Anti-fatigue effects of protein hydrolysates are often attributed to low-molecular-weight peptides which are thought to rapidly increase plasma amino acids level. Typically, these peptides are produced using time consuming and labor-intensive procedures to isolate them. Here, we found that a soy protein isolate hydrolysate (SPIH), composed of a complex mixture of peptides with an average molecular weight of 6 kDa, significantly increased endurance in a rat weight-loaded swimming test, prolonging the swimming time by 511

The hepatopancreas of crustaceans is an important immune organ involved in the secretion of immune effectors such as antimicrobial peptides. Crustacean antimicrobial peptides are very diverse and have a broad spectrum of activity, but are poorly explored. In this work, the activity of antibacterial peptides from the hepatopancreas of red king crab and snow crab was compared. Both peptides were found to be highly effective in inhibiting the growth of gram-positive bacteria. At the same time, the peptide from the red king crab was found to be highly sensitive to the presence of salt and a disulfide bond reducing agent, in contrast to the peptide from the snow crab. An approach for the chromatographic purification of the red king crab peptide from the hepatopancreas was developed. Both peptides are of interest for the development of novel antibacterial drugs.

The physicochemical properties of emulsions based on poloxamers (triblock copolymers of a hydrophobic polyoxypropylene chain and two hydrophilic polyoxyethylene chains) depend on the composition and preparation method. This study examined the impact of poloxamer P188 concentration, autoclaving mode, heating, and salt presence on the viscosity, particle size distribution, and morphology of particles using viscometric analysis, dynamic light scattering (DLS), and atomic force microscopy (AFM). It was shown that sample preparation affects the particle size and morphology but not the chemical composition of P188. The most similar properties were found for 10% P188 samples sterilized by filtration and autoclaving. The higher autoclave temperature and additional heating of the 10% P188 samples to 70 °C resulted in the formation of larger particles. For 4% P188 samples with 0.6% NaCl, samples heated at 70 °C for 15 h after sterilization filtration and autoclaving were the most similar and homogeneous. The 4% P188 sample with the higher autoclave temperature and subsequent heating had the lowest viscosity. In contrast to 10% P188, for 4% P188 in the presence of salt, the lack of heating resulted in the formation of large particles. The 4% P188 solutions with NaCl were more stable during storage than those with a higher concentration.

Crustaceans have successfully adapted to survive in their natural habitat, rich in microorganisms, due to the presence of antimicrobial peptides (AMPs) in their organism. They achieve this adaptation despite lacking the highly specific adaptive immune system found in vertebrates. One valuable source of AMPs is the hepatopancreas, a waste product from crab fishery and its processing. Applying zymographic and spectrophotometric techniques, we discovered a small peptide (approximately 5 kDa) within a low molecular weight protein fraction extracted from the acetone powder of the red king crab hepatopancreas. This peptide hydrolyzes both M. lysodeikticus cell wall and M. lysodeikticus cell wall polysaccharide, while showing no activity against gelatin. The found peptide may be of interest for application in medicine, biotechnology, and the food industry, for example as a bio-preservative.

A giant multidomain protein of striated and smooth vertebrate muscles, titin, consists of tandems of immunoglobulin (Ig)- and fibronectin type III (FnIII)-like domains representing β-sandwiches, as well as of disordered segments. Chicken smooth muscles express several titin isoforms of ~500–1500 kDa. Using various structural-analysis methods, we investigated in vitro nonspecific amyloid aggregation of the high-molecular-weight isoform of chicken smooth-muscle titin (SMTHMW, ~1500 kDa). As confirmed by X-ray diffraction analysis, under near-physiological conditions, the protein formed amorphous amyloid aggregates with a quaternary cross-β structure within a relatively short time (~60 min). As shown by circular dichroism and Fourier-transform infrared spectroscopy, the quaternary cross-β structure—unlike other amyloidogenic proteins—formed without changes in the SMTHMW secondary structure. SMTHMW aggregates partially disaggregated upon increasing the ionic strength above the physiological level. Based on the data obtained, it is not the complete protein but its particular domains/segments that are likely involved in the formation of intermolecular interactions during SMTHMW amyloid aggregation. The discovered properties of titin position this protein as an object of interest for studying amyloid aggregation in vitro and expanding our views of the fundamentals of amyloidogenesis.

Cerebral ischemia, and, as a result, insult, attacks up to 15 million people yearly in the world. In this connection, the development of effective preventive programs and methods of therapy has become one of the most urgent problems in modern angiology and pharmacology. The cytoprotective action of taxifolin (TAX) in ischemia is well known, but its limitations are also known due to its poor solubility and low capacity to pass through the hematoencephalic barrier. Molecular mechanisms underlying the protective effect of TAX in complex systems such as the brain remain poorly understood. It is known that the main cell types of the brain are neurons, astrocytes, and microglia, which regulate the activity of each other through neuroglial interactions. In this work, a comparative study of cytoprotective mechanisms of the effect of TAX and its new water-soluble form aqua taxifolin (aqTAX) was performed on cultured brain cells under ischemia-like conditions (oxygen–glucose deprivation (OGD)) followed by the reoxygenation of the culture medium. The concentration dependences of the protective effects of both taxifolin forms were determined using fluorescence microscopy, PCR analysis, and vitality tests. It was found that TAX began to effectively inhibit necrosis and the late stages of apoptosis in the concentration range of 30–100 µg/mL, with aqTAX in the range of 10–30 µg/mL. At the level of gene expression, aqTAX affected a larger number of genes than TAX; enhanced the basic and OGD/R-induced expression of genes encoding ROS-scavenging proteins with a higher efficiency, as well as anti-inflammatory and antiapoptotic proteins; and lowered the level of excitatory glutamate receptors. As a result, aqTAX significantly inhibited the OGD-induced increase in the Ca2+ levels in the cytosol ([Ca2+]i) in neurons and astrocytes under ischemic conditions. After a 40 min preincubation of cells with aqTAX under hypoxic conditions, these Ca2+ signals were completely inhibited, resulting in an almost complete suppression of necrotic death of cerebral cortical cells, which was not observed with the use of classical TAX.

The digestive gland of craboids (hepatopancreas) is rich in a huge number of various enzymes (collagenases, nucleases, hyaluronidases, proteases), which are well studied at the moment. However, little is known about crustacean lipases. In this work, using 1H NMR spectroscopy, it was found that the hepatopancreas homogenate of the red king crab Paralithodes camtschaticus demonstrates high lipolytic activity against triacetin in a wide pH range and shows moderate activity against the caprylic/capric triglyceride emulsion. Under the action of the hepatopancreas homogenate, triacylglycerols are converted into 1,2-diacylglycerol, and then into 2-monoacylglycerol and 1-monoacylglycerol. The 1-monoacylglycerol predominates in the reaction products. The use of NMR spectroscopy makes it possible to quickly detect hydrolysis products and evaluate the reaction direction.

The digestive gland of craboids (hepatopancreas) is rich in a huge number of various enzymes (collagenases, nucleases, hyaluronidases, proteases), which are well studied at the moment.However, little is known about crustacean lipases.In this work, using 1 H NMR spectroscopy, it was found that the hepatopancreas homogenate of the red king crab Paralithodes camtschaticus demonstrates high lipolytic activity against triacetin in a wide pH range and shows moderate activity against the caprylic/capric triglyceride emulsion.Under the action of the hepatopancreas homogenate, triacylglycerols are converted into 1,2-diacylglycerol, and then into 2-monoacylglycerol and 1-monoacylglycerol.The 1monoacylglycerol predominates in the reaction products.The use of NMR spectroscopy makes it possible to quickly detect hydrolysis products and evaluate the reaction direction.

This work investigated in vitro aggregation and amyloid properties of skeletal myosin binding protein-C (sMyBP-C) interacting in vivo with proteins of thick and thin filaments in the sarcomeric A-disc. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) found a rapid (5–10 min) formation of large (>2 μm) aggregates. sMyBP-C oligomers formed both at the initial 5–10 min and after 16 h of aggregation. Small angle X-ray scattering (SAXS) and DLS revealed sMyBP-C oligomers to consist of 7–10 monomers. TEM and atomic force microscopy (AFM) showed sMyBP-C to form amorphous aggregates (and, to a lesser degree, fibrillar structures) exhibiting no toxicity on cell culture. X-ray diffraction of sMyBP-C aggregates registered reflections attributed to a cross-β quaternary structure. Circular dichroism (CD) showed the formation of the amyloid-like structure to occur without changes in the sMyBP-C secondary structure. The obtained results indicating a high in vitro aggregability of sMyBP-C are, apparently, a consequence of structural features of the domain organization of proteins of this family. Formation of pathological amyloid or amyloid-like sMyBP-C aggregates in vivo is little probable due to amino-acid sequence low identity (<26%), alternating ordered/disordered regions in the protein molecule, and S–S bonds providing for general stability.

Crustacean hyaluronidases are poorly understood both in terms of their enzymatic properties and in terms of their structural features. In this work, we show that the hepatopancreas homogenate of the red king crab has a hyaluronidase activity that is an order of magnitude higher than its commercial counterpart. Zymography revealed that the molecular weight of a protein with hyalorunidase activity is 40-50 kDa. Analysis of the hepatopancreas transcriptome and results of cloning and sequencing of cDNA revealed a hyaluronidase sequence with an expected molecular weight of 42.5 kDa. Further analysis showed that hyaluronat enzymatic cleavage follows the [Formula: see text]-elimination mechanism, which is well known for bacterial hyaluronidases. The results of ion-exchange chromatography showed that the final product of hyaluronate degradation is unsaturated tetrasaccharide. Thus, we identified a new hyaluronidase of higher eukaryotes, which is not integrated into the modern classification of hyaluronidases.

Since the early 1980s, a large number of studies on enzymes from the red king crab hepatopancreas were conducted. They have been relevant both from a fundamental point of view in terms of studying the enzymes of marine organisms and in terms of rational natural resource management aimed to obtain new valuable products from the processing of crab fishing waste. Most of these works were performed by Russian scientists due to the area and amount of waste of red king crab processing in Russia (or the Soviet Union). However, the close phylogenetic kinship and the similar ecological niches of commercial crab species and the production scale of the catch provide the bases for the successful transfer of experience in the processing of the red king crab hepatopancreas to other commercial crab species caught worldwide. This review describes the value of recycled commercial crab species, discusses processing problems, and suggests possible solutions for these issues. The main emphasis is made on hepatopancreatic enzymes as the most salubrious products of red king crab waste processing.

In this study, several methods were used to analyze the hydrolysis of hyaluronic acid (HA)-based cosmetic fillers by the hepatopancreas homogenate of the Red king crab. The results show that the homogenate and commercially available hyaluronidases have similar hydrolysis activities on the fillers. Atomic force microscopy images reveal that the HA fillers consist mainly of spherical-like particles, which are converted into filamentous structures as a result of hydrolysis by the Red king crab hepatopancreas homogenate. Turbidimetric analysis of the hydrolysis process shows that HA aggregation with acidic albumin exhibits a bell-shaped dependence on reaction time. Analysis of the hydrolysis process by nuclear magnetic resonance shows that HA degradation lasts several days. The maximum rate of the reaction is detected in the 1st h of incubation. The data confirm that the purified homogenate of the Red king crab hepatopancreas exerts hyaluronidase activity on HA-based cosmetic fillers; therefore, it may be considered as a potential therapeutic agent for treating filler complications.

In this study, several methods were used to analyze the hydrolysis of hyaluronic acid (HA)based cosmetic fillers by the hepatopancreas homogenate of the Red king crab.The results show that the homogenate and commercially available hyaluronidases have similar hydrolysis activities on the fillers.Atomic force microscopy (AFM) images reveal that the HA fillers consist mainly of spherical-like particles, which are converted into filamentous structures as a result of hydrolysis by the Red king crab hepatopancreas homogenate.Turbidimetric analysis of the hydrolysis process shows that HA aggregation with acidic albumin exhibits a bell-shaped dependence on reaction time.Analysis of the hydrolysis process by nuclear magnetic resonance (NMR) shows that HA degradation lasts several days.The maximum rate of the reaction is detected in the first hours of incubation.The data confirm that the purified homogenate of the Red king crab hepatopancreas exerts hyaluronidase activity on HA-based cosmetic fillers; therefore, it may be considered as a potential therapeutic agent for treating filler complications.

The kinetics of the hydrolysis of hyaluronic acid (HA) of cosmetic fillers using thehomogenate of the red king crab hepatopancreas was studied for the first time. Turbidimetricanalysis of the reaction mixture revealed a bell–shaped time dependence of aggregation formation. The HA fillers were examined by atomic force microscopy (AFM) and it was found that they wererepresented by spherical–like structures. These structures were disrupted under the action of thehomogenate of the red king crab hepatopancreas. It was shown that the prepared homogenate hasthe activity which is similar to that observed in the commercially available hyaluronidase products.The preparation with hyaluronidase activity obtained from the red king crab hepatopancreas couldbe used as potentially safe product for treating filler complications.

The nature of renal amyloidosis involving Bence-Jones proteins in multiple myeloma is still unclear. The development of amyloidosis in neurodegenerative diseases is often associated with a high content of asparagine and glutamine residues in proteins forming amyloid deposits. To estimate the influence of Asn and Gln residues on the aggregation of Bence-Jones protein BIF, we obtained recombinant BIF and its mutants with the substitution of Tyr187→Asn (Y187N) in α-helix of CL domain, Lys170→Asn (K170N) and Ser157→Gln (S157Q) in CL domain loops, Arg109→Asn in VL-CL linker (R109N) and Asp29→Gln in VL domain loop (D29Q). The morphology of protein aggregates was studied at pH corresponding to the conditions in bloodstream (pH 7.2), distal (pH 6.5) and proximal renal tubules (pH 4.5) by atomic force microscopy (AFM) and small-angle X-ray scattering (SAXS). The Lys170→Asn replacement almost completely inhibits amyloidogenic activity. The Y187N forms fibril-like aggregates at all pH values. The Arg109→Asn replacement resulted in formation of fibril-like structures at pH 7.2 and 6.5 while the substitutions by Gln provoked formation of those structures only at pH 7.2. Therefore, the amyloidogenic properties are highly dependent on the location of Asn or Gln.

A comparative study of amyloid properties of the aggregates of smooth muscle titin (SMT) from chicken gizzard was carried out. These aggregates were formed in two solutions: 0.15 M glycine-KOH, pH 7.2–7.4 (SMT(Gly)) and 0.2 M KCl, 10 mM imidazole, pH 7.0 (SMT(KCl)). Electron microscopy data showed that SMT aggregates has an amorphous structure in both cases. The results of atomic-force microscopy demonstrated slight differences in morphology in two types of aggregates. The SMT(Gly) aggregates were represented as branching chains, composed of spherical aggregates approximately 300–500 nm in diameter and up to 35 nm in height. The SMT(KCl) aggregates formed sponge-like structures with strands of 8–10 nm in height. Structural analysis of SMT aggregates by X-ray diffraction revealed the presence of cross-β-sheet structure in the samples under study. In the presence of SMT(Gly) aggregates, thioflavine T fluorescence intensity was higher (~3-fold times) compared with that in the presence of SMT(KCl) aggregates. Congo red-stained SMT(Gly) aggregates had yellow to apple-green birefringence under polarized light, which was not observed for SMT(KCl) aggregates. Dynamic light scattering data showed the similar rate of aggregation for both types of aggregates, though SMT(KCl) aggregates were able to partially disaggregate under increased ionic strength of the solution. The ability of SMT to aggregation followed by disaggregation may be functionally significant in the cell.

The original limited proteolysis approach was proposed for large (multidomain) proteins with post-translational modifications for obtaining of their novel fragments. It was realized for human immunoglobulins representing two subclasses IgG2 and IgG3. This approach was based on two techniques: masking of protein regions which are normally susceptible to proteolytic enzymes and increasing the possibility of proteolysis for sites which are not ordinarily accessible to these enzymes. The masking of immunoglobulin part which is sensitive to proteolysis was performed by Fab fragments (Fv subfragments) of antibodies and the increase of lability of stable regions was realized by pH change and mild reduction of disulfide bonds.

The content of human sweat is studied by high-resolution NMR, and the majority of organic components most often found in sweat of conditionally healthy people are identified. Original and simple tools are designed for sweat sampling from different areas of human body. The minimal surface area needed for sampling is in the range of 50-100 cm(2). On all the surface parts of the human body examined in this work, the main constituents forming a sweat metabolic profile are lactate, glycerol, pyruvate, and serine. The only exception is the sole of the foot (planta pedis), where trace amounts of glycerol are found. An attempt is made to explain the presence of specified metabolites and their possible origin.

Two chimeric proteins, SHcapital EN, Cyrillic and SHA of the "SH3-Bergerac" family (where the beta-turn N47D48 in spectrin SH3 domain was substituted for KITVNGKTYE or KATANGKTYE sequences, respectively), were analyzed by high-resolution NMR to resolve their spatial structures and to analyze their dynamics. Although the presence of a stable beta-hairpin in the region of the insertion was confirmed, the introduced extension of the polypeptide chain in SHcapital EN, Cyrillic (approximately 17%) practically did not affect the total molecule topology. Interestingly, the introduced beta-hairpin had higher mobility in comparison with other protein regions. Finally, we performed a disorder prediction with the PONDR VSL2 algorithm and discovered that the inserted beta-hairpin in both SHH and SHA proteins exhibited significant propensity for intrinsic disorder and therefore for high mobility. In agreement with the experimental data, the predisposition for the increased intramolecular mobility was noticeably higher in SHA.