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

High-brightness electron beam evolution following laser-based cleaning of a photocathode

Laser-based techniques have been widely used for cleaning metal photocathodes to increase quantum efficiency (QE). However, the impact of laser cleaning on cathode uniformity and thereby on electron beam quality are less understood. We are evaluating whether this technique can be applied to revive photocathodes used for high-brightness electron sources in advanced x-ray free-electron laser (FEL) facilities, such as the Linac Coherent Light Source (LCLS) at the SLAC National Accelerator Laboratory. The laser-based cleaning was applied to two separate areas of the current LCLS photocathode on July 4 and July 26, 2011, respectively. The QE was increased by 8–10 times upon the laser cleaning. Since the cleaning, routine operation has exhibited a slow evolution of the QE improvement and comparatively rapid improvement of transverse emittance, with a factor of 3 QE enhancement over five months, and a significant emittance improvement over the initial 2–3 weeks following the cleaning. Currently, the QE of the LCLS photocathode is holding constant at about 1.2×10^{-4}, with a normalized injector emittance of about 0.3  μm for a 150-pC bunch charge. With the proper procedures, the laser-cleaning technique appears to be a viable tool to revive the LCLS photocathode. We present observations and analyses for the QE and emittance evolution in time following the laser-based cleaning of the LCLS photocathode, and comparison to the previous studies, the measured thermal emittance versus the QE and comparison to the theoretical model

Non-equivalence of a and b subunits within human hemoglobin in conformational relaxation and molecular oxygen rebinding

В работе изучены неэквивалентность субъединиц а и b в гемоглобине человека, конформационная релаксация и повторное связывание молекулярного кислорода

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

ПОЛУЧЕНИЕ ВЫСОКОАКТИВНОЙ ГЛУТАТИОН-S-ТРАНСФЕРАЗЫ Р1-1 ИЗ ЭРИТРОЦИТОВ ЧЕЛОВЕКА С ПОМОЩЬЮ АФФИННЫХ МЕМБРАН И СВОЙСТВА ОЧИЩЕННОГО ФЕРМЕНТА

Human glutathione S-transferase P1-1 (GSTP1-1) is an important enzyme in clinical diagnostics since its overexpression in solid tumours and lymphomas largely contributes to multiple drug resistance of cancer cells. Small amounts of the enzyme can also be isolated from erythrocytes. However, known isolation methods are disadvantageous and do not allow to obtain the purified enzyme in good yield and with high specific activity (≥ 100 U/mg protein). In order to elaborate more effective purification procedure for the erythrocyte GSTP1-1, we have for the first time synthesized and investigated glutathione-containing affinity membranes based on chemically modified cellulose paper. The membranes have been shown to successfully replace conventional glutathione-agarose affinity gels in the enzyme purification. To isolate and purify GSTP1-1 from erythrocyte hemolysate, a novel two-step method has been developed involving preliminary hemoglobin removal on a small anion exchange column and further affinity chromatography on the cartridge with glutathione-containing membranes. In terms of activity yield (76.5%), purification factor (23589-fold), and specific activity of the purified enzyme (104.5 U/mg), the method is notably superior to previously published procedures. Gel electrophoretic and MALDI-TOF mass spectrometric analyses reveal apparent homogeneity (≥ 95%) of the obtained preparation. Steady-state kinetic parameters have been determined for the purified GSTP1-1 in the conjugation reaction between glutathione and 1-chloro-2,4-dinitrobenzene at pH 6.5 and 25 °С: for the former substrate, Km = 0.19 mМ, kcat = 47.8 s–1; for the latter, Km = 0.68 mМ, kcat = 54.3 s–1. The results of the present work may be useful for screening new enzyme inhibitors with possible antitumour activity. The affinity membranes may also find application in isolating native and recombinant glutathione S-transferase (GST) isoforms from various sources, as well as fusion proteins with GST tag. Глутатион-S-трансфераза Р1-1 (GSTP1-1) человека является диагностически важным ферментом, гиперэкспрессия которого в опухолях и лимфомах приводит к формированию множественной лекарственной устойчивости опухолевых клеток. Небольшие количества фермента можно выделить из эритроцитов, однако известные методы очистки не позволяют получить препараты с высокой удельной активностью и хорошим выходом. С целью разработки более эффективной процедуры очистки эритроцитарной GSTP1-1 в настоящей работе впервые получены и охарактеризованы глутатионсодержащие аффинные мембраны на основе химически модифицированной целлюлозной бумаги. Мембраны успешно использованы для очистки фермента вместо традиционных агарозно-гелевых адсорбентов. Разработан новый метод выделения и очистки GSTP1-1 из гемолизата эритроцитов, включающий предварительное удаление гемоглобина на анионообменнике и аффинную хроматографию фермента на картридже с глутатионсодержащими мембранами. По эффективности (выход фермента 76,5 %, степень очистки 23589 раз, удельная активность 104,5 Ед/мг белка) предложенный метод значительно превосходит ранее опубликованные. Гомогенность полученного препарата, по данным гель-электрофореза и масс-спектрометрии, составляет не менее 95%. Найдены стационарные кинетические параметры очищенной GSTP1-1 в реакции конъюгации глутатиона (GSH) и 1-хлор-2,4- динитробензола (CDNB) при рН 6,5 и 25 °С: для GSH Km = 0,19 мМ, kcat = 47,8 с–1; для CDNB Km = 0,68 мМ, kcat = 54,3 с–1. Результаты работы представляют интерес для скрининга новых ингибиторов фермента с противоопухолевой активностью. Полученные аффинные мембраны могут быть использованы при выделении нативных и рекомбинантных глутатион-S-трансфераз (GST) из различных источников, а также гибридных белков с GST-доменом.

Application of cells of cord blood and umbilical cord: achievements, challenges and perspectives

This review focuses on the biological aspects of the use of cord blood as a valuable source of cells. The distinctive features of hematopoietic stem cells, cells of the immune system, mesenchymal stem cells are described. The analysis of the results of clinical research and development of therapeutic approaches using cord blood cells and umbilical cord for the treatment of various diseases has been carried out.Currently, the target area of cord blood research is hematopoietic stem cell transplantation, as well as cellular immunotherapy of tumor diseases, treatment of neurological diseases and regenerative medicine

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

Platelet-rich plasma in the treatment of chronic inflammatory diseases of lower extremities

Objectives to evaluate the effectivenes of platelet-rich plasma preparations in the treatment of chronic inflammatory diseases of lower extremities. Material and methods. The study involved patients hospitalized in the Research Institute S. V. Ochapovsky Regional Clinic Hospital No.1, Krasnodar, with trophic ulcers and chronic osteomyelitis. The patients received injections of platelet-rich plasma as platelet lysate and/or platelet gel. Results. The study involved 19 patients with trophic ulcers of the lower extremities with a duration of more than 2 months. Previously, the patients had underwent therapy without effect. In all cases, the patients received a complex of therapeutic measures, including debridement, dressing, conservative treatment, physiotherapy, antibiotic therapy. In the presence of large ulcers, 8 patients underwent autodermoplasty with complete engraftment. It has been noted that during platelet-rich plasma use the average healing time was 6-7 weeks. Four patients were observed with chronic osteomyelitis. During operation, the bone cavity was filled with platelet gel. The postoperative period was uneventful in all cases. The maximum observation period was 1.5 years. No recurrence of osteomyelitis was detected. Conclusion. The results of the study demonstrate the positive effects of platelet-rich plasma products use

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