Myocardial infarction is one of the most serious and widespread diseases in the world. In this work, a minimally invasive method for simulating myocardial infarction in mice is described in the Russian Federation for the very first time; the procedure is carried out by ligation of the coronary heart artery or by controlled electrocoagulation. As a part of the methodology, a series of anesthetic, microsurgical and revival protocols are designed, owing to which a decrease in the postoperational mortality from the initial 94.6 to 13.6% is achieved. ECG confirms the development of large-focal or surface myocardial infarction. Postmortal histological examination confirms the presence of necrosis foci in the heart muscles of 87.5% of animals. Altogether, the medical data allow us to conclude that an adequate mouse model for myocardial infarction was generated. A further study is focused on the standardization of the experimental procedure and the use of genetically modified mouse strains, with the purpose of finding the most efficient therapeutic approaches for this disease.
Метилирование является общим для всех позвоночных организмов процессом, одна из основных функций которого заключается в том, чтобы фиксировать транскрипционно неактивное состояние генов. Это может происходить, в частности, благодаря специальным белкам, которые специфично узнают метилированные районы ДНК и привлекают к ним белковые комплексы, репрессирующие транскрипцию. Белок Каизо является одним из таких специализированных белков и содержит два функциональных домена: N-концевой домен BTB/POZ и три “цинковых пальца” С2Н2-типа на С-конце, которыми он связывается с метилированной ДНК и привлекает к ней репрессионные комплексы за счет взаимодействия BTB/POZ-домена с корепрессором NCoR. В некоторых клеточных линиях позвоночных ортологи Каизо взаимодействуют с катенином р120, который, хотя и является преимущественно цитоплазматическим белком, иногда обнаруживается в ядре. Цель настоящего исследования определение параметров взаимодействия белков Каизо и катенина р120, а также изучение функциональных последствий образования такого белкового комплекса с точки зрения транскрипционного контроля метилированных генов. Мы установили, что второй и третий “цинковые пальцы” белка Каизо необходимы и достаточны для взаимодействия с катенином р120. При взаимодействии с катенином р120 происходит два молекулярных события, которые приводят к инактивации Каизо как транскрипционного фактора. Во-первых, катенин р120 маскирует сигнал ядерной локализации Каизо, и Каизо в составе комплекса с р120 мигрирует из ядра в цитоплазму; и, во-вторых, связывание с р120 делает невозможным взаимодействие Каизо с метилированной ДНК. Таким образом, С-концевой домен белка Каизо выполняет две важные функции: связывание с ДНК и взаимодействие с цитоплазматическим белком катенином р120. Подробное изучение данного взаимодействия позволит определить новые механизмы ядерно-цитоплазматических сигнальных путей в клетках млекопитающих.
Gain and loss of DNA methylation in cells is a dynamic process that tends to achieve an equilibrium. Many factors are involved in maintaining the balance between DNA methylation and demethylation. Previously, it was shown that methyl-DNA protein Kaiso may attract NCoR, SMRT repressive complexes affecting histone modifications. On the other hand, the deficiency of Kaiso resulted in reduced methylation of ICR in
Human cancer cells are subjected to hypoxic conditions in many tumours. Hypoxia causes alterations in the glycolytic pathway activation through stabilization of hypoxia-inducible factor 1. Currently, two approaches are commonly used to model hypoxia: an alternative to generating low-oxygen conditions in an incubator, cells can be treated with CoCl2. We performed RNA-seq experiments to study transcriptomes of human Caki-1 cells under real hypoxia and after CoCl2 treatment. Despite causing transcriptional changes of a much higher order of magnitude for the genes in the hypoxia regulation pathway, CoCl2 treatment fails to induce alterations in the glycolysis / gluconeogenesis pathway. Moreover, CoCl2 caused aberrant activation of other oxidoreductases in glycine, serine and threonine metabolism pathways.
Uncontrolled growth in the cell mass of malignant tumors induces intensive angiogenesis. However, the demands of the cancer cells for nutrients and oxygen remain only partially met. Hypoxia is a process that accompanies malignant transformation and evokes changes in the DNA methylation profile in solid tumors. To a certain extent, these changes, including the hypermethylation of tumor suppressor gene promoters, are related to the decrease in the activity of Tet proteins under the conditions of oxygen and free radical deficit. Stabilization, accumulation, and nuclear translocation of the transcription factor HIF1α are the key molecular events in hypoxia. We modified the clear-cell renal cancer cell line Caki1 to stabilize the HIF1α protein and characterized a model cell line that will enable the studies of the mechanisms of changes of the DNA methylation level at a constant activity of Tet proteins and a gene transcription profile characteristic of hypoxia. The CRISPR/Cas9 DNA editing system was used to edit the VHL gene. The mutant VHL protein contained a disrupted alpha-helix at the C-terminus and could not participate in the molecular pathway of proteasomal degradation of the HIF1α factor; therefore, the latter accumulated in the nucleus and activated the specific target genes. An analysis of gene transcription revealed the induction of hypoxia-associated genes in the modified cell line. The developed Сaki-1/VHLmut model can be used to discriminate between the effects evoked by oxygen-suppressed hydroxylases of the Tet family and other hypoxia-associated mechanisms of DNA methylation/demethylation.
During gastrulation and neurulation, the chordamesoderm and overlying neuroectoderm of vertebrate embryos converge under the control of a specific genetic programme to the dorsal midline, simultaneously extending along it. However, whether mechanical tensions resulting from these morphogenetic movements play a role in long-range feedback signaling that in turn regulates gene expression in the chordamesoderm and neuroectoderm is unclear. In the present work, by using a model of artificially stretched explants of
In this study, the optimized method for designing IgG-binding magnetosomes based on integration of IgG-binding fusion proteins into magnetosome membrane in vitro is presented. Fusion proteins Mbb and Mistbb consisting of magnetosome membrane protein MamC and membrane associating protein Mistic from Bacillus subtilis as anchors and BB-domains of Staphylococcus aureus protein A as IgG-binding region were used. With Response Surface Methodology (RSM) the highest level of proteins integration into magnetosome membrane was achieved under the following parameters: pH 8.78, without adding NaCl and 55 s of vortexing for Mbb; pH 9.48, 323 mM NaCl and 55 s of vortexing for Mistbb. Modified magnetosomes with Mbb and Mistbb displayed on their surface demonstrated comparable levels of IgG-binding activity, suggesting that both proteins could be efficiently used as anchor molecules. We also demonstrated that such modified magnetosomes are stable in PBS buffer during at least two weeks. IgG-binding magnetosomes obtained by this approach could serve as a multifunctional platform for displaying various types of antibodies.
VHL inactivation is a key oncogenic event for renal carcinomas. In normoxia, VHL suppresses HIF1a-mediated transcriptional response, which is characteristic to hypoxia. It has previously been shown that hypoxic conditions inhibit TET-dependent hydroxymethylation of cytosines and cause DNA hypermethylation at gene promoters. In this work, we performed VHL inactivation by CRISPR/Cas9 and studied its effects on gene expression and DNA methylation. We showed that even without hypoxia, VHL inactivation leads to hypermethylation of the genome. Hypermethylated cytosines were evenly distributed throughout the genome with a slight preference for AP-1 (JUN and FOS) binding sites. Hypermethylated cytosines tended to be enriched within the binding sites of transcription factors that showed increased gene expression after VHL inactivation. We also observed promoter hypermethylation associated with decreased gene expression for several regulators of transcription and DNA methylation including SALL3.
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