Effect of acute low oxygen exposure on the proliferation rate, viability and myogenic regulatory gene expression of C2C12 myoblasts in vitro

2020 ◽  
Vol 1 (1) ◽  
Keyword(s):  
Gene Expression ◽  
Regulatory Gene ◽  
Proliferation Rate ◽  
Low Oxygen ◽  
C2c12 Myoblasts ◽  
Oxygen Exposure

scholarly journals Effect of acute low oxygen exposure on the proliferation rate, viability and gene expression of C2C12 myoblasts in vitro

2020 ◽  
Author(s):  
Jack V Sharkey
Keyword(s):  
Gene Expression ◽  
Skeletal Muscle ◽  
Chronic Exposure ◽  
Proliferation Rate ◽  
Hypoxic Exposure ◽  
Low Oxygen ◽  
Time Points ◽  
Proliferative Phase ◽  
Hypoxic Environment

ABSTRACTINTRODUCTIONChanges in the oxygen concentration of cellular microenvironments play a significant role in regulating cell function during muscle regeneration. Generally, most in-vitro cell culture experiments have been carried out in atmospheric conditions with 21% O2, which compared to the actual micro-environment of mature skeletal muscle of between 1% and 10% pO2 is extremely hyperoxic (Li et al, 2007). Culturing skeletal muscle cells in vitro within their typical physiologically hypoxic environment in situ (2-10% pO2) has been shown to increase proliferation rate, reduce apoptosis and increase multiple MRF gene expressions, compared to culturing in a normoxic environment (21% O2). However, chronic exposure (>24 hr) to a semi-severe hypoxic environment (≤5% O2) can lead to a decrease in cell proliferation and differentiation (Chakravarthy et al, 2001). The effects of acute hypoxic exposure (24 h) has limited research and could be important in understanding the effects of hypoxia on skeletal muscle during brief exposures such as those observed within intermittent hypoxic training programmes. The purpose of this work was to examine the role of acute hypoxia (24 h) on C2C12 proliferation and relevant gene expression in 2D culture.METHODSC2C12 mouse myoblast cells were seeded into six well plates. The cells were maintained in DMEM with 20% FCS. C2C12 myoblasts were either exposed to 21% or 5% O2. The effects of acute hypoxic exposure (24hours) at different time points during the proliferative phase of myogenesis, rather than chronic exposure, on cell proliferation, cellular viability and myogenic regulatory factor gene expression was examined. At 24, 48, 72 and 96 hours RNA was extracted using the Trizol® method and mRNA expression of myogenic regulatory factors, myoD, myf5 and myogenin were detected using the 2-ΔΔCT method. Cell counts and cell viability were also quantifiedRESULTSNo significant difference was found between cells cultured in normoxic conditions (21%) and those that were exposed to low oxygen conditions for 24hours at various time points over a 96 hour culture period, with regards to proliferation rate, cell viability, and myogenic regulatory gene expression (Myf5, MyoD and Myogenin).DISCUSSIONThe effect of acute low oxygen exposure lasting 24hours appears to not be insufficient in having an effect on the proliferation rate, viability or transcription factor expression of C2C12 cells during the proliferative phase of myogenesis.

scholarly journals Gene Involved in Transcriptional Activation of the hrp Regulatory Gene hrpG in Xanthomonas oryzae pv. oryzae

2006 ◽  
Vol 188 (11) ◽  
pp. 4158-4162 ◽  
Author(s):  
Seiji Tsuge ◽  
Takeshi Nakayama ◽  
Shinsaku Terashima ◽  
Hirokazu Ochiai ◽  
Ayako Furutani ◽  
...  
Keyword(s):  
Gene Expression ◽  
Plant Pathogen ◽  
Transcriptional Activation ◽  
Regulatory Gene ◽  
Xanthomonas Oryzae ◽  
In Planta

ABSTRACT A novel regulatory gene, trh, which is involved in hrp gene expression, is identified in the plant pathogen Xanthomonas oryzae pv. oryzae. In the trh mutant, expression of HrpG, which is a key regulator for hrp gene expression, is reduced both under the in vitro hrp-inducing condition and in planta.

Different carbon sources and their concentrations change alkaloid production and gene expression in Catharanthus roseus shoots in vitro

2021 ◽  
Vol 48 (1) ◽  
pp. 40
Author(s):  
Elham Khataee ◽  
Farah Karimi ◽  
Khadijeh Razavi
Keyword(s):  
Gene Expression ◽  
Regulatory Gene ◽  
Carbon Sources ◽  
Basal Medium ◽  
Alkaloid Production ◽  
Defensive Responses ◽  
Mitogen Activated Protein ◽  
Catalase Peroxidase ◽  
Plant Alkaloids

To compare the effects of different carbon sources on physiological aspects, especially medicinal alkaloid biosynthesis and related gene expression in Catharantus roseus (L.) G.Don, we employed sucrose and sorbitol with two concentrations (87.64 mM, the equimolar concentration of sucrose in MS basal medium, and 150 mM) on the plant’s shoots in vitro in presence of 100 μM methyl jasmonate. The production of plant alkaloids including vincristine, vinblastine, ajmalicine, vindoline and catharantine and their biosynthetic and regulatory gene expression was measured. Both treatments had incremental effects on alkaloid production, upregulated the mitogen-activated protein kinase3 (MAPK3) and a downstream responsive transcription factor, ORCA3, which resulted in elevated transcript contents of the important genes in terpenoid indol alkaloids biosynthetic pathway including peroxidase1 (PRX1), geissoschizine synthase (GS), strictosidine synthase (STR) and deacetylvindoline acetyltransferase (DAT). Defensive responses such as antioxidant enzymes (catalase, peroxidase and superoxide dismutase) activities and non-enzymatic metabolites (total phenolics, flavonoids and carotenoids) contents increased under both treatments but the effects of sorbitol were stronger. Reduced fresh weight and chlorophylls contents, increased malondialdehyde (MDA) and carotenoid contents were shown after a week under all employed treatments. It seems that replacement of sucrose with sorbitol and also, increased concentrations of both carbon sources via increasing osmotic pressure make stressful conditions for the plant especially in longer times.

scholarly journals Campylobacter jejuni Gene Expression in the Chick Cecum: Evidence for Adaptation to a Low-Oxygen Environment

2005 ◽  
Vol 73 (8) ◽  
pp. 5278-5285 ◽  
Author(s):  
C. A. Woodall ◽  
M. A. Jones ◽  
P. A. Barrow ◽  
J. Hinds ◽  
G. L. Marsden ◽  
...  
Keyword(s):  
Gene Expression ◽  
Electron Transport ◽  
Campylobacter Jejuni ◽  
Metabolic Pathways ◽  
Transcriptional Profiling ◽  
Physiological State ◽  
Low Oxygen ◽  
Oxygen Environment

ABSTRACT Transcriptional profiling of Campylobacter jejuni during colonization of the chick cecum identified 59 genes that were differentially expressed in vivo compared with the genes in vitro. The data suggest that C. jejuni regulates electron transport and central metabolic pathways to alter its physiological state during establishment in the chick cecum.

scholarly journals Identifikasi homolog TcAGL-15 untuk penanda embriogenesis tanaman kakao melalui pendekatan bioinformatika Identification of TcAGL-15 homolog in the embryogenic culture from the zygotic embryo explant

2016 ◽  
Vol 74 (2) ◽  
Author(s):  
Oktaviany Ferry TRIASTANTO ◽  
Muhammad JUSUF ◽  
Djoko SANTOSO
Keyword(s):  
Gene Expression ◽  
Zygotic Embryo ◽  
Regulatory Gene ◽  
Low Frequency ◽  
Zygotic Embryos ◽  
Rt Pcr ◽  
Specific Primers ◽  
Process Understanding ◽  
Differential Pattern

Summary One of the major problems encountered in micropropagation of cacao through tissue culture is very low frequency of embryo formation. Embryogenesis is believed to have key regulatory gene determining the process. Understanding such gene may help to solve problems in the regeneration process. One of the genes reported to involve in the embryogenesis is AGAMOUS-like 15 (AGL-15). This gene has an important role in the regulation of early embryogenesis in several plants. This experiment aimed to identify AGL-15 homolog in cacao through bioinformatics approach. The first step of this experiment is to identify the AGL-15 homolog using hetero-logous primers from DNA genomic isolated from leaves of cacao plants. The sequence of the AGL-15 fragment was used in designing specific primers for longer AGL-15 fragment. These primers were then used to identify AGL-15 gene using total RNA isolated from cultured zygotic embryos. Differential pattern of AGL-15 gene expression was observed in zygotic embryos cultured for five weeks. AGL-15 heterologous primers designed from several plants could be used to identify cacao AGL-15 homolog. The putative cacao AGL-15 gene could be identified from zygotic embryos. The differential pattern of the AGL-15 gene expression is a strong indication that AGL-15 can be used as an embryogenesis marker in cacao plants.Ringkasan Salah satu kendala perbanyakan kakao melalui kultur jaringan adalah sulitnya embriogenesis, yang diduga melibatkan satu atau lebih gen kunci yang menentukan proses tersebut. Keberhasilan mengidentifikasi gen-gen kunci akan membantu menyelesaikan masalah dalam regenerasi embrio kakao. Salah satu gen yang diduga terlibat dalam proses ini adalah AGAMOUS-like 15 (AGL-15). Gen ini berperan pada regulasi selama masa awal perkembangan embrio beberapa tanaman. Penelitian ini bertujuan untuk mengidentifikasi homolog AGL-15 pada kakao melalui pen-dekatan bioinformatika dan RT-PCR. Pene-litian diawali dengan identifikasi homolog AGL-15 dari DNA genomik daun kakao meng-gunakan primer heterologus. Sekuen fragmen homolog AGL-15 yang diperoleh, kemudian digunakan untuk merancang primer spesifik AGL-15 yang berukuran lebih panjang. Primer ini selanjutnya digunakan untuk meng-identifikasi gen AGL-15 dari RNA total embrio zigotik. Pengamatan pola pita gen AGL-15 dilakukan pada kultur in vitro embrio zigotik yang berumur lima minggu. Primer hetero-logus gen AGL-15 yang berasal dari berbagai tanaman, mampu mengidentifikasi keberadaan homolog  gen  tersebut  pada  tanaman   kakao. Fragmen homolog AGL-15 putatif tanaman kakao teridentifikasi pada tingkat RNA embrio. Dengan adanya pola diferensial dari ekspresi gen AGL-15 hingga lima minggu pertama perkembangan embrio, ada indikasi kuat bahwa fragmen homolog AGL-15 dapat menjadi penanda embriogenesis pada tanaman kakao.

scholarly journals Glucose and glutamine availability regulate HepG2 transcriptional responses to low oxygen

2018 ◽  
Vol 3 ◽  
pp. 126 ◽  
Author(s):  
Alvina G. Lai ◽  
Donall Forde ◽  
Wai Hoong Chang ◽  
Fang Yuan ◽  
Xiaodong Zhuang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mass Spectrometry ◽  
Transcriptional Response ◽  
Global Changes ◽  
Low Oxygen ◽  
Transcriptional Responses ◽  
Dna Hydroxymethylation ◽  
Modified Dna ◽  
The Impact

Background: Little is known about the impact of nutrients on cellular transcriptional responses, especially in face of environmental stressors such as oxygen deprivation. Hypoxia-inducible factors (HIF) coordinate the expression of genes essential for adaptation to oxygen-deprived environments. A second family of oxygen-sensing genes known as the alpha-ketoglutarate-dependent dioxygenases are also implicated in oxygen homeostasis and epigenetic regulation. The relationship between nutritional status and cellular response to hypoxia is understudied. In vitro cell culture systems frequently propagate cells in media that contains excess nutrients, and this may directly influence transcriptional response in hypoxia. Methods: We studied the effect of glucose and glutamine concentration on HepG2 hepatoma transcriptional response to low oxygen and expression of hypoxia inducible factor-1α (HIF-1α). Mass spectrometry confirmed low oxygen perturbation of dioxygenase transcripts resulted in changes in DNA methylation. Results: Under normoxic conditions, we observed a significant upregulation of both HIF-target genes and oxygen-dependent dioxygenases in HepG2 cells cultured with physiological levels of glucose or glutamine relative to regular DMEM media, demonstrating that excess glutamine/glucose can mask changes in gene expression. Under hypoxic conditions, CA9 was the most upregulated gene in physiological glutamine media while TETs and FTO dioxygenases were downregulated in physiological glucose. Hypoxic regulation of these transcripts did not associate with changes in HIF-1α protein expression. Downregulation of TETs suggests a potential for epigenetic modulation. Mass-spectrometry quantification of modified DNA bases confirmed our transcript data. Hypoxia resulted in decreased DNA hydroxymethylation, which correlated with TETs downregulation. Additionally, we observed that TET2 expression was significantly downregulated in patients with hepatocellular carcinoma, suggesting that tumour hypoxia may deregulate TET2 expression resulting in global changes in DNA hydroxymethylation.   Conclusion: Given the dramatic effects of nutrient availability on gene expression, future in vitro experiments should be aware of how excess levels of glutamine and glucose may perturb transcriptional responses.

scholarly journals New Target Genes Controlled by the Bradyrhizobium japonicum Two-Component Regulatory System RegSR

2007 ◽  
Vol 189 (24) ◽  
pp. 8928-8943 ◽  
Author(s):  
Andrea Lindemann ◽  
Annina Moser ◽  
Gabriella Pessi ◽  
Felix Hauser ◽  
Markus Friberg ◽  
...  
Keyword(s):  
Gene Expression ◽  
Nitrogen Fixation ◽  
Bradyrhizobium Japonicum ◽  
Target Genes ◽  
Regulatory Gene ◽  
Wild Type ◽  
Transcription Profiling ◽  
Two Component ◽  
Regulated Gene Expression

ABSTRACT RegSR-like proteins, members of the family of two-component regulatory systems, are present in a large number of proteobacteria in which they globally control gene expression mostly in a redox-responsive manner. The controlled target genes feature an enormous functional diversity. In Bradyrhizobium japonicum, the facultative root nodule symbiont of soybean, RegSR activate the transcription of the nitrogen fixation regulatory gene nifA, thus forming a RegSR-NifA cascade which is part of a complex regulatory network for gene regulation in response to changing oxygen concentrations. Whole-genome transcription profiling was performed here in order to assess the full regulatory scope of RegSR. The comparative analysis of wild-type and ΔregR cells grown under oxic and microoxic conditions revealed that expression of almost 250 genes is dependent on RegR, a result that underscores the important contribution of RegR to oxygen- or redox-regulated gene expression in B. japonicum. Furthermore, transcription profiling of ΔregR bacteroids compared with wild-type bacteroids revealed expression changes for about 1,200 genes in young and mature bacteroids. Incidentally, many of these were found to be induced in symbiosis when wild-type bacteroids were compared with free-living, culture-grown wild-type cells, and they appeared to encode diverse functions possibly related to symbiosis and nitrogen fixation. We demonstrated direct RegR-mediated control at promoter regions of several selected target genes by means of DNA binding experiments and in vitro transcription assays, which revealed six novel direct RegR target promoters.

Controlling matrix formation and cross-linking by hypoxia in cardiovascular tissue engineering

2010 ◽  
Vol 109 (5) ◽  
pp. 1483-1491 ◽  
Author(s):  
Marijke A. A. van Vlimmeren ◽  
Anita Driessen-Mol ◽  
Marloes van den Broek ◽  
Carlijn V. C. Bouten ◽  
Frank P. T. Baaijens
Keyword(s):  
Gene Expression ◽  
Tissue Engineering ◽  
Tissue Development ◽  
Low Oxygen ◽  
Cardiovascular Tissue ◽  
Expression Levels ◽  
Cardiovascular Tissue Engineering ◽  
Gene Expression Levels ◽  
Oxygen Concentrations

In vivo functionality of cardiovascular tissue engineered constructs requires in vitro control of tissue development to obtain a well developed extracellular matrix (ECM). We hypothesize that ECM formation and maturation is stimulated by culturing at low oxygen concentrations. Gene expression levels of monolayers of human vascular-derived myofibroblasts, exposed to 7, 4, 2, 1, and 0.5% O2( n = 9 per group) for 24 h, were measured for vascular endothelial growth factor (VEGF), procollagen α1(I) and α1(III), elastin, and cross-link enzymes lysyl oxidase (LOX) and lysyl hydroxylase 2 (LH2). After 4 days of exposure to 7, 2, and 0.5% O2( n = 3 per group), protein synthesis was evaluated. All analyses were compared with control cultures at 21% O2. Human myofibroblasts turned to hypoxia-driven gene expression, indicated by VEGF expression, at oxygen concentrations of 4% and lower. Gene expression levels of procollagen α1(I) and α1(III) increased to 138 ± 26 and 143 ± 19%, respectively, for all oxygen concentrations below 4%. At 2% O2, LH2 and LOX gene expression levels were higher than control cultures (340 ± 53 and 136 ± 29%, respectively), and these levels increased even further with decreasing oxygen concentrations (611 ± 176 and 228 ± 45%, respectively, at 0.5% O2). Elastin gene expression levels remained unaffected. Collagen synthesis and LH2 protein levels increased at oxygen concentrations of 2% and lower. Oxygen concentrations below 4% induce enhanced ECM production by human myofibroblasts. Implementation of these results in cardiovascular tissue engineering approaches enables in vitro control of tissue development.

scholarly journals Identifikasi homolog TcAGL-15 untuk penanda embriogenesis tanaman kakao melalui pendekatan bioinformatika Identification of TcAGL-15 homolog in the embryogenic culture from the zygotic embryo explant

2016 ◽  
Vol 74 (2) ◽  
Author(s):  
Oktaviany Ferry TRIASTANTO ◽  
Muhammad JUSUF ◽  
Djoko SANTOSO
Keyword(s):  
Gene Expression ◽  
Zygotic Embryo ◽  
Regulatory Gene ◽  
Low Frequency ◽  
Zygotic Embryos ◽  
Rt Pcr ◽  
Specific Primers ◽  
Process Understanding ◽  
Differential Pattern

Summary One of the major problems encountered in micropropagation of cacao through tissue culture is very low frequency of embryo formation. Embryogenesis is believed to have key regulatory gene determining the process. Understanding such gene may help to solve problems in the regeneration process. One of the genes reported to involve in the embryogenesis is AGAMOUS-like 15 (AGL-15). This gene has an important role in the regulation of early embryogenesis in several plants. This experiment aimed to identify AGL-15 homolog in cacao through bioinformatics approach. The first step of this experiment is to identify the AGL-15 homolog using hetero-logous primers from DNA genomic isolated from leaves of cacao plants. The sequence of the AGL-15 fragment was used in designing specific primers for longer AGL-15 fragment. These primers were then used to identify AGL-15 gene using total RNA isolated from cultured zygotic embryos. Differential pattern of AGL-15 gene expression was observed in zygotic embryos cultured for five weeks. AGL-15 heterologous primers designed from several plants could be used to identify cacao AGL-15 homolog. The putative cacao AGL-15 gene could be identified from zygotic embryos. The differential pattern of the AGL-15 gene expression is a strong indication that AGL-15 can be used as an embryogenesis marker in cacao plants.Ringkasan Salah satu kendala perbanyakan kakao melalui kultur jaringan adalah sulitnya embriogenesis, yang diduga melibatkan satu atau lebih gen kunci yang menentukan proses tersebut. Keberhasilan mengidentifikasi gen-gen kunci akan membantu menyelesaikan masalah dalam regenerasi embrio kakao. Salah satu gen yang diduga terlibat dalam proses ini adalah AGAMOUS-like 15 (AGL-15). Gen ini berperan pada regulasi selama masa awal perkembangan embrio beberapa tanaman. Penelitian ini bertujuan untuk mengidentifikasi homolog AGL-15 pada kakao melalui pen-dekatan bioinformatika dan RT-PCR. Pene-litian diawali dengan identifikasi homolog AGL-15 dari DNA genomik daun kakao meng-gunakan primer heterologus. Sekuen fragmen homolog AGL-15 yang diperoleh, kemudian digunakan untuk merancang primer spesifik AGL-15 yang berukuran lebih panjang. Primer ini selanjutnya digunakan untuk meng-identifikasi gen AGL-15 dari RNA total embrio zigotik. Pengamatan pola pita gen AGL-15 dilakukan pada kultur in vitro embrio zigotik yang berumur lima minggu. Primer hetero-logus gen AGL-15 yang berasal dari berbagai tanaman, mampu mengidentifikasi keberadaan homolog  gen  tersebut  pada  tanaman   kakao. Fragmen homolog AGL-15 putatif tanaman kakao teridentifikasi pada tingkat RNA embrio. Dengan adanya pola diferensial dari ekspresi gen AGL-15 hingga lima minggu pertama perkembangan embrio, ada indikasi kuat bahwa fragmen homolog AGL-15 dapat menjadi penanda embriogenesis pada tanaman kakao.

1 EFFECT OF OXYGEN TENSION DURING IN VITRO MATURATION AND FERTILIZATION ON BOVINE EMBRYO PRODUCTION AND CUMULUS-OOCYTE-COMPLEX mRNA ABUNDANCE

2009 ◽  
Vol 21 (1) ◽  
pp. 101
Author(s):  
P. Bermejo-Alvarez ◽  
D. Rizos ◽  
P. Lonergan ◽  
A. Gutierrez-Adan
Keyword(s):  
Gene Expression ◽  
Oxygen Tension ◽  
In Vitro Maturation ◽  
Cyclin B1 ◽  
Bovine Embryo ◽  
Low Oxygen ◽  
Embryo Production ◽  
Low Oxygen Tension ◽  
Developmental Rates

Despite physiological oxygen tension being lower than in atmospheric gas, both in vitro maturation and fertilization are generally conducted under atmospheric oxygen tension (20%). The objective of this study was to evaluate the effect of two different oxygen concentrations (20 and 5%) during in vitro maturation (M) and fertilization (F) on bovine embryo production and to analyze differences in gene expression between cumulus–oocyte-complexes (COC) matured at 5% (M5) or 20% (M20) oxygen tension. A total of 1179 COC were matured, fertilized and cultured in vitro in 5 replicates divided in 4 groups according to the oxygen tension used (M5F5, M5F20, M20F5 and M20F20). Cleavage was assessed every 4 h from 24 to 48 h post-insemination (pi) and blastocyst yield was recorded from Day 6 to Day 8. For gene expression analysis, 5 pools of 10 COC (not denuded) of each group were snap frozen after maturation and stored at –80°C. After RNA extraction and RTPCR, qPCR was used to quantify relative abundance of 7 genes: the M-phase promoting factor subunit Cyclin B1, insulin-growth factor 2 (IGFR2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), lactate dehydrogenase A (LDHA), glucose-6-phosphate dehydrogenase (G6PD), glutathione peroxidase (GPX1), and the apoptosis related gene BAX. Histone 2A was used as housekeeping gene. Statistical analysis was performed analyzing two factors (M5 v. M20 and F5 v. F20) by two-way ANOVA (P ≤ 0.05) for embryo production and by one-way ANOVA (P ≤ 0.05) for gene expression. At every time point analyzed, maturation under low oxygen tension significantly improved both cleavage rate and blastocyst yield. However, the use of low oxygen tension at fertilization has a significant negative effect on developmental rates (48 hpi: M5F5: 71.6 ± 5.5; M5F20: 76.2 ± 2.7; M20F5: 40.1 ± 5.8; M20F20: 69.9 ± 0.8). (Day 6: M5F5: 12.6 ± 3; M5F20: 17.2 ± 2.4; M20F5: 2.5 ± 1.2; M20F20: 13.3 ± 4.) (Day 7: M5F5: 32.6 ± 4.9; M5F20: 38.8 ± 3.2; M20F5: 12.2 ± 1.2; M20F20: 34.2 ± 3.9.) (Day 8: M5F5: 35.5 ± 5; M5F20: 43.1 ± 5.1; M20F5: 15.6 ± 1.4; M20F20: 37.1 ± 4.2.) (Group: %Mean ± SEM). Four genes (Cyclin B1, GAPDH, IGFR2 and LDHA) were significantly upregulated in M5 COC, and three genes (GPX1, G6PD and BAX) did not show significant differences. In conclusion, low oxygen tension during maturation exerts a beneficial effect upon embryo development, whereas the opposite situation is observed during fertilization. Furthermore, the expression of a growth factor, a meiotic promoting factor and two enzymes of anaerobic glycolysis were significantly upregulated in COCs matured under low oxygen tension, which may be linked to the improvement in developmental rates.

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