Менингококковая поливакцина на основе очищенной IgAI протеазы

IgA1-protease allocated from the culture N. meningitidis serogroup A. As original materials were used three different intermediate products of vaccine production: cultural fluid, cetavlon supernatant and cetavlon precipitate. IgA1-protease was used to evaluate their protectivity and immunogenity. It was shown, that isolated IgA1 protease from the meningococcus serogroup A is able to protect mice, infected by meningococcus serogroup B.IgA1 протеаза, выделенная методами препаративной химии из промежуточных продуктов очистки менингококковой полисахаридной серогруппы А вакцины, обладает выраженной иммуногенной и протективной активностью для лабораторных мышей, обеспечивая их защиту от смертельного заражения вирулентными штаммами серогрупп А и В менингококков. Протективный эффект и нарастание анти-IgA1 антител позволяют прогнозировать вакцинные потенции изучаемого препарата, который может рассматриваться, в первую очередь, в качестве кандидата отсутствующей до настоящего времени менингококковой серогруппы В вакцины

Менингококковые вакцины: от капсульных полисахаридов до протеаз

Microbial capsular polysaccharides for many years provided a highly practical public health vaccines for preventing meningococcal, pneumococcal and Haemophilus influenza infection, and typhoid fever. Their application in the form of conjugates with protein carriers eliminate the gap in protection against these infections in children under one year. Extremely promising turned out offered us a new generation of vaccines, which have synthetic peptides conjugated to a meningococcal polysaccharide. Thus, new approaches to the solution of the problem of meningococcal disease vaccination serogroup B were open. In recent years, Russian researchers first suggested to use IgA1 protease (one of the major virulence factors of microbes and almost identical for mentioned below infections) for prevention of such diseases as meningococcal of all serogroups, pneumococcus and hemophilia infections. Patented processes for producing of the vaccine define domestic priority of its production and use.Микробные капсульные полисахариды на протяжении многих лет обеспечивали практическое здравоохранение высокоэффективными вакцинами для профилактики менингококковых, пневмококковых, гемофильных инфекций и брюшного тифа. А их применение в виде конъюгатов с белковыми носителями ликвидировало пробел при защите от этих инфекций детей в возрасте до одного года. Необычайно перспективным оказалось предложенное нами новое поколение вакцин, где уже синтетические пептиды конъюгировались с полисахаридами менингококков. Таким образом, были открыты подходы к решению проблемы вакцинопрофилактики менингококковой инфекции серогруппы В. В последние годы отечественные исследователи впервые предложили использовать для профилактики менингококковых инфекций всех серогрупп, а также инфекций, обусловленных пневмококками и гемофилами, один из главных факторов вирулентности этих микробов — IgA1 протеазу, практически идентичную для всей указанной группы инфекций. Запатентованные способы получения этой вакцины определяют отечественный приоритет ее производства и применения

1-(3-tert-butylphenyl)-2,2,2-trifluoroethanone as a potent transition-state analogue slow-binding inhibitor of human acetylcholinesterase: Kinetic, MD and QM/MM studies

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. Kinetic studies and molecular modeling of human acetylcholinesterase (AChE) inhibition by a fluorinated acetophenone derivative, 1-(3-tert-butylphenyl)-2,2,2-trifluoroethanone (TFK), were performed. Fast reversible inhibition of AChE by TFK is of competitive type with Ki = 5.15 nM. However, steady state of inhibition is reached slowly. Kinetic analysis showed that TFK is a slow-binding inhibitor (SBI) of type B with Ki * = 0.53 nM. Reversible binding of TFK provides a long residence time, τ = 20 min, on AChE. After binding, TFK acylates the active serine, forming an hemiketal. Then, disruption of hemiketal (deacylation) is slow. AChE recovers full activity in approximately 40 min. Molecular docking and MD simulations depicted the different steps. It was shown that TFK binds first to the peripheral anionic site. Then, subsequent slow induced-fit step enlarged the gorge, allowing tight adjustment into the catalytic active site. Modeling of interactions between TFK and AChE active site by QM/MM showed that the “isomerization” step of enzyme-inhibitor complex leads to a complex similar to substrate tetrahedral intermediate, a so-called “transition state analog”, followed by a labile covalent intermediate. SBIs of AChE show prolonged pharmacological efficacy. Thus, this fluoroalkylketone intended for neuroimaging, could be of interest in palliative therapy of Alzheimer’s disease and protection of central AChE against organophosphorus compounds

Steady-state kinetics of enzyme-catalyzed hydrolysis of echothiophate, a P–S bonded organophosphorus as monitored by spectrofluorimetry

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). Enzyme-catalyzed hydrolysis of echothiophate, a P–S bonded organophosphorus (OP) model, was spectrofluorimetrically monitored, using Calbiochem Probe IV as the thiol reagent. OP hydrolases were: the G117H mutant of human butyrylcholinesterase capable of hydrolyzing OPs, and a multiple mutant of Brevundimonas diminuta phosphotriesterase, GG1, designed to hydrolyze a large spectrum of OPs at high rate, including V agents. Molecular modeling of interaction between Probe IV and OP hydrolases (G117H butyrylcholinesterase, GG1, wild types of Brevundimonas diminuta and Sulfolobus solfataricus phosphotriesterases, and human paraoxonase-1) was performed. The high sensitivity of the method allowed steady-state kinetic analysis of echothiophate hydrolysis by highly purified G117H butyrylcholinesterase concentration as low as 0.85 nM. Hydrolysis was michaelian with Km = 0.20 ± 0.03 mM and kcat = 5.4 ± 1.6 min−1. The GG1 phosphotriesterase hydrolyzed echothiophate with a high efficiency (Km = 2.6 ± 0.2 mM; kcat = 53,400 min−1). With a kcat/Km = (2.6 ± 1.6) × 107 M−1min−1, GG1 fulfills the required condition of potential catalytic bioscavengers. quantum mechanics/molecular mechanics (QM/MM) and molecular docking indicate that Probe IV does not interact significantly with the selected phosphotriesterases. Moreover, results on G117H mutant show that Probe IV does not inhibit butyrylcholinesterase. Therefore, Probe IV can be recommended for monitoring hydrolysis of P–S bonded OPs by thiol-free OP hydrolases

A new sensitive spectrofluorimetric method for measurement of activity and kinetic study of cholinesterases

© 2019 Elsevier B.V. A new spectrofluorimetric method more sensitive than the Ellman method was developed for determination of both acetylcholinesterase and butyrylcholinesterase activity and for kinetic analysis of these enzymes and their mutants. Two selected mutants of human butyrylcholinesterase (E197Q and E197G) were included in this work. As for the Ellman's method, substrates are thiocholine esters, but the chromogenic reagent, DTNB (dithio-bisnitro benzoic acid) is replaced by a fluorogenic probe, “Calbiochem Probe IV”, (3-(7-Hydroxy-2-oxo-2H-chromen-3-ylcarbamoyl)acrylic acid methylester). Compared to the classical Ellman's method, the sensitivity of this new spectrofluorimetric assay is 2 orders of magnitude higher. The method allows measurement of activity in media containing <10−11 M of cholinesterase active sites at low substrate concentrations, either under first order conditions, [S] << Km, or under conditions where kinetics obeys the Michaelis-Menten model, i.e. at [S] < 1 mM for wild-type enzymes. The method adapted to titration plate reader assays is suitable for clinical and toxicological routine analyses, for high throughput screening of novel cholinesterase mutants and screening of inhibitor libraries of pharmacological interest

Slow-binding reversible inhibitor of acetylcholinesterase with long-lasting action for prophylaxis of organophosphate poisoning

© 2020, The Author(s). Organophosphorus (OP) compounds represent a serious health hazard worldwide. The dominant mechanism of their action results from covalent inhibition of acetylcholinesterase (AChE). Standard therapy of acute OP poisoning is partially effective. However, prophylactic administration of reversible or pseudo-irreversible AChE inhibitors before OP exposure increases the efficiency of standard therapy. The purpose of the study was to test the duration of the protective effect of a slow-binding reversible AChE inhibitor (C547) in a mouse model against acute exposure to paraoxon (POX). It was shown that the rate of inhibition of AChE by POX in vitro after pre-inhibition with C547 was several times lower than without C547. Ex vivo pre-incubation of mouse diaphragm with C547 significantly prevented the POX-induced muscle weakness. Then it was shown that pre-treatment of mice with C547 at the dose of 0.01 mg/kg significantly increased survival after poisoning by 2xLD50 POX. The duration of the pre-treatment was effective up to 96 h, whereas currently used drug for pre-exposure treatment, pyridostigmine at a dose of 0.15 mg/kg was effective less than 24 h. Thus, long-lasting slow-binding reversible AChE inhibitors can be considered as new potential drugs to increase the duration of pre-exposure treatment of OP poisoning

Клинико-функциональные характеристики вторичной географической атрофии на фоне экссудативной возрастной макулярной дегенерации

Background.Studies demonstrate the need for long-term follow-up of patients with wet age-related macular degeneration (AMD) treated with inhibitors of angiogenesis to monitor long-term vision outcomes and assess the safety of antiangiogenic therapy in relation to the risk of secondary geographic atrophy. Aims to determine the characteristic clinical and functional signs of secondary GA that developed against the background of wet AMD. Methods.In 22 patients (25 eyes) with wet AMD and 18 healthy subjects comparable in age and sex standard ophthalmological and instrumental studies were performed and photopic electroretinograms (ERGs) were recorded according to ISCEV standards, flicker-ERGs, multifocal ERGs and electrooculogram. Results.The appearance of the area of secondary atrophy against the background of wet AMD in eyes treated with inhibitors of angiogenesis is clinically indistinguishable from areas of geographic atrophy that developed as an outcome of dry AMD. The ERG-signs of secondary atrophy are described, which are similar to the biomarkers of primary atrophy and specifically differ from them. Secondary atrophy is characterized by the dependence of the increase in the b/a ratio on the atrophic area, reducing of the 8.3 Hz-flicker-ERG amplitude in the absence of 24 Hz-flicker ERG changes. In eyes with secondary atrophy, a significant decrease in the density of the multifocal ERG P1-peak was shown not only in the first hexagon but also in the parafoveal zone. The electrooculography results showed a sharper dark troughs decrease in with an increase in Ardens ratio in patients with secondary atrophya on the background of wet AMD, in contrast to the previously described changes in primary geographic atrophy. Conclusion.Comparison of the change in the b/a ratio with secondary atrophy area in patients with wet AMD may have clinical implications for assessing retinal dysfunction and predicting visual function. Secondary atrophy is associated with a pronounced inhibition of photoreceptor activity with better preservation of cone bipolar cells. The ERG and electrooculography data taking together indicate a more significant dysfunction of the retinal pigment epithelium in GA against the background of wet AMD and the associated deterioration of photoreceptor function than the changes characterizing primary geographic atrophy.Обоснование.Исследования демонстрируют необходимость длительного наблюдения пациентовсвлажной формой возрастной макулярной дегенерации (ВМД), получавших ингибиторы ангиогенеза,дляконтроля долгосрочных результатов зрительных функцийиоценки безопасности антиангиогенной терапии относительно риска развития вторичной географической атрофии. Цель исследования определить характерные клинико-функциональные признаки вторичной атрофии, развившейсянафоне влажной ВМД. Методы.У22 пациентов (25 глаз)свлажной ВМДи18 здоровых лиц, сопоставимыхповозрастуиполу, проводили стандартные офтальмологическиеиинструментальные исследованияирегистрировали фотопические электроретинограммы (ЭРГ)постандартам ISCEV, ритмические ЭРГ, мультифокальную ЭРГиэлектроокулограмму. Результаты.Внешний вид области вторичной атрофиинафоне влажной ВМДвглазах, получавших лечение ингибиторами ангиогенеза, клинически неотличимотобластей атрофии, развившейсякакисход сухой формы ВМД. Описаны ЭРГ-признаки вторичной атрофии, сходныесбиомаркерами первичной географической атрофиииспецифически отличающиесяотних. Вторичную атрофию характеризует возрастание индекса b/a, зависимогоотзоны атрофии, угнетение амплитуды ритмической ЭРГ частотой 8,3 Гцприотсутствии изменений ритмической ЭРГна24 Гц.Вглазахсвторичной атрофией существенно снижалась плотность пика Р1 мультифокальной ЭРГнетольковпервом гексагоне,нои взоне парафовеа. Результаты электроокулографии показали более резкое снижение темнового спадасвозрастанием отношения Арденаубольныхсатрофиейнафоне влажной ВМД вотличиеотранее описанных измененийприпервичной географической атрофии. Заключение.Сравнение изменений индекса b/aсплощадью вторичной атрофииупациентовсвлажной ВМД может иметь клиническое значениедляоценки нарушений функции сетчаткиипрогноза зрительных функций. Вторичная атрофия ассоциируетсясвыраженным угнетением активности фоторецепторовприлучшей сохранности колбочковых биполярных клеток. Данные ЭРГиэлектроокулографии говорятоболее значительном нарушении функции пигментного эпителия сетчаткиприатрофиинафоне влажной ВМДисвязанномсним ухудшением активности фоторецепторов, чем ранее описанные изменения, характеризующие первичную географическую атрофию

Diagnosis of the nutritional value of colostrum in cows of different milk yield

When forming in an organism of a newborn calf of tentative immunity, the main source of immunoglobulins, a lysozyme, the functionally active leukocytes and lymphocytes, a colostrum is. The quality of a colostrum is a very important condition for formation of full-fledged immunity. A number of scientists claim that cows of different lactic breeds considerably differ on the level of lactic efficiency, at the same time the mass fraction of immunoglobulins negatively correlates with amount of colostrum at the first milking. Studying of dynamics of quality of a colostrum depending on the size of a milk yielding of cows for a lactation was a primal problem of researches. As an object of researches served the cows of four breeds divorced in the Samara region: Black Pied breeds, Bestuzhev, Holstein and Ayrshire. It is established that the content of immunoglobulins in a colostrum of the first yield of milk at cows of different breeds changes under the influence of the level of lactic efficiency and also with the animals' age. The highest content of immunoglobulins was in a colostrum of Bestuzhev breed – 103.35-81.38 g/l, and the lowest at Holstein breed – 74.52-42.29 g/l. Immunoglobulins of a colostrum are divided into three classes – IgG, IgA, IGM. In a colostrum of the first milk yielding of cows of Black Pied breed the share of IgG makes 84.1-85.5%, Bestuzhev breed – 85.7-86.3%, Holstein – 83.9-84.4%, Ayrshire – 85.7-86.6%. The tendency of increase in a share of IgG, in process of increase in milk yields of cows for a lactation is noted. It is established that at increase in level of lactic efficiency of cows, the quality of a colostrum decreases and the number of incidence of calves increases. As a result, the size of average daily gain of young growth live weight proportionally decreases. On the basis of the received results we recommend to estimate quality of a colostrum of the first milk yield by means of an optical or digital refractometer. Carry out the targeted selection work with breeds in the direction of colostrum upgrading