Mechanism of proteolysis in matrix metalloproteinase-2 revealed by QM/MM modeling

The mechanism of enzymatic peptide hydrolysis in matrix metalloproteinase-2 (MMP-2) was studied at atomic resolution through quantum mechanics/molecular mechanics (QM/MM) simulations. An all-atom three-dimensional molecular model was constructed on the basis of a crystal structure from the Protein Data Bank (ID: 1QIB), and the oligopeptide Ace-Gln-Gly∼Ile-Ala-Gly-Nme was considered as the substrate. Two QM/MM software packages and several computational protocols were employed to calculate QM/MM energy profiles for a four-step mechanism involving an initial nucleophilic attack followed by hydrogen bond rearrangement, proton transfer, and C—N bond cleavage. These QM/MM calculations consistently yield rather low overall barriers for the chemical steps, in the range of 5–10 kcal/mol, for diverse QM treatments (PBE0, B3LYP, and BB1K density functionals as well as local coupled cluster treatments) and two MM force fields (CHARMM and AMBER). It, thus, seems likely that product release is the rate-limiting step in MMP-2 catalysis. This is supported by an exploration of various release channels through QM/MM reaction path calculations and steered molecular dynamics simulations

Comparative study of technologies for extraction of biologically active substances from the raw material of animal origin

Technologies of isolation and concentration of biologically active substances, developed in the middle of the 20th century, need adjustment and adaptation to modern conditions both to increase the activity of substances and for greater economic efficiency. The aim of the research is the comparison of dynamics of biologically active compounds extraction from porcines pancreas in two methods: the saline method based on 0.9% sodium chloride solution, and the acidic method based on 2.4% trichloroacetic acid solution. Also the purpose of research is to assess the possibilities for further optimization of technologies. The total protein concentration based on the biuret reaction in the samples taken during the extraction, as well as the calculation and analysis of the point degrees and rates of extraction are chosen as the controlled parameters. Local maxima of the protein yields into the extractant media at the 60th, 135th and 255th minute were recorded during saline extraction; and at the 75th and 135th minute during acid extraction. Also the proteomic profile of the extracts was studied. Wide range of compounds with molecular weight of less than 52 kDa was found in extracts based on physiological saline solution, and protein substances of whole presented range of molecular weights in trichloroacetic acid based extracts were considered. The predominance of low molecular weight protein fraction of interest was noted also in this method of extraction in comparison with the other methods of extraction. According to the UniProt database, we assume availability of probable compounds with a molecular weight of less than 30 kDa in the purified acidic extract. The presence of some proteins absent in the final saline extract was noted. The acidic erythrograms showed a weak degrading effect of both types of extracts on the membranes of rat erythrocytes, as well as the cytoprotective effect of acidic ultrafiltrates (less than 3 kDa). The obtained results prove a better efficiency of trichloroacetic acid extraction method used for obtaining a mixture of a wide range of compounds, including biologically active substances of low molecular weight

Evolution of in vitro digestibility techniques: a systematic review

The inability to reproduce certain digestive processes in vivo, high research costs and ethical aspects have led to the development of a large number of in vitro digestion models. These models allow us to take into account various factors of modeling complex multistage physiological processes occurring in the gastrointestinal tract, which makes them promising and widely used. A significant part of in vitro methods includes assessment by enzymatic digestion and are based on the calculation of nitrogen remaining after digestion in relation to the initial total nitrogen (according to the Dumas, Kjeldahl method, spectrophotometric or chromatographic method). There are also a number of titrometric methods (pH‑stat), which are mainly used to assess the digestibility of feed, most successfully for aquatic animals due to the simplicity of their digestive tract. Methods for assessing the digestibility of food products by enzymatic digestion have undergone various stages of evolution (since 1947) and have been widely modified by including various enzymes (pepsin, trypsin, pancreatin, erepsin, etc.) in model systems, indices for various products have been determined on their basis (pepsin-digest-residue (PDR) index, 1956; pepsin pancreatin digest (PPD) index, 1964; pepsin digest dialysate (PDD), 1989). As a result, a single protocol was formed to study the digestibility of food — INFOGEST (2014–2019), which includes three stages of digestion (oral, gastric and intestinal). It allows researchers to accurately reproduce the conditions of the human gastrointestinal tract and is widely used by scientists around the world

ОПТИЧЕСКАЯ СИСТЕМА НА ОСНОВЕ ЛАЗЕРНОГО СВЕТОДИОДА ДЛЯ КОНТРОЛЯ ДИНАМИКИ КРОВОТОКА И ФУНКЦИОНАЛЬНОГО СОСТОЯНИЯ МЫЩЦ В БИОЛОГИЧЕСКИХ ТКАНЯХ

The blood flow in the skin of the temporal region and the functional state of the muscles were investigated using the speckle-optical method. It is shown that the speckle-optical method can be used to objectify the skin blood flow and to evaluate vascular reactivity during breath-holding and hyperventilation. Obtained objective data of the tonic state of the anterior tibial muscle of healthy people can be used as indicators of the norm in the clinic for comparison with the results of examinations of patients with diseases of the motor sphere and the pathology of the neuromuscular apparatus, as well as in sports medicine.С использованием спекл-оптического метода исследованы кровоток в кожных покровах височной области и функциональное состояние мышц. Показано, что спекл-оптический метод может применяться для объективизации кожного кровотока и оценки сосудистой реактивности при задержке дыхания и гипервентиляции. Полученные объективные данные тонического состояния передней большеберцовой мышцы здоровых людей могут быть использованы в клинике в качестве показателей нормы при сравнения с результатами обследования пациентов с заболеваниями двигательной сферы и патологией нервно-мышечного аппарата, а также в спортивной медицине

Водно-солевая экстракция как метод получения смеси биологически активных соединений белковой природы из поджелудочной железы свиньи

A relevant solution to the problem of processing meat industry waste in Russia is to obtain useful biologically active compounds from abundant organs. The aim of this study was to examine the effectiveness of the saline extraction as a method for extracting a mixture of promising biologically active compounds from the porcine pancreas, as well as to determine the optimal time for the process. The study consisted of extraction of the porcine pancreas with 0,9% sodium chloride solution for 5 h 30 min with further determination of the total protein concentration and proteomic profile of the samples taken throughout the process. Based on the analysis of the dependence of the total protein content in the extractant on time, the optimal extraction time was determined to be 135–150 minutes. When studying the results of electrophoresis and the data of their processing, the optimal extraction time for the targeted isolation of the low-molecular fraction of compounds was also determined to be 90 min. At the same time, 13 protein bands with a molecular weight of 52 kDa and below were found on the electropherograms. Saline should be considered applicable for obtaining extracts rich in biologically active substances, incl. hormones, enzymes and other physiologically active compounds.Актуальным решением проблемы переработки отходов мясной промышленности в России является получение полезных биологически активных соединений из богатых ими органов. Целью настоящего исследования было изучение эффективности метода экстракции физиологическим раствором как способа извлечения смеси перспективных биологически активных соединений из поджелудочной железы свиньи, а также определение оптимального времени процесса. Исследование заключалось в проведении экстракции поджелудочной железы 0,9% раствором натрия хлорида в течение 5 ч 30 мин с дальнейшим определением общей концентрации белка биуретовым методом. Также получен протеомный профиль образцов, отбираемых на протяжении всего процесса, методом одномерного денатурирующего электрофореза по Лэммли в 12,5% полиакриламидном геле. На основе анализа зависимости содержания общего белка в экстрагенте от времени определено оптимальное время экстракции, которое составило 135–150 мин. По результатам электрофореза и данных биоинформационного анализа оптимальное время экстракции для целенаправленного выделения низкомолекулярной фракции соединений составляло 90 мин. На электрофореграммах обнаружены 13 белковых полос с молекулярной массой 52 кДа и ниже. Таким образом, 0,9% раствор натрия хлорида применим для получения экстрактов, богатых биоактивными веществами, в том числе гормонами, ферментами и другими физиологически активными соединениями

СПЕКЛ-ОПТИЧЕСКАЯ ХАРАКТЕРИСТИКА МИКРОГЕМОДИНАМИКИ КОЖНЫХ ПОКРОВОВ ВИСОЧНОЙ ОБЛАСТИ У ПАЦИЕНТОВ С АРТЕРИАЛЬНЫМИ АНЕВРИЗМАМИ

The possibility of application of the speckle-optical method for the objectivization of cutaneous blood flow and evaluation of vascular reactivity with respiratory arrest and hyperventilation is shown. 21 patients with arterial aneurysms (AA) of the brain were examined. Recording of blood flow was carried out on skin of the temporal region on both sides. Violation of vascular reactivity during respiratory tests in the form of development of paradoxical reactions or reduction of adequate responses to HF and HB on the side with AA has been established. The most informative are the power of the spectrum and the mean frequency of the spectrum.Показана возможность применения спекл-оптического метода для объективизации кожного кровотока и оценки сосудистой реактивности при задержке дыхания (ЗД) и гипервентиляции (ГВ). Обследован 21 пациент с артериальными аневризмами (АА) головного мозга. Запись кровотока проводили в кожных покровах височной области с обеих сторон. Установлено нарушение сосудистой реактивности при проведении дыхательных проб в виде развития парадоксальных реакций или снижения адекватных реакций на ЗД и ГВ на стороне с АА. Наиболее информативными являются мощность спектра и средняя частота спектра

Novel Oncogenic Transcription Factor Cooperation in RB-Deficient Cancer

The retinoblastoma tumor suppressor (RB) is a critical regulator of E2F-dependent transcription, controlling a multitude of protumorigenic networks including but not limited to cell-cycle control. Here, genome-wide assessment of E2F1 function after RB loss in isogenic models of prostate cancer revealed unexpected repositioning and cooperation with oncogenic transcription factors, including the major driver of disease progression, the androgen receptor (AR). Further investigation revealed that observed AR/E2F1 cooperation elicited novel transcriptional networks that promote cancer phenotypes, especially as related to evasion of cell death. These observations were reflected in assessment of human disease, indicating the clinical relevance of the AR/E2F1 cooperome in prostate cancer. Together, these studies reveal new mechanisms by which RB loss induces cancer progression and highlight the importance of understanding the targets of E2F1 function. SIGNIFICANCE: This study identifies that RB loss in prostate cancer drives cooperation between AR and E2F1 as coregulators of transcription, which is linked to the progression of advanced disease

Ассоциация полиморфных вариантов генов ферментов биотрансформации ксенобиотиков с восприимчивостью к заболеваемости туберкулезом легких

The objective: to investigate the relationship of polymorphic variants of genes of xenobiotic biotransformation metabolism enzymes (NAT2 (590G>A (rs1799930)), CYP2E1 (9896C>G (rs2070676)), ABCB1 (3435T>C (rs1045642)), GSTM1 (E/D), GSTT1 (E/D) with the risk of pulmonary tuberculosis.Subjects and Methods. Within the framework of this study, a population sample of unrelated 1081 individuals of Slavic nationalities (mainly Russians) living in the territory of Kursk Oblast was used.Results. The del/del (D/D) GSTM1 genotype was associated with an increased risk of developing tuberculosis, while carrying the del/del (D/D) GSTT1 genotype was associated with a lower risk of developing the disease. Polymorphism 3435T>C ABCB1 (TC genotype) was associated with increased susceptibility to pulmonary tuberculosis in the codominization model, while TT+TC genotypes had the same association in the dominance model.Цель исследования: исследовать взаимосвязь полиморфных вариантов генов ферментов метаболизма биотрансформации ксенобиотиков (NAT2 (590G>A (rs1799930)), CYP2E1 (9896C>G (rs2070676)), ABCB1 (3435T>C (rs1045642)), GSTM1 (E/D), GSTT1 (E/D) с риском развития туберкулеза легких.Материал и методы. В рамках исследования использовалась гомогенная по этническому составу популяционная выборка неродственных индивидов славянских национальностей (преимущественно русских), проживающих на территории Курской области, общей численностью 1 081 индивид.Результаты. Генотип del/del (D/D) GSTM1 ассоциировался с повышенным риском развития туберкулеза, тогда как носительство генотипа del/del (D/D) GSTT1 было ассоциировано с пониженным риском развития болезни. Полиморфизм 3435T>C ABCB1 (генотип TС) ассоциировался с повышенной восприимчивостью к туберкулезу легких в рамках модели кодоминирования, а генотипы ТТ+ТС в рамках модели доминирования