpha1-agonist cirazoline had only a weak effect. The effect of noradrenaline was fully inhibited by the beta-antagonist propranolol and was halved by the alpha1-antagonist prazosin; the alpha2-antagonist yohimbine was without effect. An increase in LPL mRNA level similar to (but not significantly exceeding) that caused by noradrenaline could also be induced by the cAMP-elevating agents forskolin and cholera toxin, and 8-Br-cAMP also increased LPL mRNA levels. The increase in LPL gene expression was not mediated via an increase in the level of an intermediary proteinaceous factor. It is concluded that the physiologically induced increase in LPL gene expression is a direct effect of noradrenaline on the brown adipocytes themselves, mediated via a dominant beta3-adrenergic pathway and an auxiliary alpha1-adrenergic pathway which converge at a regulatory point in transcriptional control. PMID:9032464

  6. Regulation of adipocyte lipolysis.

    PubMed

    Frühbeck, Gema; Méndez-Giménez, Leire; Fernández-Formoso, José-Antonio; Fernández, Secundino; Rodríguez, Amaia

    2014-06-01

    In adipocytes the hydrolysis of TAG to produce fatty acids and glycerol under fasting conditions or times of elevated energy demands is tightly regulated by neuroendocrine signals, resulting in the activation of lipolytic enzymes. Among the classic regulators of lipolysis, adrenergic stimulation and the insulin-mediated control of lipid mobilisation are the best known. Initially, hormone-sensitive lipase (HSL) was thought to be the rate-limiting enzyme of the first lipolytic step, while we now know that adipocyte TAG lipase is the key enzyme for lipolysis initiation. Pivotal, previously unsuspected components have also been identified at the protective interface of the lipid droplet surface and in the signalling pathways that control lipolysis. Perilipin, comparative gene identification-58 (CGI-58) and other proteins of the lipid droplet surface are currently known to be key regulators of the lipolytic machinery, protecting or exposing the TAG core of the droplet to lipases. The neuroendocrine control of lipolysis is prototypically exerted by catecholaminergic stimulation and insulin-induced suppression, both of which affect cyclic AMP levels and hence the protein kinase A-mediated phosphorylation of HSL and perilipin. Interestingly, in recent decades adipose tissue has been shown to secrete a large number of adipokines, which exert direct effects on lipolysis, while adipocytes reportedly express a wide range of receptors for signals involved in lipid mobilisation. Recently recognised mediators of lipolysis include some adipokines, structural membrane proteins, atrial natriuretic peptides, AMP-activated protein kinase and mitogen-activated protein kinase. Lipolysis needs to be reanalysed from the broader perspective of its specific physiological or pathological context since basal or stimulated lipolytic rates occur under diverse conditions and by different mechanisms.

  7. Regulation of adipocyte lipolysis.

    PubMed

    Frühbeck, Gema; Méndez-Giménez, Leire; Fernández-Formoso, José-Antonio; Fernández, Secundino; Rodríguez, Amaia

    2014-06-01

    In adipocytes the hydrolysis of TAG to produce fatty acids and glycerol under fasting conditions or times of elevated energy demands is tightly regulated by neuroendocrine signals, resulting in the activation of lipolytic enzymes. Among the classic regulators of lipolysis, adrenergic stimulation and the insulin-mediated control of lipid mobilisation are the best known. Initially, hormone-sensitive lipase (HSL) was thought to be the rate-limiting enzyme of the first lipolytic step, while we now know that adipocyte TAG lipase is the key enzyme for lipolysis initiation. Pivotal, previously unsuspected components have also been identified at the protective interface of the lipid droplet surface and in the signalling pathways that control lipolysis. Perilipin, comparative gene identification-58 (CGI-58) and other proteins of the lipid droplet surface are currently known to be key regulators of the lipolytic machinery, protecting or exposing the TAG core of the droplet to lipases. The neuroendocrine control of lipolysis is prototypically exerted by catecholaminergic stimulation and insulin-induced suppression, both of which affect cyclic AMP levels and hence the protein kinase A-mediated phosphorylation of HSL and perilipin. Interestingly, in recent decades adipose tissue has been shown to secrete a large number of adipokines, which exert direct effects on lipolysis, while adipocytes reportedly express a wide range of receptors for signals involved in lipid mobilisation. Recently recognised mediators of lipolysis include some adipokines, structural membrane proteins, atrial natriuretic peptides, AMP-activated protein kinase and mitogen-activated protein kinase. Lipolysis needs to be reanalysed from the broader perspective of its specific physiological or pathological context since basal or stimulated lipolytic rates occur under diverse conditions and by different mechanisms. PMID:24872083

  8. Identification of putative gene based markers of renal toxicity.

    PubMed Central

    Amin, Rupesh P; Vickers, Alison E; Sistare, Frank; Thompson, Karol L; Roman, Richard J; Lawton, Michael; Kramer, Jeffrey; Hamadeh, Hisham K; Collins, Jennifer; Grissom, Sherry; Bennett, Lee; Tucker, C Jeffrey; Wild, Stacie; Kind, Clive; Oreffo, Victor; Davis, John W; Curtiss, Sandra; Naciff, Jorge M; Cunningham, Michael; Tennant, Raymond; Stevens, James; Car, Bruce; Bertram, Timothy A; Afshari, Cynthia A

    2004-01-01

    This study, designed and conducted as part of the International Life Sciences Institute working group on the Application of Genomics and Proteomics, examined the changes in the expression profile of genes associated with the administration of three different nephrotoxicants--cisplatin, gentamicin, and puromycin--to assess the usefulness of microarrays in the understanding of mechanism(s) of nephrotoxicity. Male Sprague-Dawley rats were treated with daily doses of puromycin (5-20 mg/kg/day for 21 days), gentamicin (2-240 mg/kg/day for 7 days), or a single dose of cisplatin (0.1-5 mg/kg). Groups of rats were sacrificed at various times after administration of these compounds for standard clinical chemistry, urine analysis, and histological evaluation of the kidney. RNA was extracted from the kidney for microarray analysis. Principal component analysis and gene expression-based clustering of compound effects confirmed sample separation based on dose, time, and degree of renal toxicity. In addition, analysis of the profile components revealed some novel changes in the expression of genes that appeared to be associated with injury in specific portions of the nephron and reflected the mechanism of action of these various nephrotoxicants. For example, although puromycin is thought to specifically promote injury of the podocytes in the glomerulus, the changes in gene expression after chronic exposure of this compound suggested a pattern similar to the known proximal tubular nephrotoxicants cisplatin and gentamicin; this prediction was confirmed histologically. We conclude that renal gene expression profiling coupled with analysis of classical end points affords promising opportunities to reveal potential new mechanistic markers of renal toxicity. PMID:15033597

  9. Sida rhomboidea. Roxb Leaf Extract Down-Regulates Expression of PPARγ2 and Leptin Genes in High Fat Diet Fed C57BL/6J Mice and Retards in Vitro 3T3L1 Pre-Adipocyte Differentiation

    PubMed Central

    Thounaojam, Menaka C.; Jadeja, Ravirajsinh N.; Ramani, Umed V.; Devkar, Ranjitsinh V.; Ramachandran, A. V.

    2011-01-01

    Sida rhomboidea. Roxb leaf extract (SRLE) is being used by the populace of North-East India to alleviate symptoms of diabetes and obesity. We have previously reported its hypolipidemic and anti-diabetic properties. In this study, we report the effect of SRLE on (i) in vivo modulation of genes controlling high fat diet (HFD) induced obesity and (ii) in vitro 3T3L1 pre-adipocyte differentiation and leptin release. Supplementation with SRLE significantly prevented HFD induced increment in bodyweight, plasma lipids and leptin, visceral adiposity and adipocyte hypertrophy. Also, SRLE supplementation reduced food intake, down regulated PPARγ2, SREBP1c, FAS and LEP expressions and up-regulated CPT-1 in epididymal adipose tissue compared to obese mice. In vitro adipogenesis of 3T3L1 pre-adipocytes was significantly retarded in the presence of SRLE extract. Also decreased triglyceride accumulation, leptin release and glyceraldehyde-3-Phosphate dehydrogenase activity along with higher glycerol release without significant alteration of viability of 3T3L1 pre-adipocytes, was recorded. Our findings suggest that prevention of HFD induced visceral adiposity is primarily by down regulation of PPARγ2 and leptin gene expression coupled with attenuation of food intake in C57BL/6J mice. SRLE induced prevention of pre-adipocytes differentiation, and leptin release further substantiated these findings and scientifically validates the potential application of SRLE as a therapeutic agent against obesity. PMID:21845103

  10. Sida rhomboidea. Roxb leaf extract down-regulates expression of PPARγ2 and leptin genes in high fat diet fed C57BL/6J Mice and retards in vitro 3T3L1 pre-adipocyte differentiation.

    PubMed

    Thounaojam, Menaka C; Jadeja, Ravirajsinh N; Ramani, Umed V; Devkar, Ranjitsinh V; Ramachandran, A V

    2011-01-01

    Sida rhomboidea. Roxb leaf extract (SRLE) is being used by the populace of North-East India to alleviate symptoms of diabetes and obesity. We have previously reported its hypolipidemic and anti-diabetic properties. In this study, we report the effect of SRLE on (i) in vivo modulation of genes controlling high fat diet (HFD) induced obesity and (ii) in vitro 3T3L1 pre-adipocyte differentiation and leptin release. Supplementation with SRLE significantly prevented HFD induced increment in bodyweight, plasma lipids and leptin, visceral adiposity and adipocyte hypertrophy. Also, SRLE supplementation reduced food intake, down regulated PPARγ2, SREBP1c, FAS and LEP expressions and up-regulated CPT-1 in epididymal adipose tissue compared to obese mice. In vitro adipogenesis of 3T3L1 pre-adipocytes was significantly retarded in the presence of SRLE extract. Also decreased triglyceride accumulation, leptin release and glyceraldehyde-3-Phosphate dehydrogenase activity along with higher glycerol release without significant alteration of viability of 3T3L1 pre-adipocytes, was recorded. Our findings suggest that prevention of HFD induced visceral adiposity is primarily by down regulation of PPARγ2 and leptin gene expression coupled with attenuation of food intake in C57BL/6J mice. SRLE induced prevention of pre-adipocytes differentiation, and leptin release further substantiated these findings and scientifically validates the potential application of SRLE as a therapeutic agent against obesity.

  11. New DNA markers in the Huntington's disease gene candidate region.

    PubMed

    Lin, C S; Altherr, M; Bates, G; Whaley, W L; Read, A P; Harris, R; Lehrach, H; Wasmuth, J J; Gusella, J F; MacDonald, M E

    1991-09-01

    The search for the Huntington's disease (HD) gene has prompted construction of a complete long-range restriction map of a 2.5-Mb candidate region, distal to the DNA marker D4S10. To facilitate the procurement of cloned DNA from this candidate region, we have augmented the existing regional mapping panel of somatic cell hybrids with hybrid HHW1071 containing a t(4p16;12) chromosome from a patient with Wolf-Hirschhorn syndrome. This translocation maps between D4S180 and D4S127, subdividing the HD candidate region and setting a proximal limit to the Wolf-Hirschhorn syndrome region. Using the expanded mapping panel, we have regionally assigned 14 independently cloned cosmids, five proximal to the t(4;12) breakpoint in the same region as D4S10 and nine distal to the breakpoint. By a combination of overlap with previously mapped cosmids and pulsed-field gel analysis, each of these cosmids has been positioned on the long-range restriction map of 4p16.3, increasing the clone coverage of the candidate region to approximately 40%. Single-copy probes from mapped cosmids were used to identify eight new DNA polymorphisms spanning the HD candidate region. These new DNA markers should prove valuable for analysis of recombination and linkage disequilibrium in HD, as well as for preclinical diagnosis of the disorder.

  12. Inhibition of mitotic clonal expansion mediates fisetin-exerted prevention of adipocyte differentiation in 3T3-L1 cells.

    PubMed

    Lee, Youngyi; Bae, Eun Ju

    2013-11-01

    Adipocytes are the key player in adipose tissue inflammation and subsequent systemic insulin resistance and its development involves complex process of proliferation and differentiation of preadipocytes. Fistein, a polyphenol flavonoid, is known to exert anti-inflammatory, anti-carcinogenic and anti-diabetic effects. In this study, we aimed to investigate the effect of fisetin on adipocyte proliferation and differentiation in 3T3-L1 preadipocyte cell line and its mechanism of action. We found that fisetin inhibits adipocyte differentiation in a concentration dependent manner, which were evidenced by Oil Red O staining and the protein expression of mature adipocyte marker genes fatty acid synthase and peroxisome proliferator-activated receptor γ. Moreover, the proliferation of preadipocytes was also markedly suppressed by treatment of fisetin for 24 and 48 h in the differentiation medium. We also found that fisetin inhibition of adipocyte differentiation was largely due to the effect on mitotic clonal expansion. Fisetin suppression of preadipocyte proliferation at early stage of differentiation was accompanied by the changes of expression of a series of cell cycle regulatory proteins. Altogether, our results suggest that the inhibition of adipocyte differentiation by fisetin may be at least in part mediated by cell cycle arrest during adipogenesis.

  13. Considerations For Optimizing Microbiome Analysis Using a Marker Gene.

    PubMed

    de la Cuesta-Zuluaga, Jacobo; Escobar, Juan S

    2016-01-01

    Next-generation sequencing technologies have found a widespread use in the study of host-microbe interactions due to the increase in their throughput and their ever-decreasing costs. The analysis of human-associated microbial communities using a marker gene, particularly the 16S rRNA, has been greatly benefited from these technologies - the human gut microbiome research being a remarkable example of such analysis that has greatly expanded our understanding of microbe-mediated human health and disease, metabolism, and food absorption. 16S studies go through a series of in vitro and in silico steps that can greatly influence their outcomes. However, the lack of a standardized workflow has led to uncertainties regarding the transparency and reproducibility of gut microbiome studies. We, here, discuss the most common challenges in the archetypical 16S rRNA workflow, including the extraction of total DNA, its use as template in PCR with primers that amplify specific hypervariable regions of the gene, amplicon sequencing, the denoising and removal of low-quality reads, the detection and removal of chimeric sequences, the clustering of high-quality sequences into operational taxonomic units, and their taxonomic classification. We recommend the essential technical information that should be conveyed in publications for reproducibility of results and encourage non-experts to include procedures and available tools that mitigate most of the problems encountered in microbiome analysis.

  14. Considerations For Optimizing Microbiome Analysis Using a Marker Gene

    PubMed Central

    de la Cuesta-Zuluaga, Jacobo; Escobar, Juan S.

    2016-01-01

    Next-generation sequencing technologies have found a widespread use in the study of host–microbe interactions due to the increase in their throughput and their ever-decreasing costs. The analysis of human-associated microbial communities using a marker gene, particularly the 16S rRNA, has been greatly benefited from these technologies – the human gut microbiome research being a remarkable example of such analysis that has greatly expanded our understanding of microbe-mediated human health and disease, metabolism, and food absorption. 16S studies go through a series of in vitro and in silico steps that can greatly influence their outcomes. However, the lack of a standardized workflow has led to uncertainties regarding the transparency and reproducibility of gut microbiome studies. We, here, discuss the most common challenges in the archetypical 16S rRNA workflow, including the extraction of total DNA, its use as template in PCR with primers that amplify specific hypervariable regions of the gene, amplicon sequencing, the denoising and removal of low-quality reads, the detection and removal of chimeric sequences, the clustering of high-quality sequences into operational taxonomic units, and their taxonomic classification. We recommend the essential technical information that should be conveyed in publications for reproducibility of results and encourage non-experts to include procedures and available tools that mitigate most of the problems encountered in microbiome analysis. PMID:27551678

  15. Considerations For Optimizing Microbiome Analysis Using a Marker Gene.

    PubMed

    de la Cuesta-Zuluaga, Jacobo; Escobar, Juan S

    2016-01-01

    Next-generation sequencing technologies have found a widespread use in the study of host-microbe interactions due to the increase in their throughput and their ever-decreasing costs. The analysis of human-associated microbial communities using a marker gene, particularly the 16S rRNA, has been greatly benefited from these technologies - the human gut microbiome research being a remarkable example of such analysis that has greatly expanded our understanding of microbe-mediated human health and disease, metabolism, and food absorption. 16S studies go through a series of in vitro and in silico steps that can greatly influence their outcomes. However, the lack of a standardized workflow has led to uncertainties regarding the transparency and reproducibility of gut microbiome studies. We, here, discuss the most common challenges in the archetypical 16S rRNA workflow, including the extraction of total DNA, its use as template in PCR with primers that amplify specific hypervariable regions of the gene, amplicon sequencing, the denoising and removal of low-quality reads, the detection and removal of chimeric sequences, the clustering of high-quality sequences into operational taxonomic units, and their taxonomic classification. We recommend the essential technical information that should be conveyed in publications for reproducibility of results and encourage non-experts to include procedures and available tools that mitigate most of the problems encountered in microbiome analysis. PMID:27551678

  16. Functional clustering and lineage markers: insights into cellular differentiation and gene function from large-scale microarray studies of purified primary cell populations.

    PubMed

    Hume, David A; Summers, Kim M; Raza, Sobia; Baillie, J Kenneth; Freeman, Thomas C

    2010-06-01

    Very large microarray datasets showing gene expression across multiple tissues and cell populations provide a window on the transcriptional networks that underpin the differences in functional activity between biological systems. Clusters of co-expressed genes provide lineage markers, candidate regulators of cell function and, by applying the principle of guilt by association, candidate functions for genes of currently unknown function. We have analysed a dataset comprising pure cell populations from hemopoietic and non-hemopoietic cell types (http://biogps.gnf.org). Using a novel network visualisation and clustering approach, we demonstrate that it is possible to identify very tight expression signatures associated specifically with embryonic stem cells, mesenchymal cells and hematopoietic lineages. Selected examples validate the prediction that gene function can be inferred by co-expression. One expression cluster was enriched in phagocytes, which, alongside endosome-lysosome constituents, contains genes that may make up a 'pathway' for phagocyte differentiation. Promoters of these genes are enriched for binding sites for the ETS/PU.1 and MITF families. Another cluster was associated with the production of a specific extracellular matrix, with high levels of gene expression shared by cells of mesenchymal origin (fibroblasts, adipocytes, osteoblasts and myoblasts). We discuss the limitations placed upon such data by the presence of alternative promoters with distinct tissue specificity within many protein-coding genes.

  17. Application of resistance gene analog markers to analyses of genetic structure and diversity in rice.

    PubMed

    Ren, Juansheng; Yu, Yuchao; Gao, Fangyuan; Zeng, Lihua; Lu, Xianjun; Wu, Xianting; Yan, Wengui; Ren, Guangjun

    2013-07-01

    Plant disease resistance gene analog (RGA) markers were designed according to the conserved sequence of known RGAs and used to map resistance genes. We used genome-wide RGA markers for genetic analyses of structure and diversity in a global rice germplasm collection. Of the 472 RGA markers, 138 were polymorphic and these were applied to 178 entries selected from the USDA rice core collection. Results from the RGA markers were similar between two methods, UPGMA and STRUCTURE. Additionally, the results from RGA markers in our study were agreeable with those previously reported from SSR markers, including cluster of ancestral classification, genetic diversity estimates, genetic relatedness, and cluster of geographic origins. These results suggest that RGA markers are applicable for analyses of genetic structure and diversity in rice. However, unlike SSR markers, the RGA markers failed to differentiate temperate japonica, tropical japonica, and aromatic subgroups. The restricted way for developing RGA markers from the cDNA sequence might limit the polymorphism of RGA markers in the genome, thus limiting the discriminatory power in comparison with SSR markers. Genetic differentiation obtained using RGA markers may be useful for defining genetic diversity of a suite of random R genes in plants, as many studies show a differentiation of resistance to a wide array of pathogens. They could also help to characterize the genetic structure and geographic distribution in crops, including rice, wheat, barley, and banana.

  18. Application of resistance gene analog markers to analyses of genetic structure and diversity in rice.

    PubMed

    Ren, Juansheng; Yu, Yuchao; Gao, Fangyuan; Zeng, Lihua; Lu, Xianjun; Wu, Xianting; Yan, Wengui; Ren, Guangjun

    2013-07-01

    Plant disease resistance gene analog (RGA) markers were designed according to the conserved sequence of known RGAs and used to map resistance genes. We used genome-wide RGA markers for genetic analyses of structure and diversity in a global rice germplasm collection. Of the 472 RGA markers, 138 were polymorphic and these were applied to 178 entries selected from the USDA rice core collection. Results from the RGA markers were similar between two methods, UPGMA and STRUCTURE. Additionally, the results from RGA markers in our study were agreeable with those previously reported from SSR markers, including cluster of ancestral classification, genetic diversity estimates, genetic relatedness, and cluster of geographic origins. These results suggest that RGA markers are applicable for analyses of genetic structure and diversity in rice. However, unlike SSR markers, the RGA markers failed to differentiate temperate japonica, tropical japonica, and aromatic subgroups. The restricted way for developing RGA markers from the cDNA sequence might limit the polymorphism of RGA markers in the genome, thus limiting the discriminatory power in comparison with SSR markers. Genetic differentiation obtained using RGA markers may be useful for defining genetic diversity of a suite of random R genes in plants, as many studies show a differentiation of resistance to a wide array of pathogens. They could also help to characterize the genetic structure and geographic distribution in crops, including rice, wheat, barley, and banana. PMID:24099390

  19. Recent patents on biosafety strategies of selectable marker genes in genetically modified crops.

    PubMed

    Jiang, Yiming; Hu, Xiaoning; Huang, Haiying

    2014-01-01

    Genetically modified crops (GMCs) have been planted world wide since 1990s, but the potential insecurity of selectable marker genes raises the questions about GMC safety. Therefore, several researches have been conducted on marker gene safety issues and recently several patents have been issued on this subject. There are two main approaches to achieve this goal: seeking the biosafety selectable marker and eliminating these insecure marker genes after transformation. Results show that these two systems are quite effective. Recent patents on the two ways are discussed in this review.

  20. Markers

    ERIC Educational Resources Information Center

    Healthy Schools Network, Inc., 2011

    2011-01-01

    Dry erase whiteboards come with toxic dry erase markers and toxic cleaning products. Dry erase markers labeled "nontoxic" are not free of toxic chemicals and can cause health problems. Children are especially vulnerable to environmental health hazards; moreover, schools commonly have problems with indoor air pollution, as they are more densely…

  1. A thiostrepton resistance gene and its mutants serve as selectable markers in Geobacillus kaustophilus HTA426.

    PubMed

    Wada, Keisuke; Kobayashi, Jyumpei; Furukawa, Megumi; Doi, Katsumi; Ohshiro, Takashi; Suzuki, Hirokazu

    2016-01-01

    Effective utilization of microbes often requires complex genetic modification using multiple antibiotic resistance markers. Because a few markers have been used in Geobacillus spp., the present study was designed to identify a new marker for these thermophiles. We explored antibiotic resistance genes functional in Geobacillus kaustophilus HTA426 and identified a thiostrepton resistance gene (tsr) effective at 50 °C. The tsr gene was further used to generate the mutant tsr(H258Y) functional at 55 °C. Higher functional temperature of the mutant was attributable to the increase in thermostability of the gene product because recombinant protein produced from tsr(H258Y) was more thermostable than that from tsr. In fact, the tsr(H258Y) gene served as a selectable marker for plasmid transformation of G. kaustophilus. This new marker could facilitate complex genetic modification of G. kaustophilus and potentially other Geobacillus spp.

  2. Roles of leptin and ghrelin in adipogenesis and lipid metabolism of rainbow trout adipocytes in vitro.

    PubMed

    Salmerón, Cristina; Johansson, Marcus; Asaad, Maryam; Angotzi, Anna R; Rønnestad, Ivar; Stefansson, Sigurd O; Jönsson, Elisabeth; Björnsson, Björn Thrandur; Gutiérrez, Joaquim; Navarro, Isabel; Capilla, Encarnación

    2015-10-01

    Leptin and ghrelin are important regulators of energy homeostasis in mammals, whereas their physiological roles in fish have not been fully elucidated. In the present study, the effects of leptin and ghrelin on adipogenesis, lipolysis and on expression of lipid metabolism-related genes were examined in rainbow trout adipocytes in vitro. Leptin expression and release increased from preadipocytes to mature adipocytes in culture, but did not affect the process of adipogenesis. While ghrelin and its receptor were identified in cultured differentiated adipocytes, ghrelin did not influence either preadipocyte proliferation or differentiation, indicating that it may have other adipose-related roles. Leptin and ghrelin increased lipolysis in mature freshly isolated adipocytes, but mRNA expression of lipolysis markers was not significantly modified. Leptin significantly suppressed the fatty acid transporter-1 expression, suggesting a decrease in fatty acid uptake and storage, but did not affect expression of any of the lipogenesis or β-oxidation genes studied. Ghrelin significantly increased the mRNA levels of lipoprotein lipase, fatty acid synthase and peroxisome proliferator-activated receptor-β, and thus appears to stimulate synthesis of triglycerides as well as their mobilization. Overall, the study indicates that ghrelin, but not leptin seems to be an enhancer of lipid turn-over in adipose tissue of rainbow trout, and this regulation may at least partly be mediated through autocrine/paracrine mechanisms. The mode of action of both hormones needs to be further explored to better understand their roles in regulating adiposity in fish.

  3. Endogenous adipocyte apolipoprotein E is colocalized with caveolin at the adipocyte plasma membrane

    PubMed Central

    Yue, Lili; Mazzone, Theodore

    2011-01-01

    Apolipoprotein (apo)E is well established as a secreted protein that plays an important role in systemic lipoprotein metabolism and vascular wall homeostasis. Recently, endogenous expression of apoE in adipocytes has been shown to play an important role in adipocyte lipoprotein metabolism and gene expression consistent with a nonsecreted cellular itinerary for apoE. We designed studies to evaluate if adipocyte apoE was retained as a constituent protein in adipocytes and to identify a cellular retention compartment. Using confocal microscopy, coimmunoprecipitation, and sucrose density cellular fractionation, we establish that endogenous apoE shares a cellular itinerary with the constituent protein caveolin-1. Altering adipocyte caveolar number by modulating cellular cholesterol flux or altering caveolin expression regulates the distribution of cellular apoE between cytoplasmic and plasma membrane compartments. A mechanism for colocalization of apoE with caveolin was established by demonstrating a noncovalent interaction between an aromatic amino acid-enriched apoE N-terminal domain with the caveolin scaffolding domain. Absent apoE expression in adipocytes alters caveolar lipid composition. These observations provide evidence for an interaction between two proteins involved in cellular lipid metabolism in a cell specialized for lipid storage and flux, and rationalize a biological basis for the impact of adipocyte apoE expression on adipocyte lipoprotein metabolism. PMID:21169230

  4. Calcium-dependent intracellular signal pathways in primary cultured adipocytes and ANK3 gene variation in patients with bipolar disorder and healthy controls.

    PubMed

    Hayashi, A; Le Gal, K; Södersten, K; Vizlin-Hodzic, D; Ågren, H; Funa, K

    2015-08-01

    Bipolar disorder (BD) is a chronic psychiatric disorder of public health importance affecting >1% of the Swedish population. Despite progress, patients still suffer from chronic mood switches with potential severe consequences. Thus, early detection, diagnosis and initiation of correct treatment are critical. Cultured adipocytes from 35 patients with BD and 38 healthy controls were analysed using signal pathway reporter assays, that is, protein kinase C (PKC), protein kinase A (PKA), mitogen-activated protein kinases (extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK)), Myc, Wnt and p53. The levels of activated target transcriptional factors were measured in adipocytes before and after stimulation with lithium and escitalopram. Variations were analysed in the loci of 25 different single-nucleotide polymorphisms (SNPs). Activation of intracellular signals in several pathways analysed were significantly higher in patients than in healthy controls upon drug stimulation, especially with escitalopram stimulation of PKC, JNK and Myc, as well as lithium-stimulated PKC, whereas no meaningful difference was observed before stimulation. Univariate analyses of contingency tables for 80 categorical SNP results versus diagnoses showed a significant link with the ANK3 gene (rs10761482; likelihood ratio χ(2)=4.63; P=0.031). In a multivariate ordinal logistic fit for diagnosis, a backward stepwise procedure selected ANK3 as the remaining significant predictor. Comparison of the escitalopram-stimulated PKC activity and the ANK3 genotype showed them to add their share of the diagnostic variance, with no interaction (15% of variance explained, P<0.002). The study is cross-sectional with no longitudinal follow-up. Cohorts are relatively small with no medication-free patients, and there are no 'ill patient' controls. It takes 3 to 4 weeks of culture to expand adipocytes that may change epigenetic profiles but remove the possibility of medication effects

  5. Validation of candidate gene markers for marker-assisted selection of potato cultivars with improved tuber quality.

    PubMed

    Li, Li; Tacke, Eckhard; Hofferbert, Hans-Reinhardt; Lübeck, Jens; Strahwald, Josef; Draffehn, Astrid M; Walkemeier, Birgit; Gebhardt, Christiane

    2013-04-01

    Tuber yield, starch content, starch yield and chip color are complex traits that are important for industrial uses and food processing of potato. Chip color depends on the quantity of reducing sugars glucose and fructose in the tubers, which are generated by starch degradation. Reducing sugars accumulate when tubers are stored at low temperatures. Early and efficient selection of cultivars with superior yield, starch yield and chip color is hampered by the fact that reliable phenotypic selection requires multiple year and location trials. Application of DNA-based markers early in the breeding cycle, which are diagnostic for superior alleles of genes that control natural variation of tuber quality, will reduce the number of clones to be evaluated in field trials. Association mapping using genes functional in carbohydrate metabolism as markers has discovered alleles of invertases and starch phosphorylases that are associated with tuber quality traits. Here, we report on new DNA variants at loci encoding ADP-glucose pyrophosphorylase and the invertase Pain-1, which are associated with positive or negative effect with chip color, tuber starch content and starch yield. Marker-assisted selection (MAS) and marker validation were performed in tetraploid breeding populations, using various combinations of 11 allele-specific markers associated with tuber quality traits. To facilitate MAS, user-friendly PCR assays were developed for specific candidate gene alleles. In a multi-parental population of advanced breeding clones, genotypes were selected for having different combinations of five positive and the corresponding negative marker alleles. Genotypes combining five positive marker alleles performed on average better than genotypes with four negative alleles and one positive allele. When tested individually, seven of eight markers showed an effect on at least one quality trait. The direction of effect was as expected. Combinations of two to three marker alleles were

  6. [Progress on biosafety assessment of marker genes in genetically modified foods].

    PubMed

    Yang, Lichen; Yang, Xiaoguang

    2003-05-01

    Marker genes are useful in facilitating the detection of genetically modified organisms(GMO). These genes play an important role during the early identification stage of GMO development, but they exist in the mature genetically modified crops. So the safety assessment of these genes could not be neglected. In this paper, all the study on the biosafety assessment of marker genes were reviewed, their possible hazards and risks were appraised, and the marker genes proved safe were list too. GMO Labeling the is one important regulations for the development of genetically modified foods in the market. The accurate detecting techniques for GMO are the basis for setting up labeling regulation. In addition, some methods used to remove marker genes in genetically modified foods were introduced in the paper, which can eliminate their biosafety concern thoroughly.

  7. Adipocytes as a new source of catecholamine production.

    PubMed

    Vargovic, Peter; Ukropec, Jozef; Laukova, Marcela; Cleary, Susannah; Manz, Bernhard; Pacak, Karel; Kvetnansky, Richard

    2011-07-21

    Catecholamines are an important regulator of lipolysis in adipose tissue. Here we show that rat adipocytes, isolated from mesenteric adipose tissue, express genes of catecholamine biosynthetic enzymes and produce catecholamines de novo. Administration of tyrosine hydroxylase inhibitor, alpha-methyl-p-tyrosine, in vitro significantly reduced concentration of catecholamines in isolated adipocytes. We hypothesize that the sympathetic innervation of adipose tissues is not the only source of catecholamines, since adipocytes also have the capacity to produce both norepinephrine and epinephrine.

  8. Oleic acid enhances G protein coupled receptor 43 expression in bovine intramuscular adipocytes but not in subcutaneous adipocytes.

    PubMed

    Chung, K Y; Smith, S B; Choi, S H; Johnson, B J

    2016-05-01

    We hypothesized that fatty acids would differentially affect G protein coupled receptor (GPR) 43 mRNA expression and GPR43 protein concentrations in bovine intramuscular (IM) and subcutaneous (SC) adipocytes. The GPR43 protein was detected in bovine liver, pancreas, and semimembranosus (MUS) muscle in samples taken at slaughter. Similarly, GPR43 protein levels were similar in IM adipose tissue and SM muscle but was barely detectable in SC adipose tissue. Primary cultures of IM and SC stromal vascular cells were isolated from bovine adipose tissues. Oleic acid (100 μ) stimulated PPARγ gene expression and decreased stearoyl-CoA desaturase (SCD) gene expression but had no effect on GPR43 gene expression, which was readily detectable in both IM and SC adipocytes. Differentiation cocktail (Diff; 10 μ insulin, 4 μ dexamethasone, and 10 μ ciglitizone) stimulated CCAAT/enhancer-binding protein β (C/EBPβ) and PPARγ gene expression in SC but not IM adipocytes, but Diff increased SCD gene expression in both cell types. Linoleic acid (10 µ) increased PPARγ gene expression relative to Diff cocktail in SC adipocytes, whereas linoleic acid and α-linolenic decreased SCD gene expression relative to control adipocytes and adipocytes incubated with Diff ( < 0.05). Increasing concentrations of oleic acid (1, 10, 100, and 500 μM) increased GPR43 protein and mRNA expression in IM but not SC adipocytes. These data indicated that oleic acid alters mRNA and protein concentrations of GPR43 in bovine IM adipocytes. PMID:27285685

  9. Marker-trait association analysis of functional gene markers for provitamin A levels across diverse tropical yellow maize inbred lines

    PubMed Central

    2013-01-01

    Background Biofortification of staple crops is a cost effective and sustainable approach that can help combat vitamin A and other micronutrient deficiencies in developing countries. PCR -based DNA markers distinguishing alleles of three key genes of maize endosperm carotenoid biosynthesis (PSY1, lcyE and crtRB1) have been developed to facilitate maize provitamin A biofortification via marker assisted selection. Previous studies of these functional DNA markers revealed inconsistent effects. The germplasm previously employed for discovering and validating these functional markers was mainly of temperate origin containing low frequencies of the favourable allele of the most significant polymorphism, crtRB1-5′TE. Here, we investigate the vitamin A biofortification potential of these DNA markers in a germplasm panel of diverse tropical yellow maize inbred lines, with mixed genetic backgrounds of temperate and tropical germplasm to identify the most effective diagnostic markers for vitamin A biofortification. Results The functional DNA markers crtRB1-5′TE and crtRB1-3′TE were consistently and strongly associated with provitamin A content across the tropical maize inbred lines tested. The alleles detected by these two functional markers were in high linkage disequilibrium (R2 = 0.75) and occurred in relatively high frequency (18%). Genotypes combining the favourable alleles at the two loci (N = 20) displayed a 3.22 fold average increase in β-carotene content compared to those genotypes lacking the favourable alleles (N = 106). The PSY1 markers were monomorphic across all of the inbred lines. The functional DNA markers for lcyE were associated with lutein, and with the ratio of carotenoids in the alpha and beta branches, but not with provitamin A levels. However, the combined effects of the two genes were stronger than their individual effects on all carotenoids. Conclusions Tropical maize inbred lines harbouring the favourable alleles of the crtRB1-5

  10. Curcumin induces brown fat-like phenotype in 3T3-L1 and primary white adipocytes.

    PubMed

    Lone, Jameel; Choi, Jae Heon; Kim, Sang Woo; Yun, Jong Won

    2016-01-01

    Recent advances have been made in the understanding of pharmacological and dietary agents that contribute to browning of white adipose tissue in order to combat obesity by promoting energy expenditure. Here, we show that curcumin induces browning of 3T3-L1 and primary white adipocytes via enhanced expression of brown fat-specific genes. Curcumin-induced browning in white adipocytes was investigated by determining expression levels of brown adipocyte-specific genes/proteins by real-time reverse transcriptase polymerase chain reaction, immunoblot analysis and immunocytochemical staining. Curcumin increased mitochondrial biogenesis, as evidenced by transmission electronic microscopic detection and enhanced expression of proteins involved in fat oxidation. Cucurmin also increased protein levels of hormone-sensitive lipase and p-acyl-CoA carboxylase, suggesting its possible role in augmentation of lipolysis and suppression of lipogenesis. Increased expression of UCP1 and other brown adipocyte-specific markers was possibly mediated by curcumin-induced activation of AMP-activated protein kinase (AMPK) based on the fact that inhibition of AMPK by dorsomorphin abolished expression of PRDM16, UCP1 and peroxisome proliferator-activated receptor gamma co-activator 1-alpha while the activator 5-Aminoimidazole-4-carboxamide ribonucleotide elevated expression of these brown marker proteins. Our findings suggest that curcumin plays a dual modulatory role in inhibition of adipogenesis as well as induction of the brown fat-like phenotype and thus may have potential therapeutic implications for treatment of obesity.

  11. Targeted insertion of foreign genes into the tobacco plastid genome without physical linkage to the selectable marker gene

    SciTech Connect

    Carrer, H.; Maliga, P.

    1995-08-01

    To determine whether targeted DNA insertion into the tobacco plastid genome can be obtained without physical linkage to a selectable marker gene, we carried out biolistic transformation of chloroplasts in tobacco leaf segments with a 1:1 mix of two independently targeted antibiotic resistance genes. Plastid transformants were selected by spectinomycin resistance due to expression of an integrated aadA gene. Integration of the unselected kanamycin resistance (kan) gene into the same plastid genome was established by Southern probing in {approx}20% of the spectinomycin-selected clones. Efficient cotransformation will facilitate targeted plastid genome modification without physical linkage to a marker gene. 26 refs., 5 figs., 1 tab.

  12. Common Marker Genes Identified from Various Sample Types for Systemic Lupus Erythematosus

    PubMed Central

    Wang, Lan; Zhang, Yong-Hong; Lei, Shu-Feng; Deng, Fei-Yan

    2016-01-01

    Objective Systemic lupus erythematosus (SLE) is a complex auto-immune disease. Gene expression studies have been conducted to identify SLE-related genes in various types of samples. It is unknown whether there are common marker genes significant for SLE but independent of sample types, which may have potentials for follow-up translational research. The aim of this study is to identify common marker genes across various sample types for SLE. Methods Based on four public microarray gene expression datasets for SLE covering three representative types of blood-born samples (monocyte; peripheral blood mononuclear cell, PBMC; whole blood), we utilized three statistics (fold-change, FC; t-test p value; false discovery rate adjusted p value) to scrutinize genes simultaneously regulated with SLE across various sample types. For common marker genes, we conducted the Gene Ontology enrichment analysis and Protein-Protein Interaction analysis to gain insights into their functions. Results We identified 10 common marker genes associated with SLE (IFI6, IFI27, IFI44L, OAS1, OAS2, EIF2AK2, PLSCR1, STAT1, RNASE2, and GSTO1). Significant up-regulation of IFI6, IFI27, and IFI44L with SLE was observed in all the studied sample types, though the FC was most striking in monocyte, compared with PBMC and whole blood (8.82–251.66 vs. 3.73–74.05 vs. 1.19–1.87). Eight of the above 10 genes, except RNASE2 and GSTO1, interact with each other and with known SLE susceptibility genes, participate in immune response, RNA and protein catabolism, and cell death. Conclusion Our data suggest that there exist common marker genes across various sample types for SLE. The 10 common marker genes, identified herein, deserve follow-up studies to dissert their potentials as diagnostic or therapeutic markers to predict SLE or treatment response. PMID:27257790

  13. Identification of uterine leiomyoma-specific marker genes based on DNA methylation and their clinical application.

    PubMed

    Sato, Shun; Maekawa, Ryo; Yamagata, Yoshiaki; Tamura, Isao; Lee, Lifa; Okada, Maki; Jozaki, Kosuke; Asada, Hiromi; Tamura, Hiroshi; Sugino, Norihiro

    2016-01-01

    Differential diagnosis of uterine leiomyomas and leiomyosarcomas is needed to determine whether the uterus can be retained. Therefore, biomarkers for uterine leiomyomas, and reliable and objective diagnostic methods have been desired besides the pathological diagnosis. In the present study, we identified 12 genes specific to uterine leiomyomas based on DNA methylation. Using these marker genes specific to uterine leiomyomas, we established a hierarchical clustering system based on the DNA methylation level of the marker genes, which could completely differentiate between uterine leiomyomas and normal myometrium. Furthermore, our hierarchical clustering system completely discriminated uterine cancers and differentiated between uterine leiomyosarcomas and leiomyomas with more than 70% accuracy. In conclusion, this study identified DNA methylation-based marker genes specific to uterine leiomyomas, and our hierarchical clustering system using these marker genes was useful for differential diagnosis of uterine leiomyomas and leiomyosarcomas. PMID:27498619

  14. Identification of uterine leiomyoma-specific marker genes based on DNA methylation and their clinical application

    PubMed Central

    Sato, Shun; Maekawa, Ryo; Yamagata, Yoshiaki; Tamura, Isao; Lee, Lifa; Okada, Maki; Jozaki, Kosuke; Asada, Hiromi; Tamura, Hiroshi; Sugino, Norihiro

    2016-01-01

    Differential diagnosis of uterine leiomyomas and leiomyosarcomas is needed to determine whether the uterus can be retained. Therefore, biomarkers for uterine leiomyomas, and reliable and objective diagnostic methods have been desired besides the pathological diagnosis. In the present study, we identified 12 genes specific to uterine leiomyomas based on DNA methylation. Using these marker genes specific to uterine leiomyomas, we established a hierarchical clustering system based on the DNA methylation level of the marker genes, which could completely differentiate between uterine leiomyomas and normal myometrium. Furthermore, our hierarchical clustering system completely discriminated uterine cancers and differentiated between uterine leiomyosarcomas and leiomyomas with more than 70% accuracy. In conclusion, this study identified DNA methylation-based marker genes specific to uterine leiomyomas, and our hierarchical clustering system using these marker genes was useful for differential diagnosis of uterine leiomyomas and leiomyosarcomas. PMID:27498619

  15. Trans, trans-farnesol as a mevalonate-derived inducer of murine 3T3-F442A pre-adipocyte differentiation

    PubMed Central

    Torabi, Sheida

    2015-01-01

    Based on our finding that depletion of mevalonate-derived metabolites inhibits adipocyte differentiation, we hypothesize that trans, trans-farnesol (farnesol), a mevalonate-derived sesquiterpene, induces adipocyte differentiation. Farnesol dose-dependently (25–75 μmol/L) increased intracellular triglyceride content of murine 3T3-F442A pre-adipocytes measured by AdipoRed™ Assay and Oil Red-O staining. Concomitantly, farnesol dose-dependently increased glucose uptake and glucose transport protein 4 (GLUT4) expression without affecting cell viability. Furthermore, quantitative real-time polymerase chain reaction and Western blot showed that farnesol increased the mRNA and protein levels of peroxisome proliferator-activated receptor γ (PPARγ), a key regulator of adipocyte differentiation, and the mRNA levels of PPARγ-regulated fatty acid-binding protein 4 and adiponectin; in contrast, farnesol downregulated Pref-1 gene, a marker of pre-adipocytes. GW9662 (10 µmol/L), an antagonist of PPARγ, reversed the effects of farnesol on cellular lipid content, suggesting that PPARγ signaling pathway may mediate the farnesol effect. Farnesol (25–75 μmol/L) did not affect the mRNA level of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the rate-limiting enzyme in the mevalonate pathway. Farnesol may be the mevalonate-derived inducer of adipocyte differentiation and potentially an insulin sensitizer via activation of PPARγ and upregulation of glucose uptake. PMID:26660152

  16. Apramycin resistance as a selective marker for gene transfer in mycobacteria.

    PubMed Central

    Paget, E; Davies, J

    1996-01-01

    We have explored the potential of using the apramycin resistance gene as a marker in mycobacterial gene transfer studies. Shuttle plasmids available for both electroporation and conjugation studies have been constructed, and we have successfully validated the use of the apramycin resistance gene as a component of cloning vectors for Mycobacterium smegmatis, M. bovis BCG, and M. tuberculosis. PMID:8892841

  17. The mineralocorticoid receptor mediates aldosterone-induced differentiation of T37i cells into brown adipocytes.

    PubMed

    Penfornis, P; Viengchareun, S; Le Menuet, D; Cluzeaud, F; Zennaro, M C; Lombès, M

    2000-08-01

    By use of targeted oncogenesis, a brown adipocyte cell line was derived from a hibernoma of a transgenic mouse carrying the proximal promoter of the human mineralocorticoid receptor (MR) linked to the SV40 large T antigen. T37i cells remain capable of differentiating into brown adipocytes upon insulin and triiodothyronine treatment as judged by their ability to express uncoupling protein 1 and maintain MR expression. Aldosterone treatment of undifferentiated cells induced accumulation of intracytoplasmic lipid droplets and mitochondria. This effect was accompanied by a significant and dose-dependent increase in intracellular triglyceride content (half-maximally effective dose 10(-9) M) and involved MR, because it was unaffected by RU-38486 treatment but was totally abolished in the presence of aldosterone antagonists (spironolactone, RU-26752). The expression of early adipogenic gene markers, such as lipoprotein lipase, peroxisome proliferator-activated receptor-gamma, and adipocyte-specific fatty acid binding protein 2, was enhanced by aldosterone, confirming activation of the differentiation process. We demonstrate that, in the T37i cell line, aldosterone participates in the very early induction of brown adipocyte differentiation. Our findings may have a broader biological significance and suggest that MR is not only implicated in maintaining electrolyte homeostasis but could also play a role in metabolism and energy balance.

  18. Novel flow cytometric approach for the detection of adipocyte subpopulations during adipogenesis[S

    PubMed Central

    Durandt, Chrisna; van Vollenstee, Fiona A.; Dessels, Carla; Kallmeyer, Karlien; de Villiers, Danielle; Murdoch, Candice; Potgieter, Marnie; Pepper, Michael S.

    2016-01-01

    The ability of mesenchymal stromal cells (MSCs) to differentiate into adipocytes provides a cellular model of human origin to study adipogenesis in vitro. One of the major challenges in studying adipogenesis is the lack of tools to identify and monitor the differentiation of various subpopulations within the heterogeneous pool of MSCs. Cluster of differentiation (CD)36 plays an important role in the formation of intracellular lipid droplets, a key characteristic of adipocyte differentiation/maturation. The objective of this study was to develop a reproducible quantitative method to study adipocyte differentiation by comparing two lipophilic dyes [Nile Red (NR) and Bodipy 493/503] in combination with CD36 surface marker staining. We identified a subpopulation of adipose-derived stromal cells that express CD36 at intermediate/high levels and show that combining CD36 cell surface staining with neutral lipid-specific staining allows us to monitor differentiation of adipose-derived stromal cells that express CD36intermediate/high during adipocyte differentiation in vitro. The gradual increase of CD36intermediate/high/NRpositive cells during the 21 day adipogenesis induction period correlated with upregulation of adipogenesis-associated gene expression. PMID:26830859

  19. Cadmium modulates adipocyte functions in metallothionein-null mice

    SciTech Connect

    Kawakami, Takashige; Nishiyama, Kaori; Kadota, Yoshito; Sato, Masao; Inoue, Masahisa; Suzuki, Shinya

    2013-11-01

    Our previous study has demonstrated that exposure to cadmium (Cd), a toxic heavy metal, causes a reduction of adipocyte size and the modulation of adipokine expression. To further investigate the significance of the Cd action, we studied the effect of Cd on the white adipose tissue (WAT) of metallothionein null (MT{sup −/−}) mice, which cannot form atoxic Cd–MT complexes and are used for evaluating Cd as free ions, and wild type (MT{sup +/+}) mice. Cd administration more significantly reduced the adipocyte size of MT{sup −/−} mice than that of MT{sup +/+} mice. Cd exposure also induced macrophage recruitment to WAT with an increase in the expression level of Ccl2 (MCP-1) in the MT{sup −/−} mice. The in vitro exposure of Cd to adipocytes induce triglyceride release into culture medium, decrease in the expression levels of genes involved in fatty acid synthesis and lipid hydrolysis at 24 h, and at 48 h increase in phosphorylation of the lipid-droplet-associated protein perilipin, which facilitates the degradation of stored lipids in adipocytes. Therefore, the reduction in adipocyte size by Cd may arise from an imbalance between lipid synthesis and lipolysis. In addition, the expression levels of leptin, adiponectin and resistin decreased in adipocytes. Taken together, exposure to Cd may induce unusually small adipocytes and modulate the expression of adipokines differently from the case of physiologically small adipocytes, and may accelerate the risk of developing insulin resistance and type 2 diabetes. - Highlights: • Cd causes a marked reduction in adipocyte size in MT-null mice. • Cd enhances macrophage migration into adipose tissue and disrupt adipokine secretion. • MT gene alleviates Cd-induced adipocyte dysfunctions. • Cd enhances the degradation of stored lipids in adipocytes, mediated by perilipin. • Cd induces unusually small adipocytes and the abnormal expression of adipokines.

  20. Candidate Genes from Molecular Pathways Related to Appetite Regulatory Neural Network and Adipocyte Homeostasis and Obesity: the Coronary Artery Risk Development in Young Adults (CARDIA) Study

    PubMed Central

    Friedlander, Yechiel; Li, Guo; Fornage, Myriam; Williams, O. Dale; Lewis, Cora E.; Schreiner, Pamela; Pletcher, Mark J.; Enquobahrie, Daniel; Williams, Michelle; Siscovick, David S.

    2010-01-01

    Background Appetite regulatory neural network and adipocyte homeostasis molecular pathways are critical to long-term weight maintenance. Genetic variation in these pathways may explain variability of obesity in the general population. Aims The associations of four genes in these pathways (leptin (LEP), leptin receptor (LEPR), neuropeptide Y2 receptor (NPY2R) and peptide YY (PYY)) with obesity-related phenotypes were examined among participants in the CARDIA Study. Participants were 18-30 years old upon recruitment (1985-86). Weight, BMI and waist circumference were measured at baseline and at years 2, 5, 7, 10, 15, and 20. Genotyping was conducted using tag SNPs that characterize the common pattern of genetic variation in these genes. Race-specific linear regression models were used to examine associations of the various SNPs with obesity-related measurements, controlling for sex and age. The overall association based on the 7 repeated anthropometric measurements was tested with GEE. False discovery rate was used to adjust for multiple testing. Results In African-Americans, SNPs across the LEP gene demonstrated significant overall associations with obesity-related phenotypes. The associations between rs17151919 in LEP gene with weight tended to increase with time (SNP × time interaction p=0.0193). The difference in weight levels associated with each additional minor allele ranged from 2.6 kg at entry to 4.8 kg at year 20. Among African-American men, the global tests indicated that SNPs across the NPY2R gene were also associated with waist circumference measurements (p=0.0462). In Caucasians, SNPs across the LEP gene also tended to be associated with weight measurements (p=0.0471) and rs11684664 in PYY gene was associated with obesity-related phenotypes (p= 0.010-0.026) in women only. Conclusions Several SNPs in the LEP, NPY2R and PYY but not the LEPR genes were associated with obesity-related phenotypes in young adults. The associations were more prominent for the

  1. Oral administration of Lactobacillus plantarum 299v modulates gene expression in the ileum of pigs: prediction of crosstalk between intestinal immune cells and sub-mucosal adipocytes.

    PubMed

    Hulst, Marcel; Gross, Gabriele; Liu, Yaping; Hoekman, Arjan; Niewold, Theo; van der Meulen, Jan; Smits, Mari

    2015-05-01

    To study host-probiotic interactions in parts of the intestine only accessible in humans by surgery (jejunum, ileum and colon), pigs were used as model for humans. Groups of eight 6-week-old pigs were repeatedly orally administered with 5 × 10(12) CFU Lactobacillus plantarum 299v (L. plantarum 299v) or PBS, starting with a single dose followed by three consecutive daily dosings 10 days later. Gene expression was assessed with pooled RNA samples isolated from jejunum, ileum and colon scrapings of the eight pigs per group using Affymetrix porcine microarrays. Comparison of gene expression profiles recorded from L. plantarum 299v-treated pigs with PBS-treated pigs indicated that L. plantarum 299v affected metabolic and immunological processes, particularly in the ileum. A higher expression level of several B cell-specific transcription factors/regulators was observed, suggesting that an influx of B cells from the periphery to the ileum and/or the proliferation of progenitor B cells to IgA-committed plasma cells in the Peyer's patches of the ileum was stimulated. Genes coding for enzymes that metabolize leukotriene B4, 1,25-dihydroxyvitamin D3 and steroids were regulated in the ileum. Bioinformatics analysis predicted that these metabolites may play a role in the crosstalk between intestinal immune cells and sub-mucosal adipocytes. Together with regulation of genes that repress NFKB- and PPARG-mediated transcription, this crosstalk may contribute to tempering of inflammatory reactions. Furthermore, the enzyme adenosine deaminase, responsible for the breakdown of the anti-inflammatory mediator adenosine, was strongly down-regulated in response to L. plantarum 299v. This suggested that L. plantarum 299v-regulated production of adenosine by immune cells like regulatory T cells may also be a mechanism that tempers inflammation in the ileum, and perhaps also in other parts of the pig's body. PMID:25861755

  2. Decelerating Mature Adipocyte Dedifferentiation by Media Composition.

    PubMed

    Huber, Birgit; Kluger, Petra J

    2015-12-01

    The establishment of adipose tissue test systems is still a major challenge in the investigation of cellular and molecular interactions responsible for the pathogenesis of inflammatory diseases involving adipose tissue. Mature adipocytes are mainly involved in these pathologies, but rarely used in vitro, due to the lack of an appropriate culture medium which inhibits dedifferentiation and maintains adipocyte functionality. In our study, we showed that Dulbecco's Modified Eagle's Medium/Ham's F-12 with 10% fetal calf serum (FCS) reported for the culture of mature adipocytes favors dedifferentiation, which was accompanied by a high glycerol release, a decreasing release of leptin, and a low expression of the adipocyte marker perilipin A, but high expression of CD73 after 21 days. Optimized media containing FCS, biotin, pantothenate, insulin, and dexamethasone decelerated the dedifferentiation process. These cells showed a lower lipolysis rate, a high level of leptin release, as well as a high expression of perilipin A. CD73-positive dedifferentiated fat cells were only found in low quantity. In this work, we showed that mature adipocytes when cultured under optimized conditions could be highly valuable for adipose tissue engineering in vitro. PMID:26228997

  3. Opportunities of marker-assisted selection for rice fragrance through marker-trait association analysis of microsatellites and gene-based markers.

    PubMed

    Golestan Hashemi, F S; Rafii, M Y; Razi Ismail, M; Mohamed, M T M; Rahim, H A; Latif, M A; Aslani, F

    2015-09-01

    Developing fragrant rice through marker-assisted/aided selection (MAS) is an economical and profitable approach worldwide for the enrichment of an elite genetic background with a pleasant aroma. The PCR-based DNA markers that distinguish the alleles of major fragrance genes in rice have been synthesised to develop rice scent biofortification through MAS. Thus, the present study examined the aroma biofortification potential of these co-dominant markers in a germplasm panel of 189 F2 progeny developed from crosses between a non-aromatic variety (MR84) and a highly aromatic but low-yielding variety (MRQ74) to determine the most influential diagnostic markers for fragrance biofortification. The SSRs and functional DNA markers RM5633 (on chromosome 4), RM515, RM223, L06, NKSbad2, FMbadh2-E7, BADEX7-5, Aro7 and SCU015RM (on chromosome 8) were highly associated with the 2AP (2-acetyl-1-pyrroline) content across the population. The alleles traced via these markers were also in high linkage disequilibrium (R(2) > 0.70) and explained approximately 12.1, 27.05, 27.05, 27.05, 25.42, 25.42, 20.53, 20.43 and 20.18% of the total phenotypic variation observed for these biomarkers, respectively. F2 plants harbouring the favourable alleles of these effective markers produced higher levels of fragrance. Hence, these rice plants can be used as donor parents to increase the development of fragrance-biofortified tropical rice varieties adapted to growing conditions and consumer preferences, thus contributing to the global rice market. PMID:25865409

  4. Quality Control of Genotypes Using Heritability Estimates of Gene Content at the Marker

    PubMed Central

    Forneris, Natalia S.; Legarra, Andres; Vitezica, Zulma G.; Tsuruta, Shogo; Aguilar, Ignacio; Misztal, Ignacy; Cantet, Rodolfo J. C.

    2015-01-01

    Quality control filtering of single-nucleotide polymorphisms (SNPs) is a key step when analyzing genomic data. Here we present a practical method to identify low-quality SNPs, meaning markers whose genotypes are wrongly assigned for a large proportion of individuals, by estimating the heritability of gene content at each marker, where gene content is the number of copies of a particular reference allele in a genotype of an animal (0, 1, or 2). If there is no mutation at the marker, gene content has an additive heritability of 1 by construction. The method uses restricted maximum likelihood (REML) to estimate heritability of gene content at each SNP and also builds a likelihood-ratio test statistic to test for zero error variance in genotyping. As a by-product, estimates of the allele frequencies of markers at the base population are obtained. Using simulated data with 10% permutation error (4% actual error) in genotyping, the method had a specificity of 0.96 (4% of correct markers are rejected) and a sensitivity of 0.99 (1% of wrong markers are accepted) if markers with heritability lower than 0.975 are discarded. Checking of Mendelian errors resulted in a lower sensitivity (0.84) for the same simulation. The proposed method is further illustrated with a real data set with genotypes from 3534 animals genotyped for 50,433 markers from the Illumina PorcineSNP60 chip and a pedigree of 6473 individuals; those markers underwent very little quality control. A total of 4099 markers with P-values lower than 0.01 were discarded based on our method, with associated estimates of heritability as low as 0.12. Contrary to other techniques, our method uses all information in the population simultaneously, can be used in any population with markers and pedigree recordings, and is simple to implement using standard software for REML estimation. Scripts for its use are provided. PMID:25567991

  5. Seamless Genome Editing in Rice via Gene Targeting and Precise Marker Elimination.

    PubMed

    Nishizawa-Yokoi, Ayako; Saika, Hiroaki; Toki, Seiichi

    2016-01-01

    Positive-negative selection using hygromycin phosphotransferase (hpt) and diphtheria toxin A-fragment (DT-A) as positive and negative selection markers, respectively, allows enrichment of cells harboring target genes modified via gene targeting (GT). We have developed a successful GT system employing positive-negative selection and subsequent precise marker excision via the piggyBac transposon derived from the cabbage looper moth to introduce desired modifications into target genes in the rice genome. This approach could be applied to the precision genome editing of almost all endogenous genes throughout the genome, at least in rice. PMID:27557691

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

    SciTech Connect

    Nagasaki, Haruka; Yoshimura, Takeshi; Aoki, Naohito

    2012-04-13

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

  7. High throughput genome-specific and gene-specific molecular markers for erucic acid genes in Brassica napus (L.) for marker-assisted selection in plant breeding.

    PubMed

    Rahman, Mukhlesur; Sun, Zudong; McVetty, Peter B E; Li, Genyi

    2008-10-01

    A single base change in the Bn-FAE1.1 gene in the A genome and a two-base deletion in the Bn-FAE1.2 gene in the C genome produce the nearly zero content of erucic acid observed in canola. A BAC clone anchoring Bn-FAE1.1 from a B. rapa BAC library and a BAC clone anchoring Bn-FAE1.2 from a B. oleracea BAC library were used in this research. After sequencing the gene flanking regions, it was found that the dissimilarity of the flanking sequences of these two FAE1 homologs facilitated the design of genome-specific primers that could amplify the corresponding genome in allotetraploid B. napus. The two-base deletion in the C genome gene was detected as a sequence-characterized amplified region (SCAR) marker. To increase the throughput, one genome-specific primer was labeled with four fluorescence dyes and combined with 20 different primers to produce PCR products with different fragment sizes. Eventually, a super pool of 80 samples was detected simultaneously. This dramatically reduces the cost of marker detection. The single base change in the Bn-FAE1.1 gene was detected as single nucleotide polymorphic (SNP) marker with an ABI SNaPshot kit. A multiplexing primer set was designed by adding a polyT to the 5' primer end to increase SNP detection throughput through sample pooling. Furthermore, the Bn-FAE1.1 and Bn-FAE1.2 were integrated into the N8 and N13 linkage groups of our previously reported high-density sequence-related amplified polymorphism (SRAP) map, respectively. There were 124 SRAP markers in a N8 bin in which the Bn-FAE1.1 gene-specific SCAR marker was located and 46 SRAP markers in a N13 bin into which the Bn-FAE1.2 SNP marker was integrated. These three kinds of high throughput molecular markers have been successfully implemented in our canola/rapeseed breeding programs.

  8. Genomic distribution of AFLP markers relative to gene locations for different eukaryotic species

    PubMed Central

    2013-01-01

    Background Amplified fragment length polymorphism (AFLP) markers are frequently used for a wide range of studies, such as genome-wide mapping, population genetic diversity estimation, hybridization and introgression studies, phylogenetic analyses, and detection of signatures of selection. An important issue to be addressed for some of these fields is the distribution of the markers across the genome, particularly in relation to gene sequences. Results Using in-silico restriction fragment analysis of the genomes of nine eukaryotic species we characterise the distribution of AFLP fragments across the genome and, particularly, in relation to gene locations. First, we identify the physical position of markers across the chromosomes of all species. An observed accumulation of fragments around (peri) centromeric regions in some species is produced by repeated sequences, and this accumulation disappears when AFLP bands rather than fragments are considered. Second, we calculate the percentage of AFLP markers positioned within gene sequences. For the typical EcoRI/MseI enzyme pair, this ranges between 28 and 87% and is usually larger than that expected by chance because of the higher GC content of gene sequences relative to intergenic ones. In agreement with this, the use of enzyme pairs with GC-rich restriction sites substantially increases the above percentages. For example, using the enzyme system SacI/HpaII, 86% of AFLP markers are located within gene sequences in A. thaliana, and 100% of markers in Plasmodium falciparun. We further find that for a typical trait controlled by 50 genes of average size, if 1000 AFLPs are used in a study, the number of those within 1 kb distance from any of the genes would be only about 1–2, and only about 50% of the genes would have markers within that distance. Conclusions The high coverage of AFLP markers across the genomes and the high proportion of markers within or close to gene sequences make them suitable for genome scans and

  9. RPL13A and EEF1A1 Are Suitable Reference Genes for qPCR during Adipocyte Differentiation of Vascular Stromal Cells from Patients with Different BMI and HOMA-IR

    PubMed Central

    Gentile, Adriana-Mariel; Lhamyani, Said; Coín-Aragüez, Leticia; Oliva-Olivera, Wilfredo; Zayed, Hatem; Vega-Rioja, Antonio; Monteseirin, Javier; Romero-Zerbo, Silvana-Yanina; Tinahones, Francisco-José; Bermúdez-Silva, Francisco-Javier; El Bekay, Rajaa

    2016-01-01

    Real-time or quantitative PCR (qPCR) is a useful technique that requires reliable reference genes for data normalization in gene expression analysis. Adipogenesis is among the biological processes suitable for this technique. The selection of adequate reference genes is essential for qPCR gene expression analysis of human Vascular Stromal Cells (hVSCs) during their differentiation into adipocytes. To the best of our knowledge, there are no studies validating reference genes for the analyses of visceral and subcutaneous adipose tissue hVSCs from subjects with different Body Mass Index (BMI) and Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) index. The present study was undertaken to analyze this question. We first analyzed the stability of expression of five potential reference genes: CYC, GAPDH, RPL13A, EEF1A1, and 18S ribosomal RNA, during in vitro adipogenic differentiation, in samples from these types of patients. The expression of RPL13A and EEF1A1 was not affected by differentiation, thus being these genes the most stable candidates, while CYC, GAPDH, and 18S were not suitable for this sort of analysis. This work highlights that RPL13A and EEF1A1 are good candidates as reference genes for qPCR analysis of hVSCs differentiation into adipocytes from subjects with different BMI and HOMA-IR. PMID:27304673

  10. Development of genomic SSR markers for fingerprinting lettuce (Lactuca sativa L.) cultivars and mapping genes

    PubMed Central

    2013-01-01

    Background Lettuce (Lactuca sativa L.) is the major crop from the group of leafy vegetables. Several types of molecular markers were developed that are effectively used in lettuce breeding and genetic studies. However only a very limited number of microsattelite-based markers are publicly available. We have employed the method of enriched microsatellite libraries to develop 97 genomic SSR markers. Results Testing of newly developed markers on a set of 36 Lactuca accession (33 L. sativa, and one of each L. serriola L., L. saligna L., and L. virosa L.) revealed that both the genetic heterozygosity (UHe = 0.56) and the number of loci per SSR (Na = 5.50) are significantly higher for genomic SSR markers than for previously developed EST-based SSR markers (UHe = 0.32, Na = 3.56). Fifty-four genomic SSR markers were placed on the molecular linkage map of lettuce. Distribution of markers in the genome appeared to be random, with the exception of possible cluster on linkage group 6. Any combination of 32 genomic SSRs was able to distinguish genotypes of all 36 accessions. Fourteen of newly developed SSR markers originate from fragments with high sequence similarity to resistance gene candidates (RGCs) and RGC pseudogenes. Analysis of molecular variance (AMOVA) of L. sativa accessions showed that approximately 3% of genetic diversity was within accessions, 79% among accessions, and 18% among horticultural types. Conclusions The newly developed genomic SSR markers were added to the pool of previously developed EST-SSRs markers. These two types of SSR-based markers provide useful tools for lettuce cultivar fingerprinting, development of integrated molecular linkage maps, and mapping of genes. PMID:23339733

  11. [The adipocyte, prodigious cell].

    PubMed

    Domínguez Carmona, Manuel

    2005-01-01

    In this work, I stand out the rich endocrine role of adipocytes, that together with its function of lipidic deposit and regulating of metabolism, this confers them a central place in physiology and pathology.

  12. Markers for host-induced gene expression in Trichophyton dermatophytosis.

    PubMed

    Kaufman, Gil; Berdicevsky, Israela; Woodfolk, Judith A; Horwitz, Benjamin A

    2005-10-01

    Dermatophytes are adapted to infect keratinized tissues by their ability to utilize keratin as a nutrient source. Although there have been numerous reports that dermatophytes like Trichophyton sp. secrete proteolytic enzymes, virtually nothing is known about the patterns of gene expression in the host or even when the organisms are cultured on protein substrates in the absence of a host. We characterized the expression of an aminopeptidase gene, the Trichophyton mentagrophytes homolog of the Trichophyton rubrum Tri r 4 gene. The T. rubrum gene was originally isolated based on the ability of the protein encoded by it to induce immediate and delayed-type hypersensitivity in skin tests. T. mentagrophytes Tri m 4 is closely related to Tri r 4 (almost 94% identity at the protein level). Tri m 4 resembles other protease-encoding genes thought to be virulence factors (for example, DPP V of Aspergillus fumigatus). The Tri m 4 protein was detected immunochemically both in fungal extracts and in the culture medium. Expression of the Tri m 4 gene was induced severalfold when T. mentagrophytes was grown on keratin and elastin. Ex vivo, strong induction was observed after culture on blood plasma, but the use of homogenized skin did not result in a significant increase in Tri m 4 transcript levels. In order to identify additional genes encoding putative virulence factors, differential cDNA screening was performed. By this method, a fungal thioredoxin and a cellulase homolog were identified, and both genes were found to be strongly induced by skin extracellular matrix proteins. Induction by superficial (keratin) and deep (elastin) skin elements suggests that the products of these genes may be important in both superficial and deep dermatophytosis, and models for their function are proposed. Upregulation of several newly identified T. mentagrophytes genes on protein substrates suggests that these genes encode proteins which are relevant to the dermatophyte-skin interaction.

  13. Doses of Quercetin in the Range of Serum Concentrations Exert Delipidating Effects in 3T3-L1 Preadipocytes by Acting on Different Stages of Adipogenesis, but Not in Mature Adipocytes

    PubMed Central

    Eseberri, Itziar; Miranda, Jonatan; Lasa, Arrate; Churruca, Itziar; Portillo, María P.

    2015-01-01

    Scope. To determine whether doses of quercetin in the range of serum concentrations exert any effect on triacylglycerol accumulation in maturing preadipocytes and mature adipocytes. The influence on the expression of adipogenic markers as well as on gene expression and activity of enzymes involved in triacylglycerol metabolism were assessed. Methods and Results. 3T3-L1 preadipocytes were treated during differentiation and mature adipocytes for 24 hours with low doses (0.1–10 µM) of quercetin. Triacylglycerol content in both cell types and free fatty acid and glycerol in the incubation medium of mature adipocytes were measured spectrophotometrically. Gene and protein expression were assessed by RT-PCR and Western blot. LPL and FAS activities were quantified. During differentiation quercetin reduced triacylglycerol content at doses from 0.5 to 10 µM. 1 µM of quercetin reduced C/EBPβ gene expression, SREBP1 mature protein levels, and PPARγ gene expression. 10 µM of quercetin reduced LPL gene expression and PPARγ and SREBP1c expression. In mature adipocytes, only 10 µM of quercetin reduced triacylglycerol content. Lipogenic FAS expression and activity were reduced at this dose. Conclusion. Quercetin, in the range of serum concentrations, is able to inhibit adipogenesis, but higher doses, at least 10 µM, are needed to reduce fat accumulation in mature adipocytes. PMID:26180590

  14. SNP discovery and marker development for disease resistance candidate genes in common carp (Cyprinus carpio)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Single nucleotide polymorphisms (SNPs) in immune response genes have been reported as markers of susceptibility to infectious diseases in human and livestock. A disease caused by cyprinid herpes virus 3 (CyHV-3) is highly contagious and virulent in common carp. With the aim to investigate the gene...

  15. Establishment of codominant markers for rice blast resistance gene Pi-ta

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A single nucleotide length polymorphism (SNLP) was identified at the intron region of the Pi-ta gene to develop a codominant Pi-ta gene marker suitable for genotyping with an ABI automated machine. The DNA primer specific to the resistance Pi-ta allele was labeled with the blue dye as a forward pr...

  16. Establishment of codominant marker for rice blast resistance gene pi-ta

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A single nucleotide length polymorphism (SNLP) was identified at the intron region of the Pi-ta gene to develop a codominant Pi-ta gene marker suitable for genotyping with an ABI automated machine. The DNA primer specific to the resistance Pi-ta allele was labeled with the blue dye as a forward pr...

  17. Tropomyosin is a nice marker gene for phylogenetic analysis of molluscs.

    PubMed

    Wang, Xiaotong; Li, Li; Xu, Fei; Zhang, Guofan

    2011-10-01

    Molluscs are an extraordinarily diverse group of animals and to discriminate them based on one molecular marker/gene is very difficult because of the too fast or slow rate of nucleotide substitution. In the study, the tropomyosin cds (coding sequences) of 43 animal species were analyzed, the results of which suggested that the tropomyosin gene was a nice marker gene to phylogenetic analysis of molluscs, even for all the studied animals. In addition, InDels (insertions and deletions) in tropomyosin cds of turbo cornutus were also studied and one segment repeat, which probably happened recently and was of functional importance, was found.

  18. With current gene markers, presymptomatic diagnosis of heritable disease is still a family affair

    SciTech Connect

    Not Available

    1987-09-04

    In the last four years, genes or genetic markers have been identified for a host of disorders including Huntington's disease, cystic fibrosis, Duchenne muscular dystrophy, polycystic kidney disease, bipolar depressive disorder, retinoblastoma, Alzheimer's disease, and schizophrenia. Such discoveries have made it possible to diagnose in utero some 30 genetic diseases during the first trimester of pregnancy. Yet, while these newly discovered gene markers may be revolutionizing prenatal and presymptomatic diagnosis, they are in many respects halfway technology. Such was the opinion of several speakers at a conference sponsored by the American Medical Association in Washington, DC. At the conference, entitled DNA Probes in the Practice of Medicine, geneticists emphasized that gene markers - stretches of DNA that are usually inherited in tandem with a disease gene - are usually not sufficient for presymptomatic diagnosis of genetic disease in an individual.

  19. Dynamics of protein secretion during adipocyte differentiation.

    PubMed

    Ojima, Koichi; Oe, Mika; Nakajima, Ikuyo; Muroya, Susumu; Nishimura, Takanori

    2016-08-01

    The major functions of adipocytes include both lipid storage and the production of secretory factors. However, the type of proteins released from mouse 3T3-L1 cells during adipocyte differentiation remains poorly understood. We examined the dynamics of secreted proteins during adipocyte differentiation using mass spectrometry (MS) combined with an iTRAQ (®) labeling method that enables the simultaneous analysis of relative protein expression levels. A total of 215 proteins were identified and quantified from approximately 10 000 MS/MS spectra. Of these, approximately 38% were categorized as secreted proteins based on gene ontology classification. Adipokine secretion levels were increased with the progression of differentiation. By contrast, levels of fibril collagen components, such as subunits of type I and III collagens, were decreased during differentiation. Basement membrane components attained their peak levels at day 4 when small lipid droplets accumulated in differentiated 3T3-L1 cells. Simultaneously, peak levels of collagen microfibril components that comprise type V and VI collagen subunits were also observed. Our data demonstrated that extracellular matrix components were predominantly released during the early and middle stages of adipocyte differentiation, with a subsequent increase in the secretion of adipokines. This suggests that 3T3-L1 cells secrete adipokines after their ECM is constructed during adipocyte differentiation. PMID:27516960

  20. Insights into an adipocyte whitening program

    PubMed Central

    Hill, Bradford G

    2015-01-01

    White adipose tissue plays a critical role in regulating systemic metabolism and can remodel rapidly in response to changes in nutrient availability. Nevertheless, little is known regarding the metabolic changes occurring in adipocytes during obesity. Our laboratory recently addressed this issue in a commonly used, high-fat-diet mouse model of obesity. We found remarkable changes in adipocyte metabolism that occur prior to infiltration of macrophages in expanding adipose tissue. Results of metabolomic analyses, adipose tissue respirometry, electron microscopy, and expression analyses of key genes and proteins revealed dysregulation of several metabolic pathways, loss of mitochondrial biogenetic capacity, and apparent activation of mitochondrial autophagy which were followed in time by downregulation of numerous mitochondrial proteins important for maintaining oxidative capacity. These findings demonstrate the presence of an adipocyte whitening program that may be critical for regulating adipose tissue remodeling under conditions of chronic nutrient excess. PMID:26167407

  1. C/EBPα gene as a genetic marker for beef quality improvement.

    PubMed

    Adoligbe, C; Huangfu, Y F; Zan, L S; Wang, H

    2015-08-14

    Intramuscular fat (IMF) or intramuscular triglycerides are interspersed throughout the skeletal muscles. The IMF, also called marbling, imparts meat with flavor and juiciness and is one of the core criteria for judging carcass value. The quantity of IMF is influenced entirely by genetics. Recently, understanding the underlying genetic bases of IMF has been a focus particularly in the beef industry. In this study, with the deep insights of ameliorating the beef quality by genetic means, the role of the CCAAT/enhancer binding protein alpha (C/EBPα) gene was investigated by over-expressing C/EBPα in bovine muscle stem cells (MSCs) to initiate the adipogenic program. Prior to this, bovine MSCs were isolated and induced to differentiate into adipocytes from cells that were exposed to dexamethasone isobutylmethylxanthine and indomethacin; the presence of insulin and fetal bovine serum was examined. Either ectopic expression of C/EBPα or treatment with dexamethasone and insulin induced the accumulation of fat droplets and the expression of adipogenic induction genes (LPL, PPARγ, C/EBPβ, and C/EBPδ). The expression levels of myoblast-related genes (MyoD, Myf5, and Pax7) were also measured to assess the accuracy of the differentiation process. This study provides evidence that the C/EBPα gene is essential for cattle adipose tissue growth and development. Hence, this finding can contribute to improving beef carcass quality.

  2. Attainment of Brown Adipocyte Features in White Adipocytes of Hormone-Sensitive Lipase Null Mice

    PubMed Central

    Ström, Kristoffer; Hansson, Ola; Lucas, Stéphanie; Nevsten, Pernilla; Fernandez, Céline; Klint, Cecilia; Movérare-Skrtic, Sofia; Sundler, Frank; Ohlsson, Claes; Holm, Cecilia

    2008-01-01

    Background Hormone-sensitive lipase (HSL) is expressed predominantly in adipose tissue, where it plays an important role in catecholamine-stimulated hydrolysis of stored tri- and diglycerides, thus mobilizing fatty acids. HSL exhibits broad substrate specificity and besides acylglycerides it hydrolyzes cholesteryl esters, retinyl esters and lipoidal esters. Despite its role in fatty acid mobilization, HSL null mice have been shown to be resistant to diet-induced obesity. Methodology/Principal Findings Following a high-fat diet (HFD) regimen, energy expenditure, measured using indirect calorimetry, was increased in HSL null mice. White adipose tissue of HSL null mice was characterized by reduced mass and reduced protein expression of PPARγ, a key transcription factor in adipogenesis, and stearoyl-CoA desaturase 1, the expression of which is known to be positively correlated to the differentiation state of the adipocyte. The protein expression of uncoupling protein-1 (UCP-1), the highly specific marker of brown adipocytes, was increased 7-fold in white adipose tissue of HSL null mice compared to wildtype littermates. Transmission electron microscopy revealed an increase in the size of mitochondria of white adipocytes of HSL null mice. The mRNA expression of pRb and RIP140 was decreased in isolated white adipocytes, while the expression of UCP-1 and CPT1 was increased in HSL null mice compared to wildtype littermates. Basal oxygen consumption was increased almost 3-fold in white adipose tissue of HSL null mice and was accompanied by increased uncoupling activity. Conclusions These data suggest that HSL is involved in the determination of white versus brown adipocytes during adipocyte differentiation The exact mechanism(s) underlying this novel role of HSL remains to be elucidated, but it seems clear that HSL is required to sustain normal expression levels of pRb and RIP140, which both promote differentiation into the white, rather than the brown, adipocyte lineage

  3. Cardiac natriuretic peptides act via p38 MAPK to induce the brown fat thermogenic program in mouse and human adipocytes

    PubMed Central

    Bordicchia, Marica; Liu, Dianxin; Amri, Ez-Zoubir; Ailhaud, Gerard; Dessì-Fulgheri, Paolo; Zhang, Chaoying; Takahashi, Nobuyuki; Sarzani, Riccardo; Collins, Sheila

    2012-01-01

    The ability of mammals to resist body fat accumulation is linked to their ability to expand the number and activity of “brown adipocytes” within white fat depots. Activation of β-adrenergic receptors (β-ARs) can induce a functional “brown-like” adipocyte phenotype. As cardiac natriuretic peptides (NPs) and β-AR agonists are similarly potent at stimulating lipolysis in human adipocytes, we investigated whether NPs could induce human and mouse adipocytes to acquire brown adipocyte features, including a capacity for thermogenic energy expenditure mediated by uncoupling protein 1 (UCP1). In human adipocytes, atrial NP (ANP) and ventricular NP (BNP) activated PPARγ coactivator-1α (PGC-1α) and UCP1 expression, induced mitochondriogenesis, and increased uncoupled and total respiration. At low concentrations, ANP and β-AR agonists additively enhanced expression of brown fat and mitochondrial markers in a p38 MAPK–dependent manner. Mice exposed to cold temperatures had increased levels of circulating NPs as well as higher expression of NP signaling receptor and lower expression of the NP clearance receptor (Nprc) in brown adipose tissue (BAT) and white adipose tissue (WAT). NPR-C–/– mice had markedly smaller WAT and BAT depots but higher expression of thermogenic genes such as Ucp1. Infusion of BNP into mice robustly increased Ucp1 and Pgc-1α expression in WAT and BAT, with corresponding elevation of respiration and energy expenditure. These results suggest that NPs promote “browning” of white adipocytes to increase energy expenditure, defining the heart as a central regulator of adipose tissue biology. PMID:22307324

  4. Genetic linkage of the Huntington's disease gene to a DNA marker.

    PubMed

    Gusella, J F

    1984-11-01

    Recombinant DNA techniques have provided the means to generate large numbers of new genetic linkage markers. This technology has been used to identify a DNA marker that coinherits with the Huntington's Disease (HD) gene in family studies. The HD locus has thereby been mapped to human chromosome 4. The discovery of a genetic marker for the inheritance of HD has implications both for patient care and future research. The same approach holds considerable promise for the investigation of other genetic diseases, including Dystonia Musculorum Deformans.

  5. Cowden disease: gene marker studies and measurements of epidermal growth factor.

    PubMed Central

    Carlson, H E; Burns, T W; Davenport, S L; Luger, A M; Spence, M A; Sparkes, R S; Orth, D N

    1986-01-01

    Cowden disease (CD) is a familial syndrome characterized by tumors of the skin, oral mucosa, breast, thyroid, and intestinal epithelium. Since the syndrome is inherited as an autosomal dominant, we examined a battery of gene markers in a family with CD to detect linkage between the CD gene and known marker genes. There was no positive evidence for linkage of a CD locus with any of the markers; other investigators can add to our data to confirm and extend these findings. Additionally, we measured epidermal growth factor (EGF) in body fluids from CD patients and controls to determine if elevated EGF levels might be responsible for the widespread epithelial proliferation in CD. EGF levels in saliva, serum, plasma, and urine were similar in CD patients and control subjects. Although alterations in growth factors or their receptors may play a role in CD, excess circulating EGF is not responsible for the manifestations of the syndrome. Images Fig. 2 PMID:3487976

  6. PL1 fusion gene: a novel visual selectable marker gene that confers tolerance to multiple abiotic stresses in transgenic tomato.

    PubMed

    Jin, Feng; Li, Shu; Dang, Lijie; Chai, Wenting; Li, Pengli; Wang, Ning Ning

    2012-10-01

    Visual selectable markers, including the purple color caused by the accumulation of anthocyanins, have been proposed for use as antibiotic-free alternatives. However, the excessive accumulation of anthocyanins seriously inhibits the growth and development of transgenic plants. In our study, the AtDWF4 promoter from Arabidopsis and the tomato LeANT1 gene, encoding a MYB transcription factor, were used to construct the PL1 fusion gene to test whether it could be used as a visual selectable marker gene for tomato transformation. All the PL1 transgenic shoots exhibited intense purple color on shoot induction medium. In the transgenic tomato plants, PL1 was highly expressed in the cotyledons, but expressed only slightly in the true leaves and other organs. The expression of PL1 had no significantly adverse effects on the growth or development of the transgenic tomato plants, and conferred tolerance to multiple abiotic stresses in them. With the “cut off green shoots” method, multiple independent 35S::GFP transgenic tomato lines were successfully obtained using PL1 as the selectable marker gene. These results suggest that PL1 has potential application of visual selectable marker gene for tomato transformation.

  7. Endothelial Cell Surface Expressed Chemotaxis and Apoptosis Regulator (ECSCR) Regulates Lipolysis in White Adipocytes via the PTEN/AKT Signaling Pathway

    PubMed Central

    Kilari, Sreenivasulu; Cossette, Stephanie; Pooya, Shabnam; Bordas, Michelle; Huang, Yi-Wen

    2015-01-01

    Elevated plasma triglycerides are associated with increased susceptibility to heart disease and stroke, but the mechanisms behind this relationship are unclear. A clearer understanding of gene products which influence plasma triglycerides might help identify new therapeutic targets for these diseases. The Endothelial Cell Surface expressed Chemotaxis and apoptosis Regulator (ECSCR) was initially studied as an endothelial cell marker, but has recently been identified in white adipocytes, the primary storage cell type for triglycerides. Here we confirm ECSCR expression in white adipocytes and show that Ecscr knockout mice show elevated fasting plasma triglycerides. At a cellular level, cultured 3T3-L1 adipocytes silenced for Ecscr show a blunted Akt phosphorylation response. Additionally we show that the phosphatase and tensin homology containing (PTEN) lipid phosphatase association with ECSCR is increased by insulin stimulation. These data suggest a scenario by which ECSCR contributes to control of white adipocyte lipolysis. In this scenario, white adipocytes lacking Ecscr display elevated PTEN activity, thereby reducing AKT activation and impairing insulin-mediated suppression of lipolysis. Collectively, these results suggest that ECSCR plays a critical function in regulating lipolysis in white adipose tissue. PMID:26692198

  8. Advances in plant gene-targeted and functional markers: a review

    PubMed Central

    2013-01-01

    Public genomic databases have provided new directions for molecular marker development and initiated a shift in the types of PCR-based techniques commonly used in plant science. Alongside commonly used arbitrarily amplified DNA markers, other methods have been developed. Targeted fingerprinting marker techniques are based on the well-established practices of arbitrarily amplified DNA methods, but employ novel methodological innovations such as the incorporation of gene or promoter elements in the primers. These markers provide good reproducibility and increased resolution by the concurrent incidence of dominant and co-dominant bands. Despite their promising features, these semi-random markers suffer from possible problems of collision and non-homology analogous to those found with randomly generated fingerprints. Transposable elements, present in abundance in plant genomes, may also be used to generate fingerprints. These markers provide increased genomic coverage by utilizing specific targeted sites and produce bands that mostly seem to be homologous. The biggest drawback with most of these techniques is that prior genomic information about retrotransposons is needed for primer design, prohibiting universal applications. Another class of recently developed methods exploits length polymorphism present in arrays of multi-copy gene families such as cytochrome P450 and β-tubulin genes to provide cross-species amplification and transferability. A specific class of marker makes use of common features of plant resistance genes to generate bands linked to a given phenotype, or to reveal genetic diversity. Conserved DNA-based strategies have limited genome coverage and may fail to reveal genetic diversity, while resistance genes may be under specific evolutionary selection. Markers may also be generated from functional and/or transcribed regions of the genome using different gene-targeting approaches coupled with the use of RNA information. Such techniques have the

  9. Adaptive genetic markers discriminate migratory runs of Chinook salmon (Oncorhynchus tshawytscha) amid continued gene flow

    PubMed Central

    O'Malley, Kathleen G; Jacobson, Dave P; Kurth, Ryon; Dill, Allen J; Banks, Michael A

    2013-01-01

    Neutral genetic markers are routinely used to define distinct units within species that warrant discrete management. Human-induced changes to gene flow however may reduce the power of such an approach. We tested the efficiency of adaptive versus neutral genetic markers in differentiating temporally divergent migratory runs of Chinook salmon (Oncorhynchus tshawytscha) amid high gene flow owing to artificial propagation and habitat alteration. We compared seven putative migration timing genes to ten microsatellite loci in delineating three migratory groups of Chinook in the Feather River, CA: offspring of fall-run hatchery broodstock that returned as adults to freshwater in fall (fall run), spring-run offspring that returned in spring (spring run), and fall-run offspring that returned in spring (FRS). We found evidence for significant differentiation between the fall and federally listed threatened spring groups based on divergence at three circadian clock genes (OtsClock1b, OmyFbxw11, and Omy1009UW), but not neutral markers. We thus demonstrate the importance of genetic marker choice in resolving complex life history types. These findings directly impact conservation management strategies and add to previous evidence from Pacific and Atlantic salmon indicating that circadian clock genes influence migration timing. PMID:24478800

  10. Predictive functional profiling of microbial communities using 16S rRNA marker gene sequences

    PubMed Central

    Langille, Morgan G. I.; Zaneveld, Jesse; Caporaso, J. Gregory; McDonald, Daniel; Knights, Dan; Reyes, Joshua A.; Clemente, Jose C.; Burkepile, Deron E.; Vega Thurber, Rebecca L.; Knight, Rob; Beiko, Robert G.; Huttenhower, Curtis

    2013-01-01

    Profiling phylogenetic marker genes, such as the 16S rRNA gene, is a key tool for studies of microbial communities but does not provide direct evidence of a community’s functional capabilities. Here we describe PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States), a computational approach to predict the functional composition of a metagenome using marker gene data and a database of reference genomes. PICRUSt uses an extended ancestral-state reconstruction algorithm to predict which gene families are present and then combines gene families to estimate the composite metagenome. Using 16S information, PICRUSt recaptures key findings from the Human Microbiome Project and accurately predicts the abundance of gene families in host-associated and environmental communities, with quantifiable uncertainty. Our results demonstrate that phylogeny and function are sufficiently linked that this ‘predictive metagenomic’ approach should provide useful insights into the thousands of uncultivated microbial communities for which only marker gene surveys are currently available. PMID:23975157

  11. Glucagon Like Peptide-1 Promotes Adipocyte Differentiation via the Wnt4 Mediated Sequestering of Beta-Catenin.

    PubMed

    Liu, Rui; Li, Na; Lin, Yi; Wang, Mei; Peng, Yongde; Lewi, Keidren; Wang, Qinghua

    2016-01-01

    Glucagon-like peptide-1 (GLP-1) plays a role in the regulation of adipogenesis; however, the precise underlying molecular mechanism has not been fully defined. Wnt was recently identified as an important regulator of adipogenesis. This study aimed to investigate the involvement of the Wnt signaling pathway in the effects of GLP-1 on adipocyte differentiation. 3T3-L1 cells were induced to differentiate. The changes in the expression levels of adipogenic transcription factors and Wnts and the phosphorylation level and subcellular localization of β-catenin were quantified after GLP-1 treatment. GLP-1 stimulated adipocyte differentiation and lipid accumulation, which were accompanied by the expression of adipocyte marker genes. The expression of Wnt4 was upregulated in the process of adipocyte differentiation, which was further enhanced by treatment with GLP-1. β-catenin, an important mediator of the Wnt pathway, was immediately dephosphorylated and translocated from cytoplasm to nucleus when differentiation was induced. In the presence of GLP-1, however, β-catenin was redirected to the cell plasma membrane leading to its decreased accumulation in the nucleus. Knockdown of Wnt4 blocked the effect of GLP-1 on the cellular localization of β-catenin and expression level of adipogenic transcription factors. Our findings showed that GLP-1 promoted adipogenesis through the modulation of the Wnt4/β-catenin signaling pathway, suggesting that the GLP-1-Wntβ-catenin system might be a new target for the treatment of metabolic disease. PMID:27504979

  12. Glucagon Like Peptide-1 Promotes Adipocyte Differentiation via the Wnt4 Mediated Sequestering of Beta-Catenin

    PubMed Central

    Liu, Rui; Li, Na; Lin, Yi; Wang, Mei; Peng, Yongde; Lewi, Keidren; Wang, Qinghua

    2016-01-01

    Glucagon-like peptide-1 (GLP-1) plays a role in the regulation of adipogenesis; however, the precise underlying molecular mechanism has not been fully defined. Wnt was recently identified as an important regulator of adipogenesis. This study aimed to investigate the involvement of the Wnt signaling pathway in the effects of GLP-1 on adipocyte differentiation. 3T3-L1 cells were induced to differentiate. The changes in the expression levels of adipogenic transcription factors and Wnts and the phosphorylation level and subcellular localization of β-catenin were quantified after GLP-1 treatment. GLP-1 stimulated adipocyte differentiation and lipid accumulation, which were accompanied by the expression of adipocyte marker genes. The expression of Wnt4 was upregulated in the process of adipocyte differentiation, which was further enhanced by treatment with GLP-1. β-catenin, an important mediator of the Wnt pathway, was immediately dephosphorylated and translocated from cytoplasm to nucleus when differentiation was induced. In the presence of GLP-1, however, β-catenin was redirected to the cell plasma membrane leading to its decreased accumulation in the nucleus. Knockdown of Wnt4 blocked the effect of GLP-1 on the cellular localization of β-catenin and expression level of adipogenic transcription factors. Our findings showed that GLP-1 promoted adipogenesis through the modulation of the Wnt4/β-catenin signaling pathway, suggesting that the GLP-1-Wntβ-catenin system might be a new target for the treatment of metabolic disease. PMID:27504979

  13. Chronic activation of pattern recognition receptors suppresses brown adipogenesis of multipotent mesodermal stem cells and brown pre-adipocytes.

    PubMed

    Bae, Jiyoung; Chen, Jiangang; Zhao, Ling

    2015-06-01

    Brown adipose tissue (BAT) holds promise to combat obesity through energy-spending, non-shivering thermogenesis. Understanding of regulation of BAT development can lead to novel strategies to increase BAT mass and function for obesity treatment and prevention. Here, we report the effects of chronic activation of PRR on brown adipogenesis of multipotent mesodermal stem C3H10T1/2 cells and immortalized brown pre-adipocytes from the classical interscapular BAT of mice. Activation of NOD1, TLR4, or TLR2 by their respective synthetic ligand suppressed brown marker gene expression and lipid accumulation during differentiation of brown-like adipocytes of C3H10T1/2. Activation of the PRR only during the commitment was sufficient to suppress the differentiation. PRR activation suppressed PGC-1α mRNA, but induced PRDM16 mRNA at the commitment. Consistently, PRR activation suppressed the differentiation of immortalized brown pre-adipocytes. Activation of PRR induced NF-κB activation in both cells, which correlated with their abilities to suppress PPARγ transactivation, a critical event for brown adipogenesis. Taken together, our results demonstrate that chronic PRR activation suppressed brown adipogenesis of multipotent mesodermal stem cells and brown pre-adipocytes, possibly through suppression of PPARγ transactivation. The results suggest that anti- inflammatory therapies targeting PRRs may be beneficial for the BAT development.

  14. Evaluation of the synuclein-y (SNCG) gene as a PPARy target in murine adipocytes, dorsal root ganglia somatosensory neurons, and human adipose tissue

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Synuclein-gamma is highly expressed in both adipocytes and peripheral nervous system (PNS) somatosensory neurons. Its mRNA is induced during adipogenesis, increased in obese human white adipose tissue (WAT), may be coordinately regulated with leptin, and is decreased following treatment of murine 3T...

  15. Development of SRAP, SRAP-RGA, RAPD and SCAR markers linked with a Fusarium wilt resistance gene in eggplant.

    PubMed

    Mutlu, Nedim; Boyaci, Filiz Hatice; Göçmen, Münevver; Abak, Kazim

    2008-11-01

    Fusarium wilt (Fusarium oxysporum Schlecht. f. sp. melongenae) is a vascular disease of eggplant (Solanum melongena L.). The objectives of this work were (1) to confirm the monogenic inheritance of fusarium wilt resistance in eggplant, (2) to identify molecular markers linked to this resistance, and (3) to develop SCAR markers from most informative markers. We report the tagging of the gene for resistance to fusarium wilt (FOM) in eggplant using SRAP, RGA, SRAP-RGA and RAPD markers. Analysis of segregation data confirmed the monogenic inheritance of resistance. DNA from F(2) and BC(1) populations of eggplant segregating for fusarium wilt resistance was screened with 2,316 primer combinations to detect polymorphism. Three markers were linked within 2.6 cM of the gene. The codominant SRAP marker Me8/Em5 and dominant SRAP-RGA marker Em12/GLPL2 were tightly linked to each other and mapped 1.2 cM from the resistance gene, whereas RAPD marker H12 mapped 2.6 cM from the gene and on the same side as the other two markers. The SRAP marker was converted into two dominant SCAR markers that were confirmed to be linked to the resistance gene in the F(2,) BC(1) and F(2) of BC(3) generations of the same cross. These markers provide a starting point for mapping the eggplant FOM resistance gene in eggplant and for exploring the synteny between solanaceous crops for fusarium wilt resistance genes. The SCAR markers will be useful for identifying fusarium wilt-resistant genotypes in marker-assisted selection breeding programs using segregating progenies of the resistant eggplant progenitor used in this study.

  16. Chilean native fruit extracts inhibit inflammation linked to the pathogenic interaction between adipocytes and macrophages.

    PubMed

    Reyes-Farias, Marjorie; Vasquez, Karla; Ovalle-Marin, Angelica; Fuentes, Francisco; Parra, Claudia; Quitral, Vilma; Jimenez, Paula; Garcia-Diaz, Diego F

    2015-05-01

    Obesity is characterized by an increase in the infiltration of monocytes into the adipose tissue, causing an inflammatory condition associated with, for example, the development of insulin resistance. Thus, anti-inflammatory-based treatments could emerge as a novel and interesting approach. It has been reported that Chilean native fruits maqui (Aristotelia chilensis) and calafate (Berberis microphylla) present high contents of polyphenols, which are known for their antioxidant and anti-inflammatory properties. The aim of this study was to evaluate the ability of extracts of these fruits to block the pathogenic interaction between adipocytes and macrophages in vitro and to compare its effect with blueberry (Vaccinium corymbosum) extract treatment, which has been already described to possess several biomedical benefits. RAW264.7 macrophages were treated with 5 μg/mL lipopolysaccharides (LPS), with conditioned media (CM) from fully differentiated 3T3-L1 adipocytes, or in a coculture (CC) with 3T3-L1 adipocytes, in the presence or absence of 100 μM [total polyphenolic content] of each extract for 24 h. The gene expression and secretion profile of several inflammatory markers were evaluated. Nitric oxide secretion induced by LPS, CM, and CC was reduced by the presence of maqui (-12.2%, -45.6%, and -14.7%, respectively) and calafate (-27.6%, -43.9%, and -11.8%, respectively) extracts. Gene expression of inducible nitric oxide synthase and TNF-α was inhibited and of IL-10 was induced by maqui and calafate extract incubation. In conclusion, the extracts of these fruits present important inhibitory-like features over the inflammatory response of the interaction between adipocytes and macrophages, comprising a potential therapeutic tool against comorbidities associated with obesity development. PMID:25302660

  17. Chilean Native Fruit Extracts Inhibit Inflammation Linked to the Pathogenic Interaction Between Adipocytes and Macrophages

    PubMed Central

    Reyes-Farias, Marjorie; Vasquez, Karla; Ovalle-Marin, Angelica; Fuentes, Francisco; Parra, Claudia; Quitral, Vilma; Jimenez, Paula

    2015-01-01

    Abstract Obesity is characterized by an increase in the infiltration of monocytes into the adipose tissue, causing an inflammatory condition associated with, for example, the development of insulin resistance. Thus, anti-inflammatory-based treatments could emerge as a novel and interesting approach. It has been reported that Chilean native fruits maqui (Aristotelia chilensis) and calafate (Berberis microphylla) present high contents of polyphenols, which are known for their antioxidant and anti-inflammatory properties. The aim of this study was to evaluate the ability of extracts of these fruits to block the pathogenic interaction between adipocytes and macrophages in vitro and to compare its effect with blueberry (Vaccinium corymbosum) extract treatment, which has been already described to possess several biomedical benefits. RAW264.7 macrophages were treated with 5 μg/mL lipopolysaccharides (LPS), with conditioned media (CM) from fully differentiated 3T3-L1 adipocytes, or in a coculture (CC) with 3T3-L1 adipocytes, in the presence or absence of 100 μM [total polyphenolic content] of each extract for 24 h. The gene expression and secretion profile of several inflammatory markers were evaluated. Nitric oxide secretion induced by LPS, CM, and CC was reduced by the presence of maqui (−12.2%, −45.6%, and −14.7%, respectively) and calafate (−27.6%, −43.9%, and −11.8%, respectively) extracts. Gene expression of inducible nitric oxide synthase and TNF-α was inhibited and of IL-10 was induced by maqui and calafate extract incubation. In conclusion, the extracts of these fruits present important inhibitory-like features over the inflammatory response of the interaction between adipocytes and macrophages, comprising a potential therapeutic tool against comorbidities associated with obesity development. PMID:25302660

  18. Identification of Gene-Expression Signatures and Protein Markers for Breast Cancer Grading and Staging

    PubMed Central

    Yao, Fang; Zhang, Chi; Du, Wei; Liu, Chao; Xu, Ying

    2015-01-01

    The grade of a cancer is a measure of the cancer's malignancy level, and the stage of a cancer refers to the size and the extent that the cancer has spread. Here we present a computational method for prediction of gene signatures and blood/urine protein markers for breast cancer grades and stages based on RNA-seq data, which are retrieved from the TCGA breast cancer dataset and cover 111 pairs of disease and matching adjacent noncancerous tissues with pathologists-assigned stages and grades. By applying a differential expression and an SVM-based classification approach, we found that 324 and 227 genes in cancer have their expression levels consistently up-regulated vs. their matching controls in a grade- and stage-dependent manner, respectively. By using these genes, we predicted a 9-gene panel as a gene signature for distinguishing poorly differentiated from moderately and well differentiated breast cancers, and a 19-gene panel as a gene signature for discriminating between the moderately and well differentiated breast cancers. Similarly, a 30-gene panel and a 21-gene panel are predicted as gene signatures for distinguishing advanced stage (stages III-IV) from early stage (stages I-II) cancer samples and for distinguishing stage II from stage I samples, respectively. We expect these gene panels can be used as gene-expression signatures for cancer grade and stage classification. In addition, of the 324 grade-dependent genes, 188 and 66 encode proteins that are predicted to be blood-secretory and urine-excretory, respectively; and of the 227 stage-dependent genes, 123 and 51 encode proteins predicted to be blood-secretory and urine-excretory, respectively. We anticipate that some combinations of these blood and urine proteins could serve as markers for monitoring breast cancer at specific grades and stages through blood and urine tests. PMID:26375396

  19. Molecular beacon imaging of tumor marker gene expression in pancreatic cancer cells.

    PubMed

    Yang, Lily; Cao, Zehong; Lin, Yiming; Wood, William C; Staley, Charles A

    2005-05-01

    We have developed a fluorescence imaging-based approach to detect expression of tumor marker genes in pancreatic cancer cells using molecular beacons (MBs). MBs are short hairpin oligonucleotide probes that bind to specific oligonucleotide sequences and produce fluorescent signals. MBs targeting transcripts of two tumor marker genes, mutant K-ras and survivin, were synthesized and their specificity in detection of the expression of those genes in pancreatic cancer cells was examined. We found that K-ras MBs differentially bind to mutant K-ras mRNAs, resulting in strong fluorescent signals in pancreatic cancer cells with specific mutant K-ras genes but not in normal cells or cancer cells expressing either wild type or a different mutation of the K-ras gene. Additionally, MBs targeting survivin mRNA produced a bright fluorescent signal specifically in pancreatic cancer cells. We also demonstrated that MBs labeled with different fluorophores could detect survivin and mutant K-ras mRNAs simultaneously in single cancer cells. Furthermore, we showed that survivin and K-ras MBs have a high specificity in identifying cancer cells on frozen sections of pancreatic cancer tissues. In conclusion, molecular beacon-based imaging of expression of tumor marker genes has potential for the development of novel approaches for the detection of pancreatic cancer cells.

  20. Systems Biology in Animal Breeding: Identifying relationships among markers, genes, and phenotypes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Breeding and Genetics Symposium titled “Systems Biology in Animal Breeding: Identifying relationships among markers, genes, and phenotypes” was held at the Joint Annual Meeting of the American Dairy Science Association and the American Society of Animal Science in Phoenix, AZ, July 15 to 19, 201...

  1. Rapid and Targeted Introgression of Genes into Popular Cultivars Using Marker-Assisted Background Selection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have optimized a marker-assisted background selection (MABS)-based gene introgression approach in wheat where 97% or more of a recurrent parent genome can be recovered in just two backcrosses (BCs). A four-step MABS method was developed based on ‘Plabsim’ computer simulations and wheat genome str...

  2. Minimum entropy decomposition: Unsupervised oligotyping for sensitive partitioning of high-throughput marker gene sequences

    PubMed Central

    Eren, A Murat; Morrison, Hilary G; Lescault, Pamela J; Reveillaud, Julie; Vineis, Joseph H; Sogin, Mitchell L

    2015-01-01

    Molecular microbial ecology investigations often employ large marker gene datasets, for example, ribosomal RNAs, to represent the occurrence of single-cell genomes in microbial communities. Massively parallel DNA sequencing technologies enable extensive surveys of marker gene libraries that sometimes include nearly identical sequences. Computational approaches that rely on pairwise sequence alignments for similarity assessment and de novo clustering with de facto similarity thresholds to partition high-throughput sequencing datasets constrain fine-scale resolution descriptions of microbial communities. Minimum Entropy Decomposition (MED) provides a computationally efficient means to partition marker gene datasets into ‘MED nodes', which represent homogeneous operational taxonomic units. By employing Shannon entropy, MED uses only the information-rich nucleotide positions across reads and iteratively partitions large datasets while omitting stochastic variation. When applied to analyses of microbiomes from two deep-sea cryptic sponges Hexadella dedritifera and Hexadella cf. dedritifera, MED resolved a key Gammaproteobacteria cluster into multiple MED nodes that are specific to different sponges, and revealed that these closely related sympatric sponge species maintain distinct microbial communities. MED analysis of a previously published human oral microbiome dataset also revealed that taxa separated by less than 1% sequence variation distributed to distinct niches in the oral cavity. The information theory-guided decomposition process behind the MED algorithm enables sensitive discrimination of closely related organisms in marker gene amplicon datasets without relying on extensive computational heuristics and user supervision. PMID:25325381

  3. Identification and Utility of Markers Linked to the Zucchini Yellow Mosaic Virus Resistance Gene in Watermelon

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Zucchini yellow mosaic virus Florida stain (ZYMV-FL) is one of the most economically important viruses affecting watermelon in the United States. Inheritance of resistance to ZYMV-FL is conferred by a single recessive gene. Described here is single-reaction, polymerase chain reaction-based marker l...

  4. Impaired response of mature adipocytes of diabetic mice to hypoxia

    SciTech Connect

    Hong, Seok Jong Jin, Da P.; Buck, Donald W.; Galiano, Robert D.; Mustoe, Thomas A.

    2011-10-01

    Adipose tissue contains various cells such as infiltrated monocytes/macrophages, endothelial cells, preadipocytes, and adipocytes. Adipocytes have an endocrine function by secreting adipokines such as interleukin (IL)-6, tumor necrosis factor (TNF)-{alpha}, leptin, and adiponectin. Dysregulation of adipokines in adipose tissues leads to a chronic low-grade inflammation which could result in atherosclerosis, hypertension, and type 2 diabetes. A sustained inflammatory state, which is characterized by prolonged persistence of macrophages and neutrophils, is found in diabetic wounds. In addition, subcutaneous adipocytes are enormously increased in amount clinically in type 2 diabetes. However, the function of subcutaneous adipocytes, which play an important role in injured tissue subjected to hypoxia, has not been well characterized in vitro due to the difficulty of maintaining mature adipocytes in culture using conventional methods because of their buoyancy. In this study, we established a novel in vitro culture method of mature adipocytes by enclosing them in a hyaluronan (HA) based hydrogel to study their role in response to stress such as hypoxia. BrdU labeling and Ki67 immunostaining experiments showed that hydrogel enclosed mature adipocytes proliferate in vitro. Both mRNA and protein expression analyses for hypoxia regulated genes, such as vascular endothelial growth factor (VEGF) and heme oxygenase 1 (HO1), showed that mature adipocytes of wild type mice respond to hypoxia. In contrast, mature adipocytes of diabetic db/db and TallyHo mice did not efficiently respond to hypoxia. Our studies suggest that mature adipocytes are functionally active cells, and their abnormal function to hypoxia can be one of underlining mechanisms in type 2 diabetes.

  5. Effect of dietary conjugated linoleic acid on marbling and intramuscular adipocytes in pork.

    PubMed

    Barnes, K M; Winslow, N R; Shelton, A G; Hlusko, K C; Azain, M J

    2012-04-01

    Dietary CLA has been reported to decrease backfat and increase marbling in pigs. Our objective was to determine whether the increase in marbling involved changes in intramuscular adipocyte number or size or both. Twenty barrows (53 kg) were penned in pairs and pens were randomly assigned to receive diets containing either 1% soybean oil (SBO) or CLA (60% CLA isomers) for 6 wk. Body weight and feed intake were determined weekly. At slaughter, loin samples were obtained and flash frozen for RNA extraction and real-time reverse-transcription PCR analysis of gene expression. After a 24-h chill, loin eye area and backfat depth were measured and subjective marbling and color scores were assigned. Loin, backfat, and belly fat samples were obtained for fatty acid analysis by gas chromatography. Loin samples were also frozen in ice-cold isopentane for histological analysis of intramuscular adipocytes. Dietary CLA did not affect BW or feed intake at any point (P > 0.10), nor did treatment groups differ in HCW (P = 0.417) or loin color (P = 0.500). The CLA-fed pigs did have less (P = 0.018) backfat and smaller (P = 0.047) loin eye area than SBO-fed pigs and had a trend for an increase (P = 0.069) in marbling score. Relative gene expression for markers of preadipocytes (preadipocyte factor 1; Pref-1), differentiating adipocytes (PPARγ), and mature adipocytes [fatty acid binding protein 4 (FABP4) and perilipin (PLIN)] were determined and normalized to the expression of acidic ribosomal phosphoprotein. No significant differences were detected, but the expression of PPARγ (P = 0.265), PLIN (P = 0.265), and FABP4 (P = 0.148) was numerically greater in CLA-fed pigs than in SBO-fed pigs. Loin samples were stained with Oil Red O to identify intramuscular adipocytes. The average cell area was increased (P = 0.030) in CLA-fed pigs. The cis-9,trans-11 and trans-10,cis-12 CLA isomers were incorporated (P = 0.006) into backfat and belly fat, but only trans-10,cis-12 CLA was increased in

  6. Candidate Gene Identification with SNP Marker-Based Fine Mapping of Anthracnose Resistance Gene Co-4 in Common Bean.

    PubMed

    Burt, Andrew J; William, H Manilal; Perry, Gregory; Khanal, Raja; Pauls, K Peter; Kelly, James D; Navabi, Alireza

    2015-01-01

    Anthracnose, caused by Colletotrichum lindemuthianum, is an important fungal disease of common bean (Phaseolus vulgaris). Alleles at the Co-4 locus confer resistance to a number of races of C. lindemuthianum. A population of 94 F4:5 recombinant inbred lines of a cross between resistant black bean genotype B09197 and susceptible navy bean cultivar Nautica was used to identify markers associated with resistance in bean chromosome 8 (Pv08) where Co-4 is localized. Three SCAR markers with known linkage to Co-4 and a panel of single nucleotide markers were used for genotyping. A refined physical region on Pv08 with significant association with anthracnose resistance identified by markers was used in BLAST searches with the genomic sequence of common bean accession G19833. Thirty two unique annotated candidate genes were identified that spanned a physical region of 936.46 kb. A majority of the annotated genes identified had functional similarity to leucine rich repeats/receptor like kinase domains. Three annotated genes had similarity to 1, 3-β-glucanase domains. There were sequence similarities between some of the annotated genes found in the study and the genes associated with phosphoinositide-specific phosphilipases C associated with Co-x and the COK-4 loci found in previous studies. It is possible that the Co-4 locus is structured as a group of genes with functional domains dominated by protein tyrosine kinase along with leucine rich repeats/nucleotide binding site, phosphilipases C as well as β-glucanases. PMID:26431031

  7. Candidate Gene Identification with SNP Marker-Based Fine Mapping of Anthracnose Resistance Gene Co-4 in Common Bean

    PubMed Central

    Burt, Andrew J.; William, H. Manilal; Perry, Gregory; Khanal, Raja; Pauls, K. Peter; Kelly, James D.; Navabi, Alireza

    2015-01-01

    Anthracnose, caused by Colletotrichum lindemuthianum, is an important fungal disease of common bean (Phaseolus vulgaris). Alleles at the Co–4 locus confer resistance to a number of races of C. lindemuthianum. A population of 94 F4:5 recombinant inbred lines of a cross between resistant black bean genotype B09197 and susceptible navy bean cultivar Nautica was used to identify markers associated with resistance in bean chromosome 8 (Pv08) where Co–4 is localized. Three SCAR markers with known linkage to Co–4 and a panel of single nucleotide markers were used for genotyping. A refined physical region on Pv08 with significant association with anthracnose resistance identified by markers was used in BLAST searches with the genomic sequence of common bean accession G19833. Thirty two unique annotated candidate genes were identified that spanned a physical region of 936.46 kb. A majority of the annotated genes identified had functional similarity to leucine rich repeats/receptor like kinase domains. Three annotated genes had similarity to 1, 3-β-glucanase domains. There were sequence similarities between some of the annotated genes found in the study and the genes associated with phosphoinositide-specific phosphilipases C associated with Co-x and the COK–4 loci found in previous studies. It is possible that the Co–4 locus is structured as a group of genes with functional domains dominated by protein tyrosine kinase along with leucine rich repeats/nucleotide binding site, phosphilipases C as well as β-glucanases. PMID:26431031

  8. GFP as a marker for transient gene transfer and expression in Mycoplasma hyorhinis.

    PubMed

    Ishag, Hassan Z A; Liu, Maojun; Yang, Ruosong; Xiong, Qiyan; Feng, Zhixin; Shao, Guoqing

    2016-01-01

    Mycoplasma hyorhinis (M. hyorhinis) is an opportunistic pathogen of pigs and has been shown to transform cell cultures, which has increased the interest of researchers. The green florescence proteins (GFP) gene of Aquorea victoria, proved to be a vital marker to identify transformed cells in mixed populations. Use of GFP to observe gene transfer and expression in M. hyorhinis (strain HUB-1) has not been described. We have constructed a pMD18-O/MHRgfp plasmid containing the p97 gene promoter, origin of replication, tetracycline resistance marker and GFP gene controlled by the p97 gene promoter. The plasmid transformed into M. hyorhinis with a frequency of ~4 × 10(-3) cfu/µg plasmid DNA and could be detected by PCR amplification of the GFP gene from the total DNA of the transformant mycoplasmas. Analysis of a single clone grown on KM2-Agar containing tetracycline, showed a green fluorescence color. Conclusively, this report suggests the usefulness of GFP to monitor transient gene transfer and expression in M. hyorhinis, eventually minimizing screening procedures for gene transfer and expression. PMID:27386255

  9. ASK1 signalling regulates brown and beige adipocyte function

    PubMed Central

    Hattori, Kazuki; Naguro, Isao; Okabe, Kohki; Funatsu, Takashi; Furutani, Shotaro; Takeda, Kohsuke; Ichijo, Hidenori

    2016-01-01

    Recent studies suggest that adult humans have active brown or beige adipocytes, the activation of which might be a therapeutic strategy for the treatment of diverse metabolic diseases. Here we show that the protein kinase ASK1 regulates brown and beige adipocytes function. In brown or white adipocytes, the PKA-ASK1-p38 axis is activated in response to cAMP signalling and contributes to the cell-autonomous induction of genes, including Ucp1. Global and fat-specific ASK1 deficiency leads to impaired metabolic responses, including thermogenesis and oxygen consumption, at the cell and whole-body levels, respectively. Our data thus indicate that the ASK1 signalling axis is a regulator of brown and beige adipocyte gene expression and function. PMID:27045525

  10. Development of candidate gene markers associated to common bacterial blight resistance in common bean.

    PubMed

    Shi, Chun; Yu, Kangfu; Xie, Weilong; Perry, Gregory; Navabi, Alireza; Pauls, K Peter; Miklas, Phillip N; Fourie, Deidré

    2012-11-01

    Common bacterial blight (CBB), caused by Xanthomonas axonopodis pv. phaseoli (Xap), is a major yield-limiting factor of common bean (Phaseolus vulgaris L.) production around the world. Two major CBB-resistant quantitative trait loci (QTL), linked to the sequence characterized amplified region markers BC420 and SU91, are located at chromosomes 6 and 8, respectively. Using map-based cloning approach, four bacterial artificial chromosome (BAC) clones from the BC420-QTL locus and one BAC clone containing SU91 were sequenced by Roche 454 technique and subsequently assembled using merged assemblies from three different programs. Based on the quality of the assembly, only the sequences of BAC 32H6 and 4K7 were used for candidate gene marker (CGM) development and candidate gene (CG) selection. For the BC420-QTL locus, 21 novel genes were predicted in silico by FGENESH using Medicago gene model, whereas 16 genes were identified in the SU91-QTL locus. For each putative gene, one or more primer pairs were designed and tested in the contrasting near isogenic lines. Overall, six and nine polymorphic markers were found in the SU91- and BC420-QTL loci, respectively. Afterwards, association mapping was conducted in a breeding population of 395 dry bean lines to discover marker-trait associations. Two CGMs per each locus showed better association with CBB resistance than the BC420 and SU91 markers, which include BC420-CG10B and BC420-CG14 for BC420_QTL locus, and SU91-CG10 and SU91-CG11 for SU91_QTL locus. The strong associations between CBB resistance and the CGs 10 and 14 from BC420_QTL locus and the CGs 10 and 11 from SU91_QTL locus indicate that the genes 10 and 14 from the BC420 locus are potential CGs underlying the BC420_QTL locus, whereas the genes 10 and 11 from the SU91 locus are potential CGs underlying the SU91_QTL locus. The superiority of SU91-CG11 was further validated in a recombinant inbred line population Sanilac × OAC 09-3. Thus, co-dominant CGMs, BC420-CG14 and

  11. Development of genotyping by sequencing (GBS) and array derived SNP markers for stem rust resistance gene Sr42

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The stem rust fungus, particularly race TTKSK (Ug99), poses a serious threat to world wheat production. Gene Sr42 or SrCad (which could be the same gene or an allele of Sr42) is effective against race TTKSK. However, known genetic markers for Sr42 are mostly SSR markers which are generally labor i...

  12. Marker development for rice blast resistance gene Pi66(t) and application in USDA rice mini-core collection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Molecular markers are useful for the identification of critical genes controlling agricultural traits of interest in crop germplasm and for the utilization of these genes in crop improvement using marker assisted selection (MAS). The improvement of blast disease resistance of rice varieties is one o...

  13. SNPs in transporter and metabolizing genes as predictive markers for oxaliplatin treatment in colorectal cancer patients.

    PubMed

    Kap, Elisabeth J; Seibold, Petra; Scherer, Dominique; Habermann, Nina; Balavarca, Yesilda; Jansen, Lina; Zucknick, Manuela; Becker, Natalia; Hoffmeister, Michael; Ulrich, Alexis; Benner, Axel; Ulrich, Cornelia M; Burwinkel, Barbara; Brenner, Hermann; Chang-Claude, Jenny

    2016-06-15

    Oxaliplatin is frequently used as part of a chemotherapeutic regimen with 5-fluorouracil in the treatment of colorectal cancer (CRC). The cellular availability of oxaliplatin is dependent on metabolic and transporter enzymes. Variants in genes encoding these enzymes may cause variation in response to oxaliplatin and could be potential predictive markers. Therefore, we used a two-step procedure to comprehensively investigate 1,444 single nucleotide polymorphisms (SNPs) from these pathways for their potential as predictive markers for oxaliplatin treatment, using 623 stage II-IV CRC patients (of whom 201 patients received oxaliplatin) from a German prospective patient cohort treated with adjuvant or palliative chemotherapy. First, all genes were screened using the global test that evaluated SNP*oxaliplatin interaction terms per gene. Second, one model was created by backward elimination on all SNP*oxaliplatin interactions of the selected genes. The statistical procedure was evaluated using bootstrap analyses. Nine genes differentially associated with overall survival according to oxaliplatin treatment (unadjusted p values < 0.05) were selected. Model selection resulted in the inclusion of 14 SNPs from eight genes (six transporter genes, ABCA9, ABCB11, ABCC10, ATP1A1, ATP1B2, ATP8B3, and two metabolism genes GSTM5, GRHPR), which significantly improved model fit. Using bootstrap analysis we show an improvement of the prediction error of 3.7% in patients treated with oxaliplatin. Several variants in genes involved in metabolism and transport could thus be potential predictive markers for oxaliplatin treatment in CRC patients. If confirmed, inclusion of these variants in a predictive test could identify patients who are more likely to benefit from treatment with oxaliplatin. PMID:26835885

  14. SNPs in transporter and metabolizing genes as predictive markers for oxaliplatin treatment in colorectal cancer patients.

    PubMed

    Kap, Elisabeth J; Seibold, Petra; Scherer, Dominique; Habermann, Nina; Balavarca, Yesilda; Jansen, Lina; Zucknick, Manuela; Becker, Natalia; Hoffmeister, Michael; Ulrich, Alexis; Benner, Axel; Ulrich, Cornelia M; Burwinkel, Barbara; Brenner, Hermann; Chang-Claude, Jenny

    2016-06-15

    Oxaliplatin is frequently used as part of a chemotherapeutic regimen with 5-fluorouracil in the treatment of colorectal cancer (CRC). The cellular availability of oxaliplatin is dependent on metabolic and transporter enzymes. Variants in genes encoding these enzymes may cause variation in response to oxaliplatin and could be potential predictive markers. Therefore, we used a two-step procedure to comprehensively investigate 1,444 single nucleotide polymorphisms (SNPs) from these pathways for their potential as predictive markers for oxaliplatin treatment, using 623 stage II-IV CRC patients (of whom 201 patients received oxaliplatin) from a German prospective patient cohort treated with adjuvant or palliative chemotherapy. First, all genes were screened using the global test that evaluated SNP*oxaliplatin interaction terms per gene. Second, one model was created by backward elimination on all SNP*oxaliplatin interactions of the selected genes. The statistical procedure was evaluated using bootstrap analyses. Nine genes differentially associated with overall survival according to oxaliplatin treatment (unadjusted p values < 0.05) were selected. Model selection resulted in the inclusion of 14 SNPs from eight genes (six transporter genes, ABCA9, ABCB11, ABCC10, ATP1A1, ATP1B2, ATP8B3, and two metabolism genes GSTM5, GRHPR), which significantly improved model fit. Using bootstrap analysis we show an improvement of the prediction error of 3.7% in patients treated with oxaliplatin. Several variants in genes involved in metabolism and transport could thus be potential predictive markers for oxaliplatin treatment in CRC patients. If confirmed, inclusion of these variants in a predictive test could identify patients who are more likely to benefit from treatment with oxaliplatin.

  15. Gene markers of cellular aging in human multipotent stromal cells in culture

    PubMed Central

    2014-01-01

    Introduction Human multipotent stromal cells (MSCs) isolated from bone marrow or other tissue sources have great potential to treat a wide range of injuries and disorders in the field of regenerative medicine and tissue engineering. In particular, MSCs have inherent characteristics to suppress the immune system and are being studied in clinical studies to prevent graft-versus-host disease. MSCs can be expanded in vitro and have potential for differentiation into multiple cell lineages. However, the impact of cell passaging on gene expression and function of the cells has not been determined. Methods Commercially available human MSCs derived from bone marrow from six different donors, grown under identical culture conditions and harvested at cell passages 3, 5, and 7, were analyzed with gene-expression profiling by using microarray technology. Results The phenotype of these cells did not change as reported previously; however, a statistical analysis revealed a set of 78 significant genes that were distinguishable in expression between passages 3 and 7. None of these significant genes corresponded to the markers established by the International Society for Cellular Therapy (ISCT) for MSC identification. When the significant gene lists were analyzed through pathway analysis, these genes were involved in the top-scoring networks of cellular growth and proliferation and cellular development. A meta-analysis of the literature for significant genes revealed that the MSCs seem to be undergoing differentiation into a senescent cell type when cultured extensively. Consistent with the differences in gene expression at passage 3 and 7, MSCs exhibited a significantly greater potential for cell division at passage 3 in comparison to passage 7. Conclusions Our results identified specific gene markers that distinguish aging MSCs grown in cell culture. Confirmatory studies are needed to correlate these molecular markers with biologic attributes that may facilitate the development

  16. Retinoic acid has different effects on UCP1 expression in mouse and human adipocytes

    PubMed Central

    2013-01-01

    Background Increased adipose thermogenesis is being considered as a strategy aimed at preventing or reversing obesity. Thus, regulation of the uncoupling protein 1 (UCP1) gene in human adipocytes is of significant interest. Retinoic acid (RA), the carboxylic acid form of vitamin A, displays agonist activity toward several nuclear hormone receptors, including RA receptors (RARs) and peroxisome proliferator-activated receptor δ (PPARδ). Moreover, RA is a potent positive regulator of UCP1 expression in mouse adipocytes. Results The effects of all-trans RA (ATRA) on UCP1 gene expression in models of mouse and human adipocyte differentiation were investigated. ATRA induced UCP1 expression in all mouse white and brown adipocytes, but inhibited or had no effect on UCP1 expression in human adipocyte cell lines and primary human white adipocytes. Experiments with various RAR agonists and a RAR antagonist in mouse cells demonstrated that the stimulatory effect of ATRA on UCP1 gene expression was indeed mediated by RARs. Consistently, a PPARδ agonist was without effect. Moreover, the ATRA-mediated induction of UCP1 expression in mouse adipocytes was independent of PPARγ coactivator-1α. Conclusions UCP1 expression is differently affected by ATRA in mouse and human adipocytes. ATRA induces UCP1 expression in mouse adipocytes through activation of RARs, whereas expression of UCP1 in human adipocytes is not increased by exposure to ATRA. PMID:24059847

  17. Flanking markers bracket the neurofibromatosis type 2 (NF2) gene on chromosome 22.

    PubMed Central

    Rouleau, G A; Seizinger, B R; Wertelecki, W; Haines, J L; Superneau, D W; Martuza, R L; Gusella, J F

    1990-01-01

    Neurofibromatosis 2 or bilateral acoustic neurofibromatosis (NF2) is a severe autosomal dominant disorder characterized by the development of multiple tumors of the nervous system, including meningiomas, gliomas, neurofibromas, ependymomas, and particularly acoustic neuromas. Polymorphic DNA markers have revealed frequent loss of one copy of chromosome 22 in the tumor types associated with NF2. Family studies have demonstrated that the primary defect in NF2 is linked to DNA markers on chromosome 22, suggesting that it involves inactivation of a tumor suppressor gene. We have employed a combination of multipoint linkage analysis and examination of deletions in primary tumor specimens to precisely map the NF2 locus between flanking polymorphic DNA markers on chromosome 22. The 13-cM region bracketed by these markers corresponds to 13% of the genetic length of the long arm of chromosome 22 and is expected to contain less than 5 x 10(6) bp of DNA. The delineation of flanking markers for NF2 should permit accurate presymptomatic and prenatal diagnosis for the disorder and greatly facilitate efforts to isolate the defective gene on the basis of its location. Images Figure 1 Figure 3 PMID:2105641

  18. Molecular Screening of Blast Resistance Genes in Rice using SSR Markers

    PubMed Central

    Singh, A. K.; Singh, P. K.; Arya, Madhuri; Singh, N. K.; Singh, U. S.

    2015-01-01

    Rice Blast is the most devastating disease causing major yield losses in every year worldwide. It had been proved that using resistant rice varieties would be the most effective way to control this disease. Molecular screening and genetic diversities of major rice blast resistance genes were determined in 192 rice germplasm accessions using simple sequence repeat (SSR) markers. The genetic frequencies of the 10 major rice blast resistance genes varied from 19.79% to 54.69%. Seven accessions IC337593, IC346002, IC346004, IC346813, IC356117, IC356422 and IC383441 had maximum eight blast resistance gene, while FR13B, Hourakani, Kala Rata 1–24, Lemont, Brown Gora, IR87756-20-2-2-3, IC282418, IC356419, PKSLGR-1 and PKSLGR-39 had seven blast resistance genes. Twenty accessions possessed six genes, 36 accessions had five genes, 41 accessions had four genes, 38 accessions had three genes, 26 accessions had two genes, 13 accessions had single R gene and only one accession IC438644 does not possess any one blast resistant gene. Out of 192 accessions only 17 accessions harboured 7 to 8 blast resistance genes. PMID:25774106

  19. Biological pathways, candidate genes and molecular markers associated with quality-of-life domains: an update

    PubMed Central

    Sprangers, Mirjam A.G.; Thong, Melissa S.Y.; Bartels, Meike; Barsevick, Andrea; Ordoñana, Juan; Shi, Qiuling; Wang, Xin Shelley; Klepstad, Pål; Wierenga, Eddy A.; Singh, Jasvinder A.; Sloan, Jeff A.

    2014-01-01

    Background There is compelling evidence of a genetic foundation of patient-reported QOL. Given the rapid development of substantial scientific advances in this area of research, the current paper updates and extends reviews published in 2010. Objectives The objective is to provide an updated overview of the biological pathways, candidate genes and molecular markers involved in fatigue, pain, negative (depressed mood) and positive (well-being/happiness) emotional functioning, social functioning, and overall QOL. Methods We followed a purposeful search algorithm of existing literature to capture empirical papers investigating the relationship between biological pathways and molecular markers and the identified QOL domains. Results Multiple major pathways are involved in each QOL domain. The inflammatory pathway has the strongest evidence as a controlling mechanism underlying fatigue. Inflammation and neurotransmission are key processes involved in pain perception and the COMT gene is associated with multiple sorts of pain. The neurotransmitter and neuroplasticity theories have the strongest evidence for their relationship with depression. Oxytocin-related genes and genes involved in the serotonergic and dopaminergic pathways play a role in social functioning. Inflammatory pathways, via cytokines, also play an important role in overall QOL. Conclusions Whereas the current findings need future experiments and replication efforts, they will provide researchers supportive background information when embarking on studies relating candidate genes and/or molecular markers to QOL domains. The ultimate goal of this area of research is to enhance patients’ QOL. PMID:24604075

  20. The Drosophila melanogaster cinnabar gene is a cell autonomous genetic marker in Aedes aegypti (Diptera: Culicidae).

    PubMed

    Sethuraman, Nagaraja; O'Brochta, David A

    2005-07-01

    The cinnabar gene of Drosophila melanogaster (Meigen) encodes for kynurenine hydroxylase, an enzyme involved in ommochrome biosynthesis. This gene is commonly included as a visible genetic marker in gene vectors used to create transgenic Aedes aegypti (L.) that are homozygous for the khw allele, the mosquito homolog of cinnabar. Unexpectedly, the phenotype of cells expressing kynurenine hydroxylase in transgenic Ae. aegypti is cell autonomous as demonstrated by the recovery of insects heterozygous for the kynurenine hydroxylase transgene with mosaic eye color patterns. In addition, a transgenic gynandromorph was recovered in which one-half of the insect was expressing the kynurenine hydroxylase transgene, including one eye with red pigmentation, whereas the other half of the insect was homozygous khw and included a white eye. The cell autonomous behavior of cinnabar in transgenic Ae. aegypti is unexpected and increases the utility of this genetic marker. PMID:16119567

  1. Haploid Origin of Cork Oak Anther Embryos Detected by Enzyme and RAPD Gene Markers.

    PubMed

    Bueno; Agundez; Gomez; Carrascosa; Manzanera

    2000-05-01

    In vitro-induced cork oak (Quercus suber L.) embryos from anther cultures proved to be of haploid origin both by enzyme and RAPD gene marker analysis. The problem considered was to ascertain if embryo cultures originated either from a single haploid cell, from a microspore, or from multiple haploid cells. Therefore, a heterozygotic gene was searched for in the parent tree. The gene coding for shikimate dehydrogenase (SKDH1) proved to be heterozygous in the parental tree, and subsequently, these allozymes were screened for the embryos induced in anther cultures from the same tree. Only haploid embryos were found, confirming the microspore origin. Different genotypes were not identified inside each anther by isozyme analysis, probably because of selective pressure for one embryo early in development, but both parental SKDH1 alleles were found in the embryos of different anthers. The banding patterns detected by RAPD markers permitted the identification of multiple microspore origins inside each anther.

  2. Adipocyte telomere length associates negatively with adipocyte size, whereas adipose tissue telomere length associates negatively with the extent of fibrosis in severely obese women.

    PubMed

    el Bouazzaoui, F; Henneman, P; Thijssen, P; Visser, A; Koning, F; Lips, M A; Janssen, I; Pijl, H; Willems van Dijk, K; van Harmelen, V

    2014-05-01

    Telomere length can be considered as a biological marker for cell proliferation and aging. Obesity is associated with adipocyte hypertrophy and proliferation as well as with shorter telomeres in adipose tissue. As adipose tissue is a mixture of different cell types and the cellular composition of adipose tissue changes with obesity, it is unclear what determines telomere length of whole adipose tissue. We aimed to investigate telomere length in whole adipose tissue and isolated adipocytes in relation to adiposity, adipocyte hypertrophy and adipose tissue inflammation and fibrosis. Telomere length was measured by real-time PCR in visceral adipose tissue, and isolated adipocytes of 21 obese women with a waist ranging from 110 to 147 cm and age from 31 to 61 years. Telomere length in adipocytes was shorter than in whole adipose tissue. Telomere length of adipocytes but not whole adipose tissue correlated negatively with waist and adipocyte size, which was still significant after correction for age. Telomere length of whole adipose tissue associated negatively with fibrosis as determined by collagen content. Thus, in extremely obese individuals, adipocyte telomere length is a marker of adiposity, whereas whole adipose tissue telomere length reflects the extent of fibrosis and may indicate adipose tissue dysfunction.

  3. [The gene ontology and electro localization of ovine skin derived EST-SSR markers].

    PubMed

    Wang, Zun-Bao; Zhao, Zong-Sheng; Yu, Peng; Wu, Hong-Bin; Ban, Qian; Liang, Yao-Wei; Zheng, Wei

    2011-07-01

    Abstract: In order to study the potential gene function of ovine EST-SSR markers, nine original EST of Ovine Skin Derived polymorphic EST-SSR loci, which were developed in an early study by our lab, were ontology annotated and Electro localized. The results revealed that the original ESTs of the six loci had high homology with known genes and three of them probably played an important role in wool traits. Compared with its cDNA library, 8 loci were located on chromosomes of cattle. The homology of chromosomes between cattle and sheep was estimated based on the similarity coefficients calculated by positioning markers. Additionally, NJ clustering tree was establishedto serve for electro localization of ovine EST-SSR markers. Finally, 8 EST-SSR markers were successfully positioned on ovine chromosomes. The results from this study not only provide references for further studies on genetic mapping, in silico cloning of key genes for wool traits, but also are helpful to the researchs of chromosome evolution in animal.

  4. Development of universal genetic markers based on single-copy orthologous (COSII) genes in Poaceae.

    PubMed

    Liu, Hailan; Guo, Xiaoqin; Wu, Jiasheng; Chen, Guo-Bo; Ying, Yeqing

    2013-03-01

    KEY MESSAGE : We develop a set of universal genetic markers based on single-copy orthologous (COSII) genes in Poaceae. Being evolutionary conserved, single-copy orthologous (COSII) genes are particularly useful in comparative mapping and phylogenetic investigation among species. In this study, we identified 2,684 COSII genes based on five sequenced Poaceae genomes including rice, maize, sorghum, foxtail millet, and brachypodium, and then developed 1,072 COSII markers whose transferability and polymorphism among five bamboo species were further evaluated with 46 pairs of randomly selected primers. 91.3 % of the 46 primers obtained clear amplification in at least one bamboo species, and 65.2 % of them produced polymorphism in more than one species. We also used 42 of them to construct the phylogeny for the five bamboo species, and it might reflect more precise evolutionary relationship than the one based on the vegetative morphology. The results indicated a promising prospect of applying these markers to the investigation of genetic diversity and the classification of Poaceae. To ease and facilitate access of the information of common interest to readers, a web-based database of the COSII markers is provided ( http://www.sicau.edu.cn/web/yms/PCOSWeb/PCOS.html ).

  5. Association mapping and marker-assisted selection of the lettuce dieback resistance gene Tvr1

    PubMed Central

    2009-01-01

    Background Lettuce (Lactuca saliva L.) is susceptible to dieback, a soilborne disease caused by two viruses from the family Tombusviridae. Susceptibility to dieback is widespread in romaine and leaf-type lettuce, while modern iceberg cultivars are resistant to this disease. Resistance in iceberg cultivars is conferred by Tvr1 - a single, dominant gene that provides durable resistance. This study describes fine mapping of the resistance gene, analysis of nucleotide polymorphism and linkage disequilibrium in the Tvr1 region, and development of molecular markers for marker-assisted selection. Results A combination of classical linkage mapping and association mapping allowed us to pinpoint the location of the Tvr1 resistance gene on chromosomal linkage group 2. Nine molecular markers, based on expressed sequence tags (EST), were closely linked to Tvr1 in the mapping population, developed from crosses between resistant (Salinas and Salinas 88) and susceptible (Valmaine) cultivars. Sequencing of these markers from a set of 68 cultivars revealed a relatively high level of nucleotide polymorphism (θ = 6.7 × 10-3) and extensive linkage disequilibrium (r2 = 0.124 at 8 cM) in this region. However, the extent of linkage disequilibrium was affected by population structure and the values were substantially larger when the analysis was performed only for romaine (r2 = 0.247) and crisphead (r2 = 0.345) accessions. The association mapping approach revealed that one of the nine markers (Cntg10192) in the Tvr1 region matched exactly with resistant and susceptible phenotypes when tested on a set of 200 L. sativa accessions from all horticultural types of lettuce. The marker-trait association was also confirmed on two accessions of Lactuca serriola - a wild relative of cultivated lettuce. The combination of three single-nucleotide polymorphisms (SNPs) at the Cntg10192 marker identified four haplotypes. Three of the haplotypes were associated with resistance and one of them was always

  6. MethMarker: user-friendly design and optimization of gene-specific DNA methylation assays

    PubMed Central

    2009-01-01

    DNA methylation is a key mechanism of epigenetic regulation that is frequently altered in diseases such as cancer. To confirm the biological or clinical relevance of such changes, gene-specific DNA methylation changes need to be validated in multiple samples. We have developed the MethMarker http://methmarker.mpi-inf.mpg.de/ software to help design robust and cost-efficient DNA methylation assays for six widely used methods. Furthermore, MethMarker implements a bioinformatic workflow for transforming disease-specific differentially methylated genomic regions into robust clinical biomarkers. PMID:19804638

  7. Molecular marker assisted gene stacking for biotic and abiotic stress resistance genes in an elite rice cultivar

    PubMed Central

    Das, Gitishree; Rao, G. J. N.

    2015-01-01

    Severe yield loss due to various biotic stresses like bacterial blight (BB), gall midge (insect) and Blast (disease) and abiotic stresses like submergence and salinity are a serious constraint to the rice productivity throughout the world. The most effective and reliable method of management of the stresses is the enhancement of host resistance, through an economical and environmentally friendly approach. Through the application of marker assisted selection (MAS) technique, the present study reports a successful pyramidization of genes/QTLs to confer resistance/tolerance to blast (Pi2, Pi9), gall Midge (Gm1, Gm4), submergence (Sub1), and salinity (Saltol) in a released rice variety CRMAS2621-7-1 as Improved Lalat which had already incorporated with three BB resistance genes xa5, xa13, and Xa21 to supplement the Xa4 gene present in Improved Lalat. The molecular analysis revealed clear polymorphism between the donor and recipient parents for all the markers that are tagged to the target traits. The conventional backcross breeding approach was followed till BC3F1 generation and starting from BC1F1 onwards, marker assisted selection was employed at each step to monitor the transfer of the target alleles with molecular markers. The different BC3F1s having the target genes/QTLs were inter crossed to generate hybrids with all 10 stress resistance/tolerance genes/QTLs into a single plant/line. Homozygous plants for resistance/tolerance genes in different combinations were recovered. The BC3F3 lines were characterized for their agronomic and quality traits and promising progeny lines were selected. The SSR based background selection was done. Most of the gene pyramid lines showed a high degree of similarity to the recurrent parent for both morphological, grain quality traits and in SSR based background selection. Out of all the gene pyramids tested, two lines had all the 10 resistance/tolerance genes and showed adequate levels of resistance/tolerance against the five target

  8. Haplotype structure enables prioritization of common markers and candidate genes in autism spectrum disorder

    PubMed Central

    Vardarajan, B N; Eran, A; Jung, J-Y; Kunkel, L M; Wall, D P

    2013-01-01

    Autism spectrum disorder (ASD) is a neurodevelopmental condition that results in behavioral, social and communication impairments. ASD has a substantial genetic component, with 88–95% trait concordance among monozygotic twins. Efforts to elucidate the causes of ASD have uncovered hundreds of susceptibility loci and candidate genes. However, owing to its polygenic nature and clinical heterogeneity, only a few of these markers represent clear targets for further analyses. In the present study, we used the linkage structure associated with published genetic markers of ASD to simultaneously improve candidate gene detection while providing a means of prioritizing markers of common genetic variation in ASD. We first mined the literature for linkage and association studies of single-nucleotide polymorphisms, copy-number variations and multi-allelic markers in Autism Genetic Resource Exchange (AGRE) families. From markers that reached genome-wide significance, we calculated male-specific genetic distances, in light of the observed strong male bias in ASD. Four of 67 autism-implicated regions, 3p26.1, 3p26.3, 3q25-27 and 5p15, were enriched with differentially expressed genes in blood and brain from individuals with ASD. Of 30 genes differentially expressed across multiple expression data sets, 21 were within 10 cM of an autism-implicated locus. Among them, CNTN4, CADPS2, SUMF1, SLC9A9, NTRK3 have been previously implicated in autism, whereas others have been implicated in neurological disorders comorbid with ASD. This work leverages the rich multimodal genomic information collected on AGRE families to present an efficient integrative strategy for prioritizing autism candidates and improving our understanding of the relationships among the vast collection of past genetic studies. PMID:23715297

  9. NAT8L (N-Acetyltransferase 8-Like) Accelerates Lipid Turnover and Increases Energy Expenditure in Brown Adipocytes*

    PubMed Central

    Pessentheiner, Ariane R.; Pelzmann, Helmut J.; Walenta, Evelyn; Schweiger, Martina; Groschner, Lukas N.; Graier, Wolfgang F.; Kolb, Dagmar; Uno, Kyosuke; Miyazaki, Toh; Nitta, Atsumi; Rieder, Dietmar; Prokesch, Andreas; Bogner-Strauss, Juliane G.

    2013-01-01

    NAT8L (N-acetyltransferase 8-like) catalyzes the formation of N-acetylaspartate (NAA) from acetyl-CoA and aspartate. In the brain, NAA delivers the acetate moiety for synthesis of acetyl-CoA that is further used for fatty acid generation. However, its function in other tissues remained elusive. Here, we show for the first time that Nat8l is highly expressed in adipose tissues and murine and human adipogenic cell lines and is localized in the mitochondria of brown adipocytes. Stable overexpression of Nat8l in immortalized brown adipogenic cells strongly increases glucose incorporation into neutral lipids, accompanied by increased lipolysis, indicating an accelerated lipid turnover. Additionally, mitochondrial mass and number as well as oxygen consumption are elevated upon Nat8l overexpression. Concordantly, expression levels of brown marker genes, such as Prdm16, Cidea, Pgc1α, Pparα, and particularly UCP1, are markedly elevated in these cells. Treatment with a PPARα antagonist indicates that the increase in UCP1 expression and oxygen consumption is PPARα-dependent. Nat8l knockdown in brown adipocytes has no impact on cellular triglyceride content, lipogenesis, or oxygen consumption, but lipolysis and brown marker gene expression are increased; the latter is also observed in BAT of Nat8l-KO mice. Interestingly, the expression of ATP-citrate lyase is increased in Nat8l-silenced adipocytes and BAT of Nat8l-KO mice, indicating a compensatory mechanism to sustain the acetyl-CoA pool once Nat8l levels are reduced. Taken together, our data show that Nat8l impacts on the brown adipogenic phenotype and suggests the existence of the NAT8L-driven NAA metabolism as a novel pathway to provide cytosolic acetyl-CoA for lipid synthesis in adipocytes. PMID:24155240

  10. Incorporation of Bacterial Blight Resistance Genes Into Lowland Rice Cultivar Through Marker-Assisted Backcross Breeding.

    PubMed

    Pradhan, Sharat Kumar; Nayak, Deepak Kumar; Pandit, Elssa; Behera, Lambodar; Anandan, Annamalai; Mukherjee, Arup Kumar; Lenka, Srikanta; Barik, Durga Prasad

    2016-07-01

    Bacterial blight (BB) of rice caused by Xanthomonas oryzae pv. oryzae is a major disease of rice in many rice growing countries. Pyramided lines carrying two BB resistance gene combinations (Xa21+xa13 and Xa21+xa5) were developed in a lowland cultivar Jalmagna background through backcross breeding by integrating molecular markers. In each backcross generation, markers closely linked to the disease resistance genes were used to select plants possessing the target genes. Background selection was continued in those plants carrying resistant genes until BC(3) generation. Plants having the maximum contribution from the recurrent parent genome were selected in each generation and hybridized with the recipient parent. The BB-pyramided line having the maximum recipient parent genome recovery of 95% was selected among BC3F1 plants and selfed to isolate homozygous BC(3)F(2) plants with different combinations of BB resistance genes. Twenty pyramided lines with two resistance gene combinations exhibited high levels of tolerance against the BB pathogen. In order to confirm the resistance, the pyramided lines were inoculated with different X. oryzae pv. oryzae strains of Odisha for bioassay. The genotypes with combination of two BB resistance genes conferred high levels of resistance to the predominant X. oryzae pv. oryzae isolates prevalent in the region. The pyramided lines showed similarity with the recipient parent with respect to major agro-morphologic traits.

  11. Gene expression profile in breast cancer comprising predictive markers for metastatic risk.

    PubMed

    Sirirattanakul, S; Wannakrairot, P; Tencomnao, T; Santiyanont, R

    2015-01-01

    Quantitative multiplex reverse transcriptase-polymerase chain reaction was developed for the simultaneous detection of multiple-gene expression levels of formalin-fixed, paraffin-embedded breast cancer samples. Candidate genes were selected from previous microarray data relevant to breast cancer markers that had the potential to serve as predictive markers for metastatic risk. This multiplex gene set included 11 candidate and 3 housekeeping genes, and the aim was to predict breast cancer progression based on lymph node involvement status. Our study demonstrated that the system generated a good standard curve fit (R(2) = 0.9901-0.9998) correlated with RNA concentration. The multiplex gene expression profile indicated significantly downregulated levels of G protein-coupled receptor kinase interacting ArfGAP 2 (GIT2) and mitochondrial transcription termination factor (MTERF) genes in a lymph node-positive group of patients, with P values of 0.004 and 0.038, respectively. Therefore, this in-house method using multiple genes of interest might be an alternative tool for prediction of breast cancer metastasis. PMID:26400320

  12. Differentially expressed genes in human peripheral blood as potential markers for statin response.

    PubMed

    Won, Hong-Hee; Kim, Suk Ran; Bang, Oh Young; Lee, Sang-Chol; Huh, Wooseong; Ko, Jae-Wook; Kim, Hyung-Gun; McLeod, Howard L; O'Connell, Thomas M; Kim, Jong-Won; Lee, Soo-Youn

    2012-02-01

    There is a considerable inter-individual variation in response to statin therapy and one third of patients do not meet their treatment goals. We aimed to identify differentially expressed genes that might be involved in the effects of statin treatment and to suggest potential markers to guide statin therapy. Forty-six healthy Korean subjects received atorvastatin; their whole-genome expression profiles in peripheral blood were analyzed before and after atorvastatin administration in relation with changes in lipid profiles. The expression patterns of the differentially expressed genes were also compared with the data of familial hypercholesterolemia (FH) patients and controls. Pairwise comparison analyses revealed differentially expressed genes involved in diverse biological processes and molecular functions related with immune responses. Atorvastain mainly affected antigen binding, immune or inflammatory response including interleukin pathways. Similar expression patterns of the genes were observed in patients with FH and controls. The Charcol-Leyden crystal (CLC), CCR2, CX3CR1, LRRN3, FOS, LDLR, HLA-DRB1, ERMN, and TCN1 genes were significantly associated with cholesterol levels or statin response. Interestingly, the CLC gene, which was significantly altered by atorvastatin administration and differentially expressed between FH patients and controls, showed much bigger change in high-responsive group than in low-responsive group. We identified differentially expressed genes that might be involved in mechanisms underlying the known pleiotropic effects of atorvastatin, baseline cholesterol levels, and drug response. Our findings suggest CLC as a new candidate marker for statin response, and further validation is needed.

  13. A regulatory gene as a novel visible marker for maize transformation

    SciTech Connect

    Ludwig, S.R.; Wessler, S.R. ); Bowen, B.; Beach, L. )

    1990-01-26

    The temporal and spatial patterns of anthocyanin pigmentation in the maize plant are determined by the presence or absence of the R protein product, a presumed transcriptional activator. At least 50 unique patterns of pigmentation, conditioned by members of the R gene family, have been described. In this study, microprojectiles were used to introduce into maize cells a vector containing the transcription unit from one of these genes (Lc) fused to a constitutive promoter. This chimeric gene induces cell autonomous pigmentation in tissues that are not normally pigmented by the Lc gene. As a reporter for gene expression studies in maize, R is unique because it can be quantified in living tissue simply by counting the number of pigmented cells following bombardment. R may also be useful as a visible marker for selecting stably transformed cell lineages that can give rise to transgenic plants.

  14. Rapid and Targeted Introgression of Genes into Popular Wheat Cultivars Using Marker-Assisted Background Selection

    PubMed Central

    Kidwell, Kim; Morris, Craig F.; Chen, Xianming; Gill, Kulvinder S.

    2009-01-01

    A marker-assisted background selection (MABS)-based gene introgression approach in wheat (Triticum aestivum L.) was optimized, where 97% or more of a recurrent parent genome (RPG) can be recovered in just two backcross (BC) generations. A four-step MABS method was developed based on ‘Plabsim’ computer simulations and wheat genome structure information. During empirical optimization of this method, double recombinants around the target gene were selected in a step-wise fashion during the two BC cycles followed by selection for recurrent parent genotype on non-carrier chromosomes. The average spacing between carrier chromosome markers was <4 cM. For non-carrier chromosome markers that flanked each of the 48 wheat gene-rich regions, this distance was ∼12 cM. Employed to introgress seedling stripe rust (Puccinia striiformis f. sp. tritici) resistance gene Yr15 into the spring wheat cultivar ‘Zak’, marker analysis of 2,187 backcross-derived progeny resulted in the recovery of a BC2F2∶3 plant with 97% of the recurrent parent genome. In contrast, only 82% of the recurrent parent genome was recovered in phenotypically selected BC4F7 plants developed without MABS. Field evaluation results from 17 locations indicated that the MABS-derived line was either equal or superior to the recurrent parent for the tested agronomic characteristics. Based on these results, MABS is recommended as a strategy for rapidly introgressing a targeted gene into a wheat genotype in just two backcross generations while recovering 97% or more of the recurrent parent genotype. PMID:19484121

  15. Effect of microgrooves and fibronectin conjugation on the osteoblast marker gene expression and differentiation

    PubMed Central

    2015-01-01

    PURPOSE To determine the effect of fibronectin (FN)-conjugated, microgrooved titanium (Ti) on osteoblast differentiation and gene expression in human bone marrow-derived mesenchymal stem cells (MSCs). MATERIALS AND METHODS Photolithography was used to fabricate the microgrooved Ti, and amine functionalization (silanization) was used to immobilize fibronectin on the titanium surfaces. Osteoblast differentiation and osteoblast marker gene expression were analyzed by means of alkaline phosphatase activity assay, extracellular calcium deposition assay, and quantitative real-time PCR. RESULTS The conjugation of fibronectin on Ti significantly increased osteoblast differentiation in MSCs compared with non-conjugated Ti substrates. On the extracellular calcium deposition assays of MSCs at 21 days, an approximately two-fold increase in calcium concentration was observed on the etched 60-µm-wide/10-µm-deep microgrooved surface with fibronectin (E60/10FN) compared with the same surface without fibronectin (E60/10), and a more than four-fold increase in calcium concentration was observed on E60/10FN compared with the non-etched control (NE0) and etched control (E0) surfaces. Through a series of analyses to determine the expression of osteoblast marker genes, a significant increase in all the marker genes except type I collagen α1 mRNA was seen with E60/10FN more than with any of the other groups, as compared with NE0. CONCLUSION The FN-conjugated, microgrooved Ti substrate can provide an effective surface to promote osteoblast differentiation and osteoblast marker gene expression in MSCs. PMID:26816580

  16. Effects of adipocyte lipoprotein lipase on de novo lipogenesis and white adipose tissue browning.

    PubMed

    Bartelt, Alexander; Weigelt, Clara; Cherradi, M Lisa; Niemeier, Andreas; Tödter, Klaus; Heeren, Joerg; Scheja, Ludger

    2013-05-01

    Efficient storage of dietary and endogenous fatty acids is a prerequisite for a healthy adipose tissue function. Lipoprotein lipase (LPL) is the master regulator of fatty acid uptake from triglyceride-rich lipoproteins. In addition to LPL-mediated fatty acid uptake, adipocytes are able to synthesize fatty acids from non-lipid precursor, a process called de novo lipogenesis (DNL). As the physiological relevance of fatty acid uptake versus DNL for brown and white adipocyte function remains unclear, we studied the role of adipocyte LPL using adipocyte-specific LPL knockout animals (aLKO). ALKO mice displayed a profound increase in DNL-fatty acids, especially palmitoleate and myristoleate in brown adipose tissue (BAT) and white adipose tissue (WAT) depots while essential dietary fatty acids were markedly decreased. Consequently, we found increased expression in adipose tissues of genes encoding DNL enzymes (Fasn, Scd1, and Elovl6) as well as the lipogenic transcription factor carbohydrate response element binding protein-β. In a high-fat diet (HFD) study aLKO mice were characterized by reduced adiposity and improved plasma insulin and adipokines. However, neither glucose tolerance nor inflammatory markers were ameliorated in aLKO mice compared to controls. No signs of increased BAT activation or WAT browning were detected in aLKO mice either on HFD or after 1 week of β3-adrenergic stimulation using CL316,243. We conclude that despite a profound increase in DNL-derived fatty acids, proposed to be metabolically favorable, aLKO mice are not protected from metabolic disease per se. In addition, induction of DNL alone is not sufficient to promote browning of WAT. This article is part of a Special Issue entitled Brown and White Fat: From Signaling to Disease.

  17. Development of Agrobacterium-Mediated Virus-Induced Gene Silencing and Performance Evaluation of Four Marker Genes in Gossypium barbadense

    PubMed Central

    Pang, Jinhuan; Zhu, Yue; Li, Qing; Liu, Jinzhi; Tian, Yingchuan; Liu, Yule; Wu, Jiahe

    2013-01-01

    Gossypiumbarbadense is a cultivated cotton species and possesses many desirable traits, including high fiber quality and resistance to pathogens, especially Verticilliumdahliae (a devastating pathogen of Gossypium hirsutum, the main cultivated species). These elite traits are difficult to be introduced into G. hirsutum through classical breeding methods. In addition, genetic transformation of G. barbadense has not been successfully performed. It is therefore important to develop methods for evaluating the function and molecular mechanism of genes in G. barbadense. In this study, we had successfully introduced a virus-induced gene silencing (VIGS) system into three cultivars of G. barbadense by inserting marker genes into the tobacco rattle virus (TRV) vector. After we optimized the VIGS conditions, including light intensity, photoperiod, seedling age and Agrobacterium strain, 100% of plants agroinfiltrated with the GaPDS silencing vector showed white colored leaves. Three other marker genes, GaCLA1, GaANS and GaANR, were employed to further test this VIGS system in G. barbadense. The transcript levels of the endogenous genes in the silenced plants were reduced by more than 99% compared to control plants; these plants presented phenotypic symptoms 2 weeks after inoculation. We introduced a fusing sequence fragment of GaPDS and GaANR gene silencing vectors into a single plant, which resulted in both photobleaching and brownish coloration. The extent of silencing in plants agroinfiltrated with fusing two-gene-silencing vector was consistent with plants harboring a single gene silencing vector. The development of this VIGS system should promote analysis of gene function in G. barbadense, and help to contribute desirable traits for breeding of G. barbadense and G. hirsutum. PMID:24023833

  18. Regulation of human subcutaneous adipocyte differentiation by EID1.

    PubMed

    Vargas, Diana; Shimokawa, Noriaki; Kaneko, Ryosuke; Rosales, Wendy; Parra, Adriana; Castellanos, Ángela; Koibuchi, Noriyuki; Lizcano, Fernando

    2016-02-01

    Increasing thermogenesis in white adipose tissues can be used to treat individuals at high risk for obesity and cardiovascular disease. The objective of this study was to determine the function of EP300-interacting inhibitor of differentiation (EID1), an inhibitor of muscle differentiation, in the induction of beige adipocytes from adipose mesenchymal stem cells (ADMSCs). Subcutaneous adipose tissue was obtained from healthy women undergoing abdominoplasty. ADMSCs were isolated in vitro, grown, and transfected with EID1 or EID1 siRNA, and differentiation was induced after 48 h by administering rosiglitazone. The effects of EID1 expression under the control of the aP2 promoter (aP2-EID1) were also evaluated in mature adipocytes that were differentiated from ADMSCs. Transfection of EID1 into ADMSCs reduced triglyceride accumulation while increasing levels of thermogenic proteins, such as PGC1α, TFAM, and mitochondrial uncoupling protein 1 (UCP1), all of which are markers of energy expenditure and mitochondrial activity. Furthermore, increased expression of the beige phenotype markers CITED1 and CD137 was observed. Transfection of aP2-EID1 transfection induced the conversion of mature white adipocytes to beige adipocytes, as evidenced by increased expression of PGC1α, UCP1, TFAM, and CITED1. These results indicate that EID1 can modulate ADMSCs, inducing a brown/beige lineage. EID1 may also activate beiging in white adipocytes obtained from subcutaneous human adipose tissue. PMID:26643909

  19. Generation and evaluation of a chimeric classical swine fever virus expressing a visible marker gene.

    PubMed

    Li, Yongfeng; Wang, Xiao; Sun, Yuan; Li, Lian-Feng; Zhang, Lingkai; Li, Su; Luo, Yuzi; Qiu, Hua-Ji

    2016-03-01

    Classical swine fever virus (CSFV) is a noncytopathogenic virus, and the incorporation of an enhanced green fluorescent protein (EGFP) tag into the viral genome provides a means of direct monitoring of viral infection without immunostaining. It is well established that the 3' untranslated region (3'-UTR) of the CSFV plays an important role in viral RNA replication. Although CSFV carrying a reporter gene and chimeric CSFV have been generated and evaluated, a chimeric CSFV with a visible marker has not yet been reported. Here, we generated and evaluated a chimeric virus containing the EGFP tag and the 3'-UTR from vaccine strain HCLV (C-strain) in the genetic background of the highly virulent CSFV Shimen strain. The chimeric marker CSFV was fluorescent and had an approximately 100-fold lower viral titer, lower replication level of viral genome, and weaker fluorescence intensity than the recombinant CSFV with only the EGFP tag or the parental virus. Furthermore, the marker chimera was avirulent and displayed no viremia in inoculated pigs, which were completely protected from lethal CSFV challenge as early as 15 days post-inoculation. The chimeric marker virus was visible in vitro and attenuated in vitro and in vivo, which suggests that CSFV can be engineered to produce attenuated variants with a visible marker to facilitate in vitro studies of CSFV infection and replication and to develop of novel vaccines against CSF. PMID:26614259

  20. Millions of reads, thousands of taxa: microbial community structure and associations analyzed via marker genes.

    PubMed

    Bálint, Miklós; Bahram, Mohammad; Eren, A Murat; Faust, Karoline; Fuhrman, Jed A; Lindahl, Björn; O'Hara, Robert B; Öpik, Maarja; Sogin, Mitchell L; Unterseher, Martin; Tedersoo, Leho

    2016-09-01

    With high-throughput sequencing (HTS), we are able to explore the hidden world of microscopic organisms to an unpre-cedented level. The fast development of molecular technology and statistical methods means that microbial ecologists must keep their toolkits updated. Here, we review and evaluate some of the more widely adopted and emerging techniques for analysis of diversity and community composition, and the inference of species interactions from co-occurrence data generated by HTS of marker genes. We emphasize the importance of observational biases and statistical properties of the data and methods. The aim of the review is to critically discuss the advantages and disadvantages of established and emerging statistical methods, and to contribute to the integration of HTS-based marker gene data into community ecology.

  1. Millions of reads, thousands of taxa: microbial community structure and associations analyzed via marker genes.

    PubMed

    Bálint, Miklós; Bahram, Mohammad; Eren, A Murat; Faust, Karoline; Fuhrman, Jed A; Lindahl, Björn; O'Hara, Robert B; Öpik, Maarja; Sogin, Mitchell L; Unterseher, Martin; Tedersoo, Leho

    2016-09-01

    With high-throughput sequencing (HTS), we are able to explore the hidden world of microscopic organisms to an unpre-cedented level. The fast development of molecular technology and statistical methods means that microbial ecologists must keep their toolkits updated. Here, we review and evaluate some of the more widely adopted and emerging techniques for analysis of diversity and community composition, and the inference of species interactions from co-occurrence data generated by HTS of marker genes. We emphasize the importance of observational biases and statistical properties of the data and methods. The aim of the review is to critically discuss the advantages and disadvantages of established and emerging statistical methods, and to contribute to the integration of HTS-based marker gene data into community ecology. PMID:27358393

  2. The expression profile for the tumour suppressor gene PTEN and associated polymorphic markers

    PubMed Central

    Hamilton, J A; Stewart, L M D; Ajayi, L; Gray, I C; Gray, N E; Roberts, K G; Watson, G J; Kaisary, A V; Snary, D

    2000-01-01

    PTEN, a putative tumour suppressor gene associated with prostate and other cancers, is known to be located within the chromosomal region 10q23.3. Transcription of the PTEN gives rise to multiple mRNA species. Analyses by Northern blots, using cell lines which express PTEN together with cell lines which have lost the PTEN or carry a truncated version of the gene, has allowed us to demonstrate that the pseudogene is not transcribed. In addition, 3′ RACE studies confirmed that the multiple mRNA species arising from the gene probably result from the use of alternative polyadenylation sites. No evidence for tissue- or cell-specific patterns of transcription was found. Analysis by 5′ RACE placed the putative site for the start of transcription around 830 bp upstream of the start codon. A map of the location of the PTEN gene with a series of overlapping YAC, BAC and PACs has been constructed and the relative position of eight microsatellite markers sited. Two known and one novel marker have been positioned within the gene, the others are in flanking regions. The more accurate location of these markers should help in future studies of the extent of gene loss. Several polymorphisms were also identified, all were within introns. Four of the common polymorphisms appear to be linked. In blood, DNA from 200 individuals, including normal, BPH and prostate cancer patients, confirmed this link. Only two samples of 200 did not carry the linked haplotype, both were patients with advanced prostate cancer. It is possible that such rearrangements within PTEN could be evidence of predisposition to prostate cancer in this small number of cases. © 2000 Cancer Research Campaign PMID:10817502

  3. [The effectiveness of molecular markers for the identification of Lr28, Lr35, and Lr47 genes in common wheat].

    PubMed

    Gul'tiaeva, E I; Orina, A S; Gannibal, F B; Mitrofanova, O P; Odintsova, I G; Laĭkova, L I

    2014-02-01

    The effectiveness of molecular markers for the identification of leaf rust resistance genes Lr28, Lr35, Lr47 transferred to common wheat was assessed the using samplesof Triticum spp. and Aegilops spp. from Ae. speltoides. Markers Sr39F2/R3, BCD260F1/35R2 of gene Lr35 and PS10 of Lr47 gene were characterized by high efficiency and were revealed in a line of common wheat containing these genes, and samples of Ae. speltoides (their donor). Marker SCS421 of Lr28gene and markers Sr39#22r, Sr39#50s, BE500705 of Lr35/Sr39 genes turned out to be less specific. Marker SCS421 was amplified in the samples of the T. timopheevii species, and markers Sr39#22r, Sr39#50s--in the Ae. speltoides, Ae. tauschii, T. timopheevii, line KS90WRC010 (Lr41), the sort of common wheat In Memory of Maistrenko, obtained using synthetic hexaploid T. timopheevii x Ae. tauschii and introgressive lines obtained using Ae. speltoides. Marker BE500705, which indicates the absence of Lr35/Sr39 genes, was not revealed in lines TcLr35 and MqSr39, in Ae. speltoides, Ae. tauschii and T. boeoticum (kk-61034, 61038). Analysis of the nucleotide sequences of amplification products obtained with the markers SCS421 and Sr39#22r indicated their low homology with TcLr28 and TcLr35. Using molecular markers, we showed a different distribution of Lr28 (77%), Lr35 (100%) and Lr47 (15%) genes in 13 studied samples ofAe. speltoides. In introgressive lines derived from Ae. speltoides, contemporary Russian sorts of common wheat and triticale variants Lr28, Lr35, Lr47 genes were not revealed. PMID:25711022

  4. A unique mosaic Turner syndrome patient with androgen receptor gene derived marker chromosome.

    PubMed

    Kalkan, Rasime; Özdağ, Nermin; Bundak, Rüveyde; Çirakoğlu, Ayşe; Serakinci, Nedime

    2016-01-01

    Patients with Turner syndrome are generally characterized by having short stature with no secondary sexual characteristics. Some abnormalities, such as webbed neck, renal malformations (>50%) and cardiac defects (10%) are less common. The intelligence of these patients is considered normal. Non-mosaic monosomy X is observed in approximately 45% of postnatal patients with Turner syndrome and the rest of the patients have structural abnormalities or mosaicism involving 46,X,i(Xq), 45,X/46,XX, 45,X and other variants. The phenotype of 45,X/46,X,+mar individuals varies by the genetic continent and degree of the mosaicism. The gene content of the marker chromosome is the most important when correlating the phenotype with the genotype. Here we present an 11-year-old female who was referred for evaluation of her short stature and learning disabilities. Conventional cytogenetic investigation showed a mosaic 45,X/46,X,+mar karyotype. Fluorescence in situ hybridization showed that the marker chromosome originated from the X chromosome within the androgen receptor (AR) and X-inactive specific transcript (XIST) genes. Therefore, it is possible that aberrant activation of the marker chromosome, compromising the AR and XIST genes, may modify the Turner syndrome phenotype.

  5. Molecular marker analysis of genes controlling morphological variation in Brassica rapa (syn. campestris).

    PubMed

    Song, K; Slocum, M K; Osborn, T C

    1995-01-01

    Construction of a detailed RFLP linkage map of B. rapa (syn. campestris) made it possible, for the first time, to study individual genes controlling quantitative traits in this species. Ninety-five F2 individuals from a cross of Chinese cabbage cv 'Michihili' by Spring broccoli were analyzed for segregation at 220 RFLP loci and for variation in leaf, stem, and flowering characteristics. The number, location, and magnitude of genes underlying 28 traits were determined by using an interval mapping method. Zero to five putative quantitative trait loci (QTL) were detected for each of the traits examined. There were unequal gene effects on the expression of many traits, and the inheritance patterns of traits ranged from those controlled by a single major gene plus minor genes to those controlled by polygenes with small and similar effects. The effect of marker locus density on detection of QTL was analyzed, and the results showed that the number of QTL detected did not change when the number of marker loci used for QTL mapping was decreased from 220 to 126; however, a further reduction from 126 to 56 caused more than 15% loss of the total QTL detected. The detection of putative minor QTL by removing the masking effects of major QTL was explored.

  6. [Deletion of marker gene in transgenic goat by Cre/LoxP system].

    PubMed

    Lan, Chong; Ren, Lina; Wu, Min; Liu, Siguo; Liu, Guohui; Xu, Xujun; Chen, Jianquan; Ma, Hengdong; Cheng, Guoxiang

    2013-12-01

    In producing transgenic livestock, selectable marker genes (SMGs) are usually used to screen transgenic cells from numerous normal cells. That results in SMGs integrating into the genome and transmitting to offspring. In fact, SMGs could dramatically affect gene regulation at integration sites and also make the safety evaluation of transgenic animals complicated. In order to determine the deletion time and methods in the process of producing transgenic goats, the feasibility of deleting SMGs was explored by Cre/LoxP before or after somatic cell cloning. In addition, we compared the efficiency of protein transduction with plasmids co-transduction. We could delete 43.9% SMGs after screening out the transgenic cell clones, but these cells could not be applied to somatic cells cloning because of serious aging after two gene modifications. The SMG-free cells suitable for nuclear transfer were accessible by using the cells of transgenic goats, but this approach was more time consuming. Finally, we found that the Cre plasmid could delete SMGs with an efficiency of 7.81%, but about 30% in SMG-free cells had sequences of Cre plasmid. Compared with Cre plasmid, the integration of new exogenous gene could be avoided by TAT-CRE protein transduction, and the deletion rate of TAT-CRE transduction was between 43.9 and 72.8%. Therefore, TAT-Cre transduction could be an effective method for deleting selectable marker genes.

  7. The fur gene as a new phylogenetic marker for Vibrionaceae species identification.

    PubMed

    Machado, Henrique; Gram, Lone

    2015-04-01

    Microbial taxonomy is essential in all areas of microbial science. The 16S rRNA gene sequence is one of the main phylogenetic species markers; however, it does not provide discrimination in the family Vibrionaceae, where other molecular techniques allow better interspecies resolution. Although multilocus sequence analysis (MLSA) has been used successfully in the identification of Vibrio species, the technique has several limitations. They include the fact that several locus amplifications and sequencing have to be performed, which still sometimes lead to doubtful identifications. Using an in silico approach based on genomes from 103 Vibrionaceae strains, we demonstrate here the high resolution of the fur gene in the identification of Vibrionaceae species and its usefulness as a phylogenetic marker. The fur gene showed within-species similarity higher than 95%, and the relationships inferred from its use were in agreement with those observed for 16S rRNA analysis and MLSA. Furthermore, we developed a fur PCR sequencing-based method that allowed identification of Vibrio species. The discovery of the phylogenetic power of the fur gene and the development of a PCR method that can be used in amplification and sequencing of the gene are of general interest whether for use alone or together with the previously suggested loci in an MLSA.

  8. The fur Gene as a New Phylogenetic Marker for Vibrionaceae Species Identification

    PubMed Central

    Gram, Lone

    2015-01-01

    Microbial taxonomy is essential in all areas of microbial science. The 16S rRNA gene sequence is one of the main phylogenetic species markers; however, it does not provide discrimination in the family Vibrionaceae, where other molecular techniques allow better interspecies resolution. Although multilocus sequence analysis (MLSA) has been used successfully in the identification of Vibrio species, the technique has several limitations. They include the fact that several locus amplifications and sequencing have to be performed, which still sometimes lead to doubtful identifications. Using an in silico approach based on genomes from 103 Vibrionaceae strains, we demonstrate here the high resolution of the fur gene in the identification of Vibrionaceae species and its usefulness as a phylogenetic marker. The fur gene showed within-species similarity higher than 95%, and the relationships inferred from its use were in agreement with those observed for 16S rRNA analysis and MLSA. Furthermore, we developed a fur PCR sequencing-based method that allowed identification of Vibrio species. The discovery of the phylogenetic power of the fur gene and the development of a PCR method that can be used in amplification and sequencing of the gene are of general interest whether for use alone or together with the previously suggested loci in an MLSA. PMID:25662978

  9. Precision genome editing in plants via gene targeting and piggyBac-mediated marker excision

    PubMed Central

    Nishizawa-Yokoi, Ayako; Endo, Masaki; Ohtsuki, Namie; Saika, Hiroaki; Toki, Seiichi

    2015-01-01

    Precise genome engineering via homologous recombination (HR)-mediated gene targeting (GT) has become an essential tool in molecular breeding as well as in basic plant science. As HR-mediated GT is an extremely rare event, positive–negative selection has been used extensively in flowering plants to isolate cells in which GT has occurred. In order to utilize GT as a methodology for precision mutagenesis, the positive selectable marker gene should be completely eliminated from the GT locus. Here, we introduce targeted point mutations conferring resistance to herbicide into the rice acetolactate synthase (ALS) gene via GT with subsequent marker excision by piggyBac transposition. Almost all regenerated plants expressing piggyBac transposase contained exclusively targeted point mutations without concomitant re-integration of the transposon, resulting in these progeny showing a herbicide bispyribac sodium (BS)-tolerant phenotype. This approach was also applied successfully to the editing of a microRNA targeting site in the rice cleistogamy 1 gene. Therefore, our approach provides a general strategy for the targeted modification of endogenous genes in plants. PMID:25284193

  10. Chloroplast transformation of Platymonas (Tetraselmis) subcordiformis with the bar gene as selectable marker.

    PubMed

    Cui, Yulin; Qin, Song; Jiang, Peng

    2014-01-01

    The objective of this research was to establish a chloroplast transformation technique for Platymonas (Tetraselmis) subcordiformis. Employing the gfp gene as a reporter and the bar gene as a selectable marker, transformation vectors of P. subcordiformis chloroplast were constructed with endogenous fragments rrn16S-trnI (left) and trnA-rrn23S (right) as a recombination site of the chloroplast genome. The plasmids were transferred into P. subcordiformis via particle bombardment. Confocal laser scanning microscopy indicated that the green fluorescence protein was localized in the chloroplast of P. subcordiformis, confirming the activity of the Chlamydomonas reinhardtii promoter. Cells transformed with the bar gene were selected using the herbicide Basta. Resistant colonies were analyzed by PCR and Southern blotting, and the results indicated that the bar gene was successfully integrated into the chloroplast genome via homologous recombination. The technique will improve genetic engineering of this alga. PMID:24911932

  11. Co-transformation of grapevine somatic embryos to produce transgenic plants free of marker genes.

    PubMed

    Dutt, Manjul; Li, Zhijian T; Dhekney, Sadanand A; Gray, Dennis J

    2012-01-01

    A cotransformation system using somatic embryos was developed to produce grapevines free of selectable marker genes. This was achieved by transforming Vitis vinifera L. "Thompson Seedless" somatic embryos with a mixture of two Agrobacterium strains. The first strain contained a binary plasmid with an egfp gene of interest between the T-DNA borders. The second strain harbored the neomycin phosphotransferase (nptII) gene for positive selection and the cytosine deaminase (codA) gene for negative selection, linked together by a bidirectional dual promoter complex. Our technique included a short positive selection phase of cotransformed somatic embryos on liquid medium containing 100 mg/L kanamycin before subjecting cultures to prolonged negative selection on medium containing 250 mg/L 5-fluorocytosine. PMID:22351010

  12. Multiple endocrine neoplasia type I (MEN1): Identification of informative polymorphic markers and candidate genes

    SciTech Connect

    Taggart, R.T.; Qian, C.; An, Y.

    1994-09-01

    Linkage and tumor deletion studies have mapped this autosomal dominant disease to 11q12{yields}13. The MEN1 gene product is suspected to be a growth suppressor or regulator. We report here isolation of polymorphic PCR markers used to narrow the nonrecombinant region and localization of genomic clones encoding expressed sequences within this region. 552 genomic clones were isolated from a radiation hybrid (RH) cell line containing a 5-10 Mb region flanking the gene. We screened the RH cell line with 17 markers flanking the MEN1 region to confirm its integrity. The representation of the markers in the panel of genomic clones derived from the RH cell line indicated a 1- to 4-fold representation of the region. A set of radiation hybrids was used to sublocalized genomic and cDNA clones of interest to 3 regions: centromeric, telomeric and a 1.2 Mb nonrecombinant region. Highly polymorphic PCR markers were developed by hybridization of the clones with tetra- and trinucleotide probes 3 of 7 PCR markers (heterozygosity .46-.92) were nonrecombinant with MEN1. The PCR markers were utilized for definition of the critical region and also proved useful for presymptomatic diagnosis. Genomic clones mapped to the 1.2 Mb nonrecombinant region were used to identify expressed sequences corresponding to 5 different genes. One cDNA clone corresponded to a ubiquitously expressed gene sequence located near PYGM. Two major (3.4, 2.5 kb) and one minor transcript (1.8 kb) were found in pancreas, kidney, brain, lung, heart, skeletal muscle, and liver. DNA analysis matched with 2 anonymous cDNA clones in GenBank. The genomic and cDNA clones were used to screen Southern and Northern blots for MEN1 associated rearrangements before attempting SSCP analysis to detect point mutations. The genomic fragment used to identify the corresponding cDNA clones did not detect alterations in MEN1 patients on Southern blots, however additional fragments were identified in one MEN1 patient with the cDNA clones.

  13. The GyrA encoded gene: A pertinent marker for the phylogenetic revision of Helicobacter genus.

    PubMed

    Ménard, Armelle; Buissonnière, Alice; Prouzet-Mauléon, Valérie; Sifré, Elodie; Mégraud, Francis

    2016-03-01

    Phylogeny of Epsilonproteobacteria is based on sequencing of the 16S rRNA gene. However, this gene is not sufficiently discriminatory in Helicobacter species and alternative markers would be useful. In this study, the 16S rRNA, gyrA, hsp60, gyrB, and ureA-ureB gene sequences, as well as GyrA, HSP60 and GyrB protein sequences were analyzed as tools to support Helicobacter species phylogeny: 72 Helicobacter strains, belonging to 41 species of which 36 are validated species, were included. Results of the phylogenetic reconstructions of the GyrA gene encoded protein (approximately 730 residues) indicated the most stable trees to bootstrap resampling with a good separation of Helicobacter taxa, especially between gastric and enterohepatic species. Moreover, the GyrA tree revealed high similarity with that of the gyrB and ureA-ureB genes (restricted to urease-positive Helicobacter species). However, some differences in clustering were observed when compared to the hsp60 and 23S rRNA gene trees. Altogether, these revised phylogenies (except the 16S rRNA gene for enterohepatic Helicobacters) enabled reliable clustering of Helicobacter cinaedi and 'Flexispira' strains, determined a reliable position for Helicobacter mustelae (except the hsp60 gene) and for novel Helicobacter species proposed such as 'Helicobacter sanguini', 'Helicobacter apodemus' or 'Helicobacter winghamensis', and suggest that Helicobacter species MIT 09-6949 and MIT 05-5293 isolated from rodents constitute novel species. Although they are not commonly used to study the phylogeny of Epsilonproteobacteria, protein sequences and, in particular, the GyrA protein sequence may constitute pertinent phylogenetic markers for Helicobacter genus.

  14. Effects of Parabens on Adipocyte Differentiation

    PubMed Central

    Zhao, Ling

    2013-01-01

    Parabens are a group of alkyl esters of p-hydroxybenzoic acid that include methylparaben, ethylparaben, propylparaben, butylparaben, and benzylparaben. Paraben esters and their salts are widely used as preservatives in cosmetics, toiletries, food, and pharmaceuticals. Humans are exposed to parabens through the use of such products from dermal contact, ingestion, and inhalation. However, research on the effects of parabens on health is limited, and the effects of parabens on adipogenesis have not been systematically studied. Here, we report that (1) parabens promote adipogenesis (or adipocyte differentiation) in murine 3T3-L1 cells, as revealed by adipocyte morphology, lipid accumulation, and mRNA expression of adipocyte-specific markers; (2) the adipogenic potency of parabens is increased with increasing length of the linear alkyl chain in the following potency ranking order: methyl- < ethyl- < propyl- < butylparaben. The extension of the linear alkyl chain with an aromatic ring in benzylparaben further augments the adipogenic ability, whereas 4-hydroxybenzoic acid, the common metabolite of all parabens, and the structurally related benzoic acid (without the OH group) are inactive in promoting 3T3-L1 adipocyte differentiation; (3) parabens activate glucocorticoid receptor and/or peroxisome proliferator-activated receptor γ in 3T3-L1 preadipocytes; however, no direct binding to, or modulation of, the ligand binding domain of the glucocorticoid receptor by parabens was detected by glucocorticoid receptor competitor assays; and lastly, (4) parabens, butyl- and benzylparaben in particular, also promote adipose conversion of human adipose–derived multipotent stromal cells. Our results suggest that parabens may contribute to obesity epidemic, and the role of parabens in adipogenesis in vivo needs to be examined further. PMID:22956630

  15. Effects of parabens on adipocyte differentiation.

    PubMed

    Hu, Pan; Chen, Xin; Whitener, Rick J; Boder, Eric T; Jones, Jeremy O; Porollo, Aleksey; Chen, Jiangang; Zhao, Ling

    2013-01-01

    Parabens are a group of alkyl esters of p-hydroxybenzoic acid that include methylparaben, ethylparaben, propylparaben, butylparaben, and benzylparaben. Paraben esters and their salts are widely used as preservatives in cosmetics, toiletries, food, and pharmaceuticals. Humans are exposed to parabens through the use of such products from dermal contact, ingestion, and inhalation. However, research on the effects of parabens on health is limited, and the effects of parabens on adipogenesis have not been systematically studied. Here, we report that (1) parabens promote adipogenesis (or adipocyte differentiation) in murine 3T3-L1 cells, as revealed by adipocyte morphology, lipid accumulation, and mRNA expression of adipocyte-specific markers; (2) the adipogenic potency of parabens is increased with increasing length of the linear alkyl chain in the following potency ranking order: methyl- < ethyl- < propyl- < butylparaben. The extension of the linear alkyl chain with an aromatic ring in benzylparaben further augments the adipogenic ability, whereas 4-hydroxybenzoic acid, the common metabolite of all parabens, and the structurally related benzoic acid (without the OH group) are inactive in promoting 3T3-L1 adipocyte differentiation; (3) parabens activate glucocorticoid receptor and/or peroxisome proliferator-activated receptor γ in 3T3-L1 preadipocytes; however, no direct binding to, or modulation of, the ligand binding domain of the glucocorticoid receptor by parabens was detected by glucocorticoid receptor competitor assays; and lastly, (4) parabens, butyl- and benzylparaben in particular, also promote adipose conversion of human adipose-derived multipotent stromal cells. Our results suggest that parabens may contribute to obesity epidemic, and the role of parabens in adipogenesis in vivo needs to be examined further.

  16. Bone marrow–derived circulating progenitor cells fail to transdifferentiate into adipocytes in adult adipose tissues in mice

    PubMed Central

    Koh, Young Jun; Kang, Shinae; Lee, Hyuek Jong; Choi, Tae-Saeng; Lee, Ho Sub; Cho, Chung-Hyun; Koh, Gou Young

    2007-01-01

    Little is known about whether bone marrow–derived circulating progenitor cells (BMDCPCs) can transdifferentiate into adipocytes in adipose tissues or play a role in expanding adipocyte number during adipose tissue growth. Using a mouse bone marrow transplantation model, we addressed whether BMDCPCs can transdifferentiate into adipocytes under standard conditions as well as in the settings of diet-induced obesity, rosiglitazone treatment, and exposure to G-CSF. We also addressed the possibility of transdifferentiation to adipocytes in a murine parabiosis model. In each of these settings, our findings indicated that BMDCPCs did not transdifferentiate into either unilocular or multilocular adipocytes in adipose tissues. Most BMDCPCs became resident and phagocytic macrophages in adipose tissues — which resembled transdifferentiated multilocular adipocytes by appearance, but displayed cell surface markers characteristic for macrophages — in the absence of adipocyte marker expression. When exposed to adipogenic medium in vitro, bone marrow cells differentiated into multilocular, but not unilocular, adipocytes, but transdifferentiation was not observed in vivo, even in the contexts of adipose tissue regrowth or dermal wound healing. Our results suggest that BMDCPCs do not transdifferentiate into adipocytes in vivo and play little, if any, role in expanding the number of adipocytes during the growth of adipose tissues. PMID:18060029

  17. A single-nucleotide polymorphism in the 3'-UTR region of the adipocyte fatty acid binding protein 4 gene is associated with prognosis of triple-negative breast cancer.

    PubMed

    Wang, Wenmiao; Yuan, Peng; Yu, Dianke; Du, Feng; Zhu, Anjie; Li, Qing; Zhang, Pin; Lin, Dongxin; Xu, Binghe

    2016-04-01

    Triple-negative breast cancer (TNBC) is a subtype of breast cancer with poor prognosis and high heterogeneity. The aim of this study was to screen patients for single-nucleotide polymorphisms (SNPs) associated with the prognosis of TNBC. Database-derived SNPs (NextBio, Ensembl, NCBI and MirSNP) located in the 3'-untranslated regions (3'-UTRs) of genes that are differentially expressed in breast cancer were selected. The possible associations between 111 SNPs and progression risk among 323 TNBC patients were investigated using a two-step case-control study with a discovery cohort (n=162) and a validation cohort (n=161). We identified the rs1054135 SNP in the adipocyte fatty acid binding protein 4 (FABP4) gene as a predictor of TNBC recurrence. The G allele of rs1054135 was associated with a reduced risk of disease progression as well as a prolonged disease-free survival time (DFS), with a hazard ratio (HR) for recurrence in the combined sample of 0.269 [95%CI: 0.098-0.735;P=0.001]. Notably, for individuals having the rs1054135 SNP with the AA/AG genotype, the magnitude of increased tumour recurrence risk for overweight patients (BMI≥25kg/m2) was significantly elevated (HR2.53; 95%CI: 1.06-6.03). Immunohistochemical staining of adipocytes adjacent to TNBC tissues showed that the expression level of FABP4 was statistically significantly lower in patients with the rs1054135-GG genotype and those in the disease-free group (P=0.0004 and P=0.0091, respectively). These results suggested that the expression of a lipid metabolism-related gene and an important SNP in the 3'-UTR of FABP4 are associated with TNBC prognosis, which may aid in the screening of high-risk patients with TNBC recurrence and the development of novel chemotherapeutic agents.

  18. Expression pattern of drought stress marker genes in soybean roots under two water deficit systems

    PubMed Central

    Neves-Borges, Anna Cristina; Guimarães-Dias, Fábia; Cruz, Fernanda; Mesquita, Rosilene Oliveira; Nepomuceno, Alexandre Lima; Romano, Eduardo; Loureiro, Marcelo Ehlers; de Fátima Grossi-de-Sá, Maria; Alves-Ferreira, Márcio

    2012-01-01

    The study of tolerance mechanisms for drought stress in soybean is fundamental to the understanding and development of tolerant varieties. Using in silico analysis, four marker genes involved in the classical ABA-dependent and ABA-independent pathways of drought response were identified in the Glycine max genome in the present work. The expression profiles of the marker genes ERD1-like, GmaxRD20A-like, GmaxRD22-like and GmaxRD29B-like were investigated by qPCR in root samples of drought sensitive and tolerant soybean cultivars (BR 16 and Embrapa 48, respectively), submitted to water deficit conditions in hydroponic and pot-based systems. Among the four putative soybean homologs to Arabidopsis genes investigated herein, only GmaxRD29B-like was not regulated by water deficit stress. Distinct expression profiles and different induction levels were observed among the genes, as well as between the two drought-inducing systems. Our results showed contrasting gene expression responses for the GmaxRD20A-like and GmaxRD22-like genes. GmaxRD20A-like was highly induced by continuous drought acclimating conditions, whereas GmaxRD22-like responses decreased after abrupt water deprivation. GmaxERD1-like showed a different expression profile for the cultivars in each system. Conversely, GmaxRD20A-like and GmaxRD22-like genes exhibited similar expression levels in tolerant plants in both systems. PMID:22802707

  19. An integrated approach to gene discovery and marker development in Atlantic cod (Gadus morhua).

    PubMed

    Bowman, Sharen; Hubert, Sophie; Higgins, Brent; Stone, Cynthia; Kimball, Jennifer; Borza, Tudor; Bussey, Jillian Tarrant; Simpson, Gary; Kozera, Catherine; Curtis, Bruce A; Hall, Jennifer R; Hori, Tiago S; Feng, Charles Y; Rise, Marlies; Booman, Marije; Gamperl, A Kurt; Trippel, Edward; Symonds, Jane; Johnson, Stewart C; Rise, Matthew L

    2011-04-01

    Atlantic cod is a species that has been overexploited by the capture fishery. Programs to domesticate this species are underway in several countries, including Canada, to provide an alternative route for production. Selective breeding programs have been successfully applied in the domestication of other species, with genomics-based approaches used to augment conventional methods of animal production in recent years. Genomics tools, such as gene sequences and sets of variable markers, also have the potential to enhance and accelerate selective breeding programs in aquaculture, and to provide better monitoring tools to ensure that wild cod populations are well managed. We describe the generation of significant genomics resources for Atlantic cod through an integrated genomics/selective breeding approach. These include 158,877 expressed sequence tags (ESTs), a set of annotated putative transcripts and several thousand single nucleotide polymorphism markers that were developed from, and have been shown to be highly variable in, fish enrolled in two selective breeding programs. Our EST collection was generated from various tissues and life cycle stages. In some cases, tissues from which libraries were generated were isolated from fish exposed to stressors, including elevated temperature, or antigen stimulation (bacterial and viral) to enrich for transcripts that are involved in these response pathways. The genomics resources described here support the developing aquaculture industry, enabling the application of molecular markers within selective breeding programs. Marker sets should also find widespread application in fisheries management.

  20. The life of the gay gene: from hypothetical genetic marker to social reality.

    PubMed

    O'Riordan, Kate

    2012-01-01

    The gay gene was first identified in 1993 as a correlation between the genetic marker Xq28 and gay male sexuality. The results of this original study were never replicated, and the biological reality of such an entity remains hypothetical. However, despite such tenuous provenance, the gay gene has persisted as a reference in science news, popular science writings, and in press releases and editorials about biomedical research. An examination of the life of the gay gene in U.K. news media demonstrates that the gay gene has become an assumed back-story to genetic sexuality research over time, and that the critique of its very existence has been diminished. Latterly, the gay gene has entered into the online biomedical databases of the 21st century with the same pattern of persistence and diminishing critique. This article draws on an analysis of the U.K. press and online databases to represent the process through which the address of the gay gene has shifted and become an index of biomedicalization. The consequent unmooring of the gay gene from accountability and accuracy demonstrates that the organization of biomedical databases could benefit from greater cross-disciplinary attention. PMID:22720828

  1. The life of the gay gene: from hypothetical genetic marker to social reality.

    PubMed

    O'Riordan, Kate

    2012-01-01

    The gay gene was first identified in 1993 as a correlation between the genetic marker Xq28 and gay male sexuality. The results of this original study were never replicated, and the biological reality of such an entity remains hypothetical. However, despite such tenuous provenance, the gay gene has persisted as a reference in science news, popular science writings, and in press releases and editorials about biomedical research. An examination of the life of the gay gene in U.K. news media demonstrates that the gay gene has become an assumed back-story to genetic sexuality research over time, and that the critique of its very existence has been diminished. Latterly, the gay gene has entered into the online biomedical databases of the 21st century with the same pattern of persistence and diminishing critique. This article draws on an analysis of the U.K. press and online databases to represent the process through which the address of the gay gene has shifted and become an index of biomedicalization. The consequent unmooring of the gay gene from accountability and accuracy demonstrates that the organization of biomedical databases could benefit from greater cross-disciplinary attention.

  2. Development of IP and SCAR markers linked to the yellow seed color gene in Brassica juncea L.

    PubMed

    Huang, Zhen; Liu, Lu; Lu, Hong; Lang, Lina; Zhao, Na; Ding, Juan; Xu, Aixia

    2016-03-01

    Previous studies showed that the yellow seed color gene of a yellow mustard was located on the A09 chromosome. In this study, the sequences of the molecular markers linked to the yellow seed color gene were analyzed, the gene was primarily mapped to an interval of 23.304 to 29.402M. Twenty genes and eight markers' sequences in this region were selected to design the IP and SCAR primers. These primers were used to screen a BC8S1 population consisting of 1256 individuals. As a result, five IP and five SCAR markers were successfully developed. IP4 and Y1 were located on either side of the yellow seed color gene at a distance of 0.1 and 0.3 cM, respectively. IP1, IP2 and IP3 derived from Bra036827, Bra036828, Bra036829 separately, co-segregated with the target gene. BLAST analysis indicated that the sequences of newly developed markers showed good collinearity with those of the A09 chromosome, and that the target gene might exist between 27.079 and 27.616M. In light of annotations of the genes in this region, only Bra036828 is associated with flavonoid biosynthesis. This gene has high similarity with the TRANSPARENT TESTA6 gene, Bra036828 was hence identified as being the gene possibly responsible for yellow seed color, in our research. PMID:27162489

  3. Mechanism of Regulation of Adipocyte Numbers in Adult Organisms Through Differentiation and Apoptosis Homeostasis

    PubMed Central

    Bozec, Aline; Hannemann, Nicole

    2016-01-01

    Considering that adipose tissue (AT) is an endocrine organ, it can influence whole body metabolism. Excessive energy storage leads to the dysregulation of adipocytes, which in turn induces abnormal secretion of adipokines, triggering metabolic syndromes such as obesity, dyslipidemia, hyperglycemia, hyperinsulinemia, insulin resistance and type 2 diabetes. Therefore, investigating the molecular mechanisms behind adipocyte dysregulation could help to develop novel therapeutic strategies. Our protocol describes methods for evaluating the molecular mechanism affected by hypoxic conditions of the AT, which correlates with adipocyte apoptosis in adult mice. This protocol describes how to analyze AT in vivo through gene expression profiling as well as histological analysis of adipocyte differentiation, proliferation and apoptosis during hypoxia exposure, ascertained through staining of hypoxic cells or HIF-1α protein. Furthermore, in vitro analysis of adipocyte differentiation and its responses to various stimuli completes the characterization of the molecular pathways behind possible adipocyte dysfunction leading to metabolic syndromes. PMID:27284940

  4. Identification and physical localization of useful genes and markers to a major gene-rich region on wheat group 1S chromosomes.

    PubMed Central

    Sandhu, D; Champoux, J A; Bondareva, S N; Gill, K S

    2001-01-01

    The short arm of Triticeae homeologous group 1 chromosomes is known to contain many agronomically important genes. The objectives of this study were to physically localize gene-containing regions of the group 1 short arm, enrich these regions with markers, and study the distribution of genes and recombination. We focused on the major gene-rich region ("1S0.8 region") and identified 75 useful genes along with 93 RFLP markers by comparing 35 different maps of Poaceae species. The RFLP markers were tested by gel blot DNA analysis of wheat group 1 nullisomic-tetrasomic lines, ditelosomic lines, and four single-break deletion lines for chromosome arm 1BS. Seventy-three of the 93 markers mapped to group 1 and detected 91 loci on chromosome 1B. Fifty-one of these markers mapped to two major gene-rich regions physically encompassing 14% of the short arm. Forty-one marker loci mapped to the 1S0.8 region and 10 to 1S0.5 region. Two cDNA markers mapped in the centromeric region and the remaining 24 loci were on the long arm. About 82% of short arm recombination was observed in the 1S0.8 region and 17% in the 1S0.5 region. Less than 1% recombination was observed for the remaining 85% of the physical arm length. PMID:11290727

  5. Global Gene Expression Profiling Reveals SPINK1 as a Potential Hepatocellular Carcinoma Marker

    PubMed Central

    Kutter, Claudia; Davies, Susan; Alexander, Graeme; Odom, Duncan T.

    2013-01-01

    Background Liver cirrhosis is the most important risk factor for hepatocellular carcinoma (HCC) but the role of liver disease aetiology in cancer development remains under-explored. We investigated global gene expression profiles from HCC arising in different liver diseases to test whether HCC development is driven by expression of common or different genes, which could provide new diagnostic markers or therapeutic targets. Methodology and Principal Findings Global gene expression profiling was performed for 4 normal (control) livers as well as 8 background liver and 7 HCC from 3 patients with hereditary haemochromatosis (HH) undergoing surgery. In order to investigate different disease phenotypes causing HCC, the data were compared with public microarray repositories for gene expression in normal liver, hepatitis C virus (HCV) cirrhosis, HCV-related HCC (HCV-HCC), hepatitis B virus (HBV) cirrhosis and HBV-related HCC (HBV-HCC). Principal component analysis and differential gene expression analysis were carried out using R Bioconductor. Liver disease-specific and shared gene lists were created and genes identified as highly expressed in hereditary haemochromatosis HCC (HH-HCC) were validated using quantitative RT-PCR. Selected genes were investigated further using immunohistochemistry in 86 HCC arising in liver disorders with varied aetiology. Using a 2-fold cut-off, 9 genes were highly expressed in all HCC, 11 in HH-HCC, 270 in HBV-HCC and 9 in HCV-HCC. Six genes identified by microarray as highly expressed in HH-HCC were confirmed by RT qPCR. Serine peptidase inhibitor, Kazal type 1 (SPINK1) mRNA was very highly expressed in HH-HCC (median fold change 2291, p = 0.0072) and was detected by immunohistochemistry in 91% of HH-HCC, 0% of HH-related cirrhotic or dysplastic nodules and 79% of mixed-aetiology HCC. Conclusion HCC, arising from diverse backgrounds, uniformly over-express a small set of genes. SPINK1, a secretory trypsin inhibitor, demonstrated

  6. The Molecular Signature of HIV-1-Associated Lipomatosis Reveals Differential Involvement of Brown and Beige/Brite Adipocyte Cell Lineages.

    PubMed

    Cereijo, Rubén; Gallego-Escuredo, José Miguel; Moure, Ricardo; Villarroya, Joan; Domingo, Joan Carles; Fontdevila, Joan; Martínez, Esteban; Gutiérrez, Maria del Mar; Mateo, María Gracia; Giralt, Marta; Domingo, Pere; Villarroya, Francesc

    2015-01-01

    Highly active antiretroviral therapy has remarkably improved quality of life of HIV-1-infected patients. However, this treatment has been associated with the so-called lipodystrophic syndrome, which conveys a number of adverse metabolic effects and morphological alterations. Among them, lipoatrophy of subcutaneous fat in certain anatomical areas and hypertrophy of visceral depots are the most common. Less frequently, lipomatous enlargements of subcutaneous fat at distinct anatomic areas occur. Lipomatous adipose tissue in the dorso-cervical area ("buffalo hump") has been associated with a partial white-to-brown phenotype transition and with increased cell proliferation, but, to date, lipomatous enlargements arising in other parts of the body have not been characterized. In order to establish the main molecular events associated with the appearance of lipomatosis in HIV-1 patients, we analyzed biopsies of lipomatous tissue from "buffalo hump" and from other anatomical areas in patients, in comparison with healthy subcutaneous adipose tissue, using a marker gene expression approach. Both buffalo-hump and non-buffalo-hump lipomatous adipose tissues exhibited similar patterns of non-compromised adipogenesis, unaltered inflammation, non-fibrotic phenotype and proliferative activity. Shorter telomere length, prelamin A accumulation and SA-β-Gal induction, reminiscent of adipocyte senescence, were also common to both types of lipomatous tissues. Buffalo hump biopsies showed expression of marker genes of brown adipose tissue (e.g. UCP1) and, specifically, of "classical" brown adipocytes (e.g. ZIC1) but not of beige/brite adipocytes. No such brown fat-related gene expression occurred in lipomatous tissues at other anatomical sites. In conclusion, buffalo hump and other subcutaneous adipose tissue enlargements from HIV-1-infected patients share a similar lipomatous character. However, a distorted induction of white-to-"classical brown adipocyte" phenotype appears unique of

  7. Macrophage-secreted factors induce adipocyte inflammation and insulin resistance

    SciTech Connect

    Permana, Paska A. . E-mail: Paska.Permana@med.va.gov; Menge, Christopher; Reaven, Peter D.

    2006-03-10

    Macrophage infiltration into adipose tissue increases with obesity, a condition associated with low-grade inflammation and insulin resistance. We investigated the direct effects of macrophage-secreted factors on adipocyte inflammation and insulin resistance. 3T3-L1 adipocytes incubated with media conditioned by RAW264.7 macrophages (RAW-CM) showed dramatically increased transcription of several inflammation-related genes, greater nuclear factor kappa B (NF-{kappa}B) activity, and enhanced binding of U937 monocytes. All of these effects were prevented by co-incubation with pyrrolidinedithiocarbamate, an NF-{kappa}B inhibitor. Adipocytes incubated with RAW-CM also released more non-esterified fatty acids and this increased lipolysis was not suppressed by insulin. In addition, RAW-CM treatment decreased insulin-stimulated glucose uptake in adipocytes. Taken together, these results indicate that macrophage-secreted factors induce inflammatory responses and reduce insulin responsiveness in adipocytes. These effects of macrophage-secreted factors on adipocytes may contribute significantly to the systemic inflammation and insulin resistance associated with obesity.

  8. Differential Chemokine Signature between Human Preadipocytes and Adipocytes

    PubMed Central

    Ignacio, Rosa Mistica C.; Gibbs, Carla R.; Lee, Eun-Sook

    2016-01-01

    Obesity is characterized as an accumulation of adipose tissue mass represented by chronic, low-grade inflammation. Obesity-derived inflammation involves chemokines as important regulators contributing to the pathophysiology of obesity-related diseases such as cardiovascular disease, diabetes and some cancers. The obesity-driven chemokine network is poorly understood. Here, we identified the profiles of chemokine signature between human preadipocytes and adipocytes, using PCR arrays and qRT-PCR. Both preadipocytes and adipocytes showed absent or low levels in chemokine receptors in spite of some changes. On the other hand, the chemokine levels of CCL2, CCL7-8, CCL11, CXCL1-3, CXCL6 and CXCL10-11 were dominantly expressed in preadipocytes compared to adipocytes. Interestingly, CXCL14 was the most dominant chemokine expressed in adipocytes compared to preadipocytes. Moreover, there is significantly higher protein level of CXCL14 in conditioned media from adipocytes. In addition, we analyzed the data of the chemokine signatures in adipocytes obtained from healthy lean and obese postmenopausal women based on Gene Expression Omnibus (GEO) dataset. Adipocytes from obese individuals had significantly higher levels in chemokine signature as follows: CCL2, CCL13, CCL18-19, CCL23, CCL26, CXCL1, CXCL3 and CXCL14, as compared to those from lean ones. Also, among the chemokine networks, CXCL14 appeared to be the highest levels in adipocytes from both lean and obese women. Taken together, these results identify CXCL14 as an important chemokine induced during adipogenesis, requiring further research elucidating its potential therapeutic benefits in obesity. PMID:27340388

  9. Follistatin promotes adipocyte differentiation, browning, and energy metabolism.

    PubMed

    Braga, Melissa; Reddy, Srinivasa T; Vergnes, Laurent; Pervin, Shehla; Grijalva, Victor; Stout, David; David, John; Li, Xinmin; Tomasian, Venina; Reid, Christopher B; Norris, Keith C; Devaskar, Sherin U; Reue, Karen; Singh, Rajan

    2014-03-01

    Follistatin (Fst) functions to bind and neutralize the activity of members of the transforming growth factor-β superfamily. Fst has a well-established role in skeletal muscle, but we detected significant Fst expression levels in interscapular brown and subcutaneous white adipose tissue, and further investigated its role in adipocyte biology. Fst expression was induced during adipogenic differentiation of mouse brown preadipocytes and mouse embryonic fibroblasts (MEFs) as well as in cold-induced brown adipose tissue from mice. In differentiated MEFs from Fst KO mice, the induction of brown adipocyte proteins including uncoupling protein 1, PR domain containing 16, and PPAR gamma coactivator-1α was attenuated, but could be rescued by treatment with recombinant FST. Furthermore, Fst enhanced thermogenic gene expression in differentiated mouse brown adipocytes and MEF cultures from both WT and Fst KO groups, suggesting that Fst produced by adipocytes may act in a paracrine manner. Our microarray gene expression profiling of WT and Fst KO MEFs during adipogenic differentiation identified several genes implicated in lipid and energy metabolism that were significantly downregulated in Fst KO MEFs. Furthermore, Fst treatment significantly increases cellular respiration in Fst-deficient cells. Our results implicate a novel role of Fst in the induction of brown adipocyte character and regulation of energy metabolism. PMID:24443561

  10. Similarity of markers identified from cancer gene expression studies: observations from GEO.

    PubMed

    Shi, Xingjie; Shen, Shihao; Liu, Jin; Huang, Jian; Zhou, Yong; Ma, Shuangge

    2014-09-01

    Gene expression profiling has been extensively conducted in cancer research. The analysis of multiple independent cancer gene expression datasets may provide additional information and complement single-dataset analysis. In this study, we conduct multi-dataset analysis and are interested in evaluating the similarity of cancer-associated genes identified from different datasets. The first objective of this study is to briefly review some statistical methods that can be used for such evaluation. Both marginal analysis and joint analysis methods are reviewed. The second objective is to apply those methods to 26 Gene Expression Omnibus (GEO) datasets on five types of cancers. Our analysis suggests that for the same cancer, the marker identification results may vary significantly across datasets, and different datasets share few common genes. In addition, datasets on different cancers share few common genes. The shared genetic basis of datasets on the same or different cancers, which has been suggested in the literature, is not observed in the analysis of GEO data.

  11. Development of a SCAR (sequence-characterised amplified region) marker for acid resistance-related gene in Lactobacillus plantarum.

    PubMed

    Liu, Shu-Wen; Li, Kai; Yang, Shi-Ling; Tian, Shu-Fen; He, Ling

    2015-03-01

    A sequence characterised amplified region marker was developed to determine an acid resistance-related gene in Lactobacillus plantarum. A random amplified polymorphic DNA marker named S116-680 was reported to be closely related to the acid resistance of the strains. The DNA band corresponding to this marker was cloned and sequenced with the induction of specific designed PCR primers. The results of PCR test helped to amplify a clear specific band of 680 bp in the tested acid-resistant strains. S116-680 marker would be useful to explore the acid-resistant mechanism of L. plantarum and to screen desirable malolactic fermentation strains.

  12. Gene expression profiling of craniofacial fibrous dysplasia reveals ADAMTS2 overexpression as a potential marker.

    PubMed

    Zhou, Shang-Hui; Yang, Wen-Jun; Liu, Sheng-Wen; Li, Jiang; Zhang, Chun-Ye; Zhu, Yun; Zhang, Chen-Ping

    2014-01-01

    Fibrous dysplasia (FD) as an abnormal bone growth is one of the common fibro-osseous leasions (FOL) in oral and maxillofacial region, however, its etiology still remains unclear. Here, we performed gene expression profiling of FD using microarray analysis to explore the key molecule events in FD development, and develop potential diagnostic markers or therapeutic targets for FD. We found that 1,881 genes exhibited differential expression with more than two-fold changes in FD compared to normal bone tissues, including 1,200 upregulated genes and 681 downregulated genes. Pathway analysis indicated that obviously activated pathways are Ribosome and ECM-receptor interaction pathways; downregulated pathways are "Hepatitis C" and "cancer" signaling pathways. We further validated the expression of ADAMTS2, one of most differentiated expressed genes, by Immunohistochemistry (IHC) in 40 of FD cases. Results showed that ADAMTS2 was significantly overexpressed in FD tissues, but rarely expressed in normal bone tissues, suggesting that ADAMTS2 could be a potential biomarker for FD. Thus, this study uncovered differentially expressed candidate genes in FD, which provides pilot data for understanding FD pathogenesis, and developing novel biomarkers for diagnosis and targeting of FD.

  13. Phylogeny of the glomeromycota (arbuscular mycorrhizal fungi): recent developments and new gene markers.

    PubMed

    Redecker, Dirk; Raab, Philipp

    2006-01-01

    The fungal symbionts of arbuscular mycorrhiza form a monophyletic group in the true Fungi, the phylum Glomeromycota. Fewer than 200 described species currently are included in this group. The only member of this clade known to form a different type of symbiosis is Geosiphon pyriformis, which associates with cyanobacteria. Because none of these fungi has been cultivated without their plant hosts or cyanobacterial partners, progress in obtaining multigene phylogenies has been slow and the nuclear-encoded ribosomal RNA genes have remained the only widely accessible molecular markers. rDNA phylogenies have revealed considerable polyphyly of some glomeromycotan genera that has been used to reassess taxonomic concepts. Environmental studies using phylogenetic methods for molecular identification have recovered an amazing diversity of unknown phylotypes, suggesting considerable cryptic species diversity. Protein gene sequences that have become available recently have challenged the rDNA-supported sister group relationship of the Glomeromycota with Asco/Basidiomycota. However the number of taxa analyzed with these new markers is still too small to provide a comprehensive picture of intraphylum relationships. We use nuclear-encoded rDNA and rpb1 protein gene sequences to reassess the phylogeny of the Glomeromycota and discuss possible implications.

  14. Identification of ecotype-specific marker genes for categorization of beer-spoiling Lactobacillus brevis.

    PubMed

    Behr, Jürgen; Geissler, Andreas J; Preissler, Patrick; Ehrenreich, Armin; Angelov, Angel; Vogel, Rudi F

    2015-10-01

    The tolerance to hop compounds, which is mainly associated with inhibition of bacterial growth in beer, is a multi-factorial trait. Any approaches to predict the physiological differences between beer-spoiling and non-spoiling strains on the basis of a single marker gene are limited. We identified ecotype-specific genes related to the ability to grow in Pilsner beer via comparative genome sequencing. The genome sequences of four different strains of Lactobacillus brevis were compared, including newly established genomes of two highly hop tolerant beer isolates, one strain isolated from faeces and one published genome of a silage isolate. Gene fragments exclusively occurring in beer-spoiling strains as well as sequences only occurring in non-spoiling strains were identified. Comparative genomic arrays were established and hybridized with a set of L. brevis strains, which are characterized by their ability to spoil beer. As result, a set of 33 and 4 oligonucleotide probes could be established specifically detecting beer-spoilers and non-spoilers, respectively. The detection of more than one of these marker sequences according to a genetic barcode enables scoring of L. brevis for their beer-spoiling potential and can thus assist in risk evaluation in brewing industry.

  15. Linkage analysis of the Fanconi anemia gene FACC with chromosome 9q markers

    SciTech Connect

    Auerbach, A.D.; Shin, H.T.; Kaporis, A.G.

    1994-09-01

    Fanconi anemia (FA) is a genetically heterogeneous syndrome, with at least four different complementation groups as determined by cell fusion studies. The gene for complementation group C, FACC, has been cloned and mapped to chromosome 9q22.3 by in situ hybridization, while linkage analysis has supported the placement of another gene on chromosome 20q. We have analyzed five microsatellite markers and one RFLP on chromosome 9q in a panel of FA families from the International Fanconi Anemia Registry (IFAR) in order to place FACC on the genetic map. Polymorphisms were typed in 308 individuals from 51 families. FACC is tightly linked to both D9S151 [{Theta}{sub max}=0.025, Z{sub max}=7.75] and to D9S196 [{Theta}{sub max}=0.041, Z{sub max}=7.89]; multipoint analysis is in progress. We are currently screening a YAC clone that contains the entire FACC gene for additional microsatellite markers suitable for haplotype analysis of FA families.

  16. Identification of ecotype-specific marker genes for categorization of beer-spoiling Lactobacillus brevis.

    PubMed

    Behr, Jürgen; Geissler, Andreas J; Preissler, Patrick; Ehrenreich, Armin; Angelov, Angel; Vogel, Rudi F

    2015-10-01

    The tolerance to hop compounds, which is mainly associated with inhibition of bacterial growth in beer, is a multi-factorial trait. Any approaches to predict the physiological differences between beer-spoiling and non-spoiling strains on the basis of a single marker gene are limited. We identified ecotype-specific genes related to the ability to grow in Pilsner beer via comparative genome sequencing. The genome sequences of four different strains of Lactobacillus brevis were compared, including newly established genomes of two highly hop tolerant beer isolates, one strain isolated from faeces and one published genome of a silage isolate. Gene fragments exclusively occurring in beer-spoiling strains as well as sequences only occurring in non-spoiling strains were identified. Comparative genomic arrays were established and hybridized with a set of L. brevis strains, which are characterized by their ability to spoil beer. As result, a set of 33 and 4 oligonucleotide probes could be established specifically detecting beer-spoilers and non-spoilers, respectively. The detection of more than one of these marker sequences according to a genetic barcode enables scoring of L. brevis for their beer-spoiling potential and can thus assist in risk evaluation in brewing industry. PMID:26187837

  17. Establishment of Relational Model of Congenital Heart Disease Markers and GO Functional Analysis of the Association between Its Serum Markers and Susceptibility Genes

    PubMed Central

    Liu, Min; Zhao, Luosha; Yuan, Jiaying

    2016-01-01

    Purpose. The purpose of present study was to construct the best screening model of congenital heart disease serum markers and to provide reference for further prevention and treatment of the disease. Methods. Documents from 2006 to 2014 were collected and meta-analysis was used for screening susceptibility genes and serum markers closely related to the diagnosis of congenital heart disease. Data of serum markers were extracted from 80 congenital heart disease patients and 80 healthy controls, respectively, and then logistic regression analysis and support vector machine were utilized to establish prediction models of serum markers and Gene Ontology (GO) functional annotation. Results. Results showed that NKX2.5, GATA4, and FOG2 were susceptibility genes of congenital heart disease. CRP, BNP, and cTnI were risk factors of congenital heart disease (p < 0.05); cTnI, hs-CRP, BNP, and Lp(a) were significantly close to congenital heart disease (p < 0.01). ROC curve indicated that the accuracy rate of Lp(a) and cTnI, Lp(a) and BNP, and BNP and cTnI joint prediction was 93.4%, 87.1%, and 97.2%, respectively. But the detection accuracy rate of the markers' relational model established by support vector machine was only 85%. GO analysis suggested that NKX2.5, GATA4, and FOG2 were functionally related to Lp(a) and BNP. Conclusions. The combined markers model of BNP and cTnI had the highest accuracy rate, providing a theoretical basis for the diagnosis of congenital heart disease. PMID:27118988

  18. A flexible quantitative methodology for the analysis of gene-flow between conventionally bred maize populations using microsatellite markers.

    PubMed

    Robson, P R H; Kelly, R; Jensen, E F; Giddings, G D; Leitch, M; Davey, C; Gay, A P; Jenkins, G; Thomas, H; Donnison, I S

    2011-03-01

    Previous studies of gene-flow in agriculture have used a range of physical and biochemical markers, including transgenes. However, physical and biochemical markers are not available for all commercial varieties, and transgenes are difficult to use when trying to estimate gene flow in the field where the use of transgenes is often restricted. Here, we demonstrate the use of simple sequence repeat microsatellite markers (SSRs) to study gene flow in maize. Developing the first quantitative analysis of pooled SSR samples resulted in a high sampling efficiency which minimised the use of resources and greatly enhanced the possibility of hybrid detection. We were able to quantitatively distinguish hybrids in pools of ten samples from non-hybrid parental lines in all of the 24 pair-wise combinations of commercial varieties tested. The technique was used to determine gene flow in field studies, from which a simple model describing gene flow in maize was developed. PMID:21109994

  19. Marker production by PCR amplification with primer pairs from conserved sequences of WRKY genes in chili pepper.

    PubMed

    Kim, Hyoun-Joung; Lee, Heung-Ryul; Han, Jung-Heon; Yeom, Seon-In; Harn, Chee-Hark; Kim, Byung-Dong

    2008-04-30

    Despite increasing awareness of the importance of WRKY genes in plant defense signaling, the locations of these genes in the Capsicum genome have not been established. To develop WRKY-based markers, primer sequences were deduced from the conserved sequences of the DNA binding motif within the WRKY domains of tomato and pepper genes. These primers were derived from upstream and downstream parts of the conserved sequences of the three WRKY groups. Six primer combinations of each WRKY group were tested for polymorphisms between the mapping parents, C. annuum 'CM334' and C. annuum 'Chilsungcho'. DNA fragments amplified by primer pairs deduced from WRKY Group II genes revealed high levels of polymorphism. Using 32 primer pairs to amplify upstream and downstream parts of the WRKY domain of WRKY group II genes, 60 polymorphic bands were detected. Polymorphisms were not detected with primer pairs from downstream parts of WRKY group II genes. Half of these primers were subjected to F2 genotyping to construct a linkage map. Thirty of 41 markers were located evenly spaced on 20 of the 28 linkage groups, without clustering. This linkage map also consisted of 199 AFLP and 26 SSR markers. This WRKY-based marker system is a rapid and simple method for generating sequence-specific markers for plant gene families.

  20. Glucose starvation and hypoxia, but not the saturated fatty acid palmitic acid or cholesterol, activate the unfolded protein response in 3T3-F442A and 3T3-L1 adipocytes

    PubMed Central

    Mihai, Adina D; Schröder, Martin

    2015-01-01

    Obesity is associated with endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR) in adipose tissue. In this study we identify physiological triggers of ER stress and of the UPR in adipocytes in vitro. We show that two markers of adipose tissue remodelling in obesity, glucose starvation and hypoxia, cause ER stress in 3T3-F442A and 3T3-L1 adipocytes. Both conditions induced molecular markers of the IRE1α and PERK branches of the UPR, such as splicing of XBP1 mRNA and CHOP, as well as transcription of the ER stress responsive gene BiP. Hypoxia also induced an increase in phosphorylation of the PERK substrate eIF2α. By contrast, physiological triggers of ER stress in many other cell types, such as the saturated fatty acid palmitic acid, cholesterol, or several inflammatory cytokines including TNF-α, IL-1β, and IL-6, do not cause ER stress in 3T3-F442A and 3T3-L1 adipocytes. Our data suggest that physiological changes associated with remodelling of adipose tissue in obesity, such as hypoxia and glucose starvation, are more likely physiological ER stressors of adipocytes than the lipid overload or hyperinsulinemia associated with obesity. PMID:26257992

  1. Significance of stem cell marker Nanog gene in the diagnosis and prognosis of lung cancer

    PubMed Central

    Liu, Zeng; Zhang, Jing; Kang, Honggang; Sun, Guiming; Wang, Baozhong; Wang, Yanwen; Yang, Mengxiang

    2016-01-01

    The aim of the present study was to analyze the stem cell marker, Nanog gene, for the diagnosis and prognosis of lung cancer cases, and to study its application in the diagnosis of lung cancer. In total, 100 patients diagnosed with lung cancer between April, 2013 and May, 2015 were included in the present study. The patients were randomly divided into group A (lung cancer) and group B (squamous cell lung carcinoma). RT-PCR was used to detect the cancer and adjacent tissues, and Nanog gene expression was detected in groups A and B in cells. The results showed that, analysis of Nanog gene expression in the two groups of patients varied to different degrees. There was no significant difference between the two groups with regard to age, gender, disease stage and lymph node metastasis. Nanog gene expression in patients with carcinoma were significantly higher than that in the adjacent tissues (p<0.05). By contrast, differentiated and well-differentiated carcinoma tissue showed a significantly higher Nanog gene expression than poorly differentiated and undifferentiated carcinoma (p<0.05). The expression of Nanog in normal cells was significantly higher than that in normal lung tissues and benign lesions in lung cancer stem cells. Nanog was highly expressed in CD44+ cells, and Nanog expression in lung cancer stem cells was significantly higher (p<0.05). In conclusion, for groups A (lung cancer) and B (squamous cell lung carcinoma) the Nanog gene expression was significantly higher. The data of the present study show that the patients with stage III and IV lung cancer had a higher Nanog gene expression. In addition, there was a higher expression of Nanog in lung cancer patients. By contrast, a lower degree of cell differentiation was associated with strong Nanog gene expression in lung cancer.

  2. Mining and gene ontology based annotation of SSR markers from expressed sequence tags of Humulus lupulus.

    PubMed

    Singh, Swati; Gupta, Sanchita; Mani, Ashutosh; Chaturvedi, Anoop

    2012-01-01

    Humulus lupulus is commonly known as hops, a member of the family moraceae. Currently many projects are underway leading to the accumulation of voluminous genomic and expressed sequence tag sequences in public databases. The genetically characterized domains in these databases are limited due to non-availability of reliable molecular markers. The large data of EST sequences are available in hops. The simple sequence repeat markers extracted from EST data are used as molecular markers for genetic characterization, in the present study. 25,495 EST sequences were examined and assembled to get full-length sequences. Maximum frequency distribution was shown by mononucleotide SSR motifs i.e. 60.44% in contig and 62.16% in singleton where as minimum frequency are observed for hexanucleotide SSR in contig (0.09%) and pentanucleotide SSR in singletons (0.12%). Maximum trinucleotide motifs code for Glutamic acid (GAA) while AT/TA were the most frequent repeat of dinucleotide SSRs. Flanking primer pairs were designed in-silico for the SSR containing sequences. Functional categorization of SSRs containing sequences was done through gene ontology terms like biological process, cellular component and molecular function.

  3. Genetic transformation of Nannochloropsis oculata with a bacterial phleomycin resistance gene as dominant selective marker

    NASA Astrophysics Data System (ADS)

    Ma, Xiaolei; Pan, Kehou; Zhang, Lin; Zhu, Baohua; Yang, Guanpin; Zhang, Xiangyang

    2016-04-01

    The gene ble from Streptoalloteichus hindustanus is widely used as a selective antibiotic marker. It can control the phleomycin resistance, and significantly increase the tolerance of hosts to zeocin. The unicellular marine microalga Nannochloropsis oculata is extremely sensitive to zeocin. We selected ble as the selective marker for the genetic transformation of N. oculata. After the algal cells at a density of 2×107 cells mL-1 was digested with 4% hemicellulase and 2% driselase for 1 h, the protoplasts accounted for 90% of the total. The ble was placed at the downstream of promoter HSP70A-RUBS2 isolated from Chlamydomonas reinhardtii, yielding a recombinant expression construct pMS188. The construct was transferred into the protoplasts through electroporation (1 kV, 15 μS). The transformed protoplasts were cultured in fresh f/2 liquid medium, and selected on solid f/2 medium supplemented with 500 ng mL-1 zeocin. The PCR result proved that ble existed in the transformants. Three transformants had been cultured for at least 5 generations without losing ble. Southern blotting analysis showed that the ble has been integrated into the genome of N. oculata. The ble will serve as a new dominant selective marker in genetic engineering N. oculata.

  4. Nanoplex-Mediated Co-delivery of Fibroblast Growth Factor and Bone Morphogenetic Protein Genes Promotes Osteogenesis in Human Adipocyte-Derived Mesenchymal Stem Cells

    PubMed Central

    Atluri, Keerthi; Seabold, Denise; Hong, Liu; Elangovan, Satheesh; Salem, Aliasger K.

    2015-01-01

    This study highlights the importance of transfection mediated coordinated bone morphogenetic protein 2 (BMP-2) and fibroblast growth factor 2 (FGF-2) signaling in promoting osteogenesis. We employed plasmids independently encoding BMP-2 and FGF-2 complexed with polyethylenimine (PEI) to transfect human adipose derived mesenchymal stem cells (hADMSCs) in vitro. The nanoplexes were characterized for size, surface charge, in vitro cytotoxicity and transfection ability in hADMSCs. A significant enhancement in BMP-2 protein secretion was observed on day 7 post-transfection of hADMSCs with PEI nanoplexes loaded with both pFGF-2 and pBMP-2 (PEI/(pFGF-2 + pBMP-2)) versus transfection with PEI nanoplexes of either pFGF-2 alone or pBMP-2 alone. Osteogenic differentiation of transfected hADMSCs was determined by measuring osteocalcin and Runx-2 gene expression using real time polymerase chain reactions. A significant increase in the expression of Runx-2 and osteocalcin was observed on day 3 and day 7 post-transfection, respectively, by cells transfected with PEI/(pFGF-2 + pBMP-2) compared to cells transfected with nanoplexes containing pFGF-2 or pBMP-2 alone. Alizarin Red staining and atomic absorption spectroscopy revealed elevated levels of calcium deposition in hADMSC cultures on day 14 and day 30 post-transfection with PEI/(pFGF-2 + pBMP-2) compared to other treatments. We have shown that co-delivery of pFGF-2 and pBMP-2 results in a significant enhancement in osteogenic protein synthesis, osteogenic marker expression and subsequent mineralization. This research points to a new clinically translatable strategy for achieving efficient bone regeneration. PMID:26121311

  5. [Leptin: adipocyte hormone].

    PubMed

    Castagna, L; De Gregorio, T; Allegra, A; Buemi, M; Corsonello, A; Bonanzinga, S; Catanoso, M; Ceruso, D; Corica, F

    1998-04-01

    The authors reviewed the most recent literature on leptin, a protein produced by adipocytes which exerts its action on hypothalamus, modifying eating behavior and inhibiting the lust for food consumption. This one appeared to be the main, if not the only, physiologic action of leptin. Later leptin has been acknowledged a major role in the homeostasis. The regulation of the synthesis, and the mechanisms by which the protein modulates both food intake and energetic balance have been evaluated, and the hypotheses on the regulatory function exerted by leptin on the homeostasis, by acting on neuroendocrine system, on sexual maturity and fertility, on the sympathetic nervous system, on hemopoiesis and hydroelectrolytic balance have been discussed, some of which being already supported by experimental evidences.

  6. Ubc9 Impairs Activation of the Brown Fat Energy Metabolism Program in Human White Adipocytes.

    PubMed

    Hartig, Sean M; Bader, David A; Abadie, Kathleen V; Motamed, Massoud; Hamilton, Mark P; Long, Weiwen; York, Brian; Mueller, Michaela; Wagner, Martin; Trauner, Michael; Chan, Lawrence; Bajaj, Mandeep; Moore, David D; Mancini, Michael A; McGuire, Sean E

    2015-09-01

    Insulin resistance and type 2 diabetes mellitus (T2DM) result from an inability to efficiently store and catabolize surplus energy in adipose tissue. Subcutaneous adipocytes protect against insulin resistance and T2DM by coupling differentiation with the induction of brown fat gene programs for efficient energy metabolism. Mechanisms that disrupt these programs in adipocytes are currently poorly defined, but represent therapeutic targets for the treatment of T2DM. To gain insight into these mechanisms, we performed a high-throughput microscopy screen that identified ubiquitin carrier protein 9 (Ubc9) as a negative regulator of energy storage in human sc adipocytes. Ubc9 depletion enhanced energy storage and induced the brown fat gene program in human sc adipocytes. Induction of adipocyte differentiation resulted in decreased Ubc9 expression commensurate with increased brown fat gene expression. Thiazolidinedione treatment reduced the interaction between Ubc9 and peroxisome proliferator-activated receptor (PPAR)γ, suggesting a mechanism by which Ubc9 represses PPARγ activity. In support of this hypothesis, Ubc9 overexpression remodeled energy metabolism in human sc adipocytes by selectively inhibiting brown adipocyte-specific function. Further, Ubc9 overexpression decreased uncoupling protein 1 expression by disrupting PPARγ binding at a critical uncoupling protein 1 enhancer region. Last, Ubc9 is significantly elevated in sc adipose tissue isolated from mouse models of insulin resistance as well as diabetic and insulin-resistant humans. Taken together, our findings demonstrate a critical role for Ubc9 in the regulation of sc adipocyte energy homeostasis.

  7. Ubc9 Impairs Activation of the Brown Fat Energy Metabolism Program in Human White Adipocytes

    PubMed Central

    Bader, David A.; Abadie, Kathleen V.; Motamed, Massoud; Hamilton, Mark P.; Long, Weiwen; York, Brian; Mueller, Michaela; Wagner, Martin; Trauner, Michael; Chan, Lawrence; Bajaj, Mandeep; Moore, David D.; Mancini, Michael A.; McGuire, Sean E.

    2015-01-01

    Insulin resistance and type 2 diabetes mellitus (T2DM) result from an inability to efficiently store and catabolize surplus energy in adipose tissue. Subcutaneous adipocytes protect against insulin resistance and T2DM by coupling differentiation with the induction of brown fat gene programs for efficient energy metabolism. Mechanisms that disrupt these programs in adipocytes are currently poorly defined, but represent therapeutic targets for the treatment of T2DM. To gain insight into these mechanisms, we performed a high-throughput microscopy screen that identified ubiquitin carrier protein 9 (Ubc9) as a negative regulator of energy storage in human sc adipocytes. Ubc9 depletion enhanced energy storage and induced the brown fat gene program in human sc adipocytes. Induction of adipocyte differentiation resulted in decreased Ubc9 expression commensurate with increased brown fat gene expression. Thiazolidinedione treatment reduced the interaction between Ubc9 and peroxisome proliferator-activated receptor (PPAR)γ, suggesting a mechanism by which Ubc9 represses PPARγ activity. In support of this hypothesis, Ubc9 overexpression remodeled energy metabolism in human sc adipocytes by selectively inhibiting brown adipocyte-specific function. Further, Ubc9 overexpression decreased uncoupling protein 1 expression by disrupting PPARγ binding at a critical uncoupling protein 1 enhancer region. Last, Ubc9 is significantly elevated in sc adipose tissue isolated from mouse models of insulin resistance as well as diabetic and insulin-resistant humans. Taken together, our findings demonstrate a critical role for Ubc9 in the regulation of sc adipocyte energy homeostasis. PMID:26192107

  8. Comparative Analysis of Cartilage Marker Gene Expression Patterns during Axolotl and Xenopus Limb Regeneration.

    PubMed

    Mitogawa, Kazumasa; Makanae, Aki; Satoh, Ayano; Satoh, Akira

    2015-01-01

    Axolotls (Ambystoma mexicanum) can completely regenerate lost limbs, whereas Xenopus laevis frogs cannot. During limb regeneration, a blastema is first formed at the amputation plane. It is thought that this regeneration blastema forms a limb by mechanisms similar to those of a developing embryonic limb bud. Furthermore, Xenopus laevis frogs can form a blastema after amputation; however, the blastema results in a terminal cone-shaped cartilaginous structure called a "spike." The causes of this patterning defect in Xenopus frog limb regeneration were explored. We hypothesized that differences in chondrogenesis may underlie the patterning defect. Thus, we focused on chondrogenesis. Chondrogenesis marker genes, type I and type II collagen, were compared in regenerative and nonregenerative environments. There were marked differences between axolotls and Xenopus in the expression pattern of these chondrogenesis-associated genes. The relative deficit in the chondrogenic capacity of Xenopus blastema cells may account for the absence of total limb regenerative capacity.

  9. Massively parallel sequencing of single cells by epicPCR links functional genes with phylogenetic markers.

    PubMed

    Spencer, Sarah J; Tamminen, Manu V; Preheim, Sarah P; Guo, Mira T; Briggs, Adrian W; Brito, Ilana L; A Weitz, David; Pitkänen, Leena K; Vigneault, Francois; Juhani Virta, Marko P; Alm, Eric J

    2016-02-01

    Many microbial communities are characterized by high genetic diversity. 16S ribosomal RNA sequencing can determine community members, and metagenomics can determine the functional diversity, but resolving the functional role of individual cells in high throughput remains an unsolved challenge. Here, we describe epicPCR (Emulsion, Paired Isolation and Concatenation PCR), a new technique that links functional genes and phylogenetic markers in uncultured single cells, providing a throughput of hundreds of thousands of cells with costs comparable to one genomic library preparation. We demonstrate the utility of our technique in a natural environment by profiling a sulfate-reducing community in a freshwater lake, revealing both known sulfate reducers and discovering new putative sulfate reducers. Our method is adaptable to any conserved genetic trait and translates genetic associations from diverse microbial samples into a sequencing library that answers targeted ecological questions. Potential applications include identifying functional community members, tracing horizontal gene transfer networks and mapping ecological interactions between microbial cells. PMID:26394010

  10. Comparative Analysis of Cartilage Marker Gene Expression Patterns during Axolotl and Xenopus Limb Regeneration.

    PubMed

    Mitogawa, Kazumasa; Makanae, Aki; Satoh, Ayano; Satoh, Akira

    2015-01-01

    Axolotls (Ambystoma mexicanum) can completely regenerate lost limbs, whereas Xenopus laevis frogs cannot. During limb regeneration, a blastema is first formed at the amputation plane. It is thought that this regeneration blastema forms a limb by mechanisms similar to those of a developing embryonic limb bud. Furthermore, Xenopus laevis frogs can form a blastema after amputation; however, the blastema results in a terminal cone-shaped cartilaginous structure called a "spike." The causes of this patterning defect in Xenopus frog limb regeneration were explored. We hypothesized that differences in chondrogenesis may underlie the patterning defect. Thus, we focused on chondrogenesis. Chondrogenesis marker genes, type I and type II collagen, were compared in regenerative and nonregenerative environments. There were marked differences between axolotls and Xenopus in the expression pattern of these chondrogenesis-associated genes. The relative deficit in the chondrogenic capacity of Xenopus blastema cells may account for the absence of total limb regenerative capacity. PMID:26186213

  11. Comparative Analysis of Cartilage Marker Gene Expression Patterns during Axolotl and Xenopus Limb Regeneration

    PubMed Central

    Mitogawa, Kazumasa; Makanae, Aki; Satoh, Ayano; Satoh, Akira

    2015-01-01

    Axolotls (Ambystoma mexicanum) can completely regenerate lost limbs, whereas Xenopus laevis frogs cannot. During limb regeneration, a blastema is first formed at the amputation plane. It is thought that this regeneration blastema forms a limb by mechanisms similar to those of a developing embryonic limb bud. Furthermore, Xenopus laevis frogs can form a blastema after amputation; however, the blastema results in a terminal cone-shaped cartilaginous structure called a “spike.” The causes of this patterning defect in Xenopus frog limb regeneration were explored. We hypothesized that differences in chondrogenesis may underlie the patterning defect. Thus, we focused on chondrogenesis. Chondrogenesis marker genes, type I and type II collagen, were compared in regenerative and nonregenerative environments. There were marked differences between axolotls and Xenopus in the expression pattern of these chondrogenesis-associated genes. The relative deficit in the chondrogenic capacity of Xenopus blastema cells may account for the absence of total limb regenerative capacity. PMID:26186213

  12. Massively parallel sequencing of single cells by epicPCR links functional genes with phylogenetic markers

    PubMed Central

    Spencer, Sarah J; Tamminen, Manu V; Preheim, Sarah P; Guo, Mira T; Briggs, Adrian W; Brito, Ilana L; A Weitz, David; Pitkänen, Leena K; Vigneault, Francois; Virta, Marko PJuhani; Alm, Eric J

    2016-01-01

    Many microbial communities are characterized by high genetic diversity. 16S ribosomal RNA sequencing can determine community members, and metagenomics can determine the functional diversity, but resolving the functional role of individual cells in high throughput remains an unsolved challenge. Here, we describe epicPCR (Emulsion, Paired Isolation and Concatenation PCR), a new technique that links functional genes and phylogenetic markers in uncultured single cells, providing a throughput of hundreds of thousands of cells with costs comparable to one genomic library preparation. We demonstrate the utility of our technique in a natural environment by profiling a sulfate-reducing community in a freshwater lake, revealing both known sulfate reducers and discovering new putative sulfate reducers. Our method is adaptable to any conserved genetic trait and translates genetic associations from diverse microbial samples into a sequencing library that answers targeted ecological questions. Potential applications include identifying functional community members, tracing horizontal gene transfer networks and mapping ecological interactions between microbial cells. PMID:26394010

  13. Massively parallel sequencing of single cells by epicPCR links functional genes with phylogenetic markers.

    PubMed

    Spencer, Sarah J; Tamminen, Manu V; Preheim, Sarah P; Guo, Mira T; Briggs, Adrian W; Brito, Ilana L; A Weitz, David; Pitkänen, Leena K; Vigneault, Francois; Juhani Virta, Marko P; Alm, Eric J

    2016-02-01

    Many microbial communities are characterized by high genetic diversity. 16S ribosomal RNA sequencing can determine community members, and metagenomics can determine the functional diversity, but resolving the functional role of individual cells in high throughput remains an unsolved challenge. Here, we describe epicPCR (Emulsion, Paired Isolation and Concatenation PCR), a new technique that links functional genes and phylogenetic markers in uncultured single cells, providing a throughput of hundreds of thousands of cells with costs comparable to one genomic library preparation. We demonstrate the utility of our technique in a natural environment by profiling a sulfate-reducing community in a freshwater lake, revealing both known sulfate reducers and discovering new putative sulfate reducers. Our method is adaptable to any conserved genetic trait and translates genetic associations from diverse microbial samples into a sequencing library that answers targeted ecological questions. Potential applications include identifying functional community members, tracing horizontal gene transfer networks and mapping ecological interactions between microbial cells.

  14. Development of IP and SCAR markers linked to the yellow seed color gene in Brassica juncea L.

    PubMed Central

    Huang, Zhen; Liu, Lu; Lu, Hong; Lang, Lina; Zhao, Na; Ding, Juan; Xu, Aixia

    2016-01-01

    Previous studies showed that the yellow seed color gene of a yellow mustard was located on the A09 chromosome. In this study, the sequences of the molecular markers linked to the yellow seed color gene were analyzed, the gene was primarily mapped to an interval of 23.304 to 29.402M. Twenty genes and eight markers’ sequences in this region were selected to design the IP and SCAR primers. These primers were used to screen a BC8S1 population consisting of 1256 individuals. As a result, five IP and five SCAR markers were successfully developed. IP4 and Y1 were located on either side of the yellow seed color gene at a distance of 0.1 and 0.3 cM, respectively. IP1, IP2 and IP3 derived from Bra036827, Bra036828, Bra036829 separately, co-segregated with the target gene. BLAST analysis indicated that the sequences of newly developed markers showed good collinearity with those of the A09 chromosome, and that the target gene might exist between 27.079 and 27.616M. In light of annotations of the genes in this region, only Bra036828 is associated with flavonoid biosynthesis. This gene has high similarity with the TRANSPARENT TESTA6 gene, Bra036828 was hence identified as being the gene possibly responsible for yellow seed color, in our research. PMID:27162489

  15. Silibinin Regulates Lipid Metabolism and Differentiation in Functional Human Adipocytes

    PubMed Central

    Barbagallo, Ignazio; Vanella, Luca; Cambria, Maria T.; Tibullo, Daniele; Godos, Justyna; Guarnaccia, Laura; Zappalà, Agata; Galvano, Fabio; Li Volti, Giovanni

    2016-01-01

    Silibinin, a natural plant flavonolignan is the main active constituent found in milk thistle (Silybum marianum). It is known to have hepatoprotective, anti-neoplastic effect, and suppresses lipid accumulation in adipocytes. Objective of this study was to investigate the effect of silibinin on adipogenic differentiation and thermogenic capacity of human adipose tissue derived mesenchymal stem cells. Silibinin (10 μM) treatment, either at the beginning or at the end of adipogenic differentiation, resulted in an increase of SIRT-1, PPARα, Pgc-1α, and UCPs gene expression. Moreover, silibinin administration resulted in a decrease of PPARγ, FABP4, FAS, and MEST/PEG1 gene expression during the differentiation, confirming that this compound is able to reduce fatty acid accumulation and adipocyte size. Our data showed that silibinin regulated adipocyte lipid metabolism, inducing thermogenesis and promoting a brown remodeling in adipocyte. Taken together, our findings suggest that silibinin increases UCPs expression by stimulation of SIRT1, PPARα, and Pgc-1α, improved metabolic parameters, decreased lipid mass leading to the formation of functional adipocytes. PMID:26834634

  16. Reassessment of Blood Gene Expression Markers for the Prognosis of Relapsing-Remitting Multiple Sclerosis

    PubMed Central

    Hecker, Michael; Paap, Brigitte Katrin; Goertsches, Robert Hermann; Kandulski, Ole; Fatum, Christian; Koczan, Dirk; Hartung, Hans-Peter; Thiesen, Hans-Juergen; Zettl, Uwe Klaus

    2011-01-01

    Despite considerable advances in the treatment of multiple sclerosis, current drugs are only partially effective. Most patients show reduced disease activity with therapy, but still experience relapses, increasing disability, and new brain lesions. Since there are no reliable clinical or biological markers of disease progression, long-term prognosis is difficult to predict for individual patients. We identified 18 studies that suggested genes expressed in blood as predictive biomarkers. We validated the prognostic value of those genes with three different microarray data sets comprising 148 patients in total. Using these data, we tested whether the genes were significantly differentially expressed between patients with good and poor courses of the disease. Poor progression was defined by relapses and/or increase of disability during a two-year follow-up, independent of the administered therapy. Of 110 genes that have been proposed as predictive biomarkers, most could not be confirmed in our analysis. However, the G protein-coupled membrane receptor GPR3 was expressed at significantly lower levels in patients with poor disease progression in all data sets. GPR3 has therefore a high potential to be a biomarker for predicting future disease activity. In addition, we examined the IL17 cytokines and receptors in more detail and propose IL17RC as a new, promising, transcript-based biomarker candidate. Further studies are needed to better understand the roles of these receptors in multiple sclerosis and its treatment and to clarify the utility of GPR3 and IL17RC expression levels in the blood as markers of long-term prognosis. PMID:22216338

  17. Candidate Genes for Respiratory Disease Associated with Markers of Inflammation and Endothelial Dysfunction in Elderly Men

    PubMed Central

    Wilker, Elissa H.; Alexeeff, Stacey E.; Poon, Audrey; Litonjua, Augusto A.; Sparrow, David; Vokonas, Pantel S.; Mittleman, Murray A.; Schwartz, Joel

    2010-01-01

    Background Inflammation and endothelial dysfunction are important risk factors for cardiovascular disease (CVD). We hypothesized that candidate genes selected for a study of asthma and chronic obstructive pulmonary disorder (COPD) are associated with markers of systemic inflammation and endothelial dysfunction in an aging population. Methods Plasma levels of circulating C-reactive protein (CRP), fibrinogen, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) were obtained from 679 elderly male participants in the Normative Aging Study. Blood samples were analyzed for 202 SNPs in 25 candidate genes and included both haplotype tagSNPs and functional SNPs based on literature review. Data were stratified into discovery and replication cohorts for 2-stage analysis. In the discovery cohort, the relationship between biomarker level and genotype was analyzed using linear mixed effects with random intercepts for each subject and models were adjusted for age and BMI. A positive outcome in the discovery cohort was defined as a p-value <0.1 for the SNP. SNPs that met this criterion were analyzed in the replication cohort and confirmed for those which met a criterion of significance (p<0.025). Results In our analyses, SNPs in the CRHR1, ITPR2, and VDR genes met criteria of significant effects. Conclusions Our results suggest that genes thought to play a role in the pathogenesis of asthma and COPD may influence levels of serum markers of inflammation and endothelial dysfunction via novel SNP associations which have not previously been associated with cardiovascular disease. PMID:19409562

  18. [Molecular markers linked to mono-dominant genic male sterile gene in rapeseed (Brassica napus L.)].

    PubMed

    Wang, Dao-Jie; Guo, Ai-Guang; Li, Dian-Rong; Tian, Jian-Hua

    2006-10-01

    Bulked segregant analysis (BSA) was used to identify randomly amplified polymorphic DNA (RAPD) markers linked to the MS gene in mono-dominant GMS of rapeseed (Brassica napus L.), which was bred by Hybrid Rapeseed Research Center of Shaanxi Province. A total of 300 random 10-mer oligonucleotide primers were screened on the DNA from fertile and sterile bulks. Primer S(243) (5'CTATGCCGAC3') gave identical 1.5 kb DNA polymorphic segment OPU-03(1500) in the bulk S, but not in the bulk F (Fig.2). The DNAs from individual plants of each bulk and from their sister lines, which were generated from the same original crossing, were then screened with the primer S(243), and the same results were obtained (Figs.3,4). Other types of GMS and CMS were analyzed using primer S(243), and the specific 1.5 kb DNA segment was not found (Fig.5). Therefore, the RAPD marker OPU-03(1500) is linked to the mono-dominant GMS trait in rapeseed. This RAPD marker OPU-03(1500) was cloned into a T-easy vector and sequenced. The sequence here obtained was highly homologous to one of the Arabidopsis DNA sequences. According to this DNA conserved region in different species, we designed a pair of specific primers P1 (5'ATGTCGCTGAGGCCG-AGCAC3') and P2 (5'GGCACACTGTCACG-ATCCTTGG3') and amplified only one specific 2.3 kb DNA fragment in each bulk. There are two mutant loci between the two DNA fragments after sequencing. We designed another pair of specific primers P3 (5'CTCCAGCAGCAGCAGC-AGCCT3') and P4 (5'GCAGGAATGAGAA-CCGTAGG3') according to the DNA sequence at the mutant loci. A specific DNA segment was amplified only in the fertile line but not in the sterile line using the primers P3 and P4 (Fig.6). Therefore the RAPD marker were converted into SCAR marker. Moreover, the SCAR marker detection method was improved (Fig.7).

  19. The nuclear elongation factor-1α gene: a promising marker for phylogenetic studies of Triatominae (Hemiptera: Reduviidae).

    PubMed

    Díaz, Sebastián; Triana-Chávez, Omar; Gómez-Palacio, Andrés

    2016-09-01

    Molecular systematics is a remarkable approach for understanding the taxonomic traits and allows the exploration of the inter-population dynamics of several species in the Triatominae subfamily that are involved in Trypanosoma cruzi transmission. Compared to other relevant species that transmit vector-borne diseases, such as some species of the Diptera, there are relatively few nuclear genetic markers available for systematic studies in the Triatominae subfamily. Molecular systematic studies performed on Triatominae are based on mitochondrial gene fragments and, less frequently, on nuclear ribosomal genes or spacers. Due to the fact that these markers can occasionally present problems such as nuclear mitochondrial genes (NUMTs) or intra-genomic variation for high gene copy numbers, it is necessary to use additional nuclear markers to more reliably address the molecular evolution of Triatominae. In this study, we performed phylogenetic analysis using the nuclear elongation factor-1 alpha (EF-1α) gene in individuals from 12 species belonging to the Triatomini and Rhodniini tribes. Genetic diversities and phylogenetic topologies were compared with those obtained for the mitochondrial 16S rRNA and Cytochrome b (cyt b) genes, as well as for the D2 variable region of the ribosomal 28S rRNA gene. These results indicate that the EF-1α marker exhibits an intermediate level of diversity compared to mitochondrial and nuclear ribosomal genes, and that phylogenetic analysis based on EF-1α is highly informative for resolving deep phylogenetic relationships in Triatominae, such as tribe or genera.

  20. The nuclear elongation factor-1α gene: a promising marker for phylogenetic studies of Triatominae (Hemiptera: Reduviidae).

    PubMed

    Díaz, Sebastián; Triana-Chávez, Omar; Gómez-Palacio, Andrés

    2016-09-01

    Molecular systematics is a remarkable approach for understanding the taxonomic traits and allows the exploration of the inter-population dynamics of several species in the Triatominae subfamily that are involved in Trypanosoma cruzi transmission. Compared to other relevant species that transmit vector-borne diseases, such as some species of the Diptera, there are relatively few nuclear genetic markers available for systematic studies in the Triatominae subfamily. Molecular systematic studies performed on Triatominae are based on mitochondrial gene fragments and, less frequently, on nuclear ribosomal genes or spacers. Due to the fact that these markers can occasionally present problems such as nuclear mitochondrial genes (NUMTs) or intra-genomic variation for high gene copy numbers, it is necessary to use additional nuclear markers to more reliably address the molecular evolution of Triatominae. In this study, we performed phylogenetic analysis using the nuclear elongation factor-1 alpha (EF-1α) gene in individuals from 12 species belonging to the Triatomini and Rhodniini tribes. Genetic diversities and phylogenetic topologies were compared with those obtained for the mitochondrial 16S rRNA and Cytochrome b (cyt b) genes, as well as for the D2 variable region of the ribosomal 28S rRNA gene. These results indicate that the EF-1α marker exhibits an intermediate level of diversity compared to mitochondrial and nuclear ribosomal genes, and that phylogenetic analysis based on EF-1α is highly informative for resolving deep phylogenetic relationships in Triatominae, such as tribe or genera. PMID:27268149

  1. A novel crosstalk between Alk7 and cGMP signaling differentially regulates brown adipocyte function

    PubMed Central

    Balkow, Aileen; Jagow, Johanna; Haas, Bodo; Siegel, Franziska; Kilić, Ana; Pfeifer, Alexander

    2015-01-01

    Objective Obesity is an enormous burden for patients and health systems world-wide. Brown adipose tissue dissipates energy in response to cold and has been shown to be metabolically active in human adults. The type I transforming growth factor β (TGFβ) receptor Activin receptor-like kinase 7 (Alk7) is highly expressed in adipose tissues and is down-regulated in obese patients. Here, we studied the function of Alk7 in brown adipocytes. Methods Using pharmacological and genetic tools, Alk7 signaling pathway and its effects were studied in murine brown adipocytes. Brown adipocyte differentiation and activation was analyzed. Results Alk7 is highly upregulated during differentiation of brown adipocytes. Interestingly, Alk7 expression is increased by cGMP/protein kinase G (PKG) signaling, which enhances brown adipocyte differentiation. Activin AB effectively activates Alk7 and SMAD3 signaling. Activation of Alk7 in brown preadipocytes suppresses the master adipogenic transcription factor PPARγ and differentiation. Stimulation of Alk7 during late differentiation of brown adipocytes reduces lipid content and adipogenic marker expression but enhances UCP1 expression. Conclusions We found a so far unknown crosstalk between cGMP and Alk7 signaling pathways. Tight regulation of Alk7 is required for efficient differentiation of brown adipocytes. Alk7 has differential effects on adipogenic differentiation and the development of the thermogenic program in brown adipocytes. PMID:26266090

  2. Development of marker genes for jasmonic acid signaling in shoots and roots of wheat.

    PubMed

    Liu, Hongwei; Carvalhais, Lilia Costa; Kazan, Kemal; Schenk, Peer M

    2016-05-01

    The jasmonic acid (JA) signaling pathway plays key roles in a diverse array of plant development, reproduction, and responses to biotic and abiotic stresses. Most of our understanding of the JA signaling pathway derives from the dicot model plant Arabidopsis thaliana, while corresponding knowledge in wheat is somewhat limited. In this study, the expression of 41 genes implicated in the JA signaling pathway has been assessed on 10 day-old bread wheat seedlings, 24 h, 48 h, and 72 h after methyl-jasmonate (MeJA) treatment using quantitative real-time PCR. The examined genes have been previously reported to be involved in JA biosynthesis and catabolism, JA perception and signaling, and pathogen defense in wheat shoots and roots. This study provides evidence to suggest that the effect of MeJA treatment is more prominent in shoots than roots of wheat seedlings, and substantial regulation of the JA pathway-dependent defense genes occurs at 72 h after MeJA treatment. Results show that the expression of 22 genes was significantly affected by MeJA treatment in wheat shoots. However, only PR1.1 and PR3 were significantly differentially expressed in wheat roots, both at 24 h post-MeJA treatment, with other genes showing large variation in their gene expression in roots. While providing marker genes on JA signaling in wheat, future work may focus on elucidating the regulatory function of JA-modulated transcription factors, some of which have well-studied potential orthologs in Arabidopsis. PMID:27115051

  3. Analysis of some polymorphic markers of the CFTR gene in cystic fibrosis patients and healthy donors from the Moscow region

    SciTech Connect

    Amosenko, F.A.; Sazonova, M.A.; Kapranov, N.I.; Trubnikova, I.S.; Kalinin, V.N.

    1995-04-01

    Allelic frequencies of three polymorphic markers in the CFTR gene were estimated on chromosomes derived from cystic fibrosis (CF) patients and healthy donors from Moscow and the Moscow region. These polymorphic markers are tetranucleotide tandem repeats GATT in intron 6B, M470V in exon 10, and T854T in exon 14 (fragment A). Frequencies at allele 1 of the M470V marker, along with allele 2 of GATT and T854T, are two times higher for CF patients without {Delta}F508 mutation than for healthy donors, and there is linkage disequilibrium of these alleles of the polymorphic markers analyzed with the CF gene. Allele 1 of M470V and T854T markers, as well as allele 2 of the GATT marker (six repeats), are absolutely linked to mutation F508 of the CFTR gene. Using the polymorphic markers studied, family analysis of CF was carried out in two families. 10 refs., 1 fig., 1 tab.

  4. Production and processing studies on calpain-system gene markers for tenderness in Brahman cattle: 2. Objective meat quality.

    PubMed

    Cafe, L M; McIntyre, B L; Robinson, D L; Geesink, G H; Barendse, W; Pethick, D W; Thompson, J M; Greenwood, P L

    2010-09-01

    Effects and interactions of calpain-system tenderness gene markers on objective meat quality traits of Brahman (Bos indicus) cattle were quantified within 2 concurrent experiments at different locations. Cattle were selected for study from commercial and research herds at weaning based on their genotype for calpastatin (CAST) and calpain 3 (CAPN3) gene markers for beef tenderness. Gene marker status for mu-calpain (CAPN1-4751 and CAPN1-316) was also determined for inclusion in statistical analyses. Eighty-two heifer and 82 castrated male cattle with 0 or 2 favorable alleles for CAST and CAPN3 were studied in New South Wales (NSW), and 143 castrated male cattle with 0, 1, or 2 favorable alleles for CAST and CAPN3 were studied in Western Australia (WA). The cattle were backgrounded for 6 to 8 mo and grain-fed for 117 d (NSW) or 80 d (WA) before slaughter. One-half the cattle in each experiment were implanted with a hormonal growth promotant during feedlotting. One side of each carcass was suspended from the Achilles tendon (AT) and the other from the pelvis (tenderstretch). The M. longissimus lumborum from both sides and the M. semitendinosus from the AT side were collected; then samples of each were aged at 1 degrees C for 1 or 7 d. Favorable alleles for one or more markers reduced shear force, with little effect on other meat quality traits. The size of effects of individual markers varied with site, muscle, method of carcass suspension, and aging period. Individual marker effects were additive as evident in cattle with 4 favorable alleles for CAST and CAPN3 markers, which had shear force reductions of 12.2 N (P < 0.001, NSW) and 9.3 N (P = 0.002, WA) in AT 7 d aged M. longissimus lumborum compared with those with no favorable alleles. There was no evidence (all P > 0.05) of interactions between the gene markers, or between the hormonal growth promotant and gene markers for any meat quality traits. This study provides further evidence that selection based on the

  5. Production and processing studies on calpain-system gene markers for tenderness in Brahman cattle: 2. Objective meat quality.

    PubMed

    Cafe, L M; McIntyre, B L; Robinson, D L; Geesink, G H; Barendse, W; Pethick, D W; Thompson, J M; Greenwood, P L

    2010-09-01

    Effects and interactions of calpain-system tenderness gene markers on objective meat quality traits of Brahman (Bos indicus) cattle were quantified within 2 concurrent experiments at different locations. Cattle were selected for study from commercial and research herds at weaning based on their genotype for calpastatin (CAST) and calpain 3 (CAPN3) gene markers for beef tenderness. Gene marker status for mu-calpain (CAPN1-4751 and CAPN1-316) was also determined for inclusion in statistical analyses. Eighty-two heifer and 82 castrated male cattle with 0 or 2 favorable alleles for CAST and CAPN3 were studied in New South Wales (NSW), and 143 castrated male cattle with 0, 1, or 2 favorable alleles for CAST and CAPN3 were studied in Western Australia (WA). The cattle were backgrounded for 6 to 8 mo and grain-fed for 117 d (NSW) or 80 d (WA) before slaughter. One-half the cattle in each experiment were implanted with a hormonal growth promotant during feedlotting. One side of each carcass was suspended from the Achilles tendon (AT) and the other from the pelvis (tenderstretch). The M. longissimus lumborum from both sides and the M. semitendinosus from the AT side were collected; then samples of each were aged at 1 degrees C for 1 or 7 d. Favorable alleles for one or more markers reduced shear force, with little effect on other meat quality traits. The size of effects of individual markers varied with site, muscle, method of carcass suspension, and aging period. Individual marker effects were additive as evident in cattle with 4 favorable alleles for CAST and CAPN3 markers, which had shear force reductions of 12.2 N (P < 0.001, NSW) and 9.3 N (P = 0.002, WA) in AT 7 d aged M. longissimus lumborum compared with those with no favorable alleles. There was no evidence (all P > 0.05) of interactions between the gene markers, or between the hormonal growth promotant and gene markers for any meat quality traits. This study provides further evidence that selection based on the

  6. Development of SRAP, SNP and multiplexed SCAR molecular markers for the major seed coat color gene in Brassica rapa L.

    PubMed

    Rahman, Mukhlesur; McVetty, Peter B E; Li, Genyi

    2007-11-01

    Seed coat color inheritance in B. rapa was studied in F(1), F(2), F(3), and BC(1) progenies from a cross of a Canadian brown-seeded variety 'SPAN' and a Bangladeshi yellow sarson variety 'BARI-6'. A pollen effect was found when the yellow sarson line was used as the maternal parent. Seed coat color segregated into brown, yellow-brown and bright yellow classes. Segregation was under digenic control where the brown or yellow-brown color was dominant over bright yellow seed coat color. A sequence related amplified polymorphism (SRAP) marker linked closely to a major seed coat color gene (Br1/br1) was developed. This dominant SRAP molecular marker was successfully converted into single nucleotide polymorphism (SNP) markers and sequence characterized amplification region (SCAR) markers after the extended flanking sequence of the SRAP was obtained with chromosome walking. In total, 24 SNPs were identified with more than 2-kb sequence. A 12-bp deletion allowed the development of a SCAR marker linked closely to the Br1 gene. Using the five-fluorescence dye set supplied by ABI, four labeled M13 primers were integrated with different SCAR primers to increase the throughput of SCAR marker detection. Using multiplexed SCAR markers targeting insertions and deletions in a genome shows great potential for marker assisted selection in plant breeding.

  7. Fine mapping of the clubroot resistance gene CRb and development of a useful selectable marker in Brassica rapa.

    PubMed

    Kato, Takeyuki; Hatakeyama, Katsunori; Fukino, Nobuko; Matsumoto, Satoru

    2013-03-01

    In Chinese cabbage (Brassica rapa), the clubroot resistance (CR) gene CRb is effective against Plasmodiophora brassicae isolate No. 14, which is classified as pathotype group 3. Although markers linked to CRb have been reported, an accurate position in the genome and the gene structure are unknown. To determine the genomic location and estimate the structure of CRb, we developed 28 markers (average distance, 20.4 kb) around CRb and constructed a high-density partial map. The precise position of CRb was determined by using a population of 2,032 F2 plants generated by selfing B. rapa 'CR Shinki.' We determined that CRb is located in the 140-kb genomic region between markers KB59N07 and B1005 and found candidate resistance genes. Among other CR genes on chromosome R3, a genotype of CRa closest marker clearly matched those of CRb and Crr3 did not confer resistance to isolate No. 14. Based on the genotypes of 11 markers developed near CRb and resistance to isolate No. 14, 82 of 108 cultivars showed a strong correlation between genotypes and phenotypes. The results of this study will be useful for isolating CRb and breeding cultivars with resistance to pathotype group 3 by introducing CRb into susceptible cultivars through marker-assisted selection.

  8. Evaluation of marker-assisted selection for the stripe rust resistance gene Yr15, introgressed from wild emmer wheat

    PubMed Central

    Yaniv, Elitsur; Raats, Dina; Ronin, Yefim; Korol, Abraham B.; Grama, Adriana; Bariana, Harbans; Dubcovsky, Jorge; Schulman, Alan H.

    2016-01-01

    Stripe rust disease is caused by the fungus Puccinia striiformis f. sp. tritici and severely threatens wheat worldwide, repeatedly breaking resistance conferred by resistance genes and evolving more aggressive strains. Wild emmer wheat, Triticum dicoccoides, is an important source for novel stripe rust resistance (Yr) genes. Yr15, a major gene located on chromosome 1BS of T. dicoccoides, was previously reported to confer resistance to a broad spectrum of stripe rust isolates, at both seedling and adult plant stages. Introgressions of Yr15 into cultivated T. aestivum bread wheat and T. durum pasta wheat that began in the 1980s are widely used. In the present study, we aimed to validate SSR markers from the Yr15 region as efficient tools for marker-assisted selection (MAS) for introgression of Yr15 into wheat and to compare the outcome of gene introgression by MAS and by conventional phenotypic selection. Our findings establish the validity of MAS for introgression of Yr15 into wheat. We show that the size of the introgressed segment, defined by flanking markers, varies for both phenotypic selection and MAS. The genetic distance of the MAS marker from Yr15 and the number of backcross steps were the main factors affecting the length of the introgressed donor segments. Markers Xbarc8 and Xgwm493, which are the nearest flanking markers studied, were consistent and polymorphic in all 34 introgressions reported here and are therefore the most recommended markers for the introgression of Yr15 into wheat cultivars. Introgression directed by markers, rather than by phenotype, will facilitate simultaneous selection for multiple stripe rust resistant genes and will help to avoid escapees during the selection process.

  9. Sexually dimorphic gene expressions in eels: useful markers for early sex assessment in a conservation context

    PubMed Central

    Geffroy, Benjamin; Guilbaud, Florian; Amilhat, Elsa; Beaulaton, Laurent; Vignon, Matthias; Huchet, Emmanuel; Rives, Jacques; Bobe, Julien; Fostier, Alexis; Guiguen, Yann; Bardonnet, Agnès

    2016-01-01

    Environmental sex determination (ESD) has been detected in a range of vertebrate reptile and fish species. Eels are characterized by an ESD that occurs relatively late, since sex cannot be histologically determined before individuals reach 28 cm. Because several eel species are at risk of extinction, assessing sex at the earliest stage is a crucial management issue. Based on preliminary results of RNA sequencing, we targeted genes susceptible to be differentially expressed between ovaries and testis at different stages of development. Using qPCR, we detected testis-specific expressions of dmrt1, amh, gsdf and pre-miR202 and ovary-specific expressions were obtained for zar1, zp3 and foxn5. We showed that gene expressions in the gonad of intersexual eels were quite similar to those of males, supporting the idea that intersexual eels represent a transitional stage towards testicular differentiation. To assess whether these genes would be effective early molecular markers, we sampled juvenile eels in two locations with highly skewed sex ratios. The combined expression of six of these genes allowed the discrimination of groups according to their potential future sex and thus this appears to be a useful tool to estimate sex ratios of undifferentiated juvenile eels. PMID:27658729

  10. Efficient, Antibiotic Marker-Free Transformation of a Dicot and a Monocot Crop with Glutamate 1-Semialdehyde Aminotransferase Selectable Marker Genes.

    PubMed

    Ferradini, Nicoletta; Giancaspro, Angelica; Nicolia, Alessandro; Gadaleta, Agata; Veronesi, Fabio; Rosellini, Daniele

    2016-01-01

    Antibiotic-free, efficient in vitro selection in plant genetic engineering can improve risk perception and speed up pre-market scrutiny of genetically modified crops. We provide a protocol for genetic transformation of two important crops, durum wheat and alfalfa, using a bacterial and a plant-derived selectable marker gene encoding mutated, gabaculine-insensitive glutamate 1-semialdehyde aminotransferase (GSA) enzymes. These methods can potentially be applied, with minor adaptations, to many other monocot and dicot crop plants. PMID:26614283

  11. Efficient, Antibiotic Marker-Free Transformation of a Dicot and a Monocot Crop with Glutamate 1-Semialdehyde Aminotransferase Selectable Marker Genes.

    PubMed

    Ferradini, Nicoletta; Giancaspro, Angelica; Nicolia, Alessandro; Gadaleta, Agata; Veronesi, Fabio; Rosellini, Daniele

    2016-01-01

    Antibiotic-free, efficient in vitro selection in plant genetic engineering can improve risk perception and speed up pre-market scrutiny of genetically modified crops. We provide a protocol for genetic transformation of two important crops, durum wheat and alfalfa, using a bacterial and a plant-derived selectable marker gene encoding mutated, gabaculine-insensitive glutamate 1-semialdehyde aminotransferase (GSA) enzymes. These methods can potentially be applied, with minor adaptations, to many other monocot and dicot crop plants.

  12. NINJA-OPS: Fast Accurate Marker Gene Alignment Using Concatenated Ribosomes.

    PubMed

    Al-Ghalith, Gabriel A; Montassier, Emmanuel; Ward, Henry N; Knights, Dan

    2016-01-01

    The explosion of bioinformatics technologies in the form of next generation sequencing (NGS) has facilitated a massive influx of genomics data in the form of short reads. Short read mapping is therefore a fundamental component of next generation sequencing pipelines which routinely match these short reads against reference genomes for contig assembly. However, such techniques have seldom been applied to microbial marker gene sequencing studies, which have mostly relied on novel heuristic approaches. We propose NINJA Is Not Just Another OTU-Picking Solution (NINJA-OPS, or NINJA for short), a fast and highly accurate novel method enabling reference-based marker gene matching (picking Operational Taxonomic Units, or OTUs). NINJA takes advantage of the Burrows-Wheeler (BW) alignment using an artificial reference chromosome composed of concatenated reference sequences, the "concatesome," as the BW input. Other features include automatic support for paired-end reads with arbitrary insert sizes. NINJA is also free and open source and implements several pre-filtering methods that elicit substantial speedup when coupled with existing tools. We applied NINJA to several published microbiome studies, obtaining accuracy similar to or better than previous reference-based OTU-picking methods while achieving an order of magnitude or more speedup and using a fraction of the memory footprint. NINJA is a complete pipeline that takes a FASTA-formatted input file and outputs a QIIME-formatted taxonomy-annotated BIOM file for an entire MiSeq run of human gut microbiome 16S genes in under 10 minutes on a dual-core laptop.

  13. NINJA-OPS: Fast Accurate Marker Gene Alignment Using Concatenated Ribosomes

    PubMed Central

    Al-Ghalith, Gabriel A.; Montassier, Emmanuel; Ward, Henry N.; Knights, Dan

    2016-01-01

    The explosion of bioinformatics technologies in the form of next generation sequencing (NGS) has facilitated a massive influx of genomics data in the form of short reads. Short read mapping is therefore a fundamental component of next generation sequencing pipelines which routinely match these short reads against reference genomes for contig assembly. However, such techniques have seldom been applied to microbial marker gene sequencing studies, which have mostly relied on novel heuristic approaches. We propose NINJA Is Not Just Another OTU-Picking Solution (NINJA-OPS, or NINJA for short), a fast and highly accurate novel method enabling reference-based marker gene matching (picking Operational Taxonomic Units, or OTUs). NINJA takes advantage of the Burrows-Wheeler (BW) alignment using an artificial reference chromosome composed of concatenated reference sequences, the “concatesome,” as the BW input. Other features include automatic support for paired-end reads with arbitrary insert sizes. NINJA is also free and open source and implements several pre-filtering methods that elicit substantial speedup when coupled with existing tools. We applied NINJA to several published microbiome studies, obtaining accuracy similar to or better than previous reference-based OTU-picking methods while achieving an order of magnitude or more speedup and using a fraction of the memory footprint. NINJA is a complete pipeline that takes a FASTA-formatted input file and outputs a QIIME-formatted taxonomy-annotated BIOM file for an entire MiSeq run of human gut microbiome 16S genes in under 10 minutes on a dual-core laptop. PMID:26820746

  14. [Construction and Function Verification of a Novel Shuttle Vector Containing a Marker Gene Self-deletion System].

    PubMed

    Li, Lili; Wang, Zhan; Zhou, Yubai; Zhang, Fang; Shen, Sisi; Li, Zelin; Zeng, Yi

    2015-09-01

    For rapid and accurate screening of recombinant modified vaccinia virus Ankara (rMVA) that satisfied the quality standards of clinical trials, a novel shuttle vector that can delete the marker gene automatically during virus propagation was construted: pZL-EGFP. To construct the pZL-EGFP, the original shuttle vector pSC11 was modified by replacing the LacZ marker gene with enhanced green fluorescent protein (EGFP) and then inserting homologous sequences of TKL into the flank regions of EGFP. Baby hamster kidney (BHK)-21 cells were cotransfected with pZL-EGFP and MVA, and underwent ten passages and one plaque screening to obtain the EGFP-free rMVA carrying the exogenous gene. Resulting rMVA was tested by polymerase chain reaction and western blotting to verify pZL-EGFP function. A novel shuttle vector pZL-EGFP containing an EGFP marker gene which could be deleted automatically was constructed. This gene deletion had no effect on the activities of rMVA, and the exogenous gene could be expressed stably. These results suggest that rMVA can be packaged efficiently by homologous recombination between pZL-EGFP and MVA in BHK-21 cells, and that the carried EGFP gene can be removed automatically by intramolecular homologous recombination during virus passage. Meanwhile, the gene deletion had no influence on the activities of rMVA and the expression of exogenous target gene. This study lays a solid foundation for the future research.

  15. Adenovirus-mediated gene transfer of dominant negative ras(asn17) in 3T3L1 adipocytes does not alter insulin-stimulated P13-kinase activity or glucose transport.

    PubMed

    Gnudi, L; Frevert, E U; Houseknecht, K L; Erhardt, P; Kahn, B B

    1997-01-01

    Recent studies suggest that the ras-map kinase and PI3-kinase cascades converge. We sought to determine whether PI3-kinase is downstream of ras in insulin signaling in a classic insulin target cell. We generated a recombinant adenovirus encoding dominant negative ras by cloning the human H-ras cDNA with a ser to asn substitution at amino acid 17 (ras(asn17)) into the pACCMVpLpA vector and cotransfecting 293 cells with the pJM17 plasmid containing the adenoviral genome. Efficiency of gene transfer was assessed by infecting fully differentiated 3T3L1 adipocytes with a recombinant adenovirus expressing beta-galactosidase (beta-gal); greater than 70% of cells were infected. Infection of adipocytes with ras(asn17) resulted in 10-fold greater expression than endogenous ras. This high efficiency gene transfer allowed biochemical assays. Insulin stimulation of ras-GTP formation was inhibited in ras(asn17)-expressing cells. Map kinase gel mobility shift revealed that insulin (1 UM) or epidermal growth factor (100 ng/ml) resulted in the appearance of a hyperphosphorylated species of p42 map kinase in uninfected cells and those expressing beta-gal but not in cells expressing ras(asn17). In contrast, insulin increased IRS-1-associated PI3-kinase activity approximately 10-fold in control cells and high level overexpression of ras(asn17) did not impair this effect. Similarly, insulin and epidermal growth factor activation of total (no immunoprecipitation) PI3-kinase activity in both cytosol and total cellular membranes and insulin stimulation of glucose transport were not affected by expression of dominant negative ras. Thus, adenovirus-mediated gene transfer is effective for studying insulin signaling in fully differentiated insulin target cells. Inhibition of ras activation abolishes insulin-stimulated phosphorylation of map kinase but does not affect insulin stimulation of PI3-kinase activity. In normal cell physiology, PI3-kinase does not appear to be downstream of ras in

  16. A Rapid Molecular Test for Determining Yersinia pestis Susceptibility to Ciprofloxacin by the Quantification of Differentially Expressed Marker Genes

    PubMed Central

    Steinberger-Levy, Ida; Shifman, Ohad; Zvi, Anat; Ariel, Naomi; Beth-Din, Adi; Israeli, Ofir; Gur, David; Aftalion, Moshe; Maoz, Sharon; Ber, Raphael

    2016-01-01

    Standard antimicrobial susceptibility tests used to determine bacterial susceptibility to antibiotics are growth dependent and time consuming. The long incubation time required for standard tests may render susceptibility results irrelevant, particularly for patients infected with lethal bacteria that are slow growing on agar but progress rapidly in vivo, such as Yersinia pestis. Here, we present an alternative approach for the rapid determination of antimicrobial susceptibility, based on the quantification of the changes in the expression levels of specific marker genes following exposure to growth-inhibiting concentrations of the antibiotic, using Y. pestis and ciprofloxacin as a model. The marker genes were identified by transcriptomic DNA microarray analysis of the virulent Y. pestis Kimberley53 strain after exposure to specific concentrations of ciprofloxacin for various time periods. We identified several marker genes that were induced following exposure to growth-inhibitory concentrations of ciprofloxacin, and we confirmed the marker expression profiles at additional ciprofloxacin concentrations using quantitative RT-PCR. Eleven candidate marker transcripts were identified, of which four mRNA markers were selected for a rapid quantitative RT-PCR susceptibility test that correctly determined the Minimal Inhibitory Concentration (MIC) values and the categories of susceptibility of several Y. pestis strains and isolates harboring various ciprofloxacin MIC values. The novel molecular susceptibility test requires just 2 h of antibiotic exposure in a 7-h overall test time, in contrast to the 24 h of antibiotic exposure required for a standard microdilution test. PMID:27242774

  17. A Rapid Molecular Test for Determining Yersinia pestis Susceptibility to Ciprofloxacin by the Quantification of Differentially Expressed Marker Genes.

    PubMed

    Steinberger-Levy, Ida; Shifman, Ohad; Zvi, Anat; Ariel, Naomi; Beth-Din, Adi; Israeli, Ofir; Gur, David; Aftalion, Moshe; Maoz, Sharon; Ber, Raphael

    2016-01-01

    Standard antimicrobial susceptibility tests used to determine bacterial susceptibility to antibiotics are growth dependent and time consuming. The long incubation time required for standard tests may render susceptibility results irrelevant, particularly for patients infected with lethal bacteria that are slow growing on agar but progress rapidly in vivo, such as Yersinia pestis. Here, we present an alternative approach for the rapid determination of antimicrobial susceptibility, based on the quantification of the changes in the expression levels of specific marker genes following exposure to growth-inhibiting concentrations of the antibiotic, using Y. pestis and ciprofloxacin as a model. The marker genes were identified by transcriptomic DNA microarray analysis of the virulent Y. pestis Kimberley53 strain after exposure to specific concentrations of ciprofloxacin for various time periods. We identified several marker genes that were induced following exposure to growth-inhibitory concentrations of ciprofloxacin, and we confirmed the marker expression profiles at additional ciprofloxacin concentrations using quantitative RT-PCR. Eleven candidate marker transcripts were identified, of which four mRNA markers were selected for a rapid quantitative RT-PCR susceptibility test that correctly determined the Minimal Inhibitory Concentration (MIC) values and the categories of susceptibility of several Y. pestis strains and isolates harboring various ciprofloxacin MIC values. The novel molecular susceptibility test requires just 2 h of antibiotic exposure in a 7-h overall test time, in contrast to the 24 h of antibiotic exposure required for a standard microdilution test. PMID:27242774

  18. Development and linkage mapping of E-STS and RGA markers for functional gene homologues in apple.

    PubMed

    Naik, Suresh; Hampson, Cheryl; Gasic, Ksenija; Bakkeren, Guus; Korban, Schuyler S

    2006-08-01

    Linkage maps developed from known-function genes can be valuable in the candidate gene mapping approach. A set of 121 expressed sequence tagged site (E-STS) primer pairs were tested on a framework genetic linkage map of apple (Malus x domestica Borkh.) constructed using simple sequence repeats (SSRs) and randomly amplified polymorphic DNA (RAPD) markers. These known-function gene markers, E-STSs, were supplemented by markers for resistance gene analogues (RGAs), designed based on conserved motifs in all characterized resistance genes isolated from plant species. A total of 229 markers, including 46 apple E-STSs, 8 RGAs, 85 SSRs from apple and peach, and 88 RAPDs, were assigned to 17 linkage groups covering 832 cM of the apple genome, based on 52 individuals originating from the cross 'Antonovka debnicka' (Q12-4) x 'Summerred'. Clusters of E-STS and RGA loci were located in linkage groups previously identified to carry resistance genes, some of which confer resistance to apple scab disease caused by Venturia inaequalis (Cke.) Wint.

  19. Characterization of ESTs from black locust for gene discovery and marker development.

    PubMed

    Wang, J X; Lu, C; Yuan, C Q; Cui, B B; Qiu, Q D; Sun, P; Hu, R Y; Wu, D C; Sun, Y H; Li, Y

    2015-01-01

    Black locust (Robinia pseudoacacia L.) is an ecologically and economically important species. However, it has relatively underdeveloped genomic resources, and this limits gene discovery and marker-assisted selective breeding. In the present study, we obtained large-scale transcriptome data using a next-generation sequencing platform to compensate for the lack of black locust genomic information. Increasing the amount of transcriptome data for black locust will provide a valuable resource for multi-gene phylogenetic analyses and will facilitate research on the mechanisms whereby conserved genes and functions are maintained in the face of species divergence. We sequenced the black locust transcriptome from a cDNA library of multiple tissues and individuals on an Illumina platform, and this produced 108,229,352 clean sequence reads. The high-quality overlapping expressed sequence tags (ESTs) were assembled into 36,533 unigenes, and 4781 simple sequence repeats were characterized. A large collection of high-quality ESTs was obtained, de novo assembled, and characterized. Our results markedly expand the previous transcript catalogues of black locust and can gradually be applied to black locust breeding programs. Furthermore, our data will facilitate future research on the comparative genomics of black locust and related species. PMID:26505419

  20. Spectroscopic detection of fluorescent protein marker gene activity in genetically modified plants

    NASA Astrophysics Data System (ADS)

    Liew, O. W.; Chong, Jenny P. C.; Asundi, Anand K.

    2005-04-01

    This work focuses on developing a portable fibre optic fluorescence analyser for rapid identification of genetically modified plants tagged with a fluorescent marker gene. Independent transgenic tobacco plant lines expressing the enhanced green fluorescence protein (EGFP) gene were regenerated following Agrobacterium-mediated gene transfer. Molecular characterisation of these plant lines was carried out at the DNA level by PCR screening to confirm their transgenic status. Conventional transgene expression analysis was then carried out at the RNA level by RT-PCR and at the protein level by Western blotting using anti-GFP rabbit antiserum. The amount of plant-expressed EGFP on a Western blot was quantified against known amounts of purified EGFP by scanning densitometry. The expression level of EGFP in transformed plants was found to range from 0.1 - 0.6% of total extractable protein. A comparison between conventional western analysis of transformants and direct spectroscopic quantification using the fibre optic fluorescence analyser was made. The results showed that spectroscopic measurements of fluorescence emission from strong EGFP expressors correlated positively with Western blot data. However, the fluorescence analyser was also able to identify weakly expressing plant transformants below the detection limit of colorimetric Western blotting.

  1. Evolutionary changes of the importance of olfaction in cetaceans based on the olfactory marker protein gene.

    PubMed

    Kishida, Takushi; Thewissen, J G M

    2012-01-25

    Odontocetes and mysticetes are two extant suborders of cetaceans. It is reported that the former have no sense of olfaction, while the latter can smell in air. To explain the ecological reason why mysticetes still retain their sense of smell, two hypotheses have been proposed - the echolocation-priority hypothesis, which assumes that the acquisition of echolocation causes the reduction of the importance of olfaction, and the filter-feeder hypothesis, which assumes that olfactory ability is important for filter-feeders to locate their prey because clouds of plankton give off a peculiar odor. The olfactory marker protein (OMP) is almost exclusively expressed in vertebrate olfactory receptor neurons, and is considered to play important roles in olfactory systems. In this study, full-length open reading frames of OMP genes were identified in 6 cetacean species and we analyzed the nonsynonymous to synonymous substitution rate ratio based on the maximum likelihood method. The evolutionary changes of the selective pressures on OMP genes did fit better to the filter-feeder hypothesis than to the echolocation-priority hypothesis. In addition, no pseudogenization mutations are found in all five odontocetes OMP genes investigated in this study. It may suggest that OMP retains some function even in 'anosmic' odontocetes.

  2. Glial cell derived neurotrophic factor induces spermatogonial stem cell marker genes in chicken mesenchymal stem cells.

    PubMed

    Boozarpour, Sohrab; Matin, Maryam M; Momeni-Moghaddam, Madjid; Dehghani, Hesam; Mahdavi-Shahri, Naser; Sisakhtnezhad, Sajjad; Heirani-Tabasi, Asieh; Irfan-Maqsood, Muhammad; Bahrami, Ahmad Reza

    2016-06-01

    Mesenchymal stem cells (MSCs) are known with the potential of multi-lineage differentiation. Advances in differentiation technology have also resulted in the conversion of MSCs to other kinds of stem cells. MSCs are considered as a suitable source of cells for biotechnology purposes because they are abundant, easily accessible and well characterized cells. Nowadays small molecules are introduced as novel and efficient factors to differentiate stem cells. In this work, we examined the potential of glial cell derived neurotrophic factor (GDNF) for differentiating chicken MSCs toward spermatogonial stem cells. MSCs were isolated and characterized from chicken and cultured under treatment with all-trans retinoic acid (RA) or glial cell derived neurotrophic factor. Expression analysis of specific genes after 7days of RA treatment, as examined by RT-PCR, proved positive for some germ cell markers such as CVH, STRA8, PLZF and some genes involved in spermatogonial stem cell maintenance like BCL6b and c-KIT. On the other hand, GDNF could additionally induce expression of POU5F1, and NANOG as well as other genes which were induced after RA treatment. These data illustrated that GDNF is relatively more effective in diverting chicken MSCs towards Spermatogonial stem cell -like cells in chickens and suggests GDNF as a new agent to obtain transgenic poultry, nevertheless, exploitability of these cells should be verified by more experiments. PMID:27026484

  3. Glial cell derived neurotrophic factor induces spermatogonial stem cell marker genes in chicken mesenchymal stem cells.

    PubMed

    Boozarpour, Sohrab; Matin, Maryam M; Momeni-Moghaddam, Madjid; Dehghani, Hesam; Mahdavi-Shahri, Naser; Sisakhtnezhad, Sajjad; Heirani-Tabasi, Asieh; Irfan-Maqsood, Muhammad; Bahrami, Ahmad Reza

    2016-06-01

    Mesenchymal stem cells (MSCs) are known with the potential of multi-lineage differentiation. Advances in differentiation technology have also resulted in the conversion of MSCs to other kinds of stem cells. MSCs are considered as a suitable source of cells for biotechnology purposes because they are abundant, easily accessible and well characterized cells. Nowadays small molecules are introduced as novel and efficient factors to differentiate stem cells. In this work, we examined the potential of glial cell derived neurotrophic factor (GDNF) for differentiating chicken MSCs toward spermatogonial stem cells. MSCs were isolated and characterized from chicken and cultured under treatment with all-trans retinoic acid (RA) or glial cell derived neurotrophic factor. Expression analysis of specific genes after 7days of RA treatment, as examined by RT-PCR, proved positive for some germ cell markers such as CVH, STRA8, PLZF and some genes involved in spermatogonial stem cell maintenance like BCL6b and c-KIT. On the other hand, GDNF could additionally induce expression of POU5F1, and NANOG as well as other genes which were induced after RA treatment. These data illustrated that GDNF is relatively more effective in diverting chicken MSCs towards Spermatogonial stem cell -like cells in chickens and suggests GDNF as a new agent to obtain transgenic poultry, nevertheless, exploitability of these cells should be verified by more experiments.

  4. PDIA3 and PDIA6 gene expression as an aggressiveness marker in primary ductal breast cancer.

    PubMed

    Ramos, F S; Serino, L T R; Carvalho, C M S; Lima, R S; Urban, C A; Cavalli, I J; Ribeiro, E M S F

    2015-01-01

    Changes in the expression of the protein disulfide isomerase genes PDIA3 and PDIA6 may increase endoplasmic reticulum stress, leading to cellular instability and neoplasia. We evaluated the expression of PDIA3 and PDIA6 in invasive ductal carcinomas. Using reverse transcription-quantitative polymerase chain reaction, we compared the mRNA expression level in 45 samples of invasive ductal carcinoma with that in normal breast samples. Increased expression of the PDIA3 gene in carcinomas (P = 0.0009) was observed. In addition, PDIA3 expression was increased in tumors with lymph node metastasis (P = 0.009) and with grade III (P < 0.02). The PDIA6 gene showed higher expression levels in the presence of lymph node metastasis (U = 99.00, P = 0.0476) and lower expression for negative hormone receptors status (P = 0.0351). Our results suggest that alterations in PDIA3/6 expression levels may be involved in the breast carcinogenic process and should be further investigated as a marker of aggressiveness. PMID:26125904

  5. Conservation of endangered Spanish cattle breeds using markers of candidate genes for meat quality.

    PubMed

    Rodero, E; González, A; Avilés, C; Luque, M

    2013-01-01

    The aim was to analyze the allelic and genotypic frequencies for two genes associated with tenderness of meat (CAPN1 and CAST) and one with fat deposits (DGAT1) in three endangered Spanish cattle breeds: Berrenda en Colorado (BC), Berrenda en Negro (BN), and Cardena Andaluza (CA) to utility of their involvement in the selection of them and to help the adoption of conservation measurement. Seventy-five males and 298 females of those breeds were genotyped. Genotypic and allelic frequencies for each polymorphic locus were estimated. There were significant differences in the genotypic frequencies among breeds in CAPN1 and DGTA1 genes and in the case of the genic frequencies in CAPN1, CAST, and DGAT1 genes. The three breeds analyzed (BC, BN, and CA) presented high allelic frequencies for the favorable allele of the three markers (from 0.41 to 0.75). The association between the favorable allele and meat quality must be confirmed. In cases of association with differences in quality meat, the absence of differences in the genotypic and genic frequency distributions between the sexes is advantageous in mating planning because it implies that there is no handicap to be overcome for the conservation program and it would allow the use of sires to promote the increase in improvements within a short period of time.

  6. Hox gene clusters of early vertebrates: do they serve as reliable markers for genome evolution?

    PubMed

    Kuraku, Shigehiro

    2011-06-01

    Hox genes, responsible for regional specification along the anteroposterior axis in embryogenesis, are found as clusters in most eumetazoan genomes sequenced to date. Invertebrates possess a single Hox gene cluster with some exceptions of secondary cluster breakages, while osteichthyans (bony vertebrates) have multiple Hox clusters. In tetrapods, four Hox clusters, derived from the so-called two-round whole genome duplications (2R-WGDs), are observed. Overall, the number of Hox gene clusters has been regarded as a reliable marker of ploidy levels in animal genomes. In fact, this scheme also fits the situations in teleost fishes that experienced an additional WGD. In this review, I focus on cyclostomes and cartilaginous fishes as lineages that would fill the gap between invertebrates and osteichthyans. A recent study highlighted a possible loss of the HoxC cluster in the galeomorph shark lineage, while other aspects of cartilaginous fish Hox clusters usually mark their conserved nature. In contrast, existing resources suggest that the cyclostomes exhibit a different mode of Hox cluster organization. For this group of species, whose genomes could have differently responded to the 2R-WGDs from jawed vertebrates, therefore the number of Hox clusters may not serve as a good indicator of their ploidy level. PMID:21802046

  7. Liver X Receptor (LXR) Regulates Human Adipocyte Lipolysis*

    PubMed Central

    Stenson, Britta M.; Rydén, Mikael; Venteclef, Nicolas; Dahlman, Ingrid; Pettersson, Annie M. L.; Mairal, Aline; Åström, Gaby; Blomqvist, Lennart; Wang, Victoria; Jocken, Johan W. E.; Clément, Karine; Langin, Dominique; Arner, Peter; Laurencikiene, Jurga

    2011-01-01

    The Liver X receptor (LXR) is an important regulator of carbohydrate and lipid metabolism in humans and mice. We have recently shown that activation of LXR regulates cellular fuel utilization in adipocytes. In contrast, the role of LXR in human adipocyte lipolysis, the major function of human white fat cells, is not clear. In the present study, we stimulated in vitro differentiated human and murine adipocytes with the LXR agonist GW3965 and observed an increase in basal lipolysis. Microarray analysis of human adipocyte mRNA following LXR activation revealed an altered gene expression of several lipolysis-regulating proteins, which was also confirmed by quantitative real-time PCR. We show that expression and intracellular localization of perilipin1 (PLIN1) and hormone-sensitive lipase (HSL) are affected by GW3965. Although LXR activation does not influence phosphorylation status of HSL, HSL activity is required for the lipolytic effect of GW3965. This effect is abolished by PLIN1 knockdown. In addition, we demonstrate that upon activation, LXR binds to the proximal regions of the PLIN1 and HSL promoters. By selective knock-down of either LXR isoform, we show that LXRα is the major isoform mediating the lipolysis-related effects of LXR. In conclusion, the present study demonstrates that activation of LXRα up-regulates basal human adipocyte lipolysis. This is at least partially mediated through LXR binding to the PLIN1 promoter and down-regulation of PLIN1 expression. PMID:21030586

  8. Liver X receptor (LXR) regulates human adipocyte lipolysis.

    PubMed

    Stenson, Britta M; Rydén, Mikael; Venteclef, Nicolas; Dahlman, Ingrid; Pettersson, Annie M L; Mairal, Aline; Aström, Gaby; Blomqvist, Lennart; Wang, Victoria; Jocken, Johan W E; Clément, Karine; Langin, Dominique; Arner, Peter; Laurencikiene, Jurga

    2011-01-01

    The Liver X receptor (LXR) is an important regulator of carbohydrate and lipid metabolism in humans and mice. We have recently shown that activation of LXR regulates cellular fuel utilization in adipocytes. In contrast, the role of LXR in human adipocyte lipolysis, the major function of human white fat cells, is not clear. In the present study, we stimulated in vitro differentiated human and murine adipocytes with the LXR agonist GW3965 and observed an increase in basal lipolysis. Microarray analysis of human adipocyte mRNA following LXR activation revealed an altered gene expression of several lipolysis-regulating proteins, which was also confirmed by quantitative real-time PCR. We show that expression and intracellular localization of perilipin1 (PLIN1) and hormone-sensitive lipase (HSL) are affected by GW3965. Although LXR activation does not influence phosphorylation status of HSL, HSL activity is required for the lipolytic effect of GW3965. This effect is abolished by PLIN1 knockdown. In addition, we demonstrate that upon activation, LXR binds to the proximal regions of the PLIN1 and HSL promoters. By selective knock-down of either LXR isoform, we show that LXRα is the major isoform mediating the lipolysis-related effects of LXR. In conclusion, the present study demonstrates that activation of LXRα up-regulates basal human adipocyte lipolysis. This is at least partially mediated through LXR binding to the PLIN1 promoter and down-regulation of PLIN1 expression. PMID:21030586

  9. Assessment of genetic diversity of wheat genotypes by resistance gene analog-EST markers.

    PubMed

    Karakas, O; Gurel, F; Uncuoglu, A A

    2011-01-01

    Resistance gene analog-expressed sequence tag (RGA-EST)-based markers have been used for variety discrimination and studies of genetic diversity in wheat. Our aim is to increase the competitiveness of public wheat breeding programs through intensive use of modern selection technologies, mainly marker-assisted selection. The genetic diversity of 77 wheat nucleotide binding site (NBS)-containing RGA-ESTs was assessed. Resistant and susceptible bread wheat (Triticum aestivum) genotypes were used as sources of DNA for PCR amplifications. In our previous studies, the F₂ individuals derived from the combinations PI178383 x Harmankaya99, Izgi2001 x ES14, and Sonmez2001 x Aytin98 were evaluated for yellow rust resistance at both seedling and adult stages to identify DNA markers. We have now examined the genetic variability among the resistant and susceptible Turkish wheat cultivars for yellow rust disease and the mean genetic distance between the cultivars. The highest similarity was 0.500 between Harmankaya99 and Sonmez2001. The lowest similarity was 0.286 between Aytin98, PI178383 and Aytin98, ES14. A relatively high level (49.5%) of polymorphism was observed with 77 RGA-EST primers across the six wheat genotypes, despite the fact that all of them were local cultivars from geographically close locations. RGA-EST sequences were compared by BlastX algorithms for amino acid sequences to determine the polymorphic categories among the combinations. BlastX analyses of six RGA-ESTs that gave polymorphic patterns for all combinations were NBS-LRR class RGA, NB-ARC domain containing protein, NBS-type resistance protein RGC5, NBS-LRR-S/ TPK stem rust resistance protein, and putative MLA1 proteins, while 38 RGA-EST gave a monomorphic pattern.

  10. Genomewide gene-associated microsatellite markers for the model invasive ascidian, Ciona intestinalis species complex.

    PubMed

    Lin, Yaping; Chen, Yiyong; Xiong, Wei; Zhan, Aibin

    2016-05-01

    The vase tunicate, Ciona intestinalis species complex, has become a good model for ecological and evolutionary studies, especially those focusing on microevolution associated with rapidly changing environments. However, genomewide genetic markers are still lacking. Here, we characterized a large set of genomewide gene-associated microsatellite markers for C. intestinalis spA (=C. robusta). Bioinformatic analysis identified 4654 microsatellites from expressed sequence tags (ESTs), 2126 of which successfully assigned to chromosomes were selected for further analysis. Based on the distribution evenness on chromosomes, function annotation and suitability for primer design, we chose 545 candidate microsatellites for further characterization. After amplification validation and variation assessment, 218 loci were polymorphic in at least one of the two populations collected from the coast of Arenys de Mar, Spain (N = 24-48), and Cape Town, South Africa (N = 24-33). The number of alleles, observed heterozygosity and expected heterozygosity ranged from 2 to 11, 0 to 0.833 and 0.021 to 0.818, and from 2 to 10, 0 to 0.879 and 0.031 to 0.845 for the Spanish and African populations, respectively. When all microsatellites were tested for cross-species utility, only 60 loci (25.8%) could be successfully amplified and all loci were polymorphic in C. intestinalis spB. A high level of genomewide polymorphism is likely responsible for the low transferability. The large set of microsatellite markers characterized here is expected to provide a useful genomewide resource for ecological and evolutionary studies using C. intestinalis as a model. PMID:26505988

  11. Bisindoylmaleimide I suppresses adipocyte differentiation through stabilization of intracellular {beta}-catenin protein

    SciTech Connect

    Cho, Munju; Park, Seoyoung; Gwak, Jungsug; Kim, Dong-Eun; Yea, Sung Su; Shin, Jae-Gook; Oh, Sangtaek

    2008-02-29

    The Wnt/{beta}-catenin signaling pathway plays important roles in cell differentiation. Activation of this pathway, likely by Wnt-10b, has been shown to inhibit adipogenesis in cultured 3T3-L1 preadipocytes and mice. Here we revealed that bisindoylmaleimide I (BIM), which is widely used as a specific inhibitor of protein kinase C (PKC), inhibits adipocyte differentiation through activation of the Wnt/{beta}-catenin signaling pathway. BIM increased {beta}-catenin responsive transcription (CRT) and up-regulated intracellular {beta}-catenin levels in HEK293 cells and 3T3-L1 preadipocytes. BIM significantly decreased intracellular lipid accumulation and reduced expression of important adipocyte marker genes including peroxisome-proliferator-activated receptor {gamma} (PPAR{gamma}) and CAATT enhancer-binding protein {alpha} (C/EBP{alpha}) in 3T3-L1 preadipocytes. Taken together, our findings indicate that BIM inhibits adipogenesis by increasing the stability of {beta}-catenin protein in 3T3-L1 preadipocyte cells.

  12. Chronic hyperinsulinemia reduces insulin sensitivity and metabolic functions of brown adipocyte.

    PubMed

    Rajan, Sujith; Shankar, Kripa; Beg, Muheeb; Varshney, Salil; Gupta, Abhishek; Srivastava, Ankita; Kumar, Durgesh; Mishra, Raj K; Hussain, Zakir; Gayen, Jiaur R; Gaikwad, Anil N

    2016-09-01

    The growing pandemics of diabetes have become a real threat to world economy. Hyperinsulinemia and insulin resistance are closely associated with the pathophysiology of type 2 diabetes. In pretext of brown adipocytes being considered as the therapeutic strategy for the treatment of obesity and insulin resistance, we have tried to understand the effect of hyperinsulinemia on brown adipocyte function. We here with for the first time report that hyperinsulinemia-induced insulin resistance in brown adipocyte is also accompanied with reduced insulin sensitivity and brown adipocyte characteristics. CI treatment decreased expression of brown adipocyte-specific markers (such as PRDM16, PGC1α, and UCP1) and mitochondrial content as well as activity. CI-treated brown adipocytes showed drastic decrease in oxygen consumption rate (OCR) and spare respiratory capacity. Morphological study indicates increased accumulation of lipid droplets in CI-treated brown adipocytes. We have further validated these findings in vivo in C57BL/6 mice implanted with mini-osmotic insulin pump for 8weeks. CI treatment in mice leads to increased body weight gain, fat mass and impaired glucose intolerance with reduced energy expenditure and insulin sensitivity. CI-treated mice showed decreased BAT characteristics and function. We also observed increased inflammation and ER stress markers in BAT of CI-treated animals. The above results conclude that hyperinsulinemia has deleterious effect on brown adipocyte function, making it susceptible to insulin resistance. Thus, the above findings have greater implication in designing approaches for the treatment of insulin resistance and diabetes via recruitment of brown adipocytes. PMID:27340034

  13. Microbiological characterization of aquatic microbiomes targeting taxonomical marker genes and antibiotic resistance genes of opportunistic bacteria.

    PubMed

    Alexander, Johannes; Bollmann, Anna; Seitz, Wolfram; Schwartz, Thomas

    2015-04-15

    The dissemination of medically relevant antibiotic resistance genes (ARGs) (blaVIM-1, vanA, ampC, ermB, and mecA) and opportunistic bacteria (Enterococcus faecium/faecalis, Pseudomonas aeruginosa, Enterobacteriaceae, Staphylococcus aureus, and CNS) was determined in different anthropogenically influenced aquatic habitats in a selected region of Germany. Over a period of two years, four differently sized wastewater treatment plants (WWTPs) with and without clinical influence, three surface waters, four rain overflow basins, and three groundwater sites were analyzed by quantitative Polymerase Chain Reaction (qPCR). Results were calculated in cell equivalents per 100 ng of total DNA extracted from water samples and per 100 mL sample volume, which seems to underestimate the abundance of antibiotic resistance and opportunistic bacteria. High abundances of opportunistic bacteria and ARG were quantified in clinical wastewaters and influents of the adjacent WWTP. The removal capacities of WWTP were up to 99% for some, but not all investigated bacteria. The abundances of most ARG targets were found to be increased in the bacterial population after conventional wastewater treatment. As a consequence, downstream surface water and also some groundwater compartments displayed high abundances of all four ARGs. It became obvious that the dynamics of the ARG differed from the fate of the opportunistic bacteria. This underlines the necessity of an advanced microbial characterization of anthropogenically influenced environments.

  14. Gene expression markers in circulating tumor cells may predict bone metastasis and response to hormonal treatment in breast cancer.

    PubMed

    Wang, Haiying; Molina, Julian; Jiang, John; Ferber, Matthew; Pruthi, Sandhya; Jatkoe, Timothy; Derecho, Carlo; Rajpurohit, Yashoda; Zheng, Jian; Wang, Yixin

    2013-11-01

    Circulating tumor cells (CTCs) have recently attracted attention due to their potential as prognostic and predictive markers for the clinical management of metastatic breast cancer patients. The isolation of CTCs from patients may enable the molecular characterization of these cells, which may help establish a minimally invasive assay for the prediction of metastasis and further optimization of treatment. Molecular markers of proven clinical value may therefore be useful in predicting disease aggressiveness and response to treatment. In our earlier study, we identified a gene signature in breast cancer that appears to be significantly associated with bone metastasis. Among the genes that constitute this signature, trefoil factor 1 (TFF1) was identified as the most differentially expressed gene associated with bone metastasis. In this study, we investigated 25 candidate gene markers in the CTCs of metastatic breast cancer patients with different metastatic sites. The panel of the 25 markers was investigated in 80 baseline samples (first blood draw of CTCs) and 30 follow-up samples. In addition, 40 healthy blood donors (HBDs) were analyzed as controls. The assay was performed using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) with RNA extracted from CTCs captured by the CellSearch system. Our study indicated that 12 of the genes were uniquely expressed in CTCs and 10 were highly expressed in the CTCs obtained from patients compared to those obtained from HBDs. Among these genes, the expression of keratin 19 was highly correlated with the CTC count. The TFF1 expression in CTCs was a strong predictor of bone metastasis and the patients with a high expression of estrogen receptor β in CTCs exhibited a better response to hormonal treatment. Molecular characterization of these genes in CTCs may provide a better understanding of the mechanism underlying tumor metastasis and identify gene markers in CTCs for predicting disease progression and

  15. Gene expression markers in circulating tumor cells may predict bone metastasis and response to hormonal treatment in breast cancer

    PubMed Central

    WANG, HAIYING; MOLINA, JULIAN; JIANG, JOHN; FERBER, MATTHEW; PRUTHI, SANDHYA; JATKOE, TIMOTHY; DERECHO, CARLO; RAJPUROHIT, YASHODA; ZHENG, JIAN; WANG, YIXIN

    2013-01-01

    Circulating tumor cells (CTCs) have recently attracted attention due to their potential as prognostic and predictive markers for the clinical management of metastatic breast cancer patients. The isolation of CTCs from patients may enable the molecular characterization of these cells, which may help establish a minimally invasive assay for the prediction of metastasis and further optimization of treatment. Molecular markers of proven clinical value may therefore be useful in predicting disease aggressiveness and response to treatment. In our earlier study, we identified a gene signature in breast cancer that appears to be significantly associated with bone metastasis. Among the genes that constitute this signature, trefoil factor 1 (TFF1) was identified as the most differentially expressed gene associated with bone metastasis. In this study, we investigated 25 candidate gene markers in the CTCs of metastatic breast cancer patients with different metastatic sites. The panel of the 25 markers was investigated in 80 baseline samples (first blood draw of CTCs) and 30 follow-up samples. In addition, 40 healthy blood donors (HBDs) were analyzed as controls. The assay was performed using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) with RNA extracted from CTCs captured by the CellSearch system. Our study indicated that 12 of the genes were uniquely expressed in CTCs and 10 were highly expressed in the CTCs obtained from patients compared to those obtained from HBDs. Among these genes, the expression of keratin 19 was highly correlated with the CTC count. The TFF1 expression in CTCs was a strong predictor of bone metastasis and the patients with a high expression of estrogen receptor β in CTCs exhibited a better response to hormonal treatment. Molecular characterization of these genes in CTCs may provide a better understanding of the mechanism underlying tumor metastasis and identify gene markers in CTCs for predicting disease progression and

  16. 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.

  17. Detection of genetic diversity and selective gene introgression in coffee using RAPD markers.

    PubMed

    Orozco-Castillo, C; Chalmers, K J; Waugh, R; Powell, W

    1994-03-01

    RAPD (randomly amplified polymorphic DNA) markers generated by arbitary decamers have been successfully employed to detect genetic polymorphisms between coffee species and between Coffea arabica genotypes. The RAPD profiles were used to construct dendrograms and these were consistent with the known history and evolution of Coffea arabica. Material originating from Ethiopia and the arabica sub-groups - C. arabica var. typica and C. arabica var. bourbon - were clearly distinguished. RAPD analysis therefore reflects morphological differences between the sub-groups and the geographical origin of the coffee material. Species-specific amplification products were also identified, but, more importantly, amplification products specific to C. canephora were identified in two C. arabica genotypes, Rume Sudan and Catimor 5175. This diagnostic product is therefore indicative of interspecific gene flow in coffee and has biological implications for selective introgressive hybridisation in coffee. Our study demonstrates the power of the polymerase chain reaction technology for the generation of genetic markers for long-lived perennial tree and bush crops. PMID:24190527

  18. Tracing functional circuits using c-Fos regulated expression of marker genes targeted to neuronal projections.

    PubMed

    Murphy, Mark; Greferath, Ursula; Nag, Nupur; Nithianantharajah, Jess; Wilson, Yvette M

    2004-01-01

    We have developed novel techniques to trace functionally activated circuits and synaptic plasticity within the brain. We have generated transgenic mice, FTL, which contain a tau-lacZ fusion gene regulated by the promoter for c-fos. Following a particular nervous system stimulation in these mice, only neurons, which are functionally activated, will express LacZ, which is targeted to neuronal processes by the tau protein. In the FTL mice, we found highly inducible expression of lacZ by a range of different stimuli, and successful targeting of expression to neuronal cell bodies, axons and dendrites. To test if a functionally activated circuit could be visualized, the mice were deprived of water, which activates nuclei involved in body fluid homeostasis. LacZ was induced in these nuclei and their projections, allowing the mapping of a neuroendocrine circuit. Further studies have employed these mice in the analysis of neurons and circuits activated in vision, and learning and memory. We have also developed methods to measure markers of synaptic plasticity in the brain, and found significant experience dependent changes in the levels of these markers in different parts of the brain. We believe these techniques will aid in the identification of circuits for many different brain functions, and within those circuits, the locations of synaptic plasticity.

  19. A standard operating procedure for phylogenetic inference (SOPPI) using (rRNA) marker genes.

    PubMed

    Peplies, Jörg; Kottmann, Renzo; Ludwig, Wolfgang; Glöckner, Frank Oliver

    2008-09-01

    Phylogenetic analysis is currently used worldwide for taxonomic classification and identification of microorganisms. However, despite the countless trees that have been reconstructed and published in recent decades, so far, no user-friendly compilation of recommendations to standardize the data analysis and tree reconstruction process has been published. Consequently, this standard operating procedure for phylogenetic inference (SOPPI) offers a helping hand for working through the process from sampling in the field to phylogenetic tree reconstruction and publication. It is not meant to be authoritative or comprehensive, but should help to make phylogenetic inference and diversity analysis more reliable and comparable between different laboratories. It is mainly focused on using the ribosomal RNA as a universal phylogenetic marker, but the principles and recommendations can be applied to any valid marker gene. Feedback and suggestions from the scientific community are welcome in order to improve these guidelines further. Any updates will be made available on the SILVA webpage at http://www.arb-silva.de/projects/soppi.

  20. Development of wheat lines carrying stem rust resistance gene Sr39 with reduced Aegilops speltoides chromatin and simple PCR markers for marker-assisted selection.

    PubMed

    Mago, Rohit; Zhang, P; Bariana, H S; Verlin, D C; Bansal, U K; Ellis, J G; Dundas, I S

    2009-11-01

    The use of major resistance genes is a cost-effective strategy for preventing stem rust epidemics in wheat crops. The stem rust resistance gene Sr39 provides resistance to all currently known pathotypes of Puccinia graminis f. sp. tritici (Pgt) including Ug99 (TTKSK) and was introgressed together with leaf rust resistance gene Lr35 conferring adult plant resistance to P. triticina (Pt), into wheat from Aegilops speltoides. It has not been used extensively in wheat breeding because of the presumed but as yet undocumented negative agronomic effects associated with Ae. speltoides chromatin. This investigation reports the production of a set of recombinants with shortened Ae. speltoides segments through induction of homoeologous recombination between the wheat and the Ae. speltoides chromosome. Simple PCR-based DNA markers were developed for resistant and susceptible genotypes (Sr39#22r and Sr39#50s) and validated across a set of recombinant lines and wheat cultivars. These markers will facilitate the pyramiding of ameliorated sources of Sr39 with other stem rust resistance genes that are effective against the Pgt pathotype TTKSK and its variants. PMID:19756473

  1. AMP-Activated Kinase (AMPK) Activation by AICAR in Human White Adipocytes Derived from Pericardial White Adipose Tissue Stem Cells Induces a Partial Beige-Like Phenotype.

    PubMed

    Abdul-Rahman, Omar; Kristóf, Endre; Doan-Xuan, Quang-Minh; Vida, András; Nagy, Lilla; Horváth, Ambrus; Simon, József; Maros, Tamás; Szentkirályi, István; Palotás, Lehel; Debreceni, Tamás; Csizmadia, Péter; Szerafin, Tamás; Fodor, Tamás; Szántó, Magdolna; Tóth, Attila; Kiss, Borbála; Bacsó, Zsolt; Bai, Péter

    2016-01-01

    Beige adipocytes are special cells situated in the white adipose tissue. Beige adipocytes, lacking thermogenic cues, morphologically look quite similar to regular white adipocytes, but with a markedly different response to adrenalin. White adipocytes respond to adrenergic stimuli by enhancing lipolysis, while in beige adipocytes adrenalin induces mitochondrial biogenesis too. A key step in the differentiation and function of beige adipocytes is the deacetylation of peroxisome proliferator-activated receptor (PPARγ) by SIRT1 and the consequent mitochondrial biogenesis. AMP-activated protein kinase (AMPK) is an upstream activator of SIRT1, therefore we set out to investigate the role of AMPK in beige adipocyte differentiation using human adipose-derived mesenchymal stem cells (hADMSCs) from pericardial adipose tissue. hADMSCs were differentiated to white and beige adipocytes and the differentiation medium of the white adipocytes was supplemented with 100 μM [(2R,3S,4R,5R)-5-(4-Carbamoyl-5-aminoimidazol-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl dihydrogen phosphate (AICAR), a known activator of AMPK. The activation of AMPK with AICAR led to the appearance of beige-like morphological properties in differentiated white adipocytes. Namely, smaller lipid droplets appeared in AICAR-treated white adipocytes in a similar fashion as in beige cells. Moreover, in AICAR-treated white adipocytes the mitochondrial network was more fused than in white adipocytes; a fused mitochondrial system was characteristic to beige adipocytes. Despite the morphological similarities between AICAR-treated white adipocytes and beige cells, functionally AICAR-treated white adipocytes were similar to white adipocytes. We were unable to detect increases in basal or cAMP-induced oxygen consumption rate (a marker of mitochondrial biogenesis) when comparing control and AICAR-treated white adipocytes. Similarly, markers of beige adipocytes such as TBX1, UCP1, CIDEA, PRDM16 and TMEM26 remained the same when

  2. AMP-Activated Kinase (AMPK) Activation by AICAR in Human White Adipocytes Derived from Pericardial White Adipose Tissue Stem Cells Induces a Partial Beige-Like Phenotype

    PubMed Central

    Abdul-Rahman, Omar; Kristóf, Endre; Doan-Xuan, Quang-Minh; Vida, András; Nagy, Lilla; Horváth, Ambrus; Simon, József; Maros, Tamás; Szentkirályi, István; Palotás, Lehel; Debreceni, Tamás; Csizmadia, Péter; Szerafin, Tamás; Fodor, Tamás; Szántó, Magdolna; Tóth, Attila; Kiss, Borbála; Bacsó, Zsolt; Bai, Péter

    2016-01-01

    Beige adipocytes are special cells situated in the white adipose tissue. Beige adipocytes, lacking thermogenic cues, morphologically look quite similar to regular white adipocytes, but with a markedly different response to adrenalin. White adipocytes respond to adrenergic stimuli by enhancing lipolysis, while in beige adipocytes adrenalin induces mitochondrial biogenesis too. A key step in the differentiation and function of beige adipocytes is the deacetylation of peroxisome proliferator-activated receptor (PPARγ) by SIRT1 and the consequent mitochondrial biogenesis. AMP-activated protein kinase (AMPK) is an upstream activator of SIRT1, therefore we set out to investigate the role of AMPK in beige adipocyte differentiation using human adipose-derived mesenchymal stem cells (hADMSCs) from pericardial adipose tissue. hADMSCs were differentiated to white and beige adipocytes and the differentiation medium of the white adipocytes was supplemented with 100 μM [(2R,3S,4R,5R)-5-(4-Carbamoyl-5-aminoimidazol-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl dihydrogen phosphate (AICAR), a known activator of AMPK. The activation of AMPK with AICAR led to the appearance of beige-like morphological properties in differentiated white adipocytes. Namely, smaller lipid droplets appeared in AICAR-treated white adipocytes in a similar fashion as in beige cells. Moreover, in AICAR-treated white adipocytes the mitochondrial network was more fused than in white adipocytes; a fused mitochondrial system was characteristic to beige adipocytes. Despite the morphological similarities between AICAR-treated white adipocytes and beige cells, functionally AICAR-treated white adipocytes were similar to white adipocytes. We were unable to detect increases in basal or cAMP-induced oxygen consumption rate (a marker of mitochondrial biogenesis) when comparing control and AICAR-treated white adipocytes. Similarly, markers of beige adipocytes such as TBX1, UCP1, CIDEA, PRDM16 and TMEM26 remained the same when

  3. Characterization of a primary brown adipocyte culture system derived from human fetal interscapular fat

    PubMed Central

    Seiler, Sarah E; Xu, Dan; Ho, Jia-Pei; Lo, Kinyui Alice; Buehrer, Benjamin M; Ludlow, Y John W; Kovalik, Jean-Paul; Sun, Lei

    2015-01-01

    Brown fat has gained widespread attention as a potential therapeutic target to treat obesity and associated metabolic disorders. Indeed, the anti-obesity potential of multiple targets to stimulate both brown adipocyte differentiation and recruitment have been verified in rodent models. However, their therapeutic potential in humans is unknown due to the lack of a human primary brown adipocyte cell culture system. Likewise, the lack of a well-characterized human model has limited the discovery of novel targets for the activation of human brown fat. To address this current need, we aimed to identify and describe the first primary brown adipocyte cell culture system from human fetal interscapular brown adipose tissue. Pre-adipocytes isolated from non-viable human fetal interscapular tissue were expanded and cryopreserved. Cells were then thawed and plated alongside adult human subcutaneous and omental pre-adipocytes for subsequent differentiation and phenotypic characterization. Interscapular pre-adipocytes in cell culture differentiated into mature adipocytes that were morphologically indistinguishable from the adult white depots. Throughout differentiation, cultured human fetal interscapular adipocytes demonstrated increased expression of classical brown fat markers compared to subcutaneous and omental cells. Further, functional analysis revealed an elevation in fatty acid oxidation as well as maximal and uncoupled oxygen consumption in interscapular brown adipocytes compared to white control cells. These data collectively identify the brown phenotype of these cells. Thus, our primary cell culture system derived from non-viable human fetal interscapular brown adipose tissue provides a valuable tool for the study of human brown adipocyte biology and for the development of anti-obesity therapeutics. PMID:26451287

  4. MTHFR Gene Mutations: A Potential Marker of Late-Onset Alzheimer's Disease?

    PubMed

    Román, Gustavo C

    2015-01-01

    Recent epigenome-wide association studies have confirmed the importance of epigenetic effects mediated by DNA methylation in late-onset Alzheimer's disease (LOAD). Metabolic folate pathways and methyl donor reactions facilitated by B-group vitamins may be critical in the pathogenesis of LOAD. Methylenetetrahydrofolate reductase (MTHFR) gene mutations were studied in consecutive Alzheimer's Disease & Memory Clinic patients up to December 2014. DNA analyses of MTHFR-C667T and - A1298C homozygous and heterozygous polymorphisms in 93 consecutive elderly patients revealed high prevalence of MTHFR mutations (92.5%). Findings require confirmation in a larger series, but MTHFR mutations may become a LOAD marker, opening novel possibilities for prevention and treatment.

  5. EasyClone-MarkerFree: A vector toolkit for marker-less integration of genes into Saccharomyces cerevisiae via CRISPR-Cas9.

    PubMed

    Jessop-Fabre, Mathew M; Jakočiūnas, Tadas; Stovicek, Vratislav; Dai, Zongjie; Jensen, Michael K; Keasling, Jay D; Borodina, Irina

    2016-08-01

    Saccharomyces cerevisiae is an established industrial host for production of recombinant proteins, fuels and chemicals. To enable stable integration of multiple marker-free overexpression cassettes in the genome of S. cerevisiae, we have developed a vector toolkit EasyClone-MarkerFree. The integration of linearized expression cassettes into defined genomic loci is facilitated by CRISPR/Cas9. Cas9 is recruited to the chromosomal location by specific guide RNAs (gRNAs) expressed from a set of gRNA helper vectors. Using our genome engineering vector suite, single and triple insertions are obtained with 90-100% and 60-70% targeting efficiency, respectively. We demonstrate application of the vector toolkit by constructing a haploid laboratory strain (CEN.PK113-7D) and a diploid industrial strain (Ethanol Red) for production of 3-hydroxypropionic acid, where we tested three different acetyl-CoA supply strategies, requiring overexpression of three to six genes each. Among the tested strategies was a bacterial cytosolic pyruvate dehydrogenase complex, which was integrated into the genome in a single transformation. The publicly available EasyClone-MarkerFree vector suite allows for facile and highly standardized genome engineering, and should be of particular interest to researchers working on yeast chassis with limited markers available.

  6. Silica nanoparticles inhibit brown adipocyte differentiation via regulation of p38 phosphorylation

    NASA Astrophysics Data System (ADS)

    Son, Min Jeong; Kim, Won Kon; Kwak, Minjeong; Oh, Kyoung-Jin; Chang, Won Seok; Min, Jeong-Ki; Lee, Sang Chul; Song, Nam Woong; Bae, Kwang-Hee

    2015-10-01

    Nanoparticles are of great interest due to their wide variety of biomedical and bioengineering applications. However, they affect cellular differentiation and/or intracellular signaling when applied and exposed to target organisms or cells. The brown adipocyte is a cell type important in energy homeostasis and thus closely related to obesity. In this study, we assessed the effects of silica nanoparticles (SNPs) on brown adipocyte differentiation. The results clearly showed that brown adipocyte differentiation was significantly repressed by exposure to SNPs. The brown adipocyte-specific genes as well as mitochondrial content were also markedly reduced. Additionally, SNPs led to suppressed p38 phosphorylation during brown adipocyte differentiation. These effects depend on the size of SNPs. Taken together, these results lead us to suggest that SNP has anti-brown adipogenic effect in a size-dependent manner via regulation of p38 phosphorylation.

  7. Interactions between mesenchymal stem cells, adipocytes, and osteoblasts in a 3D tri-culture model of hyperglycemic conditions in the bone marrow microenvironment.

    PubMed

    Rinker, Torri E; Hammoudi, Taymour M; Kemp, Melissa L; Lu, Hang; Temenoff, Johnna S

    2014-03-01

    Recent studies have found that uncontrolled diabetes and consequential hyperglycemic conditions can lead to an increased incidence of osteoporosis. Osteoblasts, adipocytes, and mesenchymal stem cells (MSCs) are all components of the bone marrow microenvironment and thus may have an effect on diabetes-related osteoporosis. However, few studies have investigated the influence of these three cell types on each other, especially in the context of hyperglycemia. Thus, we developed a hydrogel-based 3D culture platform engineered to allow live-cell retrieval in order to investigate the interactions between MSCs, osteoblasts, and adipocytes in mono-, co-, and tri-culture configurations under hyperglycemic conditions for 7 days of culture. Gene expression, histochemical analysis of differentiation markers, and cell viability were measured for all cell types, and MSC-laden hydrogels were degraded to retrieve cells to assess their colony-forming capacity. Multivariate models of gene expression data indicated that primary discrimination was dependent on the neighboring cell type, validating the need for co-culture configurations to study conditions modeling this disease state. MSC viability and clonogenicity were reduced when mono- and co-cultured with osteoblasts at high glucose levels. In contrast, MSCs showed no reduction of viability or clonogenicity when cultured with adipocytes under high glucose conditions, and the adipogenic gene expression indicates that cross-talk between MSCs and adipocytes may occur. Thus, our unique culture platform combined with post-culture multivariate analysis provided a novel insight into cellular interactions within the MSC microenvironment and highlights the necessity of multi-cellular culture systems for further investigation of complex pathologies such as diabetes and osteoporosis.

  8. Interactions between Mesenchymal Stem Cells, Adipocytes, and Osteoblasts in a 3D Tri-Culture Model of Hyperglycemic Conditions in the Bone Marrow Microenvironment

    PubMed Central

    Rinker, Torri E.; Hammoudi, Taymour M.; Kemp, Melissa L.; Lu, Hang; Temenoff, Johnna S.

    2014-01-01

    Recent studies have found that uncontrolled diabetes and consequential hyperglycemic conditions can lead to increased incidence of osteoporosis. Osteoblasts, adipocytes, and mesenchymal stem cells (MSCs) are all components of the bone marrow microenvironment and thus may have an effect on diabetes-related osteoporosis. However, few studies have investigated the influence of these three cell types on each other, especially in the context of hyperglycemia. Thus, we developed a hydrogel-based 3D culture platform engineered to allow live-cell retrieval in order to investigate the interactions between MSCs, osteoblasts, and adipocytes in mono-, co-, and tri-culture configurations under hyperglycemic conditions for 7 days of culture. Gene expression, histochemical analysis of differentiation markers, and cell viability were measured for all cell types, and MSC-laden hydrogels were degraded to retrieve cells to assess colony-forming capacity. Multivariate models of gene expression data indicated that primary discrimination was dependent on neighboring cell type, validating the need for co-culture configurations to study conditions modeling this disease state. MSC viability and clonogenicity were reduced when mono- and co-cultured with osteoblasts in high glucose levels. In contrast, MSCs had no reduction of viability or clonogenicity when cultured with adipocytes in high glucose conditions and adipogenic gene expression indicated that cross-talk between MSCs and adipocytes may occur. Thus, our unique culture platform combined with post-culture multivariate analysis provided novel insight into cellular interactions within the MSC microenvironment and highlights the necessity of multi-cellular culture systems for further investigation of complex pathologies such as diabetes and osteoporosis. PMID:24463781

  9. The Molecular Signature of HIV-1-Associated Lipomatosis Reveals Differential Involvement of Brown and Beige/Brite Adipocyte Cell Lineages

    PubMed Central

    Cereijo, Rubén; Gallego-Escuredo, José Miguel; Moure, Ricardo; Villarroya, Joan; Domingo, Joan Carles; Fontdevila, Joan; Martínez, Esteban; Gutiérrez, Maria del Mar; Mateo, María Gracia; Giralt, Marta; Domingo, Pere; Villarroya, Francesc

    2015-01-01

    Highly active antiretroviral therapy has remarkably improved quality of life of HIV-1-infected patients. However, this treatment has been associated with the so-called lipodystrophic syndrome, which conveys a number of adverse metabolic effects and morphological alterations. Among them, lipoatrophy of subcutaneous fat in certain anatomical areas and hypertrophy of visceral depots are the most common. Less frequently, lipomatous enlargements of subcutaneous fat at distinct anatomic areas occur. Lipomatous adipose tissue in the dorso-cervical area (“buffalo hump”) has been associated with a partial white-to-brown phenotype transition and with increased cell proliferation, but, to date, lipomatous enlargements arising in other parts of the body have not been characterized. In order to establish the main molecular events associated with the appearance of lipomatosis in HIV-1 patients, we analyzed biopsies of lipomatous tissue from “buffalo hump” and from other anatomical areas in patients, in comparison with healthy subcutaneous adipose tissue, using a marker gene expression approach. Both buffalo-hump and non-buffalo-hump lipomatous adipose tissues exhibited similar patterns of non-compromised adipogenesis, unaltered inflammation, non-fibrotic phenotype and proliferative activity. Shorter telomere length, prelamin A accumulation and SA-β-Gal induction, reminiscent of adipocyte senescence, were also common to both types of lipomatous tissues. Buffalo hump biopsies showed expression of marker genes of brown adipose tissue (e.g. UCP1) and, specifically, of “classical” brown adipocytes (e.g. ZIC1) but not of beige/brite adipocytes. No such brown fat-related gene expression occurred in lipomatous tissues at other anatomical sites. In conclusion, buffalo hump and other subcutaneous adipose tissue enlargements from HIV-1-infected patients share a similar lipomatous character. However, a distorted induction of white-to-“classical brown adipocyte” phenotype

  10. Effects of quercetin, a natural phenolic compound, in the differentiation of human mesenchymal stem cells (MSC) into adipocytes and osteoblasts.

    PubMed

    Casado-Díaz, Antonio; Anter, Jaouad; Dorado, Gabriel; Quesada-Gómez, José Manuel

    2016-06-01

    Natural phenols may have beneficial properties against oxidative stress, which is associated with aging and major chronic aging-related diseases, such as loss of bone mineral mass (osteoporosis) and diabetes. The main aim of this study was to analyze the effect of quercetin, a major nutraceutical compound present in the "Mediterranean diet", on mesenchymal stem-cell (MSC) differentiation. Such cells were induced to differentiate into osteoblasts or adipocytes in the presence of two quercetin concentrations (0.1 and 10μM). Several physiological parameters and the expression of osteoblastogenesis and adipogenesis marker genes were monitored. Quercetin (10μM) inhibited cell proliferation, alkaline phosphatase (ALPL) activity and mineralization, down-regulating the expression of ALPL, collagen type I alpha 1 (COL1A1) and osteocalcin [bone gamma-carboxyglutamate protein (BGLAP)] osteoblastogenesis-related genes in MSC differentiating into osteoblasts. Moreover, in these cultures, CCAAT/enhancer-binding protein alpha (CEBPA) and peroxisome proliferator-activated receptor gamma 2 (PPARG2) adipogenic genes were induced, and cells differentiated into adipocytes were observed. Quercetin did not affect proliferation, but increased adipogenesis, mainly at 10-μM concentration in MSC induced to differentiate to adipocytes. β- and γ-catenin (plakoglobin) nuclear levels were reduced and increased, respectively, in quercetin-treated cultures. This suggests that the effect of high concentration of quercetin on MSC osteoblastic and adipogenic differentiation is mediated via Wnt/β-catenin inhibition. In conclusion, quercetin supplementation inhibited osteoblastic differentiation and promoted adipogenesis at the highest tested concentration. Such possible adverse effects of high quercetin concentrations should be taken into account in nutraceutical or pharmaceutical strategies using such flavonol. PMID:27142748

  11. The application of the mutated acetolactate synthase gene from rice as the selectable marker gene in the production of transgenic soybeans.

    PubMed

    Tougou, Makoto; Yamagishi, Noriko; Furutani, Noriyuki; Kaku, Koichiro; Shimizu, Tsutomu; Takahata, Yoshihito; Sakai, Jun-ichi; Kanematsu, Seiji; Hidaka, Soh

    2009-05-01

    We investigated selective culturing conditions for the production of transgenic soybeans. In this culturing system, we used the acetolactate synthase (ALS)-inhibiting herbicide-resistance gene derived from rice (Os-mALS gene) as a selectable marker gene instead of that derived from bacteria, which interfered with the cultivation and practical usage of transgenic crops. T(1) soybeans grown from one regenerated plant after selection of the ALS-targeting pyrimidinyl carboxy (PC) herbicide bispyribac-sodium (BS) exhibited herbicide resistance, and the introduction and expression of the Os-mALS gene were confirmed by genetic analysis. The selective culturing system promoted by BS herbicide, in which the Os-mALS gene was used as a selectable marker, was proved to be applicable to the production of transgenic soybeans, despite the appearance of escaped soybean plants that did not contain the Os-mALS transgene.

  12. Transgene stacking and marker elimination in transgenic rice by sequential Agrobacterium-mediated co-transformation with the same selectable marker gene.

    PubMed

    Ramana Rao, Mangu Venkata; Parameswari, Chidambaram; Sripriya, Rajasekaran; Veluthambi, Karuppannan

    2011-07-01

    Rice chitinase (chi11) and tobacco osmotin (ap24) genes, which cause disruption of fungal cell wall and cell membrane, respectively, were stacked in transgenic rice to develop resistance against the sheath blight disease. The homozygous marker-free transgenic rice line CoT23 which harboured the rice chi11 transgene was sequentially re-transformed with a second transgene ap24 by co-transformation using an Agrobacterium tumefaciens strain harbouring a single-copy cointegrate vector pGV2260::pSSJ1 and a multi-copy binary vector pBin19∆nptII-ap24 in the same cell. pGV2260::pSSJ1 T-DNA carried the hygromycin phosphotransferase (hph) and β-glucuronidase (gus) genes. pBin19∆nptII-ap24 T-DNA harboured the tobacco osmotin (ap24) gene. Co-transformation of the gene of interest (ap24) with the selectable marker gene (SMG, hph) occurred in 12 out of 18 T(0) plants (67%). Segregation of hph from ap24 was accomplished in the T(1) generation in one (line 11) of the four analysed co-transformed plants. The presence of ap24 and chi11 transgenes and the absence of the hph gene in the SMG-eliminated T(1) plants of the line 11 were confirmed by DNA blot analyses. The SMG-free transgenic plants of the line 11 harboured a single copy of the ap24 gene. Homozygous, SMG-free T(2) plants of the transgenic line 11 harboured stacked transgenes, chi11 and ap24. Northern blot analysis of the SMG-free plants revealed constitutive expression of chi11 and ap24. The transgenic plants with stacked transgenes displayed high levels of resistance against Rhizoctonia solani. Thus, we demonstrate the development of transgene-stacked and marker-free transgenic rice by sequential Agrobacterium-mediated co-transformation with the same SMG.

  13. Epidermal Wnt/β-catenin signaling regulates adipocyte differentiation via secretion of adipogenic factors

    PubMed Central

    Donati, Giacomo; Proserpio, Valentina; Lichtenberger, Beate Maria; Natsuga, Ken; Sinclair, Rodney; Fujiwara, Hironobu; Watt, Fiona M.

    2014-01-01

    It has long been recognized that the hair follicle growth cycle and oscillation in the thickness of the underlying adipocyte layer are synchronized. Although factors secreted by adipocytes are known to regulate the hair growth cycle, it is unclear whether the epidermis can regulate adipogenesis. We show that inhibition of epidermal Wnt/β-catenin signaling reduced adipocyte differentiation in developing and adult mouse dermis. Conversely, ectopic activation of epidermal Wnt signaling promoted adipocyte differentiation and hair growth. When the Wnt pathway was activated in the embryonic epidermis, there was a dramatic and premature increase in adipocytes in the absence of hair follicle formation, demonstrating that Wnt activation, rather than mature hair follicles, is required for adipocyte generation. Epidermal and dermal gene expression profiling identified keratinocyte-derived adipogenic factors that are induced by β-catenin activation. Wnt/β-catenin signaling-dependent secreted factors from keratinocytes promoted adipocyte differentiation in vitro, and we identified ligands for the bone morphogenetic protein and insulin pathways as proadipogenic factors. Our results indicate epidermal Wnt/β-catenin as a critical initiator of a signaling cascade that induces adipogenesis and highlight the role of epidermal Wnt signaling in synchronizing adipocyte differentiation with the hair growth cycle. PMID:24706781

  14. De novo generation of adipocytes from circulating progenitor cells in mouse and human adipose tissue.

    PubMed

    Gavin, Kathleen M; Gutman, Jonathan A; Kohrt, Wendy M; Wei, Qi; Shea, Karen L; Miller, Heidi L; Sullivan, Timothy M; Erickson, Paul F; Helm, Karen M; Acosta, Alistaire S; Childs, Christine R; Musselwhite, Evelyn; Varella-Garcia, Marileila; Kelly, Kimberly; Majka, Susan M; Klemm, Dwight J

    2016-03-01

    White adipocytes in adults are typically derived from tissue resident mesenchymal progenitors. The recent identification of de novo production of adipocytes from bone marrow progenitor-derived cells in mice challenges this paradigm and indicates an alternative lineage specification that adipocytes exist. We hypothesized that alternative lineage specification of white adipocytes is also present in human adipose tissue. Bone marrow from transgenic mice in which luciferase expression is governed by the adipocyte-restricted adiponectin gene promoter was adoptively transferred to wild-type recipient mice. Light emission was quantitated in recipients by in vivo imaging and direct enzyme assay. Adipocytes were also obtained from human recipients of hematopoietic stem cell transplantation. DNA was isolated, and microsatellite polymorphisms were exploited to quantify donor/recipient chimerism. Luciferase emission was detected from major fat depots of transplanted mice. No light emission was observed from intestines, liver, or lungs. Up to 35% of adipocytes in humans were generated from donor marrow cells in the absence of cell fusion. Nontransplanted mice and stromal-vascular fraction samples were used as negative and positive controls for the mouse and human experiments, respectively. This study provides evidence for a nontissue resident origin of an adipocyte subpopulation in both mice and humans.

  15. Interaction between HMGA1 and retinoblastoma protein is required for adipocyte differentiation.

    PubMed

    Esposito, Francesco; Pierantoni, Giovanna Maria; Battista, Sabrina; Melillo, Rosa Marina; Scala, Stefania; Chieffi, Paolo; Fedele, Monica; Fusco, Alfredo

    2009-09-18

    It is generally accepted that the regulation of adipogenesis prevents obesity. However, the mechanisms controlling adipogenesis have not been completely defined. We have previously demonstrated that HMGA1 proteins play a critical role in adipogenesis. In fact, suppression of HMGA1 protein synthesis by antisense technology dramatically increased growth rate and impaired adipocyte differentiation in 3T3-L1 cells. Furthermore, we showed that HMGA1 strongly potentiates the capacity of the CCAAT/enhancer-binding protein beta (C/EBPbeta) transcriptional factor to transactivate the leptin promoter, an adipocytic-specific promoter. In this study we demonstrate that HMGA1 physically interacts with retinoblastoma protein (RB), which is also required in adipocyte differentiation. Moreover, we show that RB, C/EBPbeta, and HMGA1 proteins all cooperate in controlling both Id1 and leptin gene transcriptions, which are down- and up-regulated during adipocyte differentiation, respectively. We also demonstrate that HMGA1/RB interaction regulates CDC25A and CDC6 promoter activities, which are induced by E2F-1 protein during early adipocyte differentiation, by displacing HDAC1 from the RB-E2F1 complex. Furthermore, by using Hmga1(-/-) embryonic stem cells, which failed to undergo adipocyte differentiation, we show the crucial role of HMGA1 proteins in adipocyte differentiation due to its pivotal involvement in the formation of the RB-C/EBPbeta complex. Altogether these data demonstrate a key role of the interaction between HMGA1 and RB in adipocyte differentiation. PMID:19633359

  16. Less is more: strategies to remove marker genes from transgenic plants.

    PubMed

    Yau, Yuan-Yeu; Stewart, C Neal

    2013-01-01

    Selectable marker genes (SMGs) and selection agents are useful tools in the production of transgenic plants by selecting transformed cells from a matrix consisting of mostly untransformed cells. Most SMGs express protein products that confer antibiotic- or herbicide resistance traits, and typically reside in the end product of genetically-modified (GM) plants. The presence of these genes in GM plants, and subsequently in food, feed and the environment, are of concern and subject to special government regulation in many countries. The presence of SMGs in GM plants might also, in some cases, result in a metabolic burden for the host plants. Their use also prevents the re-use of the same SMG when a second transformation scheme is needed to be performed on the transgenic host. In recent years, several strategies have been developed to remove SMGs from GM products while retaining the transgenes of interest. This review describes the existing strategies for SMG removal, including the implementation of site specific recombination systems, TALENs and ZFNs. This review discusses the advantages and disadvantages of existing SMG-removal strategies and explores possible future research directions for SMG removal including emerging technologies for increased precision for genome modification. PMID:23617583

  17. Antibiotic resistance marker genes as environmental pollutants in GMO-pristine agricultural soils in Austria.

    PubMed

    Woegerbauer, Markus; Zeinzinger, Josef; Gottsberger, Richard Alexander; Pascher, Kathrin; Hufnagl, Peter; Indra, Alexander; Fuchs, Reinhard; Hofrichter, Johannes; Kopacka, Ian; Korschineck, Irina; Schleicher, Corina; Schwarz, Michael; Steinwider, Johann; Springer, Burkhard; Allerberger, Franz; Nielsen, Kaare M; Fuchs, Klemens

    2015-11-01

    Antibiotic resistance genes may be considered as environmental pollutants if anthropogenic emission and manipulations increase their prevalence above usually occurring background levels. The prevalence of aph(3')-IIa/nptII and aph(3')-IIIa/nptIII - frequent marker genes in plant biotechnology conferring resistance to certain aminoglycosides - was determined in Austrian soils from 100 maize and potato fields not yet exposed to but eligible for GMO crop cultivation. Total soil DNA extracts were analysed by nptII/nptIII-specific TaqMan real time PCR. Of all fields 6% were positive for nptII (median: 150 copies/g soil; range: 31-856) and 85% for nptIII (1190 copies/g soil; 13-61600). The copy-number deduced prevalence of nptIII carriers was 14-fold higher compared to nptII. Of the cultivable kanamycin-resistant soil bacteria 1.8% (95% confidence interval: 0-3.3%) were positive for nptIII, none for nptII (0-0.8%). The nptII-load of the studied soils was low rendering nptII a typical candidate as environmental pollutant upon anthropogenic release into these ecosystems. PMID:26232739

  18. The effects of endothelial lipase gene (LIPG) variants on inflammation marker levels and atherosclerosis development.

    PubMed

    Dalan, Altay Burak; Toptaş, Bahar; Buğra, Zehra; Polat, Nihat; Yılmaz-Aydoğan, Hülya; Çimen, Arif; Isbir, Turgay

    2013-08-01

    Atherosclerosis is a major pathological process related with several important adverse vascular events including coronary artery disease, stroke, and peripheral arterial disease. Endothelial lipase is an enzyme the activity of which affects all of lipoproteins, whereas HDL is the main substrate. The purpose of our study was to investigate the effects of endothelial lipase gene polymorphism and inflammation markers (CRP, IL-1β, IL-6, IL-8 and TNF-α) in the atherosclerosis. 104 patients with atherosclerosis and 76 healthy individuals were included in the study. LIPG -584C/T polymorphism gene polymorphisms were assessed with PCR-RFLP method. The serum CRP levels were measured by turbidimetric method using a biochemistry autoanalyzer, whereas serum IL-1β, IL-6, IL-8, TNF-α levels were determined by enzyme-linked immunosorbent assay. In this study, we found that the frequencies of TC genotype are more prevalent in patients than controls. We found a statistically significant difference of IL-6 levels between patient and control group. Our findings suggest that T allele might play a potential role in the susceptibility to atherogenesis in the Turkish population. PMID:23673478

  19. Antibiotic resistance marker genes as environmental pollutants in GMO-pristine agricultural soils in Austria.

    PubMed

    Woegerbauer, Markus; Zeinzinger, Josef; Gottsberger, Richard Alexander; Pascher, Kathrin; Hufnagl, Peter; Indra, Alexander; Fuchs, Reinhard; Hofrichter, Johannes; Kopacka, Ian; Korschineck, Irina; Schleicher, Corina; Schwarz, Michael; Steinwider, Johann; Springer, Burkhard; Allerberger, Franz; Nielsen, Kaare M; Fuchs, Klemens

    2015-11-01

    Antibiotic resistance genes may be considered as environmental pollutants if anthropogenic emission and manipulations increase their prevalence above usually occurring background levels. The prevalence of aph(3')-IIa/nptII and aph(3')-IIIa/nptIII - frequent marker genes in plant biotechnology conferring resistance to certain aminoglycosides - was determined in Austrian soils from 100 maize and potato fields not yet exposed to but eligible for GMO crop cultivation. Total soil DNA extracts were analysed by nptII/nptIII-specific TaqMan real time PCR. Of all fields 6% were positive for nptII (median: 150 copies/g soil; range: 31-856) and 85% for nptIII (1190 copies/g soil; 13-61600). The copy-number deduced prevalence of nptIII carriers was 14-fold higher compared to nptII. Of the cultivable kanamycin-resistant soil bacteria 1.8% (95% confidence interval: 0-3.3%) were positive for nptIII, none for nptII (0-0.8%). The nptII-load of the studied soils was low rendering nptII a typical candidate as environmental pollutant upon anthropogenic release into these ecosystems.

  20. Gene-associated markers provide tools for tackling illegal fishing and false eco-certification.

    PubMed

    Nielsen, Einar E; Cariani, Alessia; Mac Aoidh, Eoin; Maes, Gregory E; Milano, Ilaria; Ogden, Rob; Taylor, Martin; Hemmer-Hansen, Jakob; Babbucci, Massimiliano; Bargelloni, Luca; Bekkevold, Dorte; Diopere, Eveline; Grenfell, Leonie; Helyar, Sarah; Limborg, Morten T; Martinsohn, Jann T; McEwing, Ross; Panitz, Frank; Patarnello, Tomaso; Tinti, Fausto; Van Houdt, Jeroen K J; Volckaert, Filip A M; Waples, Robin S; Albin, Jan E J; Vieites Baptista, Juan M; Barmintsev, Vladimir; Bautista, José M; Bendixen, Christian; Bergé, Jean-Pascal; Blohm, Dietmar; Cardazzo, Barbara; Diez, Amalia; Espiñeira, Montserrat; Geffen, Audrey J; Gonzalez, Elena; González-Lavín, Nerea; Guarniero, Ilaria; Jeráme, Marc; Kochzius, Marc; Krey, Grigorius; Mouchel, Olivier; Negrisolo, Enrico; Piccinetti, Corrado; Puyet, Antonio; Rastorguev, Sergey; Smith, Jane P; Trentini, Massimo; Verrez-Bagnis, Véronique; Volkov, Alexander; Zanzi, Antonella; Carvalho, Gary R

    2012-01-01

    Illegal, Unreported and Unregulated fishing has had a major role in the overexploitation of global fish populations. In response, international regulations have been imposed and many fisheries have been 'eco-certified' by consumer organizations, but methods for independent control of catch certificates and eco-labels are urgently needed. Here we show that, by using gene-associated single nucleotide polymorphisms, individual marine fish can be assigned back to population of origin with unprecedented high levels of precision. By applying high differentiation single nucleotide polymorphism assays, in four commercial marine fish, on a pan-European scale, we find 93-100% of individuals could be correctly assigned to origin in policy-driven case studies. We show how case-targeted single nucleotide polymorphism assays can be created and forensically validated, using a centrally maintained and publicly available database. Our results demonstrate how application of gene-associated markers will likely revolutionize origin assignment and become highly valuable tools for fighting illegal fishing and mislabelling worldwide.

  1. Less is more: strategies to remove marker genes from transgenic plants

    PubMed Central

    2013-01-01

    Selectable marker genes (SMGs) and selection agents are useful tools in the production of transgenic plants by selecting transformed cells from a matrix consisting of mostly untransformed cells. Most SMGs express protein products that confer antibiotic- or herbicide resistance traits, and typically reside in the end product of genetically-modified (GM) plants. The presence of these genes in GM plants, and subsequently in food, feed and the environment, are of concern and subject to special government regulation in many countries. The presence of SMGs in GM plants might also, in some cases, result in a metabolic burden for the host plants. Their use also prevents the re-use of the same SMG when a second transformation scheme is needed to be performed on the transgenic host. In recent years, several strategies have been developed to remove SMGs from GM products while retaining the transgenes of interest. This review describes the existing strategies for SMG removal, including the implementation of site specific recombination systems, TALENs and ZFNs. This review discusses the advantages and disadvantages of existing SMG-removal strategies and explores possible future research directions for SMG removal including emerging technologies for increased precision for genome modification. PMID:23617583

  2. The okra (Abelmoschus esculentus) transcriptome as a source for gene sequence information and molecular markers for diversity analysis.

    PubMed

    Schafleitner, Roland; Kumar, Sanjeet; Lin, Chen-Yu; Hegde, Satish Gajanana; Ebert, Andreas

    2013-03-15

    A combined leaf and pod transcriptome of okra (Abelmoschus esculentus (L.) Moench) has been produced by RNA sequencing and short read assembly. More than 150,000 unigenes were obtained, comprising some 46 million base pairs of sequence information. More than 55% of the unigenes were annotated through sequence comparison with databases. The okra transcriptome sequences were mined for simple sequence repeat (SSR) markers. From 935 non-redundant SSR motifs identified in the unigene set, 199 were chosen for testing in a germplasm set, resulting in 161 polymorphic SSR markers. From this set, 19 markers were selected for a diversity analysis on 65 okra accessions comprising three different species, revealing 58 different genotypes and resulted in clustering of the accessions according to species and geographic origin. The okra gene sequence information and the marker resource are made available to the research community for functional genomics and breeding research.

  3. FTO Obesity Variant Circuitry and Adipocyte Browning in Humans

    PubMed Central

    Claussnitzer, Melina; Dankel, Simon N.; Kim, Kyoung-Han; Quon, Gerald; Meuleman, Wouter; Haugen, Christine; Glunk, Viktoria; Sousa, Isabel S.; Beaudry, Jacqueline L.; Puviindran, Vijitha; Abdennur, Nezar A.; Liu, Jannel; Svensson, Per-Arne; Hsu, Yi-Hsiang; Drucker, Daniel J.; Mellgren, Gunnar; Hui, Chi-Chung; Hauner, Hans; Kellis, Manolis

    2016-01-01

    BACKGROUND Genomewide association studies can be used to identify disease-relevant genomic regions, but interpretation of the data is challenging. The FTO region harbors the strongest genetic association with obesity, yet the mechanistic basis of this association remains elusive. METHODS We examined epigenomic data, allelic activity, motif conservation, regulator expression, and gene coexpression patterns, with the aim of dissecting the regulatory circuitry and mechanistic basis of the association between the FTO region and obesity. We validated our predictions with the use of directed perturbations in samples from patients and from mice and with endogenous CRISPR–Cas9 genome editing in samples from patients. RESULTS Our data indicate that the FTO allele associated with obesity represses mitochondrial thermogenesis in adipocyte precursor cells in a tissue-autonomous manner. The rs1421085 T-to-C single-nucleotide variant disrupts a conserved motif for the ARID5B repressor, which leads to derepression of a potent preadipocyte enhancer and a doubling of IRX3 and IRX5 expression during early adipocyte differentiation. This results in a cell-autonomous developmental shift from energy-dissipating beige (brite) adipocytes to energy-storing white adipocytes, with a reduction in mitochondrial thermogenesis by a factor of 5, as well as an increase in lipid storage. Inhibition of Irx3 in adipose tissue in mice reduced body weight and increased energy dissipation without a change in physical activity or appetite. Knockdown of IRX3 or IRX5 in primary adipocytes from participants with the risk allele restored thermogenesis, increasing it by a factor of 7, and overexpression of these genes had the opposite effect in adipocytes from nonrisk-allele carriers. Repair of the ARID5B motif by CRISPR–Cas9 editing of rs1421085 in primary adipocytes from a patient with the risk allele restored IRX3 and IRX5 repression, activated browning expression programs, and restored thermogenesis

  4. Characterization of lipid metabolism in insulin-sensitive adipocytes differentiated from immortalized human mesenchymal stem cells

    SciTech Connect

    Prawitt, Janne; Niemeier, Andreas; Kassem, Moustapha; Beisiegel, Ulrike; Heeren, Joerg

    2008-02-15

    There is a great demand for cell models to study human adipocyte function. Here we describe the adipogenic differentiation of a telomerase-immortalized human mesenchymal stem cell line (hMSC-Tert) that maintains numerous features of terminally differentiated adipocytes even after prolonged withdrawal of the peroxisome proliferator activated receptor {gamma} (PPAR{gamma}) agonist rosiglitazone. Differentiated hMSC-Tert developed the characteristic monolocular phenotype of mature adipocytes. The expression of adipocyte specific markers was highly increased during differentiation. Most importantly, the presence of the PPAR{gamma} agonist rosiglitazone was not required for the stable expression of lipoprotein lipase, adipocyte fatty acid binding protein and perilipin on mRNA and protein levels. Adiponectin expression was post-transcriptionally down-regulated in the absence of rosiglitazone. Insulin sensitivity as measured by insulin-induced phosphorylation of Akt and S6 ribosomal protein was also independent of rosiglitazone. In addition to commonly used adipogenic markers, we investigated further PPAR{gamma}-stimulated proteins with a role in lipid metabolism. We observed an increase of lipoprotein receptor (VLDLR, LRP1) and apolipoprotein E expression during differentiation. Despite this increased expression, the receptor-mediated endocytosis of lipoproteins was decreased in differentiated adipocytes, suggesting that these proteins may have an additional function in adipose tissue beyond lipoprotein uptake.

  5. Markers Linked to the ZYMV-FL Resistance Gene and Their Use in Marker-assisted Selection in Watermelon

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Zucchini yellow mosaic virus (ZYMV) is a major pathogen that reduces yield in cucurbits. The best studied strains include ZYMV-FL and ZYMV-CH of which resistance is conferred by a single recessive gene. Bulk segregate analysis was used for watermelon populations derived from PI 595203, a line with...

  6. Ethanol extracts of chickpeas alter the total lipid content and expression levels of genes related to fatty acid metabolism in mouse 3T3-L1 adipocytes.

    PubMed

    Shinohara, Shigeo; Gu, Yuanjun; Yang, Ying; Furuta, Yasuo; Tanaka, Masahiko; Yue, Xiaohua; Wang, Weiqing; Kitano, Masaru; Kimura, Hiroshi

    2016-08-01

    Desi-type chickpeas, which have long been used as a natural treatment for diabetes, have been reported to lower visceral adiposity, dyslipidemia and insulin resistance induced by a chronic high-fat diet in rats. In this study, in order to examine the effects of chickpeas of this type in an in vitro system, we used the 3T3-L1 mouse cell line, a subclone of Swiss 3T3 cells, which can differentiate into cells with an adipocyte-like phenotype, and we used ethanol extracts of chickpeas (ECP) instead of chickpeas. Treatment of the 3T3-L1 cells with ECP led to a decrease in the lipid content in the cells. The desaturation index, defined as monounsaturated fatty acids (MUFAs)/saturated fatty acids (SFAs), was also decreased by ECP due to an increase in the cellular content of SFAs and a decrease in the content of MUFAs. The decrease in this index may reflect a decreased reaction from SFA to MUFA, which is essential for fat storage. To confirm this hypothesis, we conducted a western blot analysis, which revealed a reduction in the amount of stearoyl-CoA desaturase 1 (SCD1), a key enzyme catalyzing the reaction from SFA to MUFA. We observed simultaneous inactivations of enzymes participating in lipogenesis, i.e., liver kinase B1 (LKB1), acetyl-CoA carboxylase (ACC) and AMPK, by phosphorylation, which may lead to the suppression of reactions from acetyl-CoA to SFA via malonyl-CoA in lipogenesis. We also investigated whether lipolysis is affected by ECP. The amount of carnitine palmitoyltransferase 1 (CPT1), an enzyme important for the oxidation of fatty acids, was increased by ECP treatment. ECP also led to an increase in uncoupling protein 2 (UCP2), reported as a key protein for the oxidation of fatty acids. All of these results obtained regarding lipogenesis and fatty acid metabolism in our in vitro system are consistent with the results previously shown in rats. We also examined the effects on SCD1 and lipid contents of ethanol extracts of Kabuli

  7. Ethanol extracts of chickpeas alter the total lipid content and expression levels of genes related to fatty acid metabolism in mouse 3T3-L1 adipocytes

    PubMed Central

    Shinohara, Shigeo; Gu, Yuanjun; Yang, Ying; Furuta, Yasuo; Tanaka, Masahiko; Yue, Xiaohua; Wang, Weiqing; Kitano, Masaru; Kimura, Hiroshi

    2016-01-01

    Desi-type chickpeas, which have long been used as a natural treatment for diabetes, have been reported to lower visceral adiposity, dyslipidemia and insulin resistance induced by a chronic high-fat diet in rats. In this study, in order to examine the effects of chickpeas of this type in an in vitro system, we used the 3T3-L1 mouse cell line, a subclone of Swiss 3T3 cells, which can differentiate into cells with an adipocyte-like phenotype, and we used ethanol extracts of chickpeas (ECP) instead of chickpeas. Treatment of the 3T3-L1 cells with ECP led to a decrease in the lipid content in the cells. The desaturation index, defined as monounsaturated fatty acids (MUFAs)/saturated fatty acids (SFAs), was also decreased by ECP due to an increase in the cellular content of SFAs and a decrease in the content of MUFAs. The decrease in this index may reflect a decreased reaction from SFA to MUFA, which is essential for fat storage. To confirm this hypothesis, we conducted a western blot analysis, which revealed a reduction in the amount of stearoyl-CoA desaturase 1 (SCD1), a key enzyme catalyzing the reaction from SFA to MUFA. We observed simultaneous inactivations of enzymes participating in lipogenesis, i.e., liver kinase B1 (LKB1), acetyl-CoA carboxylase (ACC) and AMPK, by phosphorylation, which may lead to the suppression of reactions from acetyl-CoA to SFA via malonyl-CoA in lipogenesis. We also investigated whether lipolysis is affected by ECP. The amount of carnitine palmitoyltransferase 1 (CPT1), an enzyme important for the oxidation of fatty acids, was increased by ECP treatment. ECP also led to an increase in uncoupling protein 2 (UCP2), reported as a key protein for the oxidation of fatty acids. All of these results obtained regarding lipogenesis and fatty acid metabolism in our in vitro system are consistent with the results previously shown in rats. We also examined the effects on SCD1 and lipid contents of ethanol extracts of Kabuli-type chickpeas, which are

  8. Ethanol extracts of chickpeas alter the total lipid content and expression levels of genes related to fatty acid metabolism in mouse 3T3-L1 adipocytes.

    PubMed

    Shinohara, Shigeo; Gu, Yuanjun; Yang, Ying; Furuta, Yasuo; Tanaka, Masahiko; Yue, Xiaohua; Wang, Weiqing; Kitano, Masaru; Kimura, Hiroshi

    2016-08-01

    Desi-type chickpeas, which have long been used as a natural treatment for diabetes, have been reported to lower visceral adiposity, dyslipidemia and insulin resistance induced by a chronic high-fat diet in rats. In this study, in order to examine the effects of chickpeas of this type in an in vitro system, we used the 3T3-L1 mouse cell line, a subclone of Swiss 3T3 cells, which can differentiate into cells with an adipocyte-like phenotype, and we used ethanol extracts of chickpeas (ECP) instead of chickpeas. Treatment of the 3T3-L1 cells with ECP led to a decrease in the lipid content in the cells. The desaturation index, defined as monounsaturated fatty acids (MUFAs)/saturated fatty acids (SFAs), was also decreased by ECP due to an increase in the cellular content of SFAs and a decrease in the content of MUFAs. The decrease in this index may reflect a decreased reaction from SFA to MUFA, which is essential for fat storage. To confirm this hypothesis, we conducted a western blot analysis, which revealed a reduction in the amount of stearoyl-CoA desaturase 1 (SCD1), a key enzyme catalyzing the reaction from SFA to MUFA. We observed simultaneous inactivations of enzymes participating in lipogenesis, i.e., liver kinase B1 (LKB1), acetyl-CoA carboxylase (ACC) and AMPK, by phosphorylation, which may lead to the suppression of reactions from acetyl-CoA to SFA via malonyl-CoA in lipogenesis. We also investigated whether lipolysis is affected by ECP. The amount of carnitine palmitoyltransferase 1 (CPT1), an enzyme important for the oxidation of fatty acids, was increased by ECP treatment. ECP also led to an increase in uncoupling protein 2 (UCP2), reported as a key protein for the oxidation of fatty acids. All of these results obtained regarding lipogenesis and fatty acid metabolism in our in vitro system are consistent with the results previously shown in rats. We also examined the effects on SCD1 and lipid contents of ethanol extracts of Kabuli

  9. Transcriptome sequencing of sea cucumber (Apostichopus japonicus) and the identification of gene-associated markers.

    PubMed

    Zhou, Z C; Dong, Y; Sun, H J; Yang, A F; Chen, Z; Gao, S; Jiang, J W; Guan, X Y; Jiang, B; Wang, B

    2014-01-01

    Sea cucumber (Apostichopus japonicus) is an ecologically and economically important species in East and South-East Asia. This project aimed to identify large numbers of gene-associated markers and differentially expressed genes (DEGs) after lipopolysaccharides (LPS) challenge in A. japonicus using high-throughput transcriptome sequencing. A total of 162 million high-quality reads of 174 million raw reads were obtained by deep sequencing using Illumina HiSeq™ 2000 platform. Assembly of these reads generated 94 704 unigenes, with read length ranging from 200 to 16 153 bp (average length of 810 bp). A total of 36 005 were identified as coding sequences (CDSs), 32 479 of which were successfully annotated. Based on the assembly transcriptome, we identified 142 511 high-quality single nucleotide polymorphisms (SNPs). Among them, 33 775, 63 120 and 45 616 were located in sequences without predicted CDS (non-CDSs), CDSs and untranslated regions (UTRs), respectively. These putative SNPs included 82 664 transitions and 59 847 transversions. Totally, 89 375 (59.1%) were distributed in 15 473 known genes. A total of 6417 microsatellites were detected in 5970 unigenes, 3216 of which were annotated and 2481 were successfully subjected for primer design. The numbers of simple sequence repeats (SSRs) identified in non-CDSs, CDSs and UTRs were 2367, 2316 and 1734. These potential SNPs and SSRs are expected to provide abundant resources for genetic, evolutionary and ecological studies in sea cucumber. Transcriptome comparison revealed 1330, 1347 and 1291 DEGs in the coelomocytes of A. japonicus at 4 h, 24 h and 72 h after LPS challenge, respectively. Approximately 58.4% (1802) of total DEGs have been successfully annotated.

  10. Fatty acid binding protein 4 expression marks a population of adipocyte progenitors in white and brown adipose tissues.

    PubMed

    Shan, Tizhong; Liu, Weiyi; Kuang, Shihuan

    2013-01-01

    Adipose tissues regulate metabolism, reproduction, and life span. The development and growth of adipose tissue are due to increases of both adipocyte cell size and cell number; the latter is mediated by adipocyte progenitors. Various markers have been used to identify either adipocyte progenitors or mature adipocytes. The fatty acid binding protein 4 (FABP4), commonly known as adipocyte protein 2 (aP2), has been extensively used as a marker for differentiated adipocytes. However, whether aP2 is expressed in adipogenic progenitors is controversial. Using Cre/LoxP-based cell lineage tracing in mice, we have identified a population of aP2-expressing progenitors in the stromal vascular fraction (SVF) of both white and brown adipose tissues. The aP2-lineage progenitors reside in the adipose stem cell niche and express adipocyte progenitor markers, including CD34, Sca1, Dlk1, and PDGFRα. When isolated and grown in culture, the aP2-expressing SVF cells proliferate and differentiate into adipocytes upon induction. Conversely, ablation of the aP2 lineage greatly reduces the adipogenic potential of SVF cells. When grafted into wild-type mice, the aP2-lineage progenitors give rise to adipose depots in recipient mice. Therefore, the expression of aP2 is not limited to mature adipocytes, but also marks a pool of undifferentiated progenitors associated with the vasculature of adipose tissues. Our finding adds to the repertoire of adipose progenitor markers and points to a new regulator of adipose plasticity.

  11. Mapping of avirulence genes in Phytophthora infestans with amplified fragment length polymorphism markers selected by bulked segregant analysis.

    PubMed Central

    van der Lee, T; Robold, A; Testa, A; van 't Klooster, J W; Govers, F

    2001-01-01

    In this study we investigated the genetic control of avirulence in the diploid oomycete pathogen Phytophthora infestans, the causal agent of late blight on potato. The dominant avirulence (Avr) genes matched six race-specific resistance genes introgressed in potato from a wild Solanum species. AFLP markers linked to Avr genes were selected by bulked segregant analysis and used to construct two high-density linkage maps, one containing Avr4 (located on linkage group A2-a) and the other containing a cluster of three tightly linked genes, Avr3, Avr10, and Avr11 (located on linkage group VIII). Bulked segregant analysis also resulted in a marker linked to Avr1 and this allowed positioning of Avr1 on linkage group IV. No bulked segregant analysis was performed for Avr2, but linkage to a set of random markers placed Avr2 on linkage group VI. Of the six Avr genes, five were located on the most distal part of the linkage group, possibly close to the telomere. The high-density mapping was initiated to facilitate future positional cloning of P. infestans Avr genes. PMID:11238385

  12. Linkage study of nonsyndromic cleft lip with or without cleft palate using candidate genes and mapped polymorphic markers

    SciTech Connect

    Stein, J.D.; Nelson, L.D.; Conner, B.J.

    1994-09-01

    Nonsyndromic cleft lip with or without cleft palate (CL(P)) involves fusion or growth failure of facial primordia during development. Complex segregation analysis of clefting populations suggest that an autosomal dominant gene may play a role in this common craniofacial disorder. We have ascertained 16 multigenerational families with CL(P) and tested linkage to 29 candidate genes and 139 mapped short tandem repeat markers. The candidate genes were selected based on their expression in craniofacial development or were identified through murine models. These include: TGF{alpha}, TGF{beta}1, TGF{beta}2, TGF{beta}3, EGF, EGFR, GRAS, cMyc, FGFR, Jun, JunB, PDFG{alpha}, PDGF{beta}, IGF2R, GCR Hox7, Hox8, Hox2B, twirler, 5 collagen and 3 extracellular matrix genes. Linkage was tested assuming an autosomal dominant model with sex-specific decreased penetrance. Linkage to all of the candidate loci was excluded in 11 families. RARA was tested and was not informative. However, haplotype analysis of markers flanking RARA on 17q allowed exclusion of this candidate locus. We have previously excluded linkage to 61 STR markers in 11 families. Seventy-eight mapped short tandem repeat markers have recently been tested in 16 families and 30 have been excluded. The remaining are being analyzed and an exclusion map is being developed based on the entire study results.

  13. SNP discovery and development of genetic markers for mapping immune response genes in common carp (Cyprinus carpio)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Single nucleotide polymorphisms (SNPs) in immune response genes have been reported as markers for susceptibility to infectious diseases in human and livestock. A disease caused by cyprinid herpesvirus 3 (CyHV-3) is highly contagious and virulent in common carp (Cyprinus carpio). With the aim to de...

  14. Identification of markers linked to genes for sprouting tolerance (independent of grain color) in hard white winter wheat (HWWW)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Identification of markers linked to genes for sprouting tolerance (independent of grain color) in hard white winter wheat (HWWW) ABSTRACT Pre-harvest sprouting (PHS) of wheat (Triticum aestivum L.) can negatively impact end-use quality and seed viability at planting. Due to preferences for white ...

  15. The anti-obesity effects of a tuna peptide on 3T3-L1 adipocytes are mediated by the inhibition of the expression of lipogenic and adipogenic genes and by the activation of the Wnt/β-catenin signaling pathway

    PubMed Central

    KIM, YOUNG-MIN; KIM, IN-HYE; CHOI, JEONG-WOOK; LEE, MIN-KYEONG; NAM, TAEK-JEONG

    2015-01-01

    The differentiation of 3T3-L1 cells into adipocytes involves the activation of an organized system of obesity-related genes, of which those encoding CCAAT/enhancer-binding proteins (C/EBPs) and the Wnt-10b protein may play integral roles. In a previous study of ours, we found that a specific peptide found in tuna (sequence D-I-V-D-K-I-E-I; termed TP-D) inhibited 3T3-L1 cell differentiation. In the present study, we observed that the expression of expression of C/EBPs and Wnt-10b was associated with obesity. The initial step of 3T3-L1 cell differentiation involved the upregulation of C/EBP-α expression, which in turn activated various subfactors. An upstream effector of glycogen synthase kinase-3β (GSK-3β) inhibited Wnt-10b expression in 3T3-L1 adipocytes. In a previous study of ours, we sequenced the tuna peptide via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and quadrupole time-of-flight mass spectrometry (Q-TOF MS/MS) and confirmed the anti-obesity effects thereof in 3T3-L1 adipocytes. In the present study, we demonstrate that TP-D inhibits C/EBP and promotes Wnt-10b mRNA expression, thus activating the Wnt pathway. The inhibition of lipid accumulation was measured using a glucose and triglyceride (TG) assay. Our results confirmed that TP-D altered the expression levels of C/EBP-related genes in a dose-dependent manner and activated the Wnt signaling pathway. In addition, we confirmed that total adiponectin and high-molecular weight (HMW) adiponectin levels were reduced by treatment with TP-D. These data indicate that TP-D inhibits adipocyte differentiation through the inhibition of C/EBP genes and the subsequent activation of the Wnt/β-catenin signaling pathway. PMID:26046125

  16. The anti-obesity effects of a tuna peptide on 3T3-L1 adipocytes are mediated by the inhibition of the expression of lipogenic and adipogenic genes and by the activation of the Wnt/β-catenin signaling pathway.

    PubMed

    Kim, Young-Min; Kim, In-Hye; Choi, Jeong-Wook; Lee, Min-Kyeong; Nam, Taek-Jeong

    2015-08-01

    The differentiation of 3T3-L1 cells into adipocytes involves the activation of an organized system of obesity-related genes, of which those encoding CCAAT/enhancer-binding proteins (C/EBPs) and the Wnt-10b protein may play integral roles. In a previous study of ours, we found that a specific peptide found in tuna (sequence D-I-V-D-K-I-E-I; termed TP-D) inhibited 3T3-L1 cell differentiation. In the present study, we observed that the expression of expression of C/EBPs and Wnt-10b was associated with obesity. The initial step of 3T3-L1 cell differentiation involved the upregulation of C/EBP-α expression, which in turn activated various subfactors. An upstream effector of glycogen synthase kinase-3β (GSK-3β) inhibited Wnt-10b expression in 3T3-L1 adipocytes. In a previous study of ours, we sequenced the tuna peptide via sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and quadrupole time-of-flight mass spectrometry (Q-TOF MS/MS) and confirmed the anti-obesity effects thereof in 3T3-L1 adipocytes. In the present study, we demonstrate that TP-D inhibits C/EBP and promotes Wnt-10b mRNA expression, thus activating the Wnt pathway. The inhibition of lipid accumulation was measured using a glucose and triglyceride (TG) assay. Our results confirmed that TP-D altered the expression levels of C/EBP-related genes in a dose-dependent manner and activated the Wnt signaling pathway. In addition, we confirmed that total adiponectin and high-molecular weight (HMW) adiponectin levels were reduced by treatment with TP-D. These data indicate that TP-D inhibits adipocyte differentiation through the inhibition of C/EBP genes and the subsequent activation of the Wnt/β-catenin signaling pathway.

  17. A new phylogeny of the Cephalaspidea (Gastropoda: Heterobranchia) based on expanded taxon sampling and gene markers.

    PubMed

    Oskars, Trond R; Bouchet, Philippe; Malaquias, Manuel António E

    2015-08-01

    The Cephalaspidea is a diverse marine clade of euthyneuran gastropods with many groups still known largely from shells or scant anatomical data. The definition of the group and the relationships between members has been hampered by the difficulty of establishing sound synapomorphies, but the advent of molecular phylogenetics is helping to change significantly this situation. Yet, because of limited taxon sampling and few genetic markers employed in previous studies, many questions about the sister relationships and monophyletic status of several families remained open. In this study 109 species of Cephalaspidea were included covering 100% of traditional family-level diversity (12 families) and 50% of all genera (33 genera). Bayesian and maximum likelihood phylogenetics analyses based on two mitochondrial (COI, 16S rRNA) and two nuclear gene markers (28S rRNA and Histone-3) were used to infer the relationships of Cephalaspidea. The monophyly of the Cephalaspidea was confirmed. The families Cylichnidae, Diaphanidae, Haminoeidae, Philinidae, and Retusidae were found non-monophyletic. This result suggests that the family level taxonomy of the Cephalaspidea warrants a profound revision and several new family and genus names are required to reflect the new phylogenetic hypothesis presented here. We propose a new classification of the Cephalaspidea including five new families (Alacuppidae, Colinatydidae, Colpodaspididae, Mnestiidae, Philinorbidae) and one new genus (Alacuppa). Two family names (Acteocinidae, Laonidae) and two genera (Laona, Philinorbis) are reinstated as valid. An additional lineage with family rank (Philinidae "Clade 4") was unravelled, but no genus and species names are available to reflect the phylogeny and formal description will take place elsewhere. PMID:25916189

  18. The effect of missing marker genotypes on the accuracy of gene-assisted breeding value estimation: a comparison of methods.

    PubMed

    Mulder, H A; Meuwissen, T H E; Calus, M P L; Veerkamp, R F

    2010-01-01

    In livestock populations, missing genotypes on a large proportion of the animals is a major problem when implementing gene-assisted breeding value estimation for genes with known effect. The objective of this study was to compare different methods to deal with missing genotypes on accuracy of gene-assisted breeding value estimation for identified bi-allelic genes using Monte Carlo simulation. A nested full-sib half-sib structure was simulated with a mixed inheritance model with one bi-allelic quantitative trait loci (QTL) and a polygenic effect due to infinite number of polygenes. The effect of the QTL was included in gene-assisted BLUP either by random regression on predicted gene content, i.e. the number of positive alleles, or including haplotype effects in the model with an inverse IBD matrix to account for identity-by-descent relationships between haplotypes using linkage analysis information (IBD-LA). The inverse IBD matrix was constructed using segregation indicator probabilities obtained from multiple marker iterative peeling. Gene contents for unknown genotypes were predicted using either multiple marker iterative peeling or mixed model methodology. For both methods, gene-assisted breeding value estimation increased accuracies of total estimated breeding value (EBV) with 0% to 22% for genotyped animals in comparison to conventional breeding value estimation. For animals that were not genotyped, the increase in accuracy was much lower (0% to 5%), but still substantial when the heritability was 0.1 and when the QTL explained at least 15% of the genetic variance. Regression on predicted gene content yielded higher accuracies than IBD-LA. Allele substitution effects were, however, overestimated, especially when only sires and males in the last generation were genotyped. For juveniles without phenotypic records and traits measured only on females, the superiority of regression on gene content over IBD-LA was larger than when all animals had phenotypes. Missing

  19. Neural control of white, beige and brown adipocytes.

    PubMed

    Bartness, T J; Ryu, V

    2015-08-01

    Reports of brown-like adipocytes in traditionally white adipose tissue (WAT) depots occurred ~30 years ago, but interest in white adipocyte 'browning' only has gained attention more recently. We integrate some of what is known about the sympathetic nervous system (SNS) innervation of WAT and brown adipose tissue (BAT) with the few studies focusing on the sympathetic innervation of the so-called 'brite' or 'beige' adipocytes that appear when WAT sympathetic drive increases (for example, cold exposure and food deprivation). Only one brain site, the dorsomedial hypothalamic nucleus (DMH), selectively browns some (inguinal WAT (IWAT) and dorsomedial subcutaneous WAT), but not all WAT depots and only when DMH neuropeptide Y gene expression is knocked down, a browning effect is mediated by WAT SNS innervation. Other studies show that WAT sympathetic fiber density is correlated with the number of brown-like adipocytes (multilocular lipid droplets, uncoupling protein-1 immunoreactivity) at both warm and cold ambient temperatures. WAT and BAT have sensory innervation, the latter important for acute BAT cold-induced temperature increases, therefore suggesting the possible importance of sensory neural feedback from brite/beige cells for heat production. Only one report shows browned WAT capable of producing heat in vivo. Collectively, increases in WAT sympathetic drive and the phenotype of these stimulated adipocytes seems critical for the production of new and/or transdifferentiation of white to brite/beige adipocytes. Selective harnessing of WAT SNS drive to produce browning or selective browning independent of the SNS to counter increases in adiposity by increasing expenditure appears to be extremely challenging. PMID:27152173

  20. Neural control of white, beige and brown adipocytes

    PubMed Central

    Bartness, T J; Ryu, V

    2015-01-01

    Reports of brown-like adipocytes in traditionally white adipose tissue (WAT) depots occurred ~30 years ago, but interest in white adipocyte ‘browning' only has gained attention more recently. We integrate some of what is known about the sympathetic nervous system (SNS) innervation of WAT and brown adipose tissue (BAT) with the few studies focusing on the sympathetic innervation of the so-called ‘brite' or ‘beige' adipocytes that appear when WAT sympathetic drive increases (for example, cold exposure and food deprivation). Only one brain site, the dorsomedial hypothalamic nucleus (DMH), selectively browns some (inguinal WAT (IWAT) and dorsomedial subcutaneous WAT), but not all WAT depots and only when DMH neuropeptide Y gene expression is knocked down, a browning effect is mediated by WAT SNS innervation. Other studies show that WAT sympathetic fiber density is correlated with the number of brown-like adipocytes (multilocular lipid droplets, uncoupling protein-1 immunoreactivity) at both warm and cold ambient temperatures. WAT and BAT have sensory innervation, the latter important for acute BAT cold-induced temperature increases, therefore suggesting the possible importance of sensory neural feedback from brite/beige cells for heat production. Only one report shows browned WAT capable of producing heat in vivo. Collectively, increases in WAT sympathetic drive and the phenotype of these stimulated adipocytes seems critical for the production of new and/or transdifferentiation of white to brite/beige adipocytes. Selective harnessing of WAT SNS drive to produce browning or selective browning independent of the SNS to counter increases in adiposity by increasing expenditure appears to be extremely challenging. PMID:27152173

  1. Dopamine Receptors in Human Adipocytes: Expression and Functions

    PubMed Central

    Borcherding, Dana C.; Hugo, Eric R.; Idelman, Gila; De Silva, Anuradha; Richtand, Nathan W.; Loftus, Jean; Ben-Jonathan, Nira

    2011-01-01

    Introduction Dopamine (DA) binds to five receptors (DAR), classified by their ability to increase (D1R-like) or decrease (D2R-like) cAMP. In humans, most DA circulates as dopamine sulfate (DA-S), which can be de-conjugated to bioactive DA by arylsulfatase A (ARSA). The objective was to examine expression of DAR and ARSA in human adipose tissue and determine whether DA regulates prolactin (PRL) and adipokine expression and release. Methods DAR were analyzed by RT-PCR and Western blotting in explants, primary adipocytes and two human adipocyte cell lines, LS14 and SW872. ARSA expression and activity were determined by qPCR and enzymatic assay. PRL expression and release were determined by luciferase reporter and Nb2 bioassay. Analysis of cAMP, cGMP, leptin, adiponectin and interleukin 6 (IL-6) was done by ELISA. Activation of MAPK and PI3 kinase/Akt was determined by Western blotting. Results DAR are variably expressed at the mRNA and protein levels in adipose tissue and adipocytes during adipogenesis. ARSA activity in adipocyte increases after differentiation. DA at nM concentrations suppresses cAMP, stimulates cGMP, and activates MAPK in adipocytes. Acting via D2R-like receptors, DA and DA-S inhibit PRL gene expression and release. Acting via D1R/D5R receptors, DA suppresses leptin and stimulates adiponectin and IL-6 release. Conclusions This is the first report that human adipocytes express functional DAR and ARSA, suggesting a regulatory role for peripheral DA in adipose functions. We speculate that the propensity of some DAR-activating antipsychotics to increase weight and alter metabolic homeostasis is due, in part, to their direct action on adipose tissue. PMID:21966540

  2. Neural differentiation potential of human bone marrow-derived mesenchymal stromal cells: misleading marker gene expression

    PubMed Central

    Montzka, Katrin; Lassonczyk, Nina; Tschöke, Beate; Neuss, Sabine; Führmann, Tobias; Franzen, Rachelle; Smeets, Ralf; Brook, Gary A; Wöltje, Michael

    2009-01-01

    Background In contrast to pluripotent embryonic stem cells, adult stem cells have been considered to be multipotent, being somewhat more restricted in their differentiation capacity and only giving rise to cell types related to their tissue of origin. Several studies, however, have reported that bone marrow-derived mesenchymal stromal cells (MSCs) are capable of transdifferentiating to neural cell types, effectively crossing normal lineage restriction boundaries. Such reports have been based on the detection of neural-related proteins by the differentiated MSCs. In order to assess the potential of human adult MSCs to undergo true differentiation to a neural lineage and to determine the degree of homogeneity between donor samples, we have used RT-PCR and immunocytochemistry to investigate the basal expression of a range of neural related mRNAs and proteins in populations of non-differentiated MSCs obtained from 4 donors. Results The expression analysis revealed that several of the commonly used marker genes from other studies like nestin, Enolase2 and microtubule associated protein 1b (MAP1b) are already expressed by undifferentiated human MSCs. Furthermore, mRNA for some of the neural-related transcription factors, e.g. Engrailed-1 and Nurr1 were also strongly expressed. However, several other neural-related mRNAs (e.g. DRD2, enolase2, NFL and MBP) could be identified, but not in all donor samples. Similarly, synaptic vesicle-related mRNA, STX1A could only be detected in 2 of the 4 undifferentiated donor hMSC samples. More significantly, each donor sample revealed a unique expression pattern, demonstrating a significant variation of marker expression. Conclusion The present study highlights the existence of an inter-donor variability of expression of neural-related markers in human MSC samples that has not previously been described. This donor-related heterogeneity might influence the reproducibility of transdifferentiation protocols as well as contributing to the

  3. Tagging and mapping of SSR marker for rust resistance gene in lentil (Lens culinaris Medikus subsp. culinaris).

    PubMed

    Dikshit, H K; Singh, Akanksha; Singh, D; Aski, M; Jain, Neelu; Hegde, V S; Basandrai, A K; Basandrai, D; Sharma, T R

    2016-06-01

    Lentil, as an economical source of protein, minerals and vitamins, plays important role in nutritional security of the common man. Grown mainly in West Asia, North Africa (WANA) region and South Asia, it suffers from several biotic stresses such as wilt, rust, blight and broomrape. Lentil rust caused by autoecious fungus Uromyces viciae fabae (Pers.) Schroet is a serious lentil disease in Algeria, Bangladesh, Ethiopia, India, Italy, Morocco, Pakistan and Nepal. The disease symptoms are observed during flowering and early podding stages. Rust causes severe yield losses in lentil. It can only be effectively controlled by identifying the resistant source, understanding its inheritance and breeding for host resistance. The obligate parasitic nature of pathogen makes it difficult to maintain the pathogen in culture and to apply it to screen segregating progenies under controlled growth conditions. Hence, the use of molecular markers will compliment in identification of resistant types in different breeding programs. Here, we studied the inheritance of resistance to rust in lentil using F₁, F₂ and F₂:₃ from cross PL 8 (susceptible) x L 4149 (resistant) varieties. The phenotyping of lentil population was carried out at Sirmour, India. The result of genetic analysis revealed that a single dominant gene controls rust resistance in lentil genotype L 4149. The F2 population from this cross was used to tag and map the rust resistance gene using SSR and SRAP markers. Markers such as 270 SRAP and 162 SSR were studied for polymorphism and 101 SRAP and 33 SSRs were found to be polymorphic between the parents. Two SRAP and two SSR markers differentiated the resistant and susceptible bulks. SSR marker Gllc 527 was estimated to be linked to rust resistant locus at a distance of 5.9 cM. The Gllc 527 marker can be used for marker assisted selection for rust resistance; however, additional markers closer to rust resistant locus are required. The markers linked to the rust

  4. Tagging and mapping of SSR marker for rust resistance gene in lentil (Lens culinaris Medikus subsp. culinaris).

    PubMed

    Dikshit, H K; Singh, Akanksha; Singh, D; Aski, M; Jain, Neelu; Hegde, V S; Basandrai, A K; Basandrai, D; Sharma, T R

    2016-06-01

    Lentil, as an economical source of protein, minerals and vitamins, plays important role in nutritional security of the common man. Grown mainly in West Asia, North Africa (WANA) region and South Asia, it suffers from several biotic stresses such as wilt, rust, blight and broomrape. Lentil rust caused by autoecious fungus Uromyces viciae fabae (Pers.) Schroet is a serious lentil disease in Algeria, Bangladesh, Ethiopia, India, Italy, Morocco, Pakistan and Nepal. The disease symptoms are observed during flowering and early podding stages. Rust causes severe yield losses in lentil. It can only be effectively controlled by identifying the resistant source, understanding its inheritance and breeding for host resistance. The obligate parasitic nature of pathogen makes it difficult to maintain the pathogen in culture and to apply it to screen segregating progenies under controlled growth conditions. Hence, the use of molecular markers will compliment in identification of resistant types in different breeding programs. Here, we studied the inheritance of resistance to rust in lentil using F₁, F₂ and F₂:₃ from cross PL 8 (susceptible) x L 4149 (resistant) varieties. The phenotyping of lentil population was carried out at Sirmour, India. The result of genetic analysis revealed that a single dominant gene controls rust resistance in lentil genotype L 4149. The F2 population from this cross was used to tag and map the rust resistance gene using SSR and SRAP markers. Markers such as 270 SRAP and 162 SSR were studied for polymorphism and 101 SRAP and 33 SSRs were found to be polymorphic between the parents. Two SRAP and two SSR markers differentiated the resistant and susceptible bulks. SSR marker Gllc 527 was estimated to be linked to rust resistant locus at a distance of 5.9 cM. The Gllc 527 marker can be used for marker assisted selection for rust resistance; however, additional markers closer to rust resistant locus are required. The markers linked to the rust

  5. Low-Dose Bisphenol-A Impairs Adipogenesis and Generates Dysfunctional 3T3-L1 Adipocytes

    PubMed Central

    Ariemma, Fabiana; D’Esposito, Vittoria; Liguoro, Domenico; Oriente, Francesco; Cabaro, Serena; Liotti, Antonietta; Cimmino, Ilaria; Longo, Michele; Beguinot, Francesco; Formisano, Pietro; Valentino, Rossella

    2016-01-01

    Environmental endocrine disruptors (EDCs), including bisphenol-A (BPA), have been recently involved in obesity and diabetes by dysregulating adipose tissue function. Our aim was to examine whether prolonged exposure to low doses of BPA could affect adipogenesis and adipocyte metabolic functions. Therefore, 3T3-L1 pre-adipocytes were cultured for three weeks with BPA 1nM to mimic human environmental exposure. We evaluated BPA effect on cell proliferation, differentiation, gene expression and adipocyte metabolic function. BPA significantly increased pre-adipocyte proliferation (p<0.01). In 3T3-L1 adipocytes differentiated in the presence of BPA, the expression of Peroxisome proliferator-activated receptor gamma (PPARγ), Fatty Acid Binding Protein 4/Adipocyte Protein 2 (FABP4/AP2) and CCAAT/enhancer binding protein (C/EBPα) was increased by 3.5, 1.5 and 3 folds, respectively. Mature adipocytes also showed a significant increase in lipid accumulation (p<0.05) and alterations of insulin action, with significant reduction in insulin-stimulated glucose utilization (p<0.001). Moreover, in mature adipocytes, mRNA levels of Leptin, interleukin-6 (IL6) and interferon-γ (IFNγ) were significantly increased (p<0.05). In conclusion, BPA prolonged exposure at low doses, consistent with those found in the environment, may affect adipocyte differentiation program, enhancing pre-adipocyte proliferation and anticipating the expression of the master genes involved in lipid/glucose metabolism. The resulting adipocytes are hypertrophic, with impaired insulin signaling, reduced glucose utilization and increased pro-inflammatory cytokine expression. Thus, these data supported the hypothesis that BPA exposure, during critical stages of adipose tissue development, may cause adipocyte metabolic dysfunction and inflammation, thereby increasing the risk of developing obesity-related diseases. PMID:26942597

  6. Low-Dose Bisphenol-A Impairs Adipogenesis and Generates Dysfunctional 3T3-L1 Adipocytes.

    PubMed

    Ariemma, Fabiana; D'Esposito, Vittoria; Liguoro, Domenico; Oriente, Francesco; Cabaro, Serena; Liotti, Antonietta; Cimmino, Ilaria; Longo, Michele; Beguinot, Francesco; Formisano, Pietro; Valentino, Rossella

    2016-01-01

    Environmental endocrine disruptors (EDCs), including bisphenol-A (BPA), have been recently involved in obesity and diabetes by dysregulating adipose tissue function. Our aim was to examine whether prolonged exposure to low doses of BPA could affect adipogenesis and adipocyte metabolic functions. Therefore, 3T3-L1 pre-adipocytes were cultured for three weeks with BPA 1 nM to mimic human environmental exposure. We evaluated BPA effect on cell proliferation, differentiation, gene expression and adipocyte metabolic function. BPA significantly increased pre-adipocyte proliferation (p<0.01). In 3T3-L1 adipocytes differentiated in the presence of BPA, the expression of Peroxisome proliferator-activated receptor gamma (PPARγ), Fatty Acid Binding Protein 4/Adipocyte Protein 2 (FABP4/AP2) and CCAAT/enhancer binding protein (C/EBPα) was increased by 3.5, 1.5 and 3 folds, respectively. Mature adipocytes also showed a significant increase in lipid accumulation (p<0.05) and alterations of insulin action, with significant reduction in insulin-stimulated glucose utilization (p<0.001). Moreover, in mature adipocytes, mRNA levels of Leptin, interleukin-6 (IL6) and interferon-γ (IFNγ) were significantly increased (p<0.05). In conclusion, BPA prolonged exposure at low doses, consistent with those found in the environment, may affect adipocyte differentiation program, enhancing pre-adipocyte proliferation and anticipating the expression of the master genes involved in lipid/glucose metabolism. The resulting adipocytes are hypertrophic, with impaired insulin signaling, reduced glucose utilization and increased pro-inflammatory cytokine expression. Thus, these data supported the hypothesis that BPA exposure, during critical stages of adipose tissue development, may cause adipocyte metabolic dysfunction and inflammation, thereby increasing the risk of developing obesity-related diseases. PMID:26942597

  7. Gene targeting in the red alga Cyanidioschyzon merolae: single- and multi-copy insertion using authentic and chimeric selection markers.

    PubMed

    Fujiwara, Takayuki; Ohnuma, Mio; Yoshida, Masaki; Kuroiwa, Tsuneyoshi; Hirano, Tatsuya

    2013-01-01

    The unicellular red alga Cyanidioschyzon merolae is an emerging model organism for studying organelle division and inheritance: the cell is composed of an extremely simple set of organelles (one nucleus, one mitochondrion and one chloroplast), and their genomes are completely sequenced. Although a fruitful set of cytological and biochemical methods have now been developed, gene targeting techniques remain to be fully established in this organism. Thus far, only a single selection marker, URA Cm-Gs , has been available that complements the uracil-auxotrophic mutant M4. URA Cm-Gs , a chimeric URA5.3 gene of C. merolae and the related alga Galdieria sulphuraria, was originally designed to avoid gene conversion of the mutated URA5.3 allele in the parental strain M4. Although an early example of targeted gene disruption by homologous recombination was reported using this marker, the genome structure of the resultant transformants had never been fully characterized. In the current study, we showed that the use of the chimeric URA Cm-Gs selection marker caused multicopy insertion at high frequencies, accompanied by undesired recombination events at the targeted loci. The copy number of the inserted fragments was variable among the transformants, resulting in high yet uneven levels of transgene expression. In striking contrast, when the authentic URA5.3 gene (URA Cm-Cm ) was used as a selection marker, efficient single-copy insertion was observed at the targeted locus. Thus, we have successfully established a highly reliable and reproducible method for gene targeting in C. merolae. Our method will be applicable to a number of genetic manipulations in this organism, including targeted gene disruption, replacement and tagging.

  8. C/EBPα and the Corepressors CtBP1 and CtBP2 Regulate Repression of Select Visceral White Adipose Genes during Induction of the Brown Phenotype in White Adipocytes by Peroxisome Proliferator-Activated Receptor γ Agonists▿ †

    PubMed Central

    Vernochet, Cecile; Peres, Sidney B.; Davis, Kathryn E.; McDonald, Meghan E.; Qiang, Li; Wang, Hong; Scherer, Philipp E.; Farmer, Stephen R.

    2009-01-01

    White adipose tissue (WAT) stores energy in the form of triglycerides, whereas brown tissue (BAT) expends energy, primarily by oxidizing lipids. WAT also secretes many cytokines and acute-phase proteins that contribute to insulin resistance in obese subjects. In this study, we have investigated the mechanisms by which activation of peroxisome proliferator-activated receptor γ (PPARγ) with synthetic agonists induces a brown phenotype in white adipocytes in vivo and in vitro. We demonstrate that this phenotypic conversion is characterized by repression of a set of white fat genes (“visceral white”), including the resistin, angiotensinogen, and chemerin genes, in addition to induction of brown-specific genes, such as Ucp-1. Importantly, the level of expression of the “visceral white” genes is high in mesenteric and gonadal WAT depots but low in the subcutaneous WAT depot and in BAT. Mutation of critical amino acids within helix 7 of the ligand-binding domain of PPARγ prevents inhibition of visceral white gene expression by the synthetic agonists and therefore shows a direct role for PPARγ in the repression process. Inhibition of the white adipocyte genes also depends on the expression of C/EBPα and the corepressors, carboxy-terminal binding proteins 1 and 2 (CtBP1/2). The data further show that repression of resistin and angiotensinogen expression involves recruitment of CtBP1/2, directed by C/EBPα, to the minimal promoter of the corresponding genes in response to the PPARγ ligand. Developing strategies to enhance the brown phenotype in white adipocytes while reducing secretion of stress-related cytokines from visceral WAT is a means to combat obesity-associated disorders. PMID:19564408

  9. Aquaporin-10 Represents an Alternative Pathway for Glycerol Efflux from Human Adipocytes

    PubMed Central

    Laforenza, Umberto; Scaffino, Manuela F.; Gastaldi, Giulia

    2013-01-01

    Background Glycerol outflow from adipocytes has been considered for a decade to be mediated by aquaporin-7, an aquaglyceroporin highly expressed in the adipose tissue. Its involvement in glycerol metabolism has been widely studied also in humans. Recent studies in different aquaporin-7 KO mice models pose two different questions 1) the exact localization of aquaporin-7 in human white adipose tissue; 2) the existence of other aquaglyceroporins that work with aquaporin-7 to guarantee glycerol efflux and thus a normal adiposity in humans. To this purpose we investigated the expression, the localization and the functioning of aquaglyceroporin-10 in subcutaneous white adipose tissue, in isolated and cultured differentiated adipocytes. Methodology/Principal Findings Aquaporin-7 and -10 were expressed in the white adipose tissue both at mRNA and at protein level. Immunofluorescence revealed aquaporin-7 and -10 labelling in the human adipose tissue both to the plasma membrane and to a thin rim of cytoplasm of adipocytes. Aquaporin-7, but not aquaporin-10, colocalized with the endothelial marker CD34. Human cultured differentiated adipocytes showed an aquaporin-7 and -10 labelling mainly in the cytoplasm and in the lipid droplets with insulin reinforcing the lipid droplets staining and isoproterenol inducing its translocation to the plasma membrane compartment. Water and glycerol permeability measurements using adipocytes and adipose membrane vesicles confirmed the presence of functioning aquaglyceroporins. Aquaporin-10 silencing in human differentiated adipocytes resulted in a 50% decrease of glycerol and osmotic water permeability. Conclusions/Significance The results indicate that aquaporin-7, differently from mice, is present in both adipocyte and capillary plasma membranes of human adipose tissue. Aquaporin-10, on the contrary, is expressed exclusively in the adipocytes. The expression of two aquaglyceroporins in human adipose tissue is particularly important for the

  10. Adipocyte aminopeptidases in obesity and fasting.

    PubMed

    Alponti, Rafaela Fadoni; Silveira, Paulo Flavio

    2015-11-01

    This study checked the existence of a diverse array of aminopeptidase (AP) enzymes in high (HDM) and low (LDM) density microsomal and plasma membrane (MF) fractions from adipocytes of control, monosodium glutamate obese and food deprived rats. Gene expression was detected for ArgAP, AspAP, MetAP, and two AlaAP (APM and PSA). APM and PSA had the highest catalytic efficiency, whereas AspAP the highest affinity. Subcellular distribution of AP activities depended on metabolic status. Comparing catalytic levels, AspAP in HDM, LDM and MF was absent in obese and control under food deprivation; PSA in LDM was 3.5-times higher in obese than in normally fed control and control and obese under food deprivation; MetAP in MF was 4.5-times higher in obese than in food deprived obese. Data show new AP enzymes genetically expressed in subcellular compartments of adipocytes, three of them with altered catalytic levels that respond to whole-body energetic demands.

  11. [Development of a genetic transformation system for Candida tropicalis based on a reusable selection marker of URA3 gene].

    PubMed

    Xiang, Zheng; Chen, Xianzhong; Zhang, Lihua; Shen, Wei; Fan, You; Lu, Maolin

    2014-10-01

    Candida tropicalis, a diploid asporogenic yeast, is frequently utilized in industrial applications and research studies. However, the low efficiency of genetic transformation limits the strain improvement by metabolic engineering. A reliable transformation and efficient deletion of target gene are prerequisite for molecular improvement of C. tropicalis. In this study, an efficient approach for genetic transformation of C. tropicalis was developed based on the URA3 gene as a reusable selection marker and both of PDC allele genes encoding pyruvate decarboxylase were successfully deleted by this approach. Firstly, an auxotrophic mutant strain of C. tropicalis XZX which is defective in orotidine-5'-phosphate decarboxylase (URA3) was isolated by chemical mutagenesis combined with nystatin enrichment selection and 5-fluoro-orotic acid (5-FOA) resistance selection using C. tropicalis ATCC 20336 as the parent strain. Then, the first PDC deletion cassette PDC1-hisG-URA3-hisG- PDC1 (PHUHP) which contains a 1.6 kb URA3 marker gene, two copies of 1.1 kb Salmonella hisG fragments and homologous arms of target gene was constructed and transformed into C. tropicalis XZX cells. Transformants with a single copy of PDC deleted were isolated and identified by PCR and DNA sequencing, which was designated as C.tropicalis XZX02. The C.tropicalis XZX02 cells were spread on the minimal medium containing 5-FOA to generate mutant C. tropicalis XZX03 in which URA3 marker gene was excised from PHUHP fragment integrated into the PDC gene site. The second PDC gene deletion cassette PDCm-URA3-PDCm (MUM) was constructed and transformed into C. tropicalis XZX03 to generate C.tropicalis XZX04 in which both of PDC allele genes were deleted. All strains were confirmed by PCR and DNA sequencing. This efficient genetic transformation approach laid a foundation for further metabolic engineering of C. tropicalis.

  12. From Arabidopsis thaliana to Brassica napus: development of amplified consensus genetic markers (ACGM) for construction of a gene map.

    PubMed

    Fourmann, M.; Barret, P.; Froger, N.; Baron, C.; Charlot, F.; Delourme, R.; Brunel, D.

    2002-12-01

    The evolution of genomes can be studied by comparing maps of homologous genes which show changes in nucleic acid sequences and chromosome rearrangements. In this study, we developed a set of 32 amplified consensus gene markers (ACGMs) that amplified gene sequences from Arabidopsis thaliana and Brassica napus. Our methodology, based on PCR, facilitated the rapid sequencing of homologous genes from various species of the same phylogenetic family and the detection of intragenic polymorphism. We found that such polymorphism principally concerned intron sequences and we used it to attribute a Brassica oleracea or Brassica rapa origin to the B. napus sequences and to map 43 rapeseed genes. We confirm that the genetic position of homologous genes varied between B. napus and A. thaliana. ACGMs are a useful tool for genome evolution studies and for the further development of single nucleotide polymorphism suitable for use in genetic mapping and genetic diversity analyses.

  13. Sex determination in 58 bird species and evaluation of CHD gene as a universal molecular marker in bird sexing.

    PubMed

    Vucicevic, Milos; Stevanov-Pavlovic, Marija; Stevanovic, Jevrosima; Bosnjak, Jasna; Gajic, Bojan; Aleksic, Nevenka; Stanimirovic, Zoran

    2013-01-01

    The aim of this research was to test the CHD gene (Chromo Helicase DNA-binding gene) as a universal molecular marker for sexing birds of relatively distant species. The CHD gene corresponds to the aim because of its high degree of conservation and different lengths in Z and W chromosomes due to different intron sizes. DNA was isolated from feathers and the amplification of the CHD gene was performed with the following sets of polymerase chain reaction (PCR) primers: 2550F/2718R and P2/P8. Sex determination was attempted in 284 samples of 58 bird species. It was successful in 50 bird species; in 16 of those (Alopochen aegyptiacus, Ara severus, Aratinga acuticaudata, Bucorvus leadbeateri, Cereopsis novaehollandiae, Columba arquatrix, Corvus corax, C. frugilegus, Cyanoliseus patagonus, Guttera plumifera, Lamprotornis superbus, Milvus milvus, Neophron percnopterus, Ocyphaps lophotes, Podiceps cristatus, and Poicephalus senegalus), it was carried out for the first time using molecular markers and PCR. It is reasonable to assume that extensive research is necessary to define the CHD gene as a universal molecular marker for successful sex determination in all bird species (with exception of ratites). The results of this study may largely contribute to the aim. PMID:22553188

  14. Development of crop-specific transposable element (SINE) markers for studying gene flow from oilseed rape to wild radish.

    PubMed

    Prieto, J L; Pouilly, N; Jenczewski, E; Deragon, J M; Chèvre, A M

    2005-08-01

    The screening of wild populations for evidence of gene flow from a crop to a wild related species requires the unambiguous detection of crop genes within the genome of the wild species, taking into account the intraspecific variability of each species. If the crop and wild relatives share a common ancestor, as is the case for the Brassica crops and their wild relatives (subtribe Brassiceae), the species-specific markers needed to make this unambiguous detection are difficult to identify. In the model oilseed rape (Brassica napus, AACC, 2n = 38)-wild radish (Raphanus raphanistrum, RrRr, 2n = 18) system, we utilized the presence or absence of a short-interspersed element (SINE) at a given locus to develop oilseed rape-specific markers, as SINE insertions are irreversible. By means of sequence-specific amplified polymorphism (SINE-SSAP) reactions, we identified and cloned 67 bands specific to the oilseed rape genome and absent from that of wild radish. Forty-seven PCR-specific markers were developed from three combinations of primers anchored either in (1) the 5'- and 3'-genomic sequences flanking the SINE, (2) the 5'-flanking and SINE internal sequences or (3) the SINE internal and flanking 3'-sequences. Seventeen markers were monomorphic whatever the oilseed rape varieties tested, whereas 30 revealed polymorphism and behaved either as dominant (17) or co-dominant (13) markers. Polymorphic markers were mapped on 19 genomic regions assigned to ten linkage groups. The markers developed will be efficient tools to trace the occurrence and frequency of introgressions of oilseed rape genomic region within wild radish populations. PMID:15942756

  15. Purple chromoprotein gene serves as a new selection marker for transgenesis of the microalga Nannochloropsis oculata.

    PubMed

    Shih, Chen-Han; Chen, Hsiao-Yin; Lee, Hung-Chieh; Tsai, Huai-Jen

    2015-01-01

    Among the methods used to screen transgenic microalgae, antibiotics selection has raised environmental and food safety concerns, while the observation of fluorescence proteins could be influenced by the endogenous fluorescence of host chloroplasts. As an alternative, this study isolated the purple chromoprotein (CP) from Stichodacyla haddoni (shCP). A plasmid in which shCP cDNA is driven by a heat-inducible promoter was linearized and electroporated into 2.5×10(8) protoplasts of Nannochloropsis oculata. Following regeneration and cultivation on an f/2 medium plate for two weeks, we observed 26 colonies that displayed a slightly dark green coloration. After individually subculturing and performing five hours of heat shock at 42°C, a dark brown color was mosaically displayed in five of these colonies, indicating that both untransformed and transformed cells were mixed together in each colony. To obtain a uniform expression of shCP throughout the whole colony, we continuously isolated each transformed cell that exhibited brown coloration and subcultured it on a fresh plate, resulting in the generation of five transgenic lines of N. oculata which stably harbored the shCP gene for at least 22 months, as confirmed by PCR detection and observation by the naked eye. As shown by Western blot, exogenous shCP protein was expressed in these transgenic microalgae. Since shCP protein is biodegradable and originates from a marine organism, both environmental and food safety concerns have been eliminated, making this novel shCP reporter gene a simple, but effective and ecologically safe, marker for screening and isolating transgenic microalgae.

  16. Purple chromoprotein gene serves as a new selection marker for transgenesis of the microalga Nannochloropsis oculata.

    PubMed

    Shih, Chen-Han; Chen, Hsiao-Yin; Lee, Hung-Chieh; Tsai, Huai-Jen

    2015-01-01

    Among the methods used to screen transgenic microalgae, antibiotics selection has raised environmental and food safety concerns, while the observation of fluorescence proteins could be influenced by the endogenous fluorescence of host chloroplasts. As an alternative, this study isolated the purple chromoprotein (CP) from Stichodacyla haddoni (shCP). A plasmid in which shCP cDNA is driven by a heat-inducible promoter was linearized and electroporated into 2.5×10(8) protoplasts of Nannochloropsis oculata. Following regeneration and cultivation on an f/2 medium plate for two weeks, we observed 26 colonies that displayed a slightly dark green coloration. After individually subculturing and performing five hours of heat shock at 42°C, a dark brown color was mosaically displayed in five of these colonies, indicating that both untransformed and transformed cells were mixed together in each colony. To obtain a uniform expression of shCP throughout the whole colony, we continuously isolated each transformed cell that exhibited brown coloration and subcultured it on a fresh plate, resulting in the generation of five transgenic lines of N. oculata which stably harbored the shCP gene for at least 22 months, as confirmed by PCR detection and observation by the naked eye. As shown by Western blot, exogenous shCP protein was expressed in these transgenic microalgae. Since shCP protein is biodegradable and originates from a marine organism, both environmental and food safety concerns have been eliminated, making this novel shCP reporter gene a simple, but effective and ecologically safe, marker for screening and isolating transgenic microalgae. PMID:25793255

  17. Purple Chromoprotein Gene Serves as a New Selection Marker for Transgenesis of the Microalga Nannochloropsis oculata

    PubMed Central

    Shih, Chen-Han; Chen, Hsiao-Yin; Lee, Hung-Chieh; Tsai, Huai-Jen

    2015-01-01

    Among the methods used to screen transgenic microalgae, antibiotics selection has raised environmental and food safety concerns, while the observation of fluorescence proteins could be influenced by the endogenous fluorescence of host chloroplasts. As an alternative, this study isolated the purple chromoprotein (CP) from Stichodacyla haddoni (shCP). A plasmid in which shCP cDNA is driven by a heat-inducible promoter was linearized and electroporated into 2.5×108 protoplasts of Nannochloropsis oculata. Following regeneration and cultivation on an f/2 medium plate for two weeks, we observed 26 colonies that displayed a slightly dark green coloration. After individually subculturing and performing five hours of heat shock at 42°C, a dark brown color was mosaically displayed in five of these colonies, indicating that both untransformed and transformed cells were mixed together in each colony. To obtain a uniform expression of shCP throughout the whole colony, we continuously isolated each transformed cell that exhibited brown coloration and subcultured it on a fresh plate, resulting in the generation of five transgenic lines of N. oculata which stably harbored the shCP gene for at least 22 months, as confirmed by PCR detection and observation by the naked eye. As shown by Western blot, exogenous shCP protein was expressed in these transgenic microalgae. Since shCP protein is biodegradable and originates from a marine organism, both environmental and food safety concerns have been eliminated, making this novel shCP reporter gene a simple, but effective and ecologically safe, marker for screening and isolating transgenic microalgae. PMID:25793255

  18. Fine mapping and marker development for the crossability gene SKr on chromosome 5BS of hexaploid wheat (Triticum aestivum L.).

    PubMed

    Alfares, Walid; Bouguennec, Annaig; Balfourier, François; Gay, Georges; Bergès, Hélène; Vautrin, Sonia; Sourdille, Pierre; Bernard, Michel; Feuillet, Catherine

    2009-10-01

    Most elite wheat varieties cannot be crossed with related species thereby restricting greatly the germplasm that can be used for alien introgression in breeding programs. Inhibition to crossability is controlled genetically and a number of QTL have been identified to date, including the major gene Kr1 on 5BL and SKr, a strong QTL affecting crossability between wheat and rye on chromosome 5BS. In this study, we used a recombinant SSD population originating from a cross between the poorly crossable cultivar Courtot (Ct) and the crossable line MP98 to characterize the major dominant effect of SKr and map the gene at the distal end of the chromosome near the 5B homeologous GSP locus. Colinearity with barley and rice was used to saturate the SKr region with new markers and establish orthologous relationships with a 54-kb region on rice chromosome 12. In total, five markers were mapped within a genetic interval of 0.3 cM and 400 kb of BAC contigs were established on both sides of the gene to lay the foundation for map-based cloning of SKr. Two SSR markers completely linked to SKr were used to evaluate a collection of crossable wheat progenies originating from primary triticale breeding programs. The results confirm the major effect of SKr on crossability and the usefulness of the two markers for the efficient introgression of crossability in elite wheat varieties. PMID:19652174

  19. Dithizone staining of intracellular zinc: an unexpected and versatile counterscreen for auxotrophic marker genes in Saccharomyces cerevisiae.

    PubMed

    Yuan, Daniel S

    2011-01-01

    Auxotrophic marker genes such as URA3, LEU2, and HIS3 in Saccharomyces cerevisiae have long been used to select cells that have been successfully transformed with recombinant DNA. A longstanding challenge in working with these genes is that counterselection procedures are often lacking. This paper describes the unexpected discovery of a simple plate assay that imparts a bright red stain to cells experiencing nutritional stress from the lack of a marker gene. The procedure specifically stains a zinc-rich vesicular compartment analogous to the zinc-rich secretory vesicles found in insulin-secreting pancreatic islet cells and glutamate-secreting neurons. Staining was greatly diminished in zap1 mutants, which lack a homeostatic activator of zinc uptake, and in cot1 zrc1 double mutants, which lack the two yeast homologs of mammalian vesicle-specific zinc export proteins. Only one of 93 strains with temperature-sensitive alleles of essential genes exhibited an increase in dithizone staining at its non-permissive temperature, indicating that staining is not simply a sign of growth-arrested or dying cells. Remarkably, the procedure works with most commonly used marker genes, highlights subtle defects, uses no reporter constructs or expensive reagents, requires only a few hours of incubation, yields visually striking results without any instrumentation, and is not toxic to the cells. Many potential applications exist for dithizone staining, both as a versatile counterscreen for auxotrophic marker genes and as a powerful new tool for the genetic analysis of a biomedically important vesicular organelle. PMID:21998704

  20. Pulicaria jaubertii E. Gamal-Eldin reduces triacylglyceride content and modifies cellular antioxidant pathways in 3T3-L1 adipocytes.

    PubMed

    Al-Naqeb, Ghanya; Rousová, Jana; Kubátová, Alena; Picklo, Matthew J

    2016-06-25

    Levels of obesity in Middle Eastern countries are increasing. Phytochemicals have anti-obesogenic properties as evidenced by prevention of adipocyte differentiation and blocking triacylglyceride (TG) accumulation. In Yemen, Pulicaria jaubertii E. Gamal-Eldin (PJ) is a food additive and a traditional medicine. We tested the hypothesis that phytochemicals present in PJ inhibit adipocytic responses during differentiation of 3T3-L1 preadipocytes to adipocytes. Methanolic extracts of PJ did not block expression of fatty acid binding protein 4 (FABP4) a marker of differentiation but did inhibit TG accumulation. Treatment of 3T3-L1 preadipocytes increased NADPH:quinone oxidoreductase 1 (NQO1), a suppressor of TG accumulation. Further fractionation of the methanolic PJ extract with hexane and dichloromethane (DCM) demonstrated that bioactivity towards TG reduction and elevated expression of NQO1 and other antioxidant genes (glutamate cysteine ligase catalytic unit, glutathione disulfide reductase, glutathione peroxidase (GPx) 4 resided in the DCM fraction. Activity towards depleting GSH and elevating the expression of catalase and GPx3 were found in the DCM and hexane fractions. Analysis by gas chromatography and liquid chromatography coupled with mass spectrometry demonstrated the presence of catechin-like moieties in the DCM and methanolic fractions and suggest that these components were partially responsible for the bioactivity of these fractions. In summary, our data indicate that fractions derived PJ exhibit anti-adipogenic properties in part through the presence of catechin-like compounds. PMID:27163856

  1. Pulicaria jaubertii E. Gamal-Eldin reduces triacylglyceride content and modifies cellular antioxidant pathways in 3T3-L1 adipocytes.

    PubMed

    Al-Naqeb, Ghanya; Rousová, Jana; Kubátová, Alena; Picklo, Matthew J

    2016-06-25

    Levels of obesity in Middle Eastern countries are increasing. Phytochemicals have anti-obesogenic properties as evidenced by prevention of adipocyte differentiation and blocking triacylglyceride (TG) accumulation. In Yemen, Pulicaria jaubertii E. Gamal-Eldin (PJ) is a food additive and a traditional medicine. We tested the hypothesis that phytochemicals present in PJ inhibit adipocytic responses during differentiation of 3T3-L1 preadipocytes to adipocytes. Methanolic extracts of PJ did not block expression of fatty acid binding protein 4 (FABP4) a marker of differentiation but did inhibit TG accumulation. Treatment of 3T3-L1 preadipocytes increased NADPH:quinone oxidoreductase 1 (NQO1), a suppressor of TG accumulation. Further fractionation of the methanolic PJ extract with hexane and dichloromethane (DCM) demonstrated that bioactivity towards TG reduction and elevated expression of NQO1 and other antioxidant genes (glutamate cysteine ligase catalytic unit, glutathione disulfide reductase, glutathione peroxidase (GPx) 4 resided in the DCM fraction. Activity towards depleting GSH and elevating the expression of catalase and GPx3 were found in the DCM and hexane fractions. Analysis by gas chromatography and liquid chromatography coupled with mass spectrometry demonstrated the presence of catechin-like moieties in the DCM and methanolic fractions and suggest that these components were partially responsible for the bioactivity of these fractions. In summary, our data indicate that fractions derived PJ exhibit anti-adipogenic properties in part through the presence of catechin-like compounds.

  2. Gene Polymorphisms and Serum Levels of Pro- and Anti-Inflammatory Markers in Dengue Viral Infections.

    PubMed

    Feitosa, Rosimar Neris Martins; Vallinoto, Antonio Carlos Rosário; Vasconcelos, Pedro Fernando da Costa; Azevedo, Raimunda do Socorro da Silva; Azevedo, Vânia Nakauth; Machado, Luiz Fernando Almeida; Lima, Sandra Souza; Ishak, Marluísa de Oliveira Guimarães; Ishak, Ricardo

    2016-09-01

    Pro- and anti-inflammatory markers (tumor necrosis factor [TNF]-α, TNF-β, interferon [IFN]-γ, interleukin [IL]-6, IL-8, IL-10, and C-reactive protein [CRP]) were investigated in 80 patients infected with dengue viruses, 100 patients presenting with febrile illness but negative for dengue, and 99 healthy subjects. Immunoenzyme methods were used for quantitative assays in the plasma. Polymorphisms of TNF-α, TNF-β, IL-6, IL-8, and IL-10 genes were assessed by polymerase chain reaction (PCR)-restriction fragment length polymorphism and allele-specific oligonucleotide (ASO)-PCR for the IFN-γ. The highest mean serum levels of TNF-α, IFN-γ, IL-8, and CRP were observed in dengue-positive individuals. TNF-β, IL-6, and IL-10 levels were significantly higher in the dengue-negative individuals. No cytokine expression pattern was evidenced according to virus serotype. Genotypic frequency distributions were statistically significant for the polymorphisms of TNF-α and IFN-γ among positive, negative, and control dengue groups and IFN-γ among groups DENV-1, DENV-2, DENV-3, and controls. Modulation of cytokine expression and polymorphisms is a complex matter and needs further explanation considering the ethnic origins of the Brazilian population.

  3. Gene Polymorphisms and Serum Levels of Pro- and Anti-Inflammatory Markers in Dengue Viral Infections.

    PubMed

    Feitosa, Rosimar Neris Martins; Vallinoto, Antonio Carlos Rosário; Vasconcelos, Pedro Fernando da Costa; Azevedo, Raimunda do Socorro da Silva; Azevedo, Vânia Nakauth; Machado, Luiz Fernando Almeida; Lima, Sandra Souza; Ishak, Marluísa de Oliveira Guimarães; Ishak, Ricardo

    2016-09-01

    Pro- and anti-inflammatory markers (tumor necrosis factor [TNF]-α, TNF-β, interferon [IFN]-γ, interleukin [IL]-6, IL-8, IL-10, and C-reactive protein [CRP]) were investigated in 80 patients infected with dengue viruses, 100 patients presenting with febrile illness but negative for dengue, and 99 healthy subjects. Immunoenzyme methods were used for quantitative assays in the plasma. Polymorphisms of TNF-α, TNF-β, IL-6, IL-8, and IL-10 genes were assessed by polymerase chain reaction (PCR)-restriction fragment length polymorphism and allele-specific oligonucleotide (ASO)-PCR for the IFN-γ. The highest mean serum levels of TNF-α, IFN-γ, IL-8, and CRP were observed in dengue-positive individuals. TNF-β, IL-6, and IL-10 levels were significantly higher in the dengue-negative individuals. No cytokine expression pattern was evidenced according to virus serotype. Genotypic frequency distributions were statistically significant for the polymorphisms of TNF-α and IFN-γ among positive, negative, and control dengue groups and IFN-γ among groups DENV-1, DENV-2, DENV-3, and controls. Modulation of cytokine expression and polymorphisms is a complex matter and needs further explanation considering the ethnic origins of the Brazilian population. PMID:27336361

  4. Methylation of serum SST gene is an independent prognostic marker in colorectal cancer

    PubMed Central

    Liu, Yanqun; Chew, Min Hoe; Tham, Chee Kian; Tang, Choong Leong; Ong, Simon YK; Zhao, Yi

    2016-01-01

    There is an increasing demand for accurate prognostication for colorectal cancer (CRC). This study sought to assess prognostic potentials of methylation targets in the serum of CRC patients. A total of 165 CRC patients were enrolled in this prospective study. Promoter methylation levels of seven genes in pre-operative sera and matched tumor tissues were evaluated by quantitative methylation-specific PCR. Kaplan-Meier test, and univariate and multivariate Cox proportional hazards regression models were used for survival analyses. After a median follow-up of 56 months, 43 patients (28.7%) experienced tumor recurrence. In univariate survival analyses, serum methylation levels of SST and MAL were significantly predictive of cancer-specific death (P<0.005 for both). The former was also a significant predictor for tumor recurrence (P=0.007). Independent prognostic effects of serum methylation levels of SST were revealed by multivariate Cox regression model (P=0.031 and P=0.003 for cancer death and recurrence, respectively). When focusing on stage II and III patients, prognostication with serum methylated SST remained significant. Methylated SST detected in all serum samples can be traced back to the matched primary tumor tissues. We believe that methylated SST detected in the pre-operative sera of CRC patients appear to be a novel promising prognostic marker and probably can be auxiliary to tumor staging system and serum carcinoembryonic antigen towards better risk stratification. PMID:27725914

  5. Homoeologous GSL-ELONG gene replacement for manipulation of aliphatic glucosinolates in Brassica rapa L. by marker assisted selection.

    PubMed

    Hirani, Arvind H; Zelmer, Carla D; McVetty, Peter B E; Daayf, Fouad; Li, Genyi

    2013-01-01

    Aliphatic glucosinolates are the predominant sulfur-rich plant secondary metabolites in economically important Brassica crops. Glucosinolates and their hydrolysis products are involved in plant-microbe, plant-insect, plant-animal, and plant-human interactions. It is, therefore, important to manipulate glucosinolate profiles and contents in Brassica species. In this study, aliphatic glucosinolates were genetically manipulated through homoeologous recombination in backcross lines followed by marker assisted selection in B. rapa. A resynthesized B. napus line, from a cross between B. rapa and B. oleracea, was backcrossed with Chinese cabbage doubled haploid line, RI16. Marker assisted selection for non-functional gene was performed in each backcross generations. Advanced backcross progenies (BC3F2) were developed to identify homoeologous gene replacement and/or introgression. Reduction in 5C aliphatic glucosinolates (gluconapoleiferin, glucoalyssin, and glucobrassicanapin) was observed in BC3F2 progenies of the recurrent parent that carried the GSL-ELONG (-) gene. The GSL-ELONG (-) positive backcross progenies were also screened by the A-genome and BraGSL-ELONG gene specific marker, which linked with 5C aliphatic glucosinolates. The A-genome specific marker was absent in the plants of advanced backcross progenies which showed reduction in 5C aliphatic glucosinolates. The results suggest that the functional allele had been replaced by the non-functional GSL-ELONG (-) allele from B. oleracea. Some advanced backcross progenies (BC3F2) positive for the GSL-ELONG (-) allele and the A-genome specific SCAR marker BraMAM1-1 did not show reduction in 5C aliphatic glucosinolates, suggesting that GSL-ELONG (-) allele is recessive. Replacement of the functional locus in the A-genome by non-functional counterpart in the C-genome reduced the content of 5C aliphatic glucosinolates in B. rapa seeds with 20 μmol/g.

  6. Homoeologous GSL-ELONG gene replacement for manipulation of aliphatic glucosinolates in Brassica rapa L. by marker assisted selection

    PubMed Central

    Hirani, Arvind H.; Zelmer, Carla D.; McVetty, Peter B. E.; Daayf, Fouad; Li, Genyi

    2013-01-01

    Aliphatic glucosinolates are the predominant sulfur-rich plant secondary metabolites in economically important Brassica crops. Glucosinolates and their hydrolysis products are involved in plant–microbe, plant–insect, plant–animal, and plant–human interactions. It is, therefore, important to manipulate glucosinolate profiles and contents in Brassica species. In this study, aliphatic glucosinolates were genetically manipulated through homoeologous recombination in backcross lines followed by marker assisted selection in B. rapa. A resynthesized B. napus line, from a cross between B. rapa and B. oleracea, was backcrossed with Chinese cabbage doubled haploid line, RI16. Marker assisted selection for non-functional gene was performed in each backcross generations. Advanced backcross progenies (BC3F2) were developed to identify homoeologous gene replacement and/or introgression. Reduction in 5C aliphatic glucosinolates (gluconapoleiferin, glucoalyssin, and glucobrassicanapin) was observed in BC3F2 progenies of the recurrent parent that carried the GSL-ELONG- gene. The GSL-ELONG- positive backcross progenies were also screened by the A-genome and BraGSL-ELONG gene specific marker, which linked with 5C aliphatic glucosinolates. The A-genome specific marker was absent in the plants of advanced backcross progenies which showed reduction in 5C aliphatic glucosinolates. The results suggest that the functional allele had been replaced by the non-functional GSL-ELONG- allele from B. oleracea. Some advanced backcross progenies (BC3F2) positive for the GSL-ELONG- allele and the A-genome specific SCAR marker BraMAM1-1 did not show reduction in 5C aliphatic glucosinolates, suggesting that GSL-ELONG- allele is recessive. Replacement of the functional locus in the A-genome by non-functional counterpart in the C-genome reduced the content of 5C aliphatic glucosinolates in B. rapa seeds with 20 μmol/g. PMID:23532458

  7. Retransformation of marker-free potato for enhanced resistance against fungal pathogens by pyramiding chitinase and wasabi defensin genes.

    PubMed

    Khan, Raham Sher; Darwish, Nader Ahmed; Khattak, Bushra; Ntui, Valentine Otang; Kong, Kynet; Shimomae, Kazuki; Nakamura, Ikuo; Mii, Masahiro

    2014-09-01

    Multi-auto-transformation vector system has been one of the strategies to produce marker-free transgenic plants without using selective chemicals and plant growth regulators and thus facilitating transgene stacking. In the study reported here, retransformation was carried out in marker-free transgenic potato CV. May Queen containing ChiC gene (isolated from Streptomyces griseus strain HUT 6037) with wasabi defensin (WD) gene (isolated from Wasabia japonica) to pyramid the two disease resistant genes. Molecular analyses of the developed shoots confirmed the existence of both the genes of interest (ChiC and WD) in transgenic plants. Co-expression of the genes was confirmed by RT-PCR, northern blot, and western blot analyses. Disease resistance assay of in vitro plants showed that the transgenic lines co-expressing both the ChiC and WD genes had higher resistance against the fungal pathogens, Fusarium oxysporum (Fusarium wilt) and Alternaria solani (early blight) compared to the non-transformed control and the transgenic lines expressing either of the ChiC or WD genes. The disease resistance potential of the transgenic plants could be increased by transgene stacking or multiple transformations. PMID:24802621

  8. Retransformation of marker-free potato for enhanced resistance against fungal pathogens by pyramiding chitinase and wasabi defensin genes.

    PubMed

    Khan, Raham Sher; Darwish, Nader Ahmed; Khattak, Bushra; Ntui, Valentine Otang; Kong, Kynet; Shimomae, Kazuki; Nakamura, Ikuo; Mii, Masahiro

    2014-09-01

    Multi-auto-transformation vector system has been one of the strategies to produce marker-free transgenic plants without using selective chemicals and plant growth regulators and thus facilitating transgene stacking. In the study reported here, retransformation was carried out in marker-free transgenic potato CV. May Queen containing ChiC gene (isolated from Streptomyces griseus strain HUT 6037) with wasabi defensin (WD) gene (isolated from Wasabia japonica) to pyramid the two disease resistant genes. Molecular analyses of the developed shoots confirmed the existence of both the genes of interest (ChiC and WD) in transgenic plants. Co-expression of the genes was confirmed by RT-PCR, northern blot, and western blot analyses. Disease resistance assay of in vitro plants showed that the transgenic lines co-expressing both the ChiC and WD genes had higher resistance against the fungal pathogens, Fusarium oxysporum (Fusarium wilt) and Alternaria solani (early blight) compared to the non-transformed control and the transgenic lines expressing either of the ChiC or WD genes. The disease resistance potential of the transgenic plants could be increased by transgene stacking or multiple transformations.

  9. Identification of potential transcriptomic markers in developing ankylosing spondylitis: a meta-analysis of gene expression profiles.

    PubMed

    Fang, Fang; Pan, Jian; Xu, Lixiao; Li, Gang; Wang, Jian

    2015-01-01

    The goal of this study was to identify potential transcriptomic markers in developing ankylosing spondylitis by a meta-analysis of multiple public microarray datasets. Using the INMEX (integrative meta-analysis of expression data) program, we performed the meta-analysis to identify consistently differentially expressed (DE) genes in ankylosing spondylitis and further performed functional interpretation (gene ontology analysis and pathway analysis) of the DE genes identified in the meta-analysis. Three microarray datasets (26 cases and 29 controls in total) were collected for meta-analysis. 905 consistently DE genes were identified in ankylosing spondylitis, among which 482 genes were upregulated and 423 genes were downregulated. The upregulated gene with the smallest combined rank product (RP) was GNG11 (combined RP=299.64). The downregulated gene with the smallest combined RP was S100P (combined RP=335.94). In the gene ontology (GO) analysis, the most significantly enriched GO term was "immune system process" (P=3.46×10(-26)). The most significant pathway identified in the pathway analysis was antigen processing and presentation (P=8.40×10(-5)). The consistently DE genes in ankylosing spondylitis and biological pathways associated with those DE genes identified provide valuable information for studying the pathophysiology of ankylosing spondylitis.

  10. De novo characterization of Larimichthys crocea transcriptome for growth-/immune-related gene identification and massive microsatellite (SSR) marker development

    NASA Astrophysics Data System (ADS)

    Han, Zhaofang; Xiao, Shijun; Liu, Xiande; Liu, Yang; Li, Jiakai; Xie, Yangjie; Wang, Zhi Yong

    2016-04-01

    The large yellow croaker, Larimichthys crocea is an important marine fish in China with a high economic value. In the last decade, the stock conservation and aquaculture industry of this species have been facing severe challenges because of wild population collapse and degeneration of important economic traits. However, genes contributing to growth and immunity in L. crocea have not been thoroughly analyzed, and available molecular markers are still not sufficient for genetic resource management and molecular selection. In this work, we sequenced the transcriptome in L. crocea liver tissue with a Roche 454 sequencing platform and assembled the transcriptome into 93 801 transcripts. Of them, 38 856 transcripts were successfully annotated in nt, nr, Swiss-Prot, InterPro, COG, GO and KEGG databases. Based on the annotation information, 3 165 unigenes related to growth and immunity were identified. Additionally, a total of 6 391 simple sequence repeats (SSRs) were identified from the transcriptome, among which 4 498 SSRs had enough flanking regions to design primers for polymerase chain reactions (PCR). To access the polymorphism of these markers, 30 primer pairs were randomly selected for PCR amplification and validation in 30 individuals, and 12 primer pairs (40.0%) exhibited obvious length polymorphisms. This work applied RNA-Seq to assemble and analyze a live transcriptome in L. crocea. With gene annotation and sequence information, genes related to growth and immunity were identified and massive SSR markers were developed, providing valuable genetic resources for future gene functional analysis and selective breeding of L. crocea.

  11. Novel recombinant binary vectors harbouring Basta (bar) gene as a plant selectable marker for genetic transformation of plants.

    PubMed

    Nada, Reham M

    2016-04-01

    Genetic transformation is one of the most widely used technique in crop improvement. However, most of the binary vectors used in this technique, especially cloning based, contain antibiotic genes as selection marker that raise serious consumer and environmental concerns; moreover, they could be transferred to non-target hosts with deleterious effects. Therefore, the goal of this study was reconstruction of the widely used pBI121 binary vector by substituting the harmful antibiotic selection marker gene with a less-harmful selection marker, Basta (herbicide resistance gene). The generated vectors were designated as pBI121NB and pBI121CB, in which Basta gene was expressed under the control of Nos or CaMV 35S promoter, respectively. The successful integration of the new inserts into both the vectors was confirmed by PCR, restriction digestion and sequencing. Both these vectors were used in transforming Arabidopsis, Egyptian wheat and barley varieties using LBA4404 and GV3101 Agrobacterium strains. The surfactant Tween-20 resulted in an efficient transformation and the number of Arabidopsis transformants was about 6-9 %. Soaked seeds of wheat and barley were transformed with Agrobacterium to introduce the bacteria to the growing shoot apices. The percentage of transgenic lines was around 16-17 and 14-15 % for wheat and barley, respectively. The quantitative studies presented in this work showed that both LBA4404 and GV3101 strains were suitable for transforming Egyptian wheat and barley. PMID:27436915

  12. Novel recombinant binary vectors harbouring Basta (bar) gene as a plant selectable marker for genetic transformation of plants.

    PubMed

    Nada, Reham M

    2016-04-01

    Genetic transformation is one of the most widely used technique in crop improvement. However, most of the binary vectors used in this technique, especially cloning based, contain antibiotic genes as selection marker that raise serious consumer and environmental concerns; moreover, they could be transferred to non-target hosts with deleterious effects. Therefore, the goal of this study was reconstruction of the widely used pBI121 binary vector by substituting the harmful antibiotic selection marker gene with a less-harmful selection marker, Basta (herbicide resistance gene). The generated vectors were designated as pBI121NB and pBI121CB, in which Basta gene was expressed under the control of Nos or CaMV 35S promoter, respectively. The successful integration of the new inserts into both the vectors was confirmed by PCR, restriction digestion and sequencing. Both these vectors were used in transforming Arabidopsis, Egyptian wheat and barley varieties using LBA4404 and GV3101 Agrobacterium strains. The surfactant Tween-20 resulted in an efficient transformation and the number of Arabidopsis transformants was about 6-9 %. Soaked seeds of wheat and barley were transformed with Agrobacterium to introduce the bacteria to the growing shoot apices. The percentage of transgenic lines was around 16-17 and 14-15 % for wheat and barley, respectively. The quantitative studies presented in this work showed that both LBA4404 and GV3101 strains were suitable for transforming Egyptian wheat and barley.

  13. Characterization of the Miiuy Croaker (Miichthys miiuy) Transcriptome and Development of Immune-Relevant Genes and Molecular Markers

    PubMed Central

    Che, Rongbo; Sun, Yueyan; Sun, Dianqiao; Xu, Tianjun

    2014-01-01

    Background The miiuy croaker (Miichthys miiuy) is an important species of marine fish that supports capture fisheries and aquaculture. At present commercial scale aquaculture of this species is limited due to diseases caused by pathogens and parasites which restrict production and limit commercial value. The lack of transcriptomic and genomic information for the miiuy croaker limits the ability of researchers to study the pathogenesis and immune system of this species. In this study we constructed a cDNA library from liver, spleen and kidney which was sequenced using Illumina paired-end sequencing to enable gene discovery and molecular marker development. Principal Findings In our study, a total of 69,071 unigenes with an average length of 572 bp were obtained. Of these, 45,676 (66.13%) were successfully annotated in public databases. The unigenes were also annotated with Gene Ontology, Clusters of Orthologous Groups and KEGG pathways. Additionally, 498 immune-relevant genes were identified and classified. Furthermore, 14,885 putative simple sequence repeats (cSSRs) and 8,510 putative single nucleotide polymorphisms (SNPs) were identified from the 69,071 unigenes. Conclusion The miiuy croaker (Miichthys miiuy) transcriptome data provides a large resource to identify new genes involved in many processes including those involved in the response to pathogens and diseases. Furthermore, the thousands of potential cSSR and SNP markers found in this study are important resources with respect to future development of molecular marker assisted breeding programs for the miiuy croaker. PMID:24714210

  14. Regulation of pre-adipocyte proliferation and apoptosis by the small leucine-rich proteoglycans, biglycan and decorin.

    PubMed

    Ward, M; Ajuwon, K M

    2011-08-01

    Evidence for a functional role for extracellular matrix (ECM) proteins in adipose tissue is demonstrated in dynamic changes in expression of ECM genes during adipocyte differentiation and in obesity. Components of the ECM may regulate adipose cell number expansion by restricting pre-adipocyte proliferation, regulating apoptosis and inhibiting adipogenesis. Although pre-adipocytes express multiple proteoglycans, their role in pre-adipocyte proliferation up to now has remained unknown. The study described here was conducted to characterize roles of small leucine-rich proteoglycans (SLRPs) in adipocyte proliferation. Pre-adipocytes were seeded on plates coated with biglycan and decorin and were allowed to differentiate. In addition, pre-adipocytes were incubated on plates coated with biglycan, decorin, or fibronectin and measurements were made of cell proliferation and apoptosis. We are able to report that SLRPs decorin and biglycan did not affect differentiation of our 3T3-L1 cells; however, biglycan and decorin did reduce proliferation of pre-adipocytes, partly by induction of apoptosis. Furthermore, anti-proliferative capabilities of decorin and biglycan were nullified with removal of GAG side-chains suggesting that the chains played key roles in anti-proliferative effects of the SLRPs. We also found that co-treatment of decorin or biglycan with the proteoglycan fibronectin restored normal proliferation, an indication that multiple ECM proteins may act in concert to regulate overall proliferation rates of pre-adipocytes. These studies indicate that SLRPs may compose a regulatory factor in adipose tissue expansion, through hyperplasia.

  15. Standardization of Gene Expression Quantification by Absolute Real-Time qRT-PCR System Using a Single Standard for Marker and Reference Genes.

    PubMed

    Zhou, Yi-Hong; Raj, Vinay R; Siegel, Eric; Yu, Liping

    2010-08-16

    In the last decade, genome-wide gene expression data has been collected from a large number of cancer specimens. In many studies utilizing either microarray-based or knowledge-based gene expression profiling, both the validation of candidate genes and the identification and inclusion of biomarkers in prognosis-modeling has employed real-time quantitative PCR on reverse transcribed mRNA (qRT-PCR) because of its inherent sensitivity and quantitative nature. In qRT-PCR data analysis, an internal reference gene is used to normalize the variation in input sample quantity. The relative quantification method used in current real-time qRT-PCR analysis fails to ensure data comparability pivotal in identification of prognostic biomarkers. By employing an absolute qRT-PCR system that uses a single standard for marker and reference genes (SSMR) to achieve absolute quantification, we showed that the normalized gene expression data is comparable and independent of variations in the quantities of sample as well as the standard used for generating standard curves. We compared two sets of normalized gene expression data with same histological diagnosis of brain tumor from two labs using relative and absolute real-time qRT-PCR. Base-10 logarithms of the gene expression ratio relative to ACTB were evaluated for statistical equivalence between tumors processed by two different labs. The results showed an approximate comparability for normalized gene expression quantified using a SSMR-based qRT-PCR. Incomparable results were seen for the gene expression data using relative real-time qRT-PCR, due to inequality in molar concentration of two standards for marker and reference genes. Overall results show that SSMR-based real-time qRT-PCR ensures comparability of gene expression data much needed in establishment of prognostic/predictive models for cancer patients-a process that requires large sample sizes by combining independent sets of data.

  16. Autism and genetics: Clinical approach and association study with two markers of HRAS gene

    SciTech Connect

    Herault, J.; Petit, E.; Cherpi, C.

    1995-08-14

    Twin studies and familial aggregation studies indicate that genetic factors could play a role in infantile autism. In an earlier study, we identified a possible positive association between autism and a c-Harvey-ras (HRAS) oncogene marker at the 3{prime} end of the coding region. In an attempt to confirm this finding, we studied a larger population, well-characterized clinically and genetically. We report a positive association between autism and two HRAS markers, the 3{prime} marker used in the initial study and an additional marker in exon 1. 46 refs., 1 fig., 2 tabs.

  17. Transcriptome sequencing of different narrow-leafed lupin tissue types provides a comprehensive uni-gene assembly and extensive gene-based molecular markers.

    PubMed

    Kamphuis, Lars G; Hane, James K; Nelson, Matthew N; Gao, Lingling; Atkins, Craig A; Singh, Karam B

    2015-01-01

    Narrow-leafed lupin (NLL; Lupinus angustifolius L.) is an important grain legume crop that is valuable for sustainable farming and is becoming recognized as a human health food. NLL breeding is directed at improving grain production, disease resistance, drought tolerance and health benefits. However, genetic and genomic studies have been hindered by a lack of extensive genomic resources for the species. Here, the generation, de novo assembly and annotation of transcriptome datasets derived from five different NLL tissue types of the reference accession cv. Tanjil are described. The Tanjil transcriptome was compared to transcriptomes of an early domesticated cv. Unicrop, a wild accession P27255, as well as accession 83A:476, together being the founding parents of two recombinant inbred line (RIL) populations. In silico predictions for transcriptome-derived gene-based length and SNP polymorphic markers were conducted and corroborated using a survey assembly sequence for NLL cv. Tanjil. This yielded extensive indel and SNP polymorphic markers for the two RIL populations. A total of 335 transcriptome-derived markers and 66 BAC-end sequence-derived markers were evaluated, and 275 polymorphic markers were selected to genotype the reference NLL 83A:476 × P27255 RIL population. This significantly improved the completeness, marker density and quality of the reference NLL genetic map.

  18. Development of molecular markers linked to disease resistance genes in common bean based on whole genome sequence.

    PubMed

    Meziadi, Chouaïb; Richard, Manon M S; Derquennes, Amandine; Thareau, Vincent; Blanchet, Sophie; Gratias, Ariane; Pflieger, Stéphanie; Geffroy, Valérie

    2016-01-01

    Common bean (Phaseolus vulgaris) is the most important grain legume for direct human consumption in the world, particularly in developing countries where it constitutes the main source of protein. Unfortunately, common bean yield stability is constrained by a number of pests and diseases. As use of resistant genotypes is the most economic and ecologically safe means for controlling plant diseases, efforts have been made to genetically characterize resistance genes (R genes) in common bean. Despite its agronomic importance, genomic resources available in common bean were limited until the recent sequencing of common bean genome (Andean genotype G19833). Besides allowing the annotation of Nucleotide Binding-Leucine Rich Repeat (NB-LRR) encoding gene family, which is the prevalent class of disease R genes in plants, access to the whole genome sequence of common bean can be of great help for intense selection to increase the overall efficiency of crop improvement programs using marker-assisted selection (MAS). This review presents the state of the art of common bean NB-LRR gene clusters, their peculiar location in subtelomeres and correlation with genetically characterized monogenic R genes, as well as how the availability of the whole genome sequence can boost the development of molecular markers for MAS.

  19. Genetic mapping, marker assisted selection and allelic relationships for the Pu 6 gene conferring rust resistance in sunflower.

    PubMed

    Bulos, Mariano; Vergani, Pablo Nicolas; Altieri, Emiliano

    2014-09-01

    Rust resistance in the sunflower line P386 is controlled by Pu 6 , a gene which was reported to segregate independently from other rust resistant genes, such as R 4 . The objectives of this work were to map Pu 6 , to provide and validate molecular tools for its identification, and to determine the linkage relationship of Pu 6 and R 4 . Genetic mapping of Pu 6 with six markers covered 24.8 cM of genetic distance on the lower end of linkage Group 13 of the sunflower consensus map. The marker most closely linked to Pu 6 was ORS316 at 2.5 cM in the distal position. ORS316 presented five alleles when was assayed with a representative set of resistant and susceptible lines. Allelism test between Pu 6 and R 4 indicated that both genes are linked at a genetic distance of 6.25 cM. This is the first confirmation based on an allelism test that at least two members of the R adv /R 4 /R 11 / R 13a /R 13b /Pu 6 cluster of genes are at different loci. A fine elucidation of the architecture of this complex locus will allow designing and constructing completely new genomic regions combining genes from different resistant sources and the elimination of the linkage drag around each resistant gene.

  20. Androgen Receptor Splice Variants Contribute to Prostate Cancer Aggressiveness through Induction of EMT and Expression of Stem Cell Marker Genes

    PubMed Central

    Kong, Dejuan; Sethi, Seema; Li, Yiwei; Chen, Wei; Sakr, Wael A.; Heath, Elisabeth; Sarkar, Fazlul H.

    2014-01-01

    Background The mechanism(s) by which androgen receptor (AR) splice variants contribute to castration-resistant prostate cancer (CRPC) is still lacking. Methods Expressions of Epithelial-to-Mesenchymal Transition (EMT) and stem cell markers were molecularly tested using prostate cancer (PCa) cells transfected with AR and AR3 (also known as AR-V7) plasmids or siRNA, and also cultured cells under androgen deprivation therapy (ADT) condition. Cell migration, clonogenicity, sphere forming capacity was assessed using PCa cells under all experimental conditions and 3, 3′-diindolylmethane (DIM; BR-DIM) treatment. Human PCa samples from BR-DIM untreated or treated patients were also used for assessing the expression of AR3 and stem cell markers. Results Overexpression of AR led to the induction of EMT phenotype, while overexpression of AR3 not only induced EMT but also led to the expression of stem cell signature genes. More importantly, ADT enhanced the expression of AR and AR3 concomitant with up-regulated expression of EMT and stem cell marker genes. Dihydrotestosterone (DHT) treatment decreased the expression of AR and AR3, and reversed the expression of these EMT and stem cell marker genes. BR-DIM administered to PCa patients prior to radical prostatectomy inhibited the expression of cancer stem cell markers consistent with inhibition of self-renewal of PCa cells after BR-DIM treatment. Conclusion AR variants could contribute to PCa progression through induction of EMT and acquisition of stem cell characteristics, which could be attenuated by BR-DIM, suggesting that BR-DIM could become a promising agent for the prevention of CRPC and/or for the treatment of PCa PMID:25307492

  1. Direct action of capsaicin in brown adipogenesis and activation of brown adipocytes.

    PubMed

    Kida, Ryosuke; Yoshida, Hirofumi; Murakami, Masaru; Shirai, Mitsuyuki; Hashimoto, Osamu; Kawada, Teruo; Matsui, Tohru; Funaba, Masayuki

    2016-01-01

    The ingestion of capsaicin, the principle pungent component of red and chili peppers, induces thermogenesis, in part, through the activation of brown adipocytes expressing genes related to mitochondrial biogenesis and uncoupling such as peroxisome proliferator-activated receptor (Ppar) γ coactivator-1α (Pgc-1α) and uncoupling protein 1 (Ucp1). Capsaicin has been suggested to induce the activation of brown adipocytes, which is mediated by the stimulation of sympathetic nerves. However, capsaicin may directly affect the differentiation of brown preadipocytes, brown adipocyte function, or both, through its significant absorption. We herein demonstrated that Trpv1, a capsaicin receptor, is expressed in brown adipose tissue, and that its expression level is increased during the differentiation of HB2 brown preadipocytes. Furthermore, capsaicin induced calcium influx in brown preadipocytes. A treatment with capsaicin in the early stage of brown adipogenesis did not affect lipid accumulation or the expression levels of Fabp4 (a gene expressed in mature adipocytes), Pparγ2 (a master regulator of adipogenesis) or brown adipocyte-selective genes. In contrast, a treatment with capsaicin in the late stage of brown adipogenesis slightly increased the expression levels of Fabp4, Pparγ2 and Pgc-1α. Although capsaicin did not affect the basal expression level of Ucp1, Ucp1 induction by forskolin was partially inhibited by capsaicin, irrespective of the dose of capsaicin. The results of the present study suggest the direct effects of capsaicin on brown adipocytes or in the late stage of brown adipogenesis. PMID:26781688

  2. Resistin regulates the expression of plasminogen activator inhibitor-1 in 3T3-L1 adipocytes.

    PubMed

    Ikeda, Yoshito; Tsuchiya, Hiroyuki; Hama, Susumu; Kajimoto, Kazuaki; Kogure, Kentaro

    2014-05-30

    Resistin and plasminogen activator inhibitor-1 (PAI-1) are adipokines, which are secreted from adipocytes. Increased plasma resistin and PAI-1 levels aggravate metabolic syndrome through exacerbation of insulin resistance and induction of chronic inflammation. However, the relationship between resistin and PAI-1 gene expression remains unclear. Previously, we found that resistin regulates lipid metabolism via carbohydrate responsive element-binding protein (ChREBP) during adipocyte maturation (Ikeda et al., 2013) [6]. In this study, to clarify the relationship between expression of resistin and PAI-1, PAI-1 expression in differentiated 3T3-L1 adipocytes was measured after transfection with anti-resistin siRNA. We found that PAI-1 gene expression and secreted PAI-1 protein were significantly decreased by resistin knockdown. Furthermore, phosphorylation of Akt, which can inhibit PAI-1 expression, was accelerated and the activity of protein phosphatase 2A (PP2A) was suppressed in resistin knockdown 3T3-L1 adipocytes. In addition, the expression of glucose transporter type 4, a ChREBP target gene, was reduced and was associated with inhibition of PP2A. The addition of culture medium collected from COS7 cells transfected with a resistin expression plasmid rescued the suppression of PAI-1 expression in resistin knockdown 3T3-L1 adipocytes. Our findings suggest that resistin regulates PAI-1 expression in 3T3-L1 adipocytes via Akt phosphorylation.

  3. Metformin limits the adipocyte tumor-promoting effect on ovarian cancer.

    PubMed

    Tebbe, Calvin; Chhina, Jasdeep; Dar, Sajad A; Sarigiannis, Kalli; Giri, Shailendra; Munkarah, Adnan R; Rattan, Ramandeep

    2014-07-15

    Omental adipocytes promote ovarian cancer by secretion of adipokines, cytokines and growth factors, and acting as fuel depots. We investigated if metformin modulates the ovarian cancer promoting effects of adipocytes. Effect of conditioned media obtained from differentiated mouse 3T3L1 preadipoctes on the proliferation and migration of a mouse ovarian surface epithelium cancer cell line (ID8) was estimated. Conditioned media from differentiated adipocytes increased the proliferation and migration of ID8 cells, which was attenuated by metformin. Metformin inhibited adipogenesis by inhibition of key adipogenesis regulating transcription factors (CEBPα, CEBPß, and SREBP1), and induced AMPK. A targeted Cancer Pathway Finder RT-PCR (real-time polymerase chain reaction) based gene array revealed 20 up-regulated and 2 down-regulated genes in ID8 cells exposed to adipocyte conditioned media, which were altered by metformin. Adipocyte conditioned media also induced bio-energetic changes in the ID8 cells by pushing them into a highly metabolically active state; these effects were reversed by metformin. Collectively, metformin treatment inhibited the adipocyte mediated ovarian cancer cell proliferation, migration, expression of cancer associated genes and bio-energetic changes. Suggesting, that metformin could be a therapeutic option for ovarian cancer at an early stage, as it not only targets ovarian cancer, but also modulates the environmental milieu.

  4. Direct action of capsaicin in brown adipogenesis and activation of brown adipocytes.

    PubMed

    Kida, Ryosuke; Yoshida, Hirofumi; Murakami, Masaru; Shirai, Mitsuyuki; Hashimoto, Osamu; Kawada, Teruo; Matsui, Tohru; Funaba, Masayuki

    2016-01-01

    The ingestion of capsaicin, the principle pungent component of red and chili peppers, induces thermogenesis, in part, through the activation of brown adipocytes expressing genes related to mitochondrial biogenesis and uncoupling such as peroxisome proliferator-activated receptor (Ppar) γ coactivator-1α (Pgc-1α) and uncoupling protein 1 (Ucp1). Capsaicin has been suggested to induce the activation of brown adipocytes, which is mediated by the stimulation of sympathetic nerves. However, capsaicin may directly affect the differentiation of brown preadipocytes, brown adipocyte function, or both, through its significant absorption. We herein demonstrated that Trpv1, a capsaicin receptor, is expressed in brown adipose tissue, and that its expression level is increased during the differentiation of HB2 brown preadipocytes. Furthermore, capsaicin induced calcium influx in brown preadipocytes. A treatment with capsaicin in the early stage of brown adipogenesis did not affect lipid accumulation or the expression levels of Fabp4 (a gene expressed in mature adipocytes), Pparγ2 (a master regulator of adipogenesis) or brown adipocyte-selective genes. In contrast, a treatment with capsaicin in the late stage of brown adipogenesis slightly increased the expression levels of Fabp4, Pparγ2 and Pgc-1α. Although capsaicin did not affect the basal expression level of Ucp1, Ucp1 induction by forskolin was partially inhibited by capsaicin, irrespective of the dose of capsaicin. The results of the present study suggest the direct effects of capsaicin on brown adipocytes or in the late stage of brown adipogenesis.

  5. Development of genomic SSR markers for fingerprinting lettuce (Lactuca sativa L.) cultivars and mapping genes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Lettuce (Lactuca sativa L.) is the major vegetable from the group of leafy vegetables. Several types of molecular markers were developed that are effictively used in lettuce breeding and genetic studies. However only a very limited number of microsattelite-based markers are publicly avai...

  6. Minimum information about a marker gene sequence (MIMARKS) and minimum information about any (x) sequence (MIxS) specifications.

    SciTech Connect

    Yilmaz, P.; Kottmann, R.; Field, D.; Knight, R.; Cole, J. R.; Amaral-Zettler, L.; Gilbert, J. A.

    2011-05-01

    Here we present a standard developed by the Genomic Standards Consortium (GSC) for reporting marker gene sequences - the minimum information about a marker gene sequence (MIMARKS). We also introduce a system for describing the environment from which a biological sample originates. The 'environmental packages' apply to any genome sequence of known origin and can be used in combination with MIMARKS and other GSC checklists. Finally, to establish a unified standard for describing sequence data and to provide a single point of entry for the scientific community to access and learn about GSC checklists, we present the minimum information about any (x) sequence (MIxS). Adoption of MIxS will enhance our ability to analyze natural genetic diversity documented by massive DNA sequencing efforts from myriad ecosystems in our ever-changing biosphere.

  7. Minimum information about a marker gene sequence (MIMARKS) and minimum information about any (x) sequence (MIxS) specifications.

    PubMed

    Yilmaz, Pelin; Kottmann, Renzo; Field, Dawn; Knight, Rob; Cole, James R; Amaral-Zettler, Linda; Gilbert, Jack A; Karsch-Mizrachi, Ilene; Johnston, Anjanette; Cochrane, Guy; Vaughan, Robert; Hunter, Christopher; Park, Joonhong; Morrison, Norman; Rocca-Serra, Philippe; Sterk, Peter; Arumugam, Manimozhiyan; Bailey, Mark; Baumgartner, Laura; Birren, Bruce W; Blaser, Martin J; Bonazzi, Vivien; Booth, Tim; Bork, Peer; Bushman, Frederic D; Buttigieg, Pier Luigi; Chain, Patrick S G; Charlson, Emily; Costello, Elizabeth K; Huot-Creasy, Heather; Dawyndt, Peter; DeSantis, Todd; Fierer, Noah; Fuhrman, Jed A; Gallery, Rachel E; Gevers, Dirk; Gibbs, Richard A; San Gil, Inigo; Gonzalez, Antonio; Gordon, Jeffrey I; Guralnick, Robert; Hankeln, Wolfgang; Highlander, Sarah; Hugenholtz, Philip; Jansson, Janet; Kau, Andrew L; Kelley, Scott T; Kennedy, Jerry; Knights, Dan; Koren, Omry; Kuczynski, Justin; Kyrpides, Nikos; Larsen, Robert; Lauber, Christian L; Legg, Teresa; Ley, Ruth E; Lozupone, Catherine A; Ludwig, Wolfgang; Lyons, Donna; Maguire, Eamonn; Methé, Barbara A; Meyer, Folker; Muegge, Brian; Nakielny, Sara; Nelson, Karen E; Nemergut, Diana; Neufeld, Josh D; Newbold, Lindsay K; Oliver, Anna E; Pace, Norman R; Palanisamy, Giriprakash; Peplies, Jörg; Petrosino, Joseph; Proctor, Lita; Pruesse, Elmar; Quast, Christian; Raes, Jeroen; Ratnasingham, Sujeevan; Ravel, Jacques; Relman, David A; Assunta-Sansone, Susanna; Schloss, Patrick D; Schriml, Lynn; Sinha, Rohini; Smith, Michelle I; Sodergren, Erica; Spo, Aymé; Stombaugh, Jesse; Tiedje, James M; Ward, Doyle V; Weinstock, George M; Wendel, Doug; White, Owen; Whiteley, Andrew; Wilke, Andreas; Wortman, Jennifer R; Yatsunenko, Tanya; Glöckner, Frank Oliver

    2011-05-01

    Here we present a standard developed by the Genomic Standards Consortium (GSC) for reporting marker gene sequences--the minimum information about a marker gene sequence (MIMARKS). We also introduce a system for describing the environment from which a biological sample originates. The 'environmental packages' apply to any genome sequence of known origin and can be used in combination with MIMARKS and other GSC checklists. Finally, to establish a unified standard for describing sequence data and to provide a single point of entry for the scientific community to access and learn about GSC checklists, we present the minimum information about any (x) sequence (MIxS). Adoption of MIxS will enhance our ability to analyze natural genetic diversity documented by massive DNA sequencing efforts from myriad ecosystems in our ever-changing biosphere.

  8. Minimum information about a marker gene sequence (MIMARKS) and minimum information about any (x) sequence (MIxS) specifications

    PubMed Central

    Yilmaz, Pelin; Kottmann, Renzo; Field, Dawn; Knight, Rob; Cole, James R; Amaral-Zettler, Linda; Gilbert, Jack A; Karsch-Mizrachi, Ilene; Johnston, Anjanette; Cochrane, Guy; Vaughan, Robert; Hunter, Christopher; Park, Joonhong; Morrison, Norman; Rocca-Serra, Philippe; Sterk, Peter; Arumugam, Manimozhiyan; Bailey, Mark; Baumgartner, Laura; Birren, Bruce W; Blaser, Martin J; Bonazzi, Vivien; Booth, Tim; Bork, Peer; Bushman, Frederic D; Buttigieg, Pier Luigi; Chain, Patrick S G; Charlson, Emily; Costello, Elizabeth K; Huot-Creasy, Heather; Dawyndt, Peter; DeSantis, Todd; Fierer, Noah; Fuhrman, Jed A; Gallery, Rachel E; Gevers, Dirk; Gibbs, Richard A; Gil, Inigo San; Gonzalez, Antonio; Gordon, Jeffrey I; Guralnick, Robert; Hankeln, Wolfgang; Highlander, Sarah; Hugenholtz, Philip; Jansson, Janet; Kau, Andrew L; Kelley, Scott T; Kennedy, Jerry; Knights, Dan; Koren, Omry; Kuczynski, Justin; Kyrpides, Nikos; Larsen, Robert; Lauber, Christian L; Legg, Teresa; Ley, Ruth E; Lozupone, Catherine A; Ludwig, Wolfgang; Lyons, Donna; Maguire, Eamonn; Methé, Barbara A; Meyer, Folker; Muegge, Brian; Nakielny, Sara; Nelson, Karen E; Nemergut, Diana; Neufeld, Josh D; Newbold, Lindsay K; Oliver, Anna E; Pace, Norman R; Palanisamy, Giriprakash; Peplies, Jörg; Petrosino, Joseph; Proctor, Lita; Pruesse, Elmar; Quast, Christian; Raes, Jeroen; Ratnasingham, Sujeevan; Ravel, Jacques; Relman, David A; Assunta-Sansone, Susanna; Schloss, Patrick D; Schriml, Lynn; Sinha, Rohini; Smith, Michelle I; Sodergren, Erica; Spor, Aymé; Stombaugh, Jesse; Tiedje, James M; Ward, Doyle V; Weinstock, George M; Wendel, Doug; White, Owen; Whiteley, Andrew; Wilke, Andreas; Wortman, Jennifer R; Yatsunenko, Tanya; Glöckner, Frank Oliver

    2012-01-01

    Here we present a standard developed by the Genomic Standards Consortium (GSC) for reporting marker gene sequences—the minimum information about a marker gene sequence (MIMARKS). We also introduce a system for describing the environment from which a biological sample originates. The ‘environmental packages’ apply to any genome sequence of known origin and can be used in combination with MIMARKS and other GSC checklists. Finally, to establish a unified standard for describing sequence data and to provide a single point of entry for the scientific community to access and learn about GSC checklists, we present the minimum information about any (x) sequence (MIxS). Adoption of MIxS will enhance our ability to analyze natural genetic diversity documented by massive DNA sequencing efforts from myriad ecosystems in our ever-changing biosphere. PMID:21552244

  9. Heteroduplex analysis can increase the informativeness of PCR-amplified VNTR markers: Application using a marker tightly linked to the COL2A1 gene

    SciTech Connect

    Wilkin, D.J.; Cohn, D.H. UCLA School of Medicine, Los Angeles, CA ); Koprivnikar, K.E. )

    1993-02-01

    Variable number of tandem repeat (VNTR) polymorphism provide a high degree of informativeness in linkage studies. Whether performed by standard methods or by polymerase chain reaction (PCR), analysis of these markers involves assessment of the length of each allele. VNTR alleles usually differ in the number of tandem repeats. During PCR amplification of a VNTR closely linked to the type II collagen gene (COL2A1), we identified allelic microheterogeneity through the analysis of unique heteroduplexes between amplified strands of the two alleles. In one large pedigree, heteroduplex analysis identified only three distinct alleles. The identification of these heteroduplexes allowed the determination of the COL2A1 inheritance pattern in the family, which otherwise would have been noninformative. 26 refs., 3 figs.

  10. Consequence of Menin Deficiency in Mouse Adipocytes Derived by In Vitro Differentiation

    PubMed Central

    Parekh, Vaishali I.; Modali, Sita D.; Desai, Shruti S.; Agarwal, Sunita K.

    2015-01-01

    Lipoma in patients with the multiple endocrine neoplasia type 1 (MEN1) syndrome is a type of benign fat-cell tumor that has biallelic inactivation of MEN1 that encodes menin and could serve as a model to investigate normal and pathologic fat-cell (adipocyte) proliferation and function. The role of menin and its target genes in adipocytes is not known. We used in vitro differentiation to derive matched normal and menin-deficient adipocytes from wild type (WT) and menin-null (Men1-KO) mouse embryonic stem cells (mESCs), respectively, or 3T3-L1 cells without or with menin knockdown to investigate cell size, lipid content, and gene expression changes. Adipocytes derived from Men1-KO mESCs or after menin knockdown in 3T3-L1 cells showed a 1.5–1.7-fold increase in fat-cell size. Global gene expression analysis of mESC-derived adipocytes showed that lack of menin downregulated the expression of many differentially methylated genes including the tumor suppressor long noncoding RNA Meg3 but upregulated gene expression from the prolactin gene family locus. Our results show that menin deficiency leads to fat-cell hypertrophy and provide model systems that could be used to study the regulation of fat-cell size. PMID:26229531

  11. A conservative region of the mercuric reductase gene (mera) as a molecular marker of bacterial mercury resistance

    PubMed Central

    Sotero-Martins, Adriana; de Jesus, Michele Silva; Lacerda, Michele; Moreira, Josino Costa; Filgueiras, Ana Luzia Lauria; Barrocas, Paulo Rubens Guimarães

    2008-01-01

    The most common bacterial mercury resistance mechanism is based on the reduction of Hg(II) to Hg0, which is dependent of the mercuric reductase enzyme (MerA) activity. The use of a 431 bp fragment of a conservative region of the mercuric reductase (merA) gene was applied as a molecular marker of this mechanism, allowing the identification of mercury resistant bacterial strains. PMID:24031221

  12. De Novo Assembly, Gene Annotation and Marker Development Using Illumina Paired-End Transcriptome Sequences in Celery (Apium graveolens L.)

    PubMed Central

    Fu, Nan; Wang, Qian; Shen, Huo-Lin

    2013-01-01

    Background Celery is an increasing popular vegetable species, but limited transcriptome and genomic data hinder the research to it. In addition, a lack of celery molecular markers limits the process of molecular genetic breeding. High-throughput transcriptome sequencing is an efficient method to generate a large transcriptome sequence dataset for gene discovery, molecular marker development and marker-assisted selection breeding. Principal Findings Celery transcriptomes from four tissues were sequenced using Illumina paired-end sequencing technology. De novo assembling was performed to generate a collection of 42,280 unigenes (average length of 502.6 bp) that represent the first transcriptome of the species. 78.43% and 48.93% of the unigenes had significant similarity with proteins in the National Center for Biotechnology Information (NCBI) non-redundant protein database (Nr) and Swiss-Prot database respectively, and 10,473 (24.77%) unigenes were assigned to Clusters of Orthologous Groups (COG). 21,126 (49.97%) unigenes harboring Interpro domains were annotated, in which 15,409 (36.45%) were assigned to Gene Ontology(GO) categories. Additionally, 7,478 unigenes were mapped onto 228 pathways using the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG). Large numbers of simple sequence repeats (SSRs) were indentified, and then the rate of successful amplication and polymorphism were investigated among 31 celery accessions. Conclusions This study demonstrates the feasibility of generating a large scale of sequence information by Illumina paired-end sequencing and efficient assembling. Our results provide a valuable resource for celery research. The developed molecular markers are the foundation of further genetic linkage analysis and gene localization, and they will be essential to accelerate the process of breeding. PMID:23469050

  13. Integration of gene-based markers in a pearl millet genetic map for identification of candidate genes underlying drought tolerance quantitative trait loci

    PubMed Central

    2012-01-01

    Background Identification of genes underlying drought tolerance (DT) quantitative trait loci (QTLs) will facilitate understanding of molecular mechanisms of drought tolerance, and also will accelerate genetic improvement of pearl millet through marker-assisted selection. We report a map based on genes with assigned functional roles in plant adaptation to drought and other abiotic stresses and demonstrate its use in identifying candidate genes underlying a major DT-QTL. Results Seventy five single nucleotide polymorphism (SNP) and conserved intron spanning primer (CISP) markers were developed from available expressed sequence tags (ESTs) using four genotypes, H 77/833-2, PRLT 2/89-33, ICMR 01029 and ICMR 01004, representing parents of two mapping populations. A total of 228 SNPs were obtained from 30.5 kb sequenced region resulting in a SNP frequency of 1/134 bp. The positions of major pearl millet linkage group (LG) 2 DT-QTLs (reported from crosses H 77/833-2 × PRLT 2/89-33 and 841B × 863B) were added to the present consensus function map which identified 18 genes, coding for PSI reaction center subunit III, PHYC, actin, alanine glyoxylate aminotransferase, uridylate kinase, acyl-CoA oxidase, dipeptidyl peptidase IV, MADS-box, serine/threonine protein kinase, ubiquitin conjugating enzyme, zinc finger C- × 8-C × 5-C × 3-H type, Hd3, acetyl CoA carboxylase, chlorophyll a/b binding protein, photolyase, protein phosphatase1 regulatory subunit SDS22 and two hypothetical proteins, co-mapping in this DT-QTL interval. Many of these candidate genes were found to have significant association with QTLs of grain yield, flowering time and leaf rolling under drought stress conditions. Conclusions We have exploited available pearl millet EST sequences to generate a mapped resource of seventy five new gene-based markers for pearl millet and demonstrated its use in identifying candidate genes underlying a major DT-QTL in this species. The reported gene-based markers represent

  14. Decaffeinated Green Tea and Voluntary Exercise Induce Gene Changes Related to Beige Adipocyte Formation in High Fat-Fed Obese Mice*

    PubMed Central

    Sae-tan, Sudathip; Rogers, Connie J.; Lambert, Joshua D.

    2015-01-01

    We have previously reported that decaffeinated green tea extract (GTE) in combination with voluntary exercise (Ex) reduces metabolic syndrome in high fat-fed C57BL/6J mice. Here, we examined for the first time the effect of treatment with 77 mg/g GTE, Ex, or both (GTE + Ex) on genes related to the conversion of white adipose tissue (WAT) to brown fat-like adipose tissue (BLAT) in this model. GTE+Ex induced genes related to lipolysis (hormone sensitive lipase [3.0-fold] and patatin-like phospholipase domain-containing protein 2 [2-fold]), mitochondrial β-oxidation (NADH dehydrogenase 5 [2.3-fold], cytochrome B [2.0-fold], and cytochrome C oxidase III [1.9-fold increase]), and adipose tissue browning (peroxisome proliferator-activated receptor-γ coactivator-1α [1.8-fold], bone morphogenetic protein 4 [2.6-fold], and phosphatase and tensin homolog [2.6-fold]) in visceral WAT compared to HF-fed mice. These results suggest that GTE+Ex function in part by inducing the conversion of WAT to BLAT and provides novel mechanistic insight into this combination. PMID:25844091

  15. HER-2 gene amplification, serum nucleosomes, CEA and CA15.3 tumor markers in breast cancer patients.

    PubMed

    Zeiwar, M M; Zaki, Seham M; Mohammad, Lamiaa A; Zidan, Amal A; El Nagar, Mona Roshdy

    2007-01-01

    Breast cancer is the most frequently diagnosed cancer in women in the world, for which tumor markers are needed for early detection, clinical prognistication and monitoring. The study was designed to assess the usefulness of HER-2 gene amplification, serum nucleosomes, CEA and CA15.3 tumor markers in the diagnosis of invasive ductal carcinoma and analyze whether their levels correlate with the clinicopathological features. The study was carried out on fifty patients with invasive ductal carcinoma and 25 age matched women with benign breast diseases (BBD). Cancer patients were categorised into three subgroups according to absence (-) or presence (+) of axillary lymph nodes (N) or presence of distant metastasis (M+) into: subgroup I (N-) included 15 patients, subgroup II (N+) included 20 patients and subgroup III (M+) included 15 patients. All individuals were subjected to CBC, fasting blood sugar, liver & kidney function tests, CEA and CA15.3 by electrochemiluminescence, serum nucleosomes by cell death detection ELISA and amplification of HER-2 gene by differential PCR. The HER-2 gene PCR results were + ve in 28% of cancer patients; 20% of subgroup I, 25% of subgroup II and 40% of subgroup III, but in none of the BBD patients. HER-2 gene amplification results showed significant positive correlation with tumor grade. Serum nucleosomes showed significant increase in cancer patients as compared to that of BBD group, significant negative correlation with HER-2 gene amplification and significant positive correlation with CA15.3. Serum nucleosomes was the most sensitive marker (76% versus 32% and 50% for CEA & CA15.3 respectively) but the least specific (72% versus 92% and 96% for CEA & CA15.3 respectively). Elevated CEA and CA15.3 levels were detected in 13.3% and 33.3% respectively in node negative patients, these percentage increased in node positive patients to 20% and 40% and in metastatic patients to 66.7% and 80% respectively. In conclusion, serum nucleosomes is

  16. [Genetic structure of the Iranian-speaking population of Azerbaijan from data on frequencies of immunologic and biochemical gene markers].

    PubMed

    Asadova, P Sh; Shneĭder, Iu V; Shil'nikova, I N; Zhukova, O V

    2003-11-01

    The data on the genetic studies of Iranian-speaking populations from Azerbaijan (Talyshs and Tats) are presented. In these populations gene frequency distributions for the immunological (AB0, MN, Rhesus-D, -C, -E, P, Lewis, and Kell-Chellano) and biochemical (HP, GC, C'3, TF, 6PGD, GLO1, ESD, ACP1, and PGM1) gene markers were determined. Comparison of the genetic structure of the populations examined with the other Iranian-speaking populations (Persians and Kurds from Iran, Ossetins and Tajiks) and Azerbaijanis showed that Iranian-speaking populations from Azerbaijan were more close to Azerbaijanis, than to Iranian-speaking populations inhabiting other world regions. PMID:14714471

  17. A new species of Docosia Winnertz from Central Europe, with DNA barcoding based on four gene markers (Diptera, Mycetophilidae)

    PubMed Central

    Ševčík, Jan; Kaspřák, David; Rulik, Björn

    2016-01-01

    Abstract A new species of Docosia Winnertz, Docosia dentata sp. n., is described and illustrated, based on a single male specimen collected in Muránska planina National Park in Central Slovakia. DNA sequences (COI, COII, CytB, and ITS2) are included and compared for 13 species of Docosia. There was found only little congruence between the molecular results and previous scarce data about interspecific relationships based on morphology. The COI and CytB gene markers showed the highest interspecific gene distances while ITS2 showed the lowest ones. An updated key to the 23 Central European species of Docosia is also presented. PMID:26843833

  18. Molecular characterization and expression pattern of a germ cell marker gene dnd in gibel carp (Carassius gibelio).

    PubMed

    Li, Shi-Zhu; Liu, Wei; Li, Zhi; Wang, Yang; Zhou, Li; Yi, Mei-Sheng; Gui, Jian-Fang

    2016-10-10

    As a germ cell marker gene, Dead end (dnd) has been identified and characterized in many vertebrates. Recently, we created a complete germ cell-depleted gonad model by the dnd-specific morpholino-mediated knockdown approach, and revealed sex-biased gene expression alteration through utilizing unisexual gynogenetic superiority in polyploid gibel carp. However, dnd and its expression pattern are still unclear in the gibel carp. In this study, we further analyzed molecular characterization of gibel carp dnd and its dynamic expression pattern during gametogenesis and embryogenesis. Similar to other homologs in vertebrates, gibel carp dnd contains a conserved RRM motif and five other motifs, and is highly evolutionary conserved in genomic organization and neighborhood gene synteny. RT-PCR and Western blot analyses showed its gonad-specific expression intensively in testis and ovary. Section in situ hybridization (SISH) and immunofluorescence localization revealed its dynamic expression pattern specific to oogenic cells and spermatogenetic cells during oogenesis and spermatogenesis. Moreover, its temporal and spatial distribution specific to PGCs were also demonstrated by RT-PCR and whole mount in situ hybridization (WISH) during embryogenesis. Therefore, gibel carp Dnd is a conserved germ cell marker during gametogenesis, and its maternal transcript is also a useful marker for tracing PGC specification and migration. PMID:27418526

  19. Identification of resistance to new virulent races of rust in sunflowers and validation of DNA markers in the gene pool.

    PubMed

    Qi, Lili; Gulya, Tom; Seiler, Gerald J; Hulke, Brent S; Vick, Brady A

    2011-02-01

    Sunflower rust, caused by Puccinia helianthi, is a prevalent disease in many countries throughout the world. The U.S. Department of Agriculture (USDA)-Agricultural Research Service, Sunflower Research Unit has released rust resistant breeding materials for several decades. However, constantly coevolving rust populations have formed new virulent races to which current hybrids have little resistance. The objectives of this study were to identify resistance to race 336, the predominant race in North America, and to race 777, the most virulent race currently known, and to validate molecular markers known to be linked to rust resistance genes in the sunflower gene pool. A total of 104 entries, including 66 released USDA inbred lines, 14 USDA interspecific germplasm lines, and 24 foreign germplasms, all developed specifically for rust resistance, were tested for their reaction to races 336 and 777. Only 13 of the 104 entries tested were resistant to both races, whereas another six were resistant only to race 336. The interspecific germplasm line, Rf ANN-1742, was resistant to both races and was identified as a new rust resistance source. A selection of 24 lines including 19 lines resistant to races 777 and/or 336 was screened with DNA markers linked to rust resistance genes R(1), R(2), R(4u), and R(5). The results indicated that the existing resistant lines are diverse in rust resistance genes. Durable genetic resistance through gene pyramiding will be effective for the control of rust.

  20. Heat shock induced excision of selectable marker genes in transgenic banana by the Cre-lox site-specific recombination system.

    PubMed

    Chong-Pérez, Borys; Kosky, Rafael G; Reyes, Maritza; Rojas, Luis; Ocaña, Bárbara; Tejeda, Marisol; Pérez, Blanca; Angenon, Geert

    2012-06-30

    Selectable marker genes are indispensable for efficient production of transgenic events, but are no longer needed after the selection process and may cause public concern and technological problems. Although several gene excision systems exist, few have been optimized for vegetatively propagated crops. Using a Cre-loxP auto-excision strategy, we obtained transgenic banana plants cv. Grande Naine (Musa AAA) devoid of the marker gene used for selection. We used T-DNA vectors with the cre recombinase gene under control of a heat shock promoter and selectable marker gene cassettes placed between two loxP sites in direct orientation, and a gene of interest inserted outside of the loxP sites. Heat shock promoters pGmHSP17.6-L and pHSP18.2, from soybean and Arabidopsis respectively, were tested. A transient heat shock treatment of primary transgenic embryos was sufficient for inducing cre and excising cre and the marker genes. Excision efficiency, as determined by PCR and Southern hybridization was 59.7 and 40.0% for the GmHSP17.6-L and HSP18.2 promoters, respectively. Spontaneous excision was not observed in 50 plants derived from untreated transgenic embryos. To our knowledge this is the first report describing an efficient marker gene removal system for banana. The method described is simple and might be generally applicable for the production of marker-free transgenic plants of many crop species.

  1. Anti-Inflammatory Effect of Spirulina platensis in Macrophages Is Beneficial for Adipocyte Differentiation and Maturation by Inhibiting Nuclear Factor-κB Pathway in 3T3-L1 Adipocytes.

    PubMed

    Pham, Tho X; Lee, Ji-Young

    2016-06-01

    We previously showed that the organic extract of a blue-green alga, Spirulina platensis (SPE), had potent anti-inflammatory effects in macrophages. As the interplay between macrophages and adipocytes is critical for adipocyte functions, we investigated the contribution of the anti-inflammatory effects of SPE in macrophages to adipogenesis/lipogenesis in 3T3-L1 adipocytes. 3T3-L1 preadipocytes were treated with 10% conditioned medium from lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages (CMC) or LPS-stimulated, but SPE-pretreated, macrophages (CMS) at different stages of adipocyte differentiation. The expression of adipocyte differentiation markers, such as CCAAT/enhancer-binding protein α, peroxisome proliferator-activated receptor γ, and perilipin, was significantly repressed by CMC when added on day 3, while the repression was attenuated by CMS. Oil Red O staining confirmed that adipocyte maturation in CMS-treated cells, but not in CMC-treated cells, was equivalent to that of control cells. Nuclear translocation of nuclear factor κB (NF-κB) p65 was decreased by CMS compared to CMC. In lipid-laden adipocytes, CMC promoted the loss of lipid droplets, while CMS had minimal effects. Histone deacetylase 9 mRNA and protein levels were increased during adipocyte maturation, which were decreased by CMC. In conclusion, by cross-talking with adipocytes, the anti-inflammatory effects of SPE in macrophages promoted adipocyte differentiation/maturation, at least in part, by repressing the activation of NF-κB inflammatory pathways, which otherwise can be compromised in inflammatory conditions. PMID:27206252

  2. CNMS: The preferred genic markers for comparative genomic, molecular phylogenetic, functional genetic diversity and differential gene regulatory expression analyses in chickpea.

    PubMed

    Bajaj, Deepak; Das, Shouvik; Parida, Swarup K

    2015-09-01

    The intra/inter-genomic comparative mapping-based phylogenetic footprinting identified 5 paralogous and 656 orthologous genome-wide CNMS markers in the upstream sequences of chickpea genes. These CNMS markers revealed a high-degree of gene-based syntenic relationship between chickpea and Medicago genomes while minimum between chickpea and Vitis genomes. The time of divergence and duplication estimated using CNMS markers highlight the expected phylogenetic relationships between chickpea and six dicot (legume) species as well as occurrence of ancient genome (approximately 53 Mya) with small-scale recent segmental (approximately 10 Mya) duplication events in chickpea. A wider level of functional molecular diversity (14 to 88 percent) and admixed population genetic structure was detected among desi, kabuli and wild genotypes by genic CNMS markers at a genome-wide scale suggesting their utility in large-scale genetic analysis in chickpea. The subfunctionalization at the cis-regulatory element region and TFBS (transcription factor binding site) motif levels in the upstream sequences of CNMS marker-associated orthologous genes than the paralogues was predominant. Functional constraint might have considerable effect on these CNMScontaining regulatory elements controlling consistent orthologous gene expression in dicots. A rapid subfunctionalization based on diverge differential expression of paralogous CNMS marker-associated genes particularly those that underwent recent small-scale segmental duplication events in chickpea was apparent. The differential regulation of expression and subfunctionalization potential of Ultra CNMS marker-associated genes suggest their utility in deciphering the complex gene regulatory function as well as identification and targeted mapping of potential genes/QTLs governing vital agronomic traits in chickpea. The gene-based CNMS markers with desirable inherent genetic attributes like higher degree of comparative genome mapping, functional

  3. Uric Acid-Induced Adipocyte Dysfunction Is Attenuated by HO-1 Upregulation: Potential Role of Antioxidant Therapy to Target Obesity

    PubMed Central

    Sodhi, Komal; Hilgefort, Jordan; Banks, George; Gilliam, Chelsea; Stevens, Sarah; Ansinelli, Hayden A.; Getty, Morghan; Abraham, Nader G.; Shapiro, Joseph I.

    2016-01-01

    Increased uric acid levels have been implicated in the pathogenesis of metabolic syndrome. To examine the mechanisms by which this occurs, we hypothesized that an increase in heme oxygenase 1, a potent antioxidant gene, will decrease uric acid levels and adipocyte dysfunction via suppression of ROS and xanthine oxidase (XO) levels. We examined the effect of uric acid on adipogenesis in human mesenchymal stem cells (MSCs) in the presence and absence of cobalt protoporphyrin (CoPP), an HO-1 inducer, and tin mesoporphyrin (SnMP), an HO activity inhibitor. Uric acid increased adipogenesis by increasing NADPH oxidase expression and elevation in the adipogenesis markers C/EBPα, PPARγ, and Mest, while decreasing small lipid droplets and Wnt10b levels. We treated MSCs with fructose, a fuel source that increases uric acid levels. Our results showed that fructose increased XO expression as compared to the control and concomitant treatment with CoPP significantly decreased XO expression and uric acid levels. These beneficial effects of CoPP were reversed by SnMP, supporting a role for HO activity in mediating these effects. These findings demonstrate that increased levels of HO-1 appear crucial in modulating the phenotype of adipocytes exposed to uric acid and in downregulating XO and NADPH oxidase levels. PMID:26681956

  4. The use of small interfering RNAs to inhibit adipocyte differentiation in human preadipocytes and fetal-femur-derived mesenchymal cells

    SciTech Connect

    Xu, Y.; Mirmalek-Sani, S.-H.; Yang, X.; Zhang, J.; Oreffo, R.O.C. . E-mail: roco@soton.ac.uk

    2006-06-10

    RNA interference (RNAi) has been used in functional genomics and offers innovative approaches in the development of novel therapeutics. Human mesenchymal stem cells offer a unique cell source for tissue engineering/regeneration strategies. The current study examined the potential of small interfering RNAs (siRNA) against human peroxisome proliferator activated receptor gamma (PPAR{gamma}) to suppress adipocyte differentiation (adipogenesis) in human preadipocytes and fetal-femur-derived mesenchymal cells. Adipogenesis was investigated using cellular and biochemical analysis. Transient transfection with PPAR{gamma}-siRNA using a liposomal-based strategy resulted in a significant inhibition of adipogenesis in human preadipocytes and fetal-femur-derived mesenchymal cells, compared to controls (cell, liposomal and negative siRNA). The inhibitory effect of PPAR{gamma}-siRNA was supported by testing human PPAR{gamma} mRNA and adipogenic associated genes using reverse transcription polymerase chain reaction (RT-PCR) to adiponectin receptor 1 and 2 as well as examination of fatty acid binding protein 3 (FABP{sub 3}) expression, an adipocyte-specific marker. The current studies indicate that PPAR{gamma}-siRNA is a useful tool to study adipogenesis in human cells, with potential applications both therapeutic and in the elucidation of mesenchymal cell differentiation in the modulation of cell differentiation in human mesenchymal cells.

  5. microRNA-320/RUNX2 axis regulates adipocytic differentiation of human mesenchymal (skeletal) stem cells

    PubMed Central

    Hamam, D; Ali, D; Vishnubalaji, R; Hamam, R; Al-Nbaheen, M; Chen, L; Kassem, M; Aldahmash, A; Alajez, N M

    2014-01-01

    The molecular mechanisms promoting lineage-specific commitment of human mesenchymal (skeletal or stromal) stem cells (hMSCs) into adipocytes (ADs) are not fully understood. Thus, we performed global microRNA (miRNA) and gene expression profiling during adipocytic differentiation of hMSC, and utilized bioinformatics as well as functional and biochemical assays, and identified several novel miRNAs differentially expressed during adipogenesis. Among these, miR-320 family (miR-320a, 320b, 320c, 320d and 320e) were ~2.2–3.0-fold upregulated. Overexpression of miR-320c in hMSC enhanced adipocytic differentiation and accelerated formation of mature ADs in ex vivo cultures. Integrated analysis of bioinformatics and global gene expression profiling in miR-320c overexpressing cells and during adipocytic differentiation of hMSC identified several biologically relevant gene targets for miR-320c including RUNX2, MIB1 (mindbomb E3 ubiquitin protein ligase 1), PAX6 (paired box 6), YWHAH and ZWILCH. siRNA-mediated silencing of those genes enhanced adipocytic differentiation of hMSC, thus corroborating an important role for those genes in miR-320c-mediated adipogenesis. Concordant with that, lentiviral-mediated stable expression of miR-320c at physiological levels (~1.5-fold) promoted adipocytic and suppressed osteogenic differentiation of hMSC. Luciferase assay validated RUNX2 (Runt-related transcription factor 2) as a bona fide target for miR-320 family. Therefore, our data suggest miR-320 family as possible molecular switch promoting adipocytic differentiation of hMSC. Targeting miR-320 may have therapeutic potential in vivo through regulation of bone marrow adipogenesis. PMID:25356868

  6. Biosafety and risk assessment framework for selectable marker genes in transgenic crop plants: a case of the science not supporting the politics.

    PubMed

    Ramessar, Koreen; Peremarti, Ariadna; Gómez-Galera, Sonia; Naqvi, Shaista; Moralejo, Marian; Muñoz, Pilar; Capell, Teresa; Christou, Paul

    2007-06-01

    Selectable marker gene systems are vital for the development of transgenic crops. Since the creation of the first transgenic plants in the early 1980s and their subsequent commercialization worldwide over almost an entire decade, antibiotic and herbicide resistance selectable marker gene systems have been an integral feature of plant genetic modification. Without them, creating transgenic crops is not feasible on purely economic and practical terms. These systems allow the relatively straightforward identification and selection of plants that have stably incorporated not only the marker genes but also genes of interest, for example herbicide tolerance and pest resistance. Bacterial antibiotic resistance genes are also crucial in molecular biology manipulations in the laboratory. An unprecedented debate has accompanied the development and commercialization of transgenic crops. Divergent policies and their implementation in the European Union on one hand and the rest of the world on the other (industrialized and developing countries alike), have resulted in disputes with serious consequences on agricultural policy, world trade and food security. A lot of research effort has been directed towards the development of marker-free transformation or systems to remove selectable markers. Such research has been in a large part motivated by perceived problems with antibiotic resistance selectable markers; however, it is not justified from a safety point of view. The aim of this review is to discuss in some detail the currently available scientific evidence that overwhelmingly argues for the safety of these marker gene systems. Our conclusion, supported by numerous studies, most of which are commissioned by some of the very parties that have taken a position against the use of antibiotic selectable marker gene systems, is that there is no scientific basis to argue against the use and presence of selectable marker genes as a class in transgenic plants.

  7. Establishment of lipofection for studying miRNA function in human adipocytes.

    PubMed

    Enlund, Eveliina; Fischer, Simon; Handrick, René; Otte, Kerstin; Debatin, Klaus-Michael; Wabitsch, Martin; Fischer-Posovszky, Pamela

    2014-01-01

    miRNA dysregulation has recently been linked to human obesity and its related complications such as type 2 diabetes. In order to study miRNA function in human adipocytes, we aimed for the modulation of mature miRNA concentration in these cells. Adipocytes, however, tend to be resistant to transfection and there is often a need to resort to viral transduction or electroporation. Our objective therefore was to identify an efficient, non-viral transfection reagent capable of delivering small RNAs into these cells. To achieve this, we compared the efficiencies of three transfection agents, Lipofectamine 2000, ScreenFect A and BPEI 1.2 k in delivering fluorescent-labelled siRNA into human Simpson-Golabi-Behmel syndrome (SGBS) preadipocytes and adipocytes. Downregulation of a specific target gene in response to miRNA mimic overexpression was assayed in SGBS cells and also in ex vivo differentiated primary human adipocytes. Our results demonstrated that while all three transfection agents were able to internalize the oligos, only lipofection resulted in the efficient downregulation of a specific target gene both in SGBS cells and in primary human adipocytes. Lipofectamine 2000 outperformed ScreenFect A in preadipocytes, but in adipocytes the two reagents gave comparable results making ScreenFect A a notable new alternative for the gold standard Lipofectamine 2000. PMID:24849298

  8. Gene Mapping of a Mutant Mungbean (Vigna radiata L.) Using New Molecular Markers Suggests a Gene Encoding a YUC4-like Protein Regulates the Chasmogamous Flower Trait

    PubMed Central

    Chen, Jingbin; Somta, Prakit; Chen, Xin; Cui, Xiaoyan; Yuan, Xingxing; Srinives, Peerasak

    2016-01-01

    Mungbean (Vigna radiata L.) is a cleistogamous plant in which flowers are pollinated before they open, which prevents yield improvements through heterosis. We previously generated a chasmogamous mutant (CM) mungbean in which open flowers are pollinated. In this study, we developed insertion/deletion (indel) markers based on the transcriptome differences between CM and Sulu-1 (i.e., normal flowering) plants. An F2 population derived from a cross between CM and Sulu-1 was used for gene mapping. Segregation analyses revealed that a single recessive gene regulates the production of chasmogamous flowers. Using newly developed indel and simple sequence repeat markers, the cha gene responsible for the chasmogamous flower trait was mapped to a 277.1-kb segment on chromosome 6. Twelve candidate genes were detected in this segment, including Vradi06g12650, which encodes a YUCCA family protein associated with floral development. A single base pair deletion producing a frame-shift mutation and a premature stop codon in Vradi06g12650 was detected only in CM plants. This suggested that Vradi06g12650 is a cha candidate gene. Our results provide important information for the molecular breeding of chasmogamous mungbean lines, which may serve as new genetic resources for hybrid cultivar development. PMID:27375671

  9. Linkage disequilibrium between the juvenile neuronal ceroid lipofuscinosis gene and marker loci on chromosome 16p12. 1

    SciTech Connect

    Lerner, T.J.; MacCormack, K.; Gleitsman, J.; Schlumpf, K.; Breakefield, X.O.; Gusella, J.F.; Haines, J.L. )

    1994-01-01

    The neuronal ceroid lipofuscinoses (NCL; Batten disease) are a collection of autosomal recessive disorders characterized by the accumulation of autofluorescent lipopigments in the neurons and other cell types. Clinically, these disorders are characterized by progressive encephalopathy, loss of vision, and seizures. CLN3, the gene responsible for juvenile NCL, has been mapped to a 15-cM region flanked by the marker loci D16S148 and D16S150 on human chromosome 16. CLN2, the gene causing the late-infantile form of NCL (LNCL), is not yet mapped. The authors have used highly informative dinucleoide repeat markers mapping between D16S148 and D16S150 to refine the localization of CLN3 and to test for linkage to CLN2. The authors find significant linkage disequilibrium between CLN3 and the dinucleotide repeat marker loci D16S288 (X[sup 2](7) = 46.5, P < .005), D16S298 (X[sup 2](6) = 36.6, P < .005), and D16S299 (X[sup 2](7) = 73.8, P < .005), and also a novel RFLP marker at the D16S272 locus (X[sup 2](1) = 5.7, P = .02). These markers all map to 16p12.1. The D16S298/D16S299 haplotype [open quotes]5/4[close quotes] is highly overrepresented, accounting for 54% of CLN3 chromosomes as compared with 8% of control chromosomes (X[sup 2] = 117, df = 1, P < .001). Examination of the haplotypes suggests that the CLN3 locus can be narrowed to the region immediately surrounding these markers in 16p12.1. Analysis of D16S299 in LNCL pedigrees supports the previous finding that CLN3 and CLN2 are different genetic loci. This study also indicates that dinucleotide repeat markers play a valuable role in disequilibrium studies. 23 refs., 1 fig., 4 tabs.

  10. Identification of Single Nucleotide Polymorphism Marker and Association Analysis of Marbling Score in Fas Gene of Hanwoo.

    PubMed

    Kim, Seung-Chang; Lee, Seung-Hwan; Lee, Ji-Woong; Kim, Tae-Hun; Choi, Bong-Hwan

    2016-01-01

    The Fas (APO-1, TNFRSF6) gene known as a member of the tumor necrosis factor receptor superfamily was selected for DNA marker development in Korean cattle. It is a cell membrane protein and mediates programmed cell death (apoptosis). We discovered single nucleotide polymorphisms (SNPs) within Fas gene in order to develop novel DNA markers related to economical traits at the genomic level. The sequences of whole exon and 1 kb range of both front and back of the gene were determined by direct-sequencing methods using 24 cattle. A total of 55 SNPs were discovered and we selected 31 common polymorphic sites considering their allele frequencies, haplotype-tagging status and linkage disequilibrium (LD) for genotyping in larger-scale subjects. The SNPs were confirmed genotype through the SNaPshot method (n = 274) and were examined for a possible genetic association between Fas polymorphisms and marbling score. So, the SNPs that were identified significant are g.30256G>C, g.31474C>A, g.31940A>G, and g.32982G>A. These results suggest that SNPs of Fas gene were associated with intramuscular fat content of meat quality traits in Korean cattle.

  11. Comparison of 16S rRNA and protein-coding genes as molecular markers for assessing microbial diversity (Bacteria and Archaea) in ecosystems.

    PubMed

    Roux, Simon; Enault, François; Bronner, Gisèle; Debroas, Didier

    2011-12-01

    PCR amplification of the rRNA gene is the most popular method for assessing microbial diversity. However, this molecular marker is often present in multiple copies in cells presenting, in addition, an intragenomic heterogeneity. In this context, housekeeping genes may be used as taxonomic markers for ecological studies. However, the efficiency of these protein-coding genes compared to 16S rRNA genes has not been tested on environmental data. For this purpose, five protein marker genes for which primer sets are available, were selected (rplB, pyrG, fusA, leuS and rpoB) and compared with 16S rRNA gene results from PCR amplification or metagenomic data from aquatic ecosystems. Analysis of the major groups found in these ecosystems, such as Actinobacteria, Bacteroides, Proteobacteria and Cyanobacteria, showed good agreement between the protein markers and the results given by 16S rRNA genes from metagenomic reads. However, with the markers it was possible to detect minor groups among the microbial assemblages, providing more details compared to 16S rRNA results from PCR amplification. In addition, the use of a set of protein markers made it possible to deduce a mean copy number of rRNA operons. This average estimate is essentially lower than the one estimated in sequenced genomes. PMID:22066608

  12. An analysis of linkage disequilibrium in the interleukin-1 gene cluster, using a novel grouping method for multiallelic markers.

    PubMed Central

    Cox, A; Camp, N J; Nicklin, M J; di Giovine, F S; Duff, G W

    1998-01-01

    In population- and family-based association studies, it is useful to have some knowledge of the patterns of linkage disequilibrium that exist between markers in candidate regions. When such studies are carried out with multiallelic markers, it is often convenient to group the alleles into a biallelic system, for analysis. In this study, we specifically examined the interleukin-1 (IL-1) gene cluster on chromosome 2, a region containing candidates for many inflammatory and autoimmune disorders. Data were collected on eight markers, four of which were multiallelic. Using these data, we investigated the effect of three allele-grouping strategies, including a novel method, on the detection of linkage disequilibrium. The novel approach, termed the "delta method," measures the deviation from the expected haplotype frequencies under linkage equilibrium, for each allelic combination. This information is then used to group the alleles, in an attempt to avoid the grouping together of alleles at one locus that are in opposite disequilibrium with the same allele at the second locus. The estimate haplotype frequencies (EH) program was used to estimate haplotype frequencies and the disequilibrium measure. In our data it was found that the delta method compared well with the other two strategies. Using this method, we found that there was a reasonable correlation between disequilibrium and physical distance in the region (r=-.540, P=.001, one-tailed). We also identified a common, eight-locus haplotype of the IL-1 gene cluster. PMID:9545388

  13. PECAM-1 gene polymorphism (rs668) and subclinical markers of carotid atherosclerosis in patients with type 2 diabetes mellitus

    PubMed Central

    Nikolajević Starčević, J; Šantl Letonja, M; Makuc, J; Cokan Vujkovac, A; Reschner, H; Bregar, D; Petrovič, D

    2016-01-01

    ABSTRACT The platelet endothelial cell adhesion molecule 1 (PECAM-1) plays an important role in many inflammatory processes, including the development of atherosclerosis. Polymorphism rs668 of the PECAM-1 gene (373C/G) is functional, and it was reported to be associated with increased serum levels of PECAM-1. We investigated the association between the rs668 polymorphism of PECAM-1 and subclinical markers of carotid atherosclerosis in subjects with type 2 diabetes mellitus (T2DM). Five hundred and ninety-five T2DM subjects and 200 control subjects were enrolled. The carotid intima-media thickness (CIMT) and plaque characteristics (presence and structure) were assessed ultrasonographically. Biochemical analyses were performed using standard biochemical methods. Geno-typing of the PECAM-1 gene polymorphism (rs668) was performed using KASPar assays. The control examinations were performed 3.8 ± 0.5 years after the initial examination. Higher CIMT was found in patients with T2DM in comparison with subjects without T2DM. Statistically sig-nificantly faster progression of the atherosclerotic markers was shown in subjects with T2DM in comparison with the control group. When adjusted to other risk factors, the rs668 GG genotype was associated with an increased risk of carotid plaques in subjects with T2DM. We concluded that our study demonstrated a minor effect of the rs668 PECAM-1 on markers of carotid atherosclerosis in subjects with T2DM.

  14. Mapping of the vaccinia virus DNA polymerase gene by marker rescue and cell-free translation of selected RNA

    SciTech Connect

    Jones, E.V.; Moss, B.

    1984-01-01

    The previous demonstration that a phosphonoacetate (PAA)-resistant (PAA/sup r/) vaccinia virus mutant synthesized an altered DNA polymerase provided the key to mapping this gene. Marker rescue was performed in cells infected with wild-type PAA-sensitive (PAA/sup s/) vaccinia by transfecting with calcium phosphate-precipitated DNA from a PAA/sup r/ mutant virus. Formation of PAA/sup r/ recombinants was measured by plaque assay in the presence of PAA. Of the 12 HindIII fragments cloned in plasmid or cosmid vectors, only fragment E conferred the PAA/sup r/ phenotype. Successive subcloning of the 15-kilobase HindIII fragment E localized the marker within a 7.5-kilobase BamHI-HindIII fragment and then within a 2.9-kilobase EcoRI fragment. The location of the DNA polymerase gene, about 57 kilobases from the left end of the genome, was confirmed by cell-free translation of mRNA selected by hybridization to plasmids containing regions of PAA/sup r/ vaccinia DNA active in marker rescue. A 100,000-dalton polypeptide that comigrated with authentic DNA polymerase was synthesized. Correspondence of the in vitro translation product with purified vaccinia DNA polymerase was established by peptide mapping.

  15. The PPAR alpha gene is associated with triglyceride, low-density cholesterol and inflammation marker response to fenofibrate intervention: the GOLDN study

    Technology Transfer Automated Retrieval System (TEKTRAN)

    As a peroxisome proliferator-activated receptor alpha (PPAR Alpha) agonist, fenofibrate favorably modulates dyslipidemia and inflammation markers, which are associated with cardiovascular risk. To determine whether variation in the PPAR Alpha receptor gene was associated with lipid and inflammatory ...

  16. Effects of arachidonic acid on the concentration of hydroxyeicosatetraenoic acids in culture media of mesenchymal stromal cells differentiating into adipocytes or osteoblasts.

    PubMed

    Casado-Díaz, Antonio; Ferreiro-Vera, Carlos; Priego-Capote, Feliciano; Dorado, Gabriel; Luque-de-Castro, María Dolores; Quesada-Gómez, José Manuel

    2014-01-01

    Metabolites derived from the polyunsaturated fatty acids (PUFA) may modulate the mesenchymal stromal cell (MSC) differentiation. Such cells can differentiate into different cellular types, including adipocytes and osteoblasts. Aging favors the bone marrow MSC differentiation toward the former, causing a loss of bone density associated with pathologies like osteoporosis. The omega-6 arachidonic acid (AA) favors MSC adipogenesis to a greater extent than omega-3 eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). In this work, we study the joint action of both PUFA. Thus, not induced and induced to adipocyte or osteoblast MSC were treated with 20 μM of each PUFA (either AA, AA + DHA or AA + EPA). The expression of osteogenic and adipogenic molecular markers, the alox15b lipoxygenase gene expression and the 5-, 8-, 11-, 12- and 15-hydroxyeicosatetraenoic acids (HETE) derived from the AA metabolism in the culture media were determined. The results show that the adipogenesis induction of AA is not suppressed by the joint presence of EPA and DHA. In fact, both increased the adipogenic effect of AA on MSC differentiated into osteoblasts. The different HETE concentrations increased in cultures supplemented with AA, albeit such concentrations were lower in the cultures induced to differentiate, mainly at day 21 after the induction. Furthermore, the reduction in the HETE concentration was correlated with a higher expression of the alox15b gene. These results highlight the PUFA metabolism differences between uninduced and induced MSC to differentiate into adipocytes and osteoblasts, besides the relevant role of the lipoxygenase gene expression in adipogenesis induction.

  17. The endocrine disruptor diethylstilbestrol induces adipocyte differentiation and promotes obesity in mice

    SciTech Connect

    Hao, Chan-Juan; Cheng, Xue-Jia; Xia, Hong-Fei Ma, Xu

    2012-08-15

    Epidemiology studies indicate that exposure to endocrine disruptors during developmental “window” contributes to adipogenesis and the development of obesity. Implication of endocrine disruptor such as diethylstilbestrol (DES) on adipose tissue development has been poorly investigated. Here we evaluated the effects of DES on adipocyte differentiation in vitro and in vivo, and explored potential mechanism involved in its action. DES induced 3T3-L1 preadipocyte differentiation in a dose-dependent manner, and activated the expression of estrogen receptor (ER) and peroxisome proliferator-acivated receptor (PPAR) γ as well as its target genes required for adipogenesis in vitro. ER mediated the enhancement of DES-induced PPARγ activity. Moreover, DES perturbed key regulators of adipogenesis and lipogenic pathway in vivo. In utero exposure to low dose of DES significantly increased body weight, liver weight and fat mass in female offspring at postnatal day (PND) 60. In addition, serum triglyceride and glucose levels were also significantly elevated. These results suggest that perinatal exposure to DES may be expected to increase the incidence of obesity in a sex-dependent manner and can act as a potential chemical stressor for obesity and obesity-related disorders. -- Highlights: ► DES induced adipocyte differentiation in a dose-dependent manner in 3T3-L1 cells. ► DES activated adipogenic critical regulators and markers in vitro and in vivo. ► Perinatal exposure to DES led to the obese phenotype in female offspring. ► DES might be a potential chemical stressor for obesity and obesity-related disorders.

  18. ATF3 inhibits PPARγ-stimulated transactivation in adipocyte cells

    SciTech Connect

    Jang, Min-Kyung; Jung, Myeong Ho

    2015-01-02

    Highlights: • ATF3 inhibits PPARγ-stimulated transcriptional activation. • ATF3 interacts with PPARγ. • ATF3 suppresses p300-mediated transcriptional coactivation. • ATF3 decreases the binding of PPARγ and recruitment of p300 to PPRE. - Abstract: Previously, we reported that activating transcription factor 3 (ATF3) downregulates peroxisome proliferator activated receptor (PPARγ) gene expression and inhibits adipocyte differentiation in 3T3-L1 cells. Here, we investigated another role of ATF3 on the regulation of PPARγ activity. ATF3 inhibited PPARγ-stimulated transactivation of PPARγ responsive element (PPRE)-containing reporter or GAL4/PPARγ chimeric reporter. Thus, ATF3 effectively repressed rosiglitazone-stimulated expression of adipocyte fatty acid binding protein (aP2), PPARγ target gene, in 3T3-L1 cells. Coimmunoprecipitation and GST pulldown assay demonstrated that ATF3 interacted with PPARγ. Accordingly, ATF3 prevented PPARγ from binding to PPRE on the aP2 promoter. Furthermore, ATF3 suppressed p300-mediated transcriptional coactivation of PPRE-containing reporter. Chromatin immunoprecipitation assay showed that overexpression of ATF3 blocked both binding of PPARγ and recruitment of p300 to PPRE on aP2 promoter induced by rosiglitazone treatment in 3T3-L1 cells. Taken together, these results suggest that ATF3 interacts with PPARγ and represses PPARγ-mediated transactivation through suppression of p300-stimulated coactivation in 3T3-L1 cells, which may play a role in inhibition of adipocyte differentiation.

  19. Annotated genetic linkage maps of Pinus pinaster Ait. from a Central Spain population using microsatellite and gene based markers

    PubMed Central

    2012-01-01

    Background Pinus pinaster Ait. is a major resin producing species in Spain. Genetic linkage mapping can facilitate marker-assisted selection (MAS) through the identification of Quantitative Trait Loci and selection of allelic variants of interest in breeding populations. In this study, we report annotated genetic linkage maps for two individuals (C14 and C15) belonging to a breeding program aiming to increase resin production. We use different types of DNA markers, including last-generation molecular markers. Results We obtained 13 and 14 linkage groups for C14 and C15 maps, respectively. A total of 211 and 215 markers were positioned on each map and estimated genome length was between 1,870 and 2,166 cM respectively, which represents near 65% of genome coverage. Comparative mapping with previously developed genetic linkage maps for P. pinaster based on about 60 common markers enabled aligning linkage groups to this reference map. The comparison of our annotated linkage maps and linkage maps reporting QTL information revealed 11 annotated SNPs in candidate genes that co-localized with previously reported QTLs for wood properties and water use efficiency. Conclusions This study provides genetic linkage maps from a Spanish population that shows high levels of genetic divergence with French populations from which segregating progenies have been previously mapped. These genetic maps will be of interest to construct a reliable consensus linkage map for the species. The importance of developing functional genetic linkage maps is highlighted, especially when working with breeding populations for its future application in MAS for traits of interest. PMID:23036012

  20. Marker genes that are less conserved in their sequences are useful for predicting genome-wide similarity levels between closely related prokaryotic strains

    DOE PAGES

    Lan, Yemin; Rosen, Gail; Hershberg, Ruth

    2016-05-03

    The 16s rRNA gene is so far the most widely used marker for taxonomical classification and separation of prokaryotes. Since it is universally conserved among prokaryotes, it is possible to use this gene to classify a broad range of prokaryotic organisms. At the same time, it has often been noted that the 16s rRNA gene is too conserved to separate between prokaryotes at finer taxonomic levels. In this paper, we examine how well levels of similarity of 16s rRNA and 73 additional universal or nearly universal marker genes correlate with genome-wide levels of gene sequence similarity. We demonstrate that themore » percent identity of 16s rRNA predicts genome-wide levels of similarity very well for distantly related prokaryotes, but not for closely related ones. In closely related prokaryotes, we find that there are many other marker genes for which levels of similarity are much more predictive of genome-wide levels of gene sequence similarity. Finally, we show that the identities of the markers that are most useful for predicting genome-wide levels of similarity within closely related prokaryotic lineages vary greatly between lineages. However, the most useful markers are always those that are least conserved in their sequences within each lineage. In conclusion, our results show that by choosing markers that are less conserved in their sequences within a lineage of interest, it is possible to better predict