Comparative study of biocorrective protein-peptide agent to improve quality and safety of livestock products

Water with modified isotopic composition (D/H = 40 ppm) as a solubilizing agent for biologically active substances extraction from immune organs Sus Scrofa increases protein yield in thymus to 20%, in spleen up to 38%, mesenteric lymph nodes up to 35% in comparison with distilled water (D/H = 150 ppm). It was found a significant amount of neurotransmitter amino acids, such as aspartic and glutamic acids, glycine (thymus - 10.5; 13.7; 7.6%; spleen - 12.2; 10.7; 7.8% lymph nodes - 11.0; 13.3; 11.1%, respectively) having the immunological and adaptogenic activity, in extracts of Sus scrofa immunocompetent organs (thymus, spleen, mesenteric lymph nodes). Application of water with modified isotopic composition as solubilizing agent (D / H = 40 ppm) for extraction of immunocompetent organs' biologically active compounds led to increased amino acid content, including hydrophilic amino acids, in thymus extracts - up to 22%, in spleen extracts - up to 15% , in lymph nodes exctracts - up to 8%, in comparison to distilled water (D / H = 150 ppm). Peptide profile analysis revealed positive effect of water (D/H = 40 ppm) on a quality protein content extracts in molecular weight range from 10 to 20 kDa and from 30 to 45 kDa and peptide composition (2 kDa), at the same time quantitative content of compounds adaptogenic and immunocorrective action increased. Reducing of deuterium content in the solubilizing agent enhances quantitative amino acid content, i.e. extraction in an aqueous- salt solution based on deuterium depleted water of animal tissues with a high content of amino acids with hydrophilic radicals proceeded more completely

Solvent water interactions within the active site of the membrane type I matrix metalloproteinase

Matrix metalloproteinases (MMP) are an important family of proteases which catalyze the degradation of extracellular matrix components. While the mechanism of peptide cleavage is well established, the process of enzyme regeneration, which represents the rate limiting step of the catalytic cycle, remains unresolved. This step involves the loss of the newly formed N-terminus (amine) and C-terminus (carboxylate) protein fragments from the site of catalysis coupled with the inclusion of one or more solvent waters. Here we report a novel crystal structure of membrane type I MMP (MT1-MMP or MMP-14), which includes a small peptide bound at the catalytic Zn site via its C-terminus. This structure models the initial product state formed immediately after peptide cleavage but before the final proton transfer to the bound amine; the amine is not present in our system and as such proton transfer cannot occur. Modeling of the protein, including earlier structural data of Bertini and coworkers [I. Bertini, et al., Angew. Chem., Int. Ed., 2006, 45, 7952–7955], suggests that the C-terminus of the peptide is positioned to form an H-bond network to the amine site, which is mediated by a single oxygen of the functionally important Glu240 residue, facilitating efficient proton transfer. Additional quantum chemical calculations complemented with magneto-optical and magnetic resonance spectroscopies clarify the role of two additional, non-catalytic first coordination sphere waters identified in the crystal structure. One of these auxiliary waters acts to stabilize key intermediates of the reaction, while the second is proposed to facilitate C-fragment release, triggered by protonation of the amine. Together these results complete the enzymatic cycle of MMPs and provide new design criteria for inhibitors with improved efficacy.Financial support was provided by the Max Planck Gesellschaft and the Cluster of Excellence RESOLV (EXC 1069) funded by the Deutsche Forschungsgemeinschaft. I. S. is supported by the Binational Science Foundation (BSF) and Israel Science Foundation (ISF). I. S. and M. H. are also supported by the ERC Advanced Grant 695437 THz-Calorimetry. M. G. is an Awardee of the Weizmann Institute of Science National Postdoctoral Award Program for Advancing Women in Science and a recipient of an A. v. Humboldt Fellowship. N. C. acknowledges the support of the Australian Research Council: Future Fellowship (FT140100834). Open Access funding provided by the Max Planck Society

МЕТОДОЛОГИЯ ИССЛЕДОВАНИЯ БЕЛКОВО-ПЕПТИДНЫХ КОМПОНЕНТОВ ЭКСТРАКТОВ ТКАНЕЙ SUS SCROFA

The article presents a comparative analysis of four methods for quantifying the protein-peptide complexes content in extracts obtained from animal raw materials, as well as the low- and highmolecular weight extract fractions: the direct spectrophotometric determination at wavelengths of 260 and 280 nm with subsequent calculation by the Kalckar formula; the biuret reaction by the Kingsley-Weichselbaum method; the method with Bradford reagent and the standard Lowry method. Experimental data analysis demonstrates that in case of the extract that contains protein-peptidic complexes in different molecular weights range, the Kingsley-Weichselbaum method shows the highest quality of protein concentration determination; while studying highmolecular weight fraction (more than 30 kDa), it is possible to obtain more information by combining the spectrophotometric method and the Kingsley-Weichselbaum method. Low-molecular weight fractions (less than 30 kD) should be investigated by complex methods including the spectrophotometric method, Lowry and Bradford methods. These methods make it possible to presumably estimate protein molecules size ranges (by amount of peptide bonds), and also to determine hydrophobic and aromatic amino acids presence.В данной статье приведены данные сравнительного анализа четырех методов количественной оценки содержания белково-пептидных комплексов в  экстрактах, полученных на основе сырья животного происхождения, а  также низкомолекулярных и высокомолекулярных фракциях экстракта: прямого спектрофотометрического определения при длинах волн 260 и 280 нм с последующим расчетом по формуле Калькара; биуретовой реакции по методу Кингслея — Вейксельбаума; с использованием реактива Брэдфорда и по стандартному методу Лоури. Анализ полученных экспериментальных данных показал, что в  случае с  экстрактом, содержащим белково-пептидные комплексы в  различном диапазоне молекулярных масс наиболее качественно показывает концентрацию белка метод Кингслея-Вейксельбаума, при исследовании высокомолекулярной фракции (более 30 кДа) больше информации возможно получить при сочетании спектрофотометрического метода и также метода Кингслея-Вейксельбаума. Низкомолекулярные (менее 30 кДа) фракции следует исследовать комплексно, с  применением спектрофотометрического метода, методов Лоури и  Брэдфорда. Данные методы дают возможность предположительно оценить диапазоны размеров белковых молекул (по числу пептидных связей), а также определить наличие гидрофобных и ароматических аминокислот

ПРИМЕНЕНИЕ АЛЬТЕРНАТИВНЫХ МЕТОДОВ БИОМОДЕЛИРОВАНИЯ ДЛЯ ИЗУЧЕНИЯ ЭФФЕКТИВНОСТИ ФУНКЦИОНАЛЬНЫХ ПРОДУКТОВ ПИТАНИЯ

The article describes in vitro methods basic principles, authors analyzed cell and tissue cultures used to assess toxicity and specific biological activity, including metabolic processes, include antihypertensive and cytoprotective properties analysis, antioxidant activity (in vitro and ex vivo) used to study ingredients functional properties based on animal origin raw materials, as well as meat products.В статье рассмотрены основные принципы методов in vitro, проанализированы применяемые культуры клеток и тканей, используемые для оценки токсичности и специфической биологической активности, включая исследования метаболических процессов, анализ гипотензивных и цитопротективных свойств, антиоксидантной активности (in vitro и ex vivo), используемые для оценки функциональных свойств ингредиентов на основе сырья животного происхождения, а также мясных продуктов

Albiglutide and cardiovascular outcomes in patients with type 2 diabetes and cardiovascular disease (Harmony Outcomes): a double-blind, randomised placebo-controlled trial

Background: Glucagon-like peptide 1 receptor agonists differ in chemical structure, duration of action, and in their effects on clinical outcomes. The cardiovascular effects of once-weekly albiglutide in type 2 diabetes are unknown. We aimed to determine the safety and efficacy of albiglutide in preventing cardiovascular death, myocardial infarction, or stroke. Methods: We did a double-blind, randomised, placebo-controlled trial in 610 sites across 28 countries. We randomly assigned patients aged 40 years and older with type 2 diabetes and cardiovascular disease (at a 1:1 ratio) to groups that either received a subcutaneous injection of albiglutide (30–50 mg, based on glycaemic response and tolerability) or of a matched volume of placebo once a week, in addition to their standard care. Investigators used an interactive voice or web response system to obtain treatment assignment, and patients and all study investigators were masked to their treatment allocation. We hypothesised that albiglutide would be non-inferior to placebo for the primary outcome of the first occurrence of cardiovascular death, myocardial infarction, or stroke, which was assessed in the intention-to-treat population. If non-inferiority was confirmed by an upper limit of the 95% CI for a hazard ratio of less than 1·30, closed testing for superiority was prespecified. This study is registered with ClinicalTrials.gov, number NCT02465515. Findings: Patients were screened between July 1, 2015, and Nov 24, 2016. 10 793 patients were screened and 9463 participants were enrolled and randomly assigned to groups: 4731 patients were assigned to receive albiglutide and 4732 patients to receive placebo. On Nov 8, 2017, it was determined that 611 primary endpoints and a median follow-up of at least 1·5 years had accrued, and participants returned for a final visit and discontinuation from study treatment; the last patient visit was on March 12, 2018. These 9463 patients, the intention-to-treat population, were evaluated for a median duration of 1·6 years and were assessed for the primary outcome. The primary composite outcome occurred in 338 (7%) of 4731 patients at an incidence rate of 4·6 events per 100 person-years in the albiglutide group and in 428 (9%) of 4732 patients at an incidence rate of 5·9 events per 100 person-years in the placebo group (hazard ratio 0·78, 95% CI 0·68–0·90), which indicated that albiglutide was superior to placebo (p<0·0001 for non-inferiority; p=0·0006 for superiority). The incidence of acute pancreatitis (ten patients in the albiglutide group and seven patients in the placebo group), pancreatic cancer (six patients in the albiglutide group and five patients in the placebo group), medullary thyroid carcinoma (zero patients in both groups), and other serious adverse events did not differ between the two groups. There were three (<1%) deaths in the placebo group that were assessed by investigators, who were masked to study drug assignment, to be treatment-related and two (<1%) deaths in the albiglutide group. Interpretation: In patients with type 2 diabetes and cardiovascular disease, albiglutide was superior to placebo with respect to major adverse cardiovascular events. Evidence-based glucagon-like peptide 1 receptor agonists should therefore be considered as part of a comprehensive strategy to reduce the risk of cardiovascular events in patients with type 2 diabetes. Funding: GlaxoSmithKline

RESEARCH METHODOLOGY OF SUS SCROFA TISSUE EXTRACTS PROTEIN-PEPTIDE COMPONENTS

The article presents a comparative analysis of four methods for quantifying the protein-peptide complexes content in extracts obtained from animal raw materials, as well as the low- and highmolecular weight extract fractions: the direct spectrophotometric determination at wavelengths of 260 and 280 nm with subsequent calculation by the Kalckar formula; the biuret reaction by the Kingsley-Weichselbaum method; the method with Bradford reagent and the standard Lowry method. Experimental data analysis demonstrates that in case of the extract that contains protein-peptidic complexes in different molecular weights range, the Kingsley-Weichselbaum method shows the highest quality of protein concentration determination; while studying highmolecular weight fraction (more than 30 kDa), it is possible to obtain more information by combining the spectrophotometric method and the Kingsley-Weichselbaum method. Low-molecular weight fractions (less than 30 kD) should be investigated by complex methods including the spectrophotometric method, Lowry and Bradford methods. These methods make it possible to presumably estimate protein molecules size ranges (by amount of peptide bonds), and also to determine hydrophobic and aromatic amino acids presence

Effects of tissue-specific biomolecules on piglets after-weaning period

Background and Aim: Now-a-days antibiotics are the main tool for correcting the pathological conditions of pigs; unfortunately, antibiotics are a potential threat to the environment, as they lead to the spread of antibiotic-resistant infections. This study aimed to study the immunomodulatory encapsulated biomolecules on piglets in the post-weaning period. Materials and Methods: An immunomodulator based on biomolecules obtained from animal raw materials included in alginate capsules to improve absorption has been developed. The study presents the results of a study on 25 weaned piglets (25-30 days old) which received biomolecules at a dose of 200 mg/piglet for 14 days, followed by 400 mg/piglet from days 15 to 28. Blood was taken from animals for analysis (biochemical, hematological, cytometric, and enzyme immunoassay) and the integral index of blood serum antimicrobial activity was determined. Results: Experimental animals, whose initial weight was 1.6 times less than that of the control animals, were able to bridge this gap and, on the 28th day, there were no differences in weight. Stimulation of the production of cytokines interleukin (IL)-2 and IL-4 was observed and the antimicrobial resistance of blood serum to Escherichia coli also increased. A positive effect on the metabolism of piglets was noted, which helped them adapt to a change in diet (from colostrum to solid food). Conclusion: The results show that the immunomodulation at the dose of 150 mg/kg body weight has a great potential for improving weaned pigs

Suspension Cell Culture of <i>Dioscorea deltoidea</i>—A Renewable Source of Biomass and Furostanol Glycosides for Food and Pharmaceutical Industry

Dioscorea deltoidea is a medicinal plant valued for its high content of steroidal glycosides (SG)—bioactive compounds with cardioprotective and immunomodulation actions, also used to treat reproductive system disorders. To overcome the limitations of natural resources of this species, a suspension cell culture of D. deltoidea was developed as a renewable and ecologically sustainable source of raw biomass and SG. Cell culture demonstrated stable and intensive growth in the laboratory (20 L) and industrial (630 L) bioreactors operated under a semi-continuous regime (specific growth rate 0.11–1.12 day−1, growth index 3.5–3.7). Maximum dry weight accumulation (8.5–8.8 g/L) and SG content (47–57 mg/g DW) were recorded during the stationary phase. Bioreactor-produced cell biomass contained inorganic macro (K, Ca, Mg, Na) and micro (Zn, Mn, Fe, B, Al, Cu, Cr, Se, Co, Ni) elements in concentrations within the safe range of dietary recommendations. Acute toxicity test showed no or insignificant changes in organ weight, hematological panel and blood biochemistry of laboratory animals fed with 2000 and 5000 mg/kg dry biomass. The results suggest that cell culture of D. deltoidea grown in bioreactors has great potential to be used as functional foods and a component of specialized dietary supplements in complex therapy of reproductive system disorders and mineral deficiency