Получение и характеристика рекомбинантной глутатионтрансферазы Р1 человека и скрининг новых ингибиторов фермента

Human glutathione transferase P1 (GSTP1) plays an important role in the second phase of xenobiotic biotransformation and in the regulation of apoptotic signal pathways. Directed screening of new enzyme inhibitors is an actual task since selective suppression of GSTP1 activity in tumor cells may substantially increase their sensitivity to chemotherapy. Known methods to obtain recombinant GSTP1 with a hexahistidine tag in the structure are complex, laborious, and suffer from significant losses of the enzyme activity. With the aim to create a simple and effective bacterial expression system for tagless GSTP1 posessing native structure and high activity, in the present work the full-length gstp1 gene was cloned into the pTXB1 plasmid vector, followed by transformation of E. coli cells. The optimized expression level amounted to 30–32 mg of the enzyme per liter of broth. Using glutathione-containing affinity membranes, the purified enzyme was isolated from bacterial lysate with the yield of 75.7 % and specific activity of 102.6 U/mg protein. The enzyme homogeneity was confirmed by gel-electrophoretic and mass-spectrometric data. Physico-chemical and catalytic properties of recombinant GSTP1 practically coincided with those of the native erythrocytary enzyme. The results of in silico and in vitro screening allowed to reveal structural factors and interactions determining the efficiency of the enzyme inhibition by carbocyclic and N-heterocyclic ligands. The preferable orientation of “good” inhibitors in the GSTP1 H-site was also established. Three strong enzyme inhibitors were found: 1,10-phenanthroline-5,6-dione, Alizarin Red S, and indigo carmine, with their respective IC50 values of 31, 16 and 2.3 μM. The new inhibitors are of certain interest for the development of novel lead structures with potential antitumor activity.Глутатионтрансфераза Р1 человека (GSTP1) играет важную роль во второй фазе биотрансформации ксенобиотиков и в регуляции апоптотических сигнальных путей. Направленный скрининг новых ингибиторов фермента является актуальной задачей, так как избирательное подавление активности GSTP1 в опухолевых клетках может существенно повысить их чувствительность к химиотерапии. Известные методы получения рекомбинантной GSTP1 с гексагистидиновым тэгом в структуре отличаются сложностью, трудоемкостью и значительными потерями активности фермента. С целью создать простую и эффективную систему бактериальной экспрессии бестэговой GSTP1 с нативной структурой и высокой активностью, в настоящей работе проведено клонирование полноразмерного гена gstp1 в плазмидный вектор pTXB1 с последующей трансформацией клеток E. coli. Оптимизированный уровень экспрессии составил 30–32 мг фермента/л среды. С использованием глутатионсодержащих аффинных мембран из бактериального лизата выделен очищенный фермент (выход 75,7 %, удельная активность 102,6 Ед/мг белка). Гомогенность препарата подтверждена данными гель-электрофореза и масс-спектрометрии. Физико-химические и каталитические свойства рекомбинантной GSTP1 практически совпали с таковыми для нативного фермента из эритроцитов. По результатам скрининга in silico и in vitro выявлены структурные факторы и взаимодействия, определяющие эффективность ингибирования фермента карбо- и N-гетероциклическими лигандами. Установлена предпочтительная ориентация «хороших» ингибиторов в Н-сайте GSTP1. Обнаружены новые ингибиторы фермента: 1,10-фенантролин-5,6-дион, ализариновый красный С и индигокармин с величиной IC50 соответственно 31, 16 и 2,3 мкМ. Найденные соединения представляют интерес для создания новых лидерных структур с потенциальной противоопухолевой активностью

Evidence of High Harmonics from Echo-Enabled Harmonic Generation for Seeding X-Ray Free Electron Lasers

Echo-enabled harmonic generation free electron lasers hold great promise for the generation of fully coherent radiation in x-ray wavelengths. Here we report the first evidence of high harmonics from the echo-enabled harmonic generation technique in the realistic scenario where the laser energy modulation is comparable to the beam slice energy spread. In this experiment, coherent radiation at the seventh harmonic of the second seed laser is generated when the energy modulation amplitude is about 2-3 times the slice energy spread. The experiment confirms the underlying physics of echo-enabled harmonic generation and may have a strong impact on emerging seeded x-ray free electron lasers that are capable of generating laserlike x rays which will advance many areas of science

Surgical aspects of full-thickness skin autograft engraftment on a granulating wound

Background. The paper presents the clinical results of treating patients using full-thickness skin autografts for granulating wounds.Objective. To study the surgical aspects of full-thickness skin autograft engraftment on a granulating wound.Material and Methods. In traumatology and burn research, to achieve the best cosmetic results, transplantation methods of free full-thickness skin autografts are used. In the Regional Clinical Hospital no. 1, a method of skin grafting with a full-thickness autograft was developed, which allows to close the defect in the conditions of a purulent wound: granulations are excised before skin transplantation, and a vacuum is applied after grafting.Results. Annually, the specialists of the Burn Center of the Scientific Research Institute – Regional Clinical Hospital no. 1 assist more than 1300 patients. Of these, from 20 to 25 cases are face burns. 132 patients with burns of the face have been admitted to the hospital over the past 10 years. 37 patients underwent plastic surgery with a full-thickness skin autograft. The authors presented the technology for the surgical treatment of deep burns on the face. After cleansing the wound from areas of necrosis and granulations, usually 20 days after the injury, the upper layers of granulation tissue are removed with a dermatome parallel to the skin surface, to a depth of 1–2 mm. Then, plastic surgery with the free full-thickness skin autograft is carried out on the skin of the face with the formation of cuts for the nose, mouth, eyes. The wound is tightly bandaged with 5–7 rounds of a medical bandage or a vacuum-assisted closure is applied.Conclusion. In case of traumatic skin detachment, plastic surgery according to Krasovitov should be performed in the first hours from the moment of injury. Our study allows transplantation of a full-thickness skin graft to granulating wounds as well. After 5 days, the condition of skin autografts is assessed at the first dressing. Their engraftment is observed on the 7th day. In the postoperative period, scar tissue does not form

A proof-of-principle echo-enabled harmonic generation experiment at SLAC

In this paper we describe the technical design of an ongoing proof-of-principle echo-enabled harmonic generation (EEHG) experiment at the Next Linear Collider Test Accelerator (NLCTA) at SLAC. We present the design considerations and the technical details of the experiment

Effective Scenario of Loop Quantum Cosmology

Semiclassical states in isotropic loop quantum cosmology are employed to show that the improved dynamics has the correct classical limit. The effective Hamiltonian for the quantum cosmological model with a massless scalar field is thus obtained, which incorporates also the next to leading order quantum corrections. The possibility that the higher order correction terms may lead to significant departure from the leading order effective scenario is revealed. If the semiclassicality of the model is maintained in the large scale limit, there are great possibilities for $k=0$ Friedmann expanding universe to undergo a collapse in the future due to the quantum gravity effect. Thus the quantum bounce and collapse may contribute a cyclic universe in the new scenario.Comment: 4 pages, 2 figures; version published in PR

Characterization of Stem-Like Cells in Mucoepidermoid Tracheal Paediatric Tumor

Stem cells contribute to regeneration of tissues and organs. Cells with stem cell-like properties have been identified in tumors from a variety of origins, but to our knowledge there are yet no reports on tumor-related stem cells in the human upper respiratory tract. In the present study, we show that a tracheal mucoepidermoid tumor biopsy obtained from a 6 year-old patient contained a subpopulation of cells with morphology, clonogenicity and surface markers that overlapped with bone marrow mesenchymal stromal cells (BM-MSCs). These cells, designated as MEi (mesenchymal stem cell-like mucoepidermoid tumor) cells, could be differentiated towards mesenchymal lineages both with and without induction, and formed spheroids in vitro. The MEi cells shared several multipotent characteristics with BM-MSCs. However, they displayed differences to BM-MSCs in growth kinectics and gene expression profiles relating to cancer pathways and tube development. Despite this, the MEi cells did not possess in vivo tumor-initiating capacity, as proven by the absence of growth in situ after localized injection in immunocompromised mice. Our results provide an initial characterization of benign tracheal cancer-derived niche cells. We believe that this report could be of importance to further understand tracheal cancer initiation and progression as well as therapeutic development

CLINICAL EFFECTS OF MESENCHYMAL STROMAL CELLS IN LYMPHOMA PATIENTS WITH AUTOLOGOUS HEMATOPOIETIC STEM CELL TRANSPLANTATION

The clinical and laboratory effects of bone-marrow derived mesenchymal stromal cells (MSC) in patients with malignant lymphomas following autologous hematopoietic stem cell transplantation (auto-HSCT) have been investigated. Co-transplantation of MSC in average dose of 0,178 106/kg was conducted in 74 patients with auto-HSCT. The control group included 83 patients eligible for standard HSCT. We revealed the decreasing of the period of neutropenia and thrombocytopenia when hematopoietic stem cells were co-transplanted with low doses ex vivo expanded autologous MSC. Patients with MSC co-transplantation were differed by more effective early lymphocyte recovery. At the same time MSC co-transplantation did not increase the incidence of infectious complications and cases of renal and. hepatic toxicity. Patients with MSC co-transplantation did not differ from opposite group by 5-year overall survival, but were characterized by significantly better progression-free survival

THE COMPARATIVE ANALYSIS OF GRAFT CELL SUBTYPES AND ITS CYTOKINE PRODUCTION OF LYMPHOMA AND LEUCOSIS PATIENTS

Cell subtypes and cytokine profile of apheresis products collected from lymphoma and acute leucosis patients were analyzed. It was shown, that acute leucosis patients' grafts contained higher relative numbers of naïve T-cells, CD4+CD25high T-cells, T-lymphocytes (non-significant trend) and lower counts of granulocytes. Significant increase of relative numbers of dividing CD34+ cells (in S, G2/M phases of the cell cycle) was demonstrated, in acute leucosis patients' grafts. In lymphoma grafts the levels of CD34+ cells in G0/G1 phases were found, to be increased. Cells isolated from grafts of acute leucosis patients characterized by higher levels of proinflammatory cytokines production, such as IL-1, IL-6, MIP-1β, TNF-α, IL-8, IFN-γ (the last two ones— non-significant trend) and cytokines, essential for humoral immune response (IL-4 and — in trend — IL-10). The existing differences didn't influence on effectiveness of early lymphocyte recovery