scholarly journals Cybrid Models of Pathological Cell Processes in Different Diseases

2018 ◽  
Vol 2018 ◽  
pp. 1-6 ◽  
Author(s):  
Margarita A. Sazonova ◽  
Vasily V. Sinyov ◽  
Anastasia I. Ryzhkova ◽  
Elena V. Galitsyna ◽  
Alexandra A. Melnichenko ◽  
...  
Keyword(s):  
Mitochondrial Genome ◽  
Ethidium Bromide ◽  
Cell Cultures ◽  
21St Century ◽  
Standard Technique ◽  
Therapeutic Approaches ◽  
Cell Processes ◽  
Mtdna Replication ◽  
Cybrid Cell ◽  
In Cells

Modelling of pathological processes in cells is one of the most sought-after technologies of the 21st century. Using models of such processes may help to study the pathogenetic mechanisms of various diseases. The aim of the present study was to analyse the literature, dedicated to obtaining and investigating cybrid models. Besides, the possibility of modeling pathological processes in cells and treatment of different diseases using the models was evaluated. Methods of obtaining Rho0 cell cultures showed that, during their creation, mainly a standard technique, based on the use of mtDNA replication inhibitors (ethidium bromide), was applied. Cybrid lines were usually obtained by PEG fusion. Most frequently, platelets acted as donors of mitochondria. According to the analysis of the literature data, cybrid cell cultures can be modeled to study the dysfunction of the mitochondrial genome and molecular cellular pathological processes. Such models can be very promising for the development of therapeutic approaches to the treatment of various human diseases.

scholarly journals Creation of Cybrid Cultures Containing mtDNA Mutations m.12315G>A and m.1555G>A, Associated with Atherosclerosis

Biomolecules ◽  
2019 ◽  
Vol 9 (9) ◽  
pp. 499 ◽  
Author(s):  
Margarita A. Sazonova ◽  
Vasily V. Sinyov ◽  
Anastasia I. Ryzhkova ◽  
Marina D. Sazonova ◽  
Zukhra B. Khasanova ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Drug Therapy ◽  
Mitochondrial Genome ◽  
Cell Lines ◽  
Threshold Value ◽  
Mtdna Mutation ◽  
Single Nucleotide ◽  
Mtdna Mutations ◽  
Cybrid Cell ◽  
In Cells

In the present work, a pilot creation of four cybrid cultures with high heteroplasmy level was performed using mitochondrial genome mutations m.12315G>A and m.1555G>A. According to data of our preliminary studies, the threshold heteroplasmy level of mutation m.12315G>A is associated with atherosclerosis. At the same time, for a mutation m.1555G>A, such a heteroplasmy level is associated with the absence of atherosclerosis. Cybrid cultures were created by fusion of rho0-cells and mitochondria from platelets with a high heteroplasmy level of the investigated mutations. To create rho0-cells, THP-1 culture of monocytic origin was taken. According to the results of the study, two cybrid cell lines containing mutation m.12315G>A with the heteroplasmy level above the threshold value (25% and 44%, respectively) were obtained. In addition, two cybrid cell lines containing mutation m.1555G>A with a high heteroplasmy level (24%) were obtained. Cybrid cultures with mtDNA mutation m.12315G>A can be used to model both the occurrence and development of atherosclerosis in cells and the titration of drug therapy for patients with atherosclerosis. With the help of cybrid cultures containing single nucleotide replacement of mitochondrial genome m.1555G>A, it is possible to develop approaches to the gene therapy of atherosclerosis.

scholarly journals Creation of cybrid cell cultures containing a single-nucleotide substitution associated with atherosclerosis in the MT-TL2 gene

2020 ◽  
pp. 140-147
Author(s):  
М.А. Сазонова ◽  
В.В. Синёв ◽  
А.И. Рыжкова ◽  
М.Д. Сазонова ◽  
Н.А. Дорощук ◽  
...  
Keyword(s):  
Mitochondrial Genome ◽  
Mitochondrial Dysfunction ◽  
Cell Lines ◽  
Cell Cultures ◽  
Gradient Centrifugation ◽  
Sybr Green ◽  
Sybr Green I ◽  
Cellular Mechanisms ◽  
Study Participants ◽  
Cybrid Cell

Введение. Цибридные клеточные модели наиболее перспективны для изучения патогенеза различных заболеваний. Авторами статьи впервые были созданы такие модели для изучения митохондриальной дисфункции и патологических процессов, развивающихся при атеросклерозе. Цель работы - создание цибридных культур с высоким уровнем гетероплазмии по мутации митохондриального генома m.12315G>A. В предварительных исследованиях авторами статьи было установлено, что пороговый уровень гетероплазмии мутации m.12315G>A ассоциирован с атеросклерозом. Методика. Цибридные культуры создавали путем слияния безмитохондриальных клеток (rho0) и митохондрий из тромбоцитов участников исследования с высоким уровнем гетероплазмии исследуемых мутаций. Для создания rho0-клеток была взята культура моноцитарного происхождения THP-1. Безмитохондриальные клетки были получены с помощью метода M. Kинга и Г. Аттарди. Тромбоциты выделяли из цельной крови участников исследования. Для этого был применен метод центрифугирования в градиенте плотности фиколла-урографина. Для получения цибридных культур клеток была использована методика «ПЭГ-слияния». В созданных безмитохондриальных и цибридных клеточных культурах был проведен количественный анализ копий митохондриального генома. Согласно результатам данного анализа было подтверждено либо отсутствие митохондрий (rho0-клетки), либо их наличие (цибриды). Количество копий мтДНК детектировалось с помощью реал-тайм ПЦР в присутствии красителя SYBR Green I. Результаты. Получены 4 цибридные клеточные линии, содержащие мутацию m.12315G>A с уровнем гетероплазмии выше порогового значения. Заключение. Созданы 4 цибридные культуры с высоким уровнем гетероплазмии по мутации митохондриального генома m.12315G>A. Полученные цибридные клеточные линии могут служить моделями для изучения молекулярно-клеточных механизмов митохондриальной дисфункции при атеросклерозе и других сердечно-сосудистых заболеваниях. Цибридные культуры можно использовать для моделирования атерогенеза, а также для подбора патогенетически обоснованной лекарственной терапии при атеросклерозе. Introduction. Cybrid cell models are most promising for studying pathological mechanisms in different diseases. The authors for the first time created such models for studying mitochondrial dysfunction and pathological processes underlying atherosclerosis. Aim. Creation of cybrid cultures with a high heteroplasmy level for mitochondrial genome mutation m.12315G>A. A preliminary study by the authors showed that the heteroplasmy level of mutation m.12315G>A was associated with atherosclerosis. Methods. Cybrid cultures were created by fusing non-mitochondrial cells (rho0) and mitochondria from platelets of study participants with a high heteroplasmy level of the mutations under study. A THP-1 culture of monocytic origin was used to create rho0 cells. Non-mitochondrial cells were obtained using the M. King and G. Attardi method. Platelets were extracted from whole blood of study participants with Ficoll-Urografin density gradient centrifugation. Cybrid cell cultures were obtained by the PEG-mediated fusion method. In the created non-mitochondrial and cybrid cell cultures, quantitative analysis of mitochondrial genome copies was performed. This analysis confirmed either the absence of mitochondria (rho0-cells) or their presence (cybrids). The mtDNA copies were quantified using real-time PCR in the presence of the SYBR Green I stain. Results. Four cybrid cell lines were obtained, which contained the m.12315G>A mutation with heteroplasmy levels higher than the threshold level. Conclusion. Four cybrid cultures were created with a high heteroplasmy level for the mitochondrial genome mutation m.12315G>A. The obtained cell lines can be used as models for studying molecular cellular mechanisms of mitochondrial dysfunction in atherosclerosis and cardiovascular diseases. In addition, they may be useful for modeling atherogenesis in cells and for selecting therapy for patients with atherosclerosis.

scholarly journals Changes in phenolic acids and stilbenes induced in embryogenic cell cultures of Norway spruce by two fractions of Sirococcus strobilinus mycelian

2011 ◽  
Vol 57 (No. 1) ◽  
pp. 1-7 ◽  
Author(s):  
J. Malá ◽  
M. Hrubcová ◽  
P. Máchová ◽  
H. Cvrčková ◽  
O. Martincová ◽  
...  
Keyword(s):  
Cell Wall ◽  
Norway Spruce ◽  
Phenolic Acids ◽  
Cell Cultures ◽  
Total Content ◽  
Cell Suspension Cultures ◽  
Moderate Increase ◽  
Embryogenic Cell ◽  
Defence Responses ◽  
In Cells

We examined defence responses in embryogenic cell suspension cultures of Norway spruce (Picea abies [L.] Karst) elicited by intracellular protein and cell wall fractions (PF and WF, respectively) prepared from mycelia of the fungus Sirococcus strobilinus Preuss focusing on changes in (soluble and cell wall-bound) phenolic and stilbene concentrations. Treatment with both preparations induced an increase in the total contents of phenolic acids in Norway spruce cells and variations in the levels of stilbene glycosides. More rapid and intense induction of defence response was observed in cells after WF application. The contents of soluble phenolic acids (especially benzoic acid derivatives) and cell wall-bound phenolic acids (especially ferulic acid) started to increase (relative to controls) within 4 h after the addition of the WF preparation and remained high in elicited cells for 8–12 h. A moderate increase in phenolic acids in cells exposed to the PF preparation was observed within 8 h after application. However, after 24 h of WF treatment a decline of total phenolics was observed, while in PF elicited Norway spruce cells the phenolic content continued to increase. Significantly decreased concentrations of stilbene glycosides, isorhapontin, astringin and piceid, were determined in PF and WF treated Norway spruce cell cultures. The total content of stilbene glycosides decreased within 8 h after WF application to 68% of the amount determined in the control and within 12 h to 73% of the control in PF-treated cells. These results demonstrate that both PF and WF prepared from the Sirococcus strobilinus mycelium elicit changes in the metabolism of phenylpropanoids, which are involved in the defence responses of plants to pathogens.

The effect of double-strength standard saline citrate on silver staining. I. Nucleoli and micronucleoli in the somatic and germ line of the grasshopper Arcyptera fusca (Orthoptera)

1986 ◽  
Vol 28 (2) ◽  
pp. 219-226 ◽  
Author(s):  
J. Gosàlvez ◽  
J. de la Torre ◽  
C. Garcia de la Vega ◽  
C. López-Fernández
Keyword(s):  
Silver Nitrate ◽  
Silver Staining ◽  
Germ Line ◽  
Standard Technique ◽  
Previous Treatment ◽  
Nucleolar Activity ◽  
Standard Saline Citrate ◽  
Nucleolar Proteins ◽  
In Cells

The nucleolar activity in cells from both somatic and germ line of the grasshopper Arcyptera fusca has been analyzed by means of silver staining using a standard technique with and without a previous treatment of the slides with double-strength standard saline citrate (2 × SSC). Results show that the treatment with 2 × SSC improves the silver staining of the main nucleoli and in some tissues reliably uncovers other sites along the chromosomes with attached silver precipitates (micronucleoli). The parallel origin of the main nucleoli and the micronucleoli, the possible enlargement of rDNA activity in some tissues, and the ability of the silver nitrate to stain certain nucleolar proteins are discussed.Key words: Arcyptera, Orthoptera, nucleolus, micronucleus, silver staining.

Relaxin and prostaglandin on oxytocin secretion from bovine luteal cells during different stages of gestation

1990 ◽  
Vol 122 (3) ◽  
pp. 396-402 ◽  
Author(s):  
Al-Hassan I. Musah ◽  
Christian Schwabe ◽  
Lloyd L. Anderson
Keyword(s):  
Cell Cultures ◽  
Culture Media ◽  
Hormone Replacement ◽  
Corpora Lutea ◽  
Luteal Cells ◽  
Oxytocin Release ◽  
Prostaglandin F ◽  
Dose Dependent ◽  
Estradiol 17Β ◽  
In Cells

Abstract The objective of this study was to determine whether bovine luteal cells from different stages of gestation secrete oxytocin and whether relaxin, cloprostenol (a potent analogue of prostaglandin F2α), estradiol-17β, and LH can acutely alter oxytocin secretion. Bovine luteal cells (105) were cultured for 24 h without treatment and with medium-hormone replacement every 24 h. Oxytocin was quantified by radioimmunoassay of the culture media. Basal oxytocin secretion was similar (22-31 pmol/l, p>0.05) for all stages of gestation (days 100, 145, 160, 185, 200, 210, and 240). Relaxin induced a dose-dependent suppression of oxytocin release. After 24 h of incubation, addition of 0, 16.7, 83.5, and 167 nmol/l porcine relaxin (3000 U/mg) induced 54 ± 4, 105 ± 16, 47 ± 4, and 38 ± 4 pmol/l of oxytocin in cells from 160-day-old corpora lutea and 138 ± 12, 21 ± 2, 19 ± 3, and 15 ± 2 pmol/l oxytocin in cells from 240-day-old corpora lutea. From luteal cells of 160- and 240-day-old corpora lutea, 2 μmol/l cloprostenol induced a marked increase (p<0.01) of 208 ± 39 and 371 ± 34 pmol/l oxytocin, respectively. Addition of 167 nmol/l relaxin did not prevent cloprostenol-induced oxytocin secretion during the first 48 h, but a decrease (p<0.05) in oxytocin occurred in day 3 cell cultures. These results indicate that cultured luteal cells obtained from different stages of gestation in cattle can secrete oxytocin and suggest a role for relaxin in the regulation of oxytocin release.

scholarly journals The Molecular ‘Myc-anisms’ behind Myc-Driven Tumorigenesis and the Relevant Myc-Directed Therapeutics

2020 ◽  
Vol 21 (24) ◽  
pp. 9486
Author(s):  
Jessica McAnulty ◽  
Analisa DiFeo
Keyword(s):  
Cell Cycle ◽  
Protein Stability ◽  
Cell Cycle Regulation ◽  
Binding Pocket ◽  
Therapeutic Strategies ◽  
Drug Binding ◽  
Therapeutic Approaches ◽  
Cell Processes ◽  
Cycle Regulation

MYC, a well-studied proto-oncogene that is overexpressed in >20% of tumors across all cancers, is classically known as “undruggable” due to its crucial roles in cell processes and its lack of a drug binding pocket. Four decades of research and creativity led to the discovery of a myriad of indirect (and now some direct!) therapeutic strategies targeting Myc. This review explores the various mechanisms in which Myc promotes cancer and highlights five key therapeutic approaches to disrupt Myc, including transcription, Myc-Max dimerization, protein stability, cell cycle regulation, and metabolism, in order to develop more specific Myc-directed therapies.

Epilogue

2019 ◽  
pp. 244-248
Author(s):  
Geoffrey E. Hill
Keyword(s):  
Sexual Selection ◽  
Mitochondrial Genome ◽  
21St Century ◽  
Evolutionary Ecology ◽  
Early Years ◽  
Mitochondrial Genomes ◽  
Respiratory Enzymes ◽  
The Core ◽  
The Past ◽  
Full Consideration

Evolutionary ecology is at the precipice of a paradigm shift. For many years and through the early years of the 21st century, mitochondrial genomes were dismissed as unimportant to the evolution of complex life. Variation within mitochondrial genomes was proposed to be functionally neutral. These conceptions about mitochondrial genomes and mitonuclear genomic interactions have begun to change within the past decade, but currently accepted theories of sexual selection and speciation were proposed before the discovery of the mitochondrial genome. Evolutionary ecology has yet to fully appreciate the fundamental implications of two genomes coding for the core respiratory enzymes of eukaryotes. This chapter promotes a fundamental rethinking of key theories in evolutionary ecology with full consideration of the necessity of coadaptation of mitochondrial and nuclear genes.

Changes in PPAR-γ Expression Are Associated with microRNA Profiles during Fetal Programming due to Maternal Overweight and Obesity

2021 ◽  
pp. 1-12
Author(s):  
Noemí Gaytán-Pacheco ◽  
Victoria Lima-Rogel ◽  
Alejandro Méndez-Mancilla ◽  
Francisco Escalante-Padrón ◽  
Juan Carlos Toro-Ortíz ◽  
...  
Keyword(s):  
Cell Cultures ◽  
Maternal Obesity ◽  
Normal Weight ◽  
Overweight And Obesity ◽  
Epigenetic Changes ◽  
Maternal Weight ◽  
Ppar Γ ◽  
Microrna Profile ◽  
In Cells

<b><i>Background:</i></b> There has been a global increase in the prevalence of obesity in pregnant women in recent years. Animal studies have shown that intrauterine environment associated with maternal obesity leads to epigenetic changes. However, the effects of epigenetic changes occurring before birth in response to maternal conditions have not been clearly characterized in humans. <b><i>Objective:</i></b> The aim of the study was to analyze peroxisome proliferator-activated receptor (PPAR)-γ expression in cell cultures from newborns from mothers with overweight and obesity, in response to in vitro metabolic challenges and their relationship with microRNA profile and cytokine expression. <b><i>Methods/Study design:</i></b> The profile of circulating microRNAs from 72 mother-child pairs (including healthy infants born to normal weight [<i>n</i> = 35], overweight [<i>n</i> = 25], and obese [<i>n</i> = 12] mothers) was determined through real-time PCR, and the PPAR-γ expression in peripheral blood mononuclear cell cultures from offspring was analyzed after in vitro challenges. <b><i>Results:</i></b> miR-146a, miR-155, and miR-378a were upregulated in overweight mothers, while miR-378a was upregulated in obese mothers compared to normal weight mothers. In children from overweight mothers, miR-155 and miR-221 were downregulated and miR-146a was upregulated, while offspring of mothers with obesity showed downregulation of miR-155, miR-221, and miR-1301. These microRNAs have direct or indirect relation with PPAR-γ expression. In vitro exposure to high triglyceride and exposure to miR-378a induced a higher expression of PPAR-γ in cells from offspring of mothers with overweight and obesity. In contrast, cells from offspring of mothers with obesity cultured with high glucose concentrations showed PPAR-γ downregulation. IL-1ß, IL-6, and TNF-α expression in cells of offspring of overweight and obese mothers differed from that of offspring of normal weight mothers. Limitation of our study is the small sample size. <b><i>Conclusion:</i></b> The blood microRNA profile, and in vitro PPAR-γ and inflammatory cytokine expression in cells of newborn infants are associated with maternal obesity indicating that epigenetic marks may be established during intrauterine development. <b><i>Key Message:</i></b> Neonatal microRNA profile is associated with maternal weight. Neonatal microRNA profile is independent of maternal microRNA profile. PPAR-γ expression in newborn cell cultures is affected by maternal weight

scholarly journals Tetramethylpyrazine blocks TFAM degradation and up-regulates mitochondrial DNA copy number by interacting with TFAM

2017 ◽  
Vol 37 (3) ◽  
Author(s):  
Linhua Lan ◽  
Miaomiao Guo ◽  
Yong Ai ◽  
Fuhong Chen ◽  
Ya Zhang ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Lon Protease ◽  
Mitochondrial Transcription Factor ◽  
Mitochondrial Dna Copy Number ◽  
Antitumour Agent ◽  
Small Molecule Compound ◽  
Mtdna Replication ◽  
Mitochondrial Transcription Factor A ◽  
In Cells

The natural small molecule compound: 2,3,5,6-tetramethylpyrazine (TMP), is a major component of the Chinese medicine Chuanxiong, which has wide clinical applications in dilating blood vessels, inhibiting platelet aggregation and treating thrombosis. Recent work suggests that TMP is also an antitumour agent. Despite its chemotherapeutic potential, the mechanism(s) underlying TMP action are unknown. Herein, we demonstrate that TMP binds to mitochondrial transcription factor A (TFAM) and blocks its degradation by the mitochondrial Lon protease. TFAM is a key regulator of mtDNA replication, transcription and transmission. Our previous work showed that when TFAM is not bound to DNA, it is rapidly degraded by the ATP-dependent Lon protease, which is essential for mitochondrial proteostasis. In cultured cells, TMP specifically blocks Lon-mediated degradation of TFAM, leading to TFAM accumulation and subsequent up-regulation of mtDNA content in cells with substantially low levels of mtDNA. In vitro protease assays show that TMP does not directly inhibit mitochondrial Lon, rather interacts with TFAM and blocks degradation. Pull-down assays show that biotinylated TMP interacts with TFAM. These findings suggest a novel mechanism whereby TMP stabilizes TFAM and confers resistance to Lon-mediated degradation, thereby promoting mtDNA up-regulation in cells with low mtDNA content.

scholarly journals Novel Lines of Evidence for the Asymmetric Strand Displacement Model of Mitochondrial DNA Replication

2018 ◽  
Vol 39 (2) ◽  
Author(s):  
Chih-Lin Hsieh
Keyword(s):  
Mitochondrial Dna ◽  
Mitochondrial Genome ◽  
Nuclear Genome ◽  
Strand Displacement ◽  
Single Stranded Dna ◽  
Quantitative Evidence ◽  
Qualitative And Quantitative ◽  
Mtdna Replication ◽  
Displacement Model

ABSTRACT The mitochondrial genome, which consists of 16,569 bp of DNA with a cytosine-rich light (L) strand and a heavy (H) strand, exists as a multicopy closed circular genome within the mitochondrial matrix. The machinery for replication of the mammalian mitochondrial genome is distinct from that for replication of the nuclear genome. Three models have been proposed for mitochondrial DNA (mtDNA) replication, and one of the key differences among them is whether extensive single-stranded regions exist on the H strand. Here, three different methods that can detect single-stranded DNA (ssDNA) are utilized to identify the presence, location, and abundance of ssDNA on mtDNA. Importantly, none of these newly described methods involve the complication of prior mtDNA fractionation. The H strand was found to have extensive single-stranded regions with a profile consistent with the strand displacement model of mtDNA replication, whereas single strandedness was predominantly absent on the L strand. These findings are consistent with the in vivo occupancy of mitochondrial single-stranded DNA binding protein reported previously and provide strong new qualitative and quantitative evidence for the asymmetric strand displacement model of mtDNA replication.

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