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Five new non-holostane di- and trisulfated triterpene pentaosides, conicospermiumosides A 3 -1 ( 1 ), A 3 -2 ( 2 ), A 3 -3 ( 3 ), A 7 -1 ( 4 ), and A 7 -2 ( 5 ) were isolated from the Far Eastern sea cucumber Cucumaria conicospermium Levin et Stepanov (Cucumariidae, Dendrochirotida). Twelve known glycosides found earlier in other Cucumaria species were also obtained and identified. The structures of new compounds were established on the basis of extensive analysis of the 1D and 2D NMR spectra, as well as by the HR-ESI-MS data. The aglycones of 1 - 5 differed by side chains structures. Additionally, conicospermiumoside A 7 -1 ( 4 ) had a 9(11)-double bond in the aglycone, while the remaining glycosides contained a 7(8)-intranuclear double bond. Eight types of carbohydrate chains known earlier from the glycosides of the sea cucumbers of the Cucumaria genus were found as part of the glycosides of C. conicospermium . The set of sugar chains of the glycosides from C. conicospermium was similar to that from C. okhotensis . The raw biogenetic series of aglycones, leading to the formation of hexa-nor-lanostane derivatives in the process of biosynthesis and a sort of functionally-structural division that was realized due to separation of biosynthetic pathways of holostane and lanostane derivatives, can be traced when the structures of the glycosides isolated from C. conicospermium are compared. The cytotoxic action against three human breast cancer cell lines (MCF-7, T-47D, MDA-MB-231), and non-tumor MCF-10A and hemolytic activity of compounds 1 - 5 , as well as seven known glycosides were tested. Conicospermiumosides A 3 -3 ( 3 ) and A 7 -1 ( 4 ), having a 22-oxo-23(24)-en fragment, were strongly hemolytic despite lacking a lactone in their aglycones. Moreover, both compounds demonstrated a promising suppressing action against triple negative breast cancer cells. The cells of the MDA-MB-231 line were most sensitive to the cytotoxic action of the glycosides, while the MCF-7 cell line was most sustainable. Six glycosides were selected for further study of some aspects of anticancer action against MDA-MB-231. The selective action of the compounds 4 and 8 on the MDA-MB-231 cells without significant toxicity against the MCF-10A cells was noticeable. More importantly, the selectivity of the compounds was changed over time and maximal selectivity to cancer cells was demonstrated by glycoside 1 at 48 h of exposition. The glycosides 1 , 3 and the desulfated derivative 7a strongly inhibited colony formation and growth of the TNBC cells until the process stops completely. Okhotoside B 1 ( 8 ), DS-okhotoside A 1 -1 ( 7a ), and conicospermiumoside A 3 -3 ( 3 ) showed a potent cell migration-inhibiting capacity. Quantitative structure-activity relationships (QSARs) calculated on the basis of a correlational analysis of the physicochemical properties and structural features of the glycosides and their cytotoxic activity against different cell lines showed some structural features influenced differently, sometimes even in opposite ways, on the activity of glycosides toward diverse cells (erythrocytes, MCF-10A, and TNBC MDA-MB-231 cells). This observation indicated that glycosides obviously target different membrane components, such as lipids of erythrocytes and some receptors on the surface of mammary normal or tumor cells.

Four new glycosides of polyhydroxysteroids, ceramasterosides A, B, D, and E ( 1 - 4 ), and two previously known compounds, ceramasteroside C 1 ( 5 ) and attenuatoside B-I ( 6 ), were isolated from an extract of a deep-sea sea star species, the orange cookie star Ceramaster patagonicus . The structures of 1 - 4 were elucidated by the extensive NMR and ESIMS methods. Steroid monoglycosides 1 and 2 had a common 3β,6α,8,15β,16β-pentahydroxysteroid nucleus and a C-29 oxidized stigmastane side chain and differed from each other only in monosaccharide residues. Ceramasteroside A ( 1 ) contained 3- O -methyl-4- O -sulfated β-D-xylopyranose, while ceramasteroside B ( 2 ) had 3- O -methyl-4- O -sulfated β-D-glucopyranose, recorded from starfish-derived steroid glycosides for the first time. Their biological activity was studied using a model of lipopolysaccharide-induced (LPS) inflammation in a SIM-A9 murine microglial cell line. During the LPS-induced activation of microglial cells, 1 , 3 , and 5 , at a non-toxic concentration of 1 µM, showed the highest efficiency in reducing the production of intracellular NO, while 4 proved to be most efficient in reducing the extracellular nitrite production. All the test compounds reduced the LPS-induced malondialdehyde (MDA) production. The in vitro experiments have demonstrated, for the first time, the antioxidant activity of the compounds under study.

Stonikacidin A ( 1 ), the first representative of a new class of 4-bromopyrrole alkaloids containing an aldonic acid core, was isolated from the marine sponge Lissodendoryx papillosa . The compound is named in honor of Prof. Valentin A. Stonik, who is one of the outstanding investigators in the field of marine natural chemistry. The structure of 1 was determined using NMR, MS analysis, and chemical correlations. The L-idonic acid core was established by the comparison of GC, NMR, MS, and optical rotation data of methyl-pentaacetyl-aldonates obtained from the hydrolysis products of 1 and standard hexoses. The L-form of the idonic acid residue in 1 was confirmed by GC analysis of pentaacetate of ( S )-2-butyl ester of the hydrolysis product from 1 and compared with corresponding derivatives of L- and D-idonic acids. The biosynthetic pathway for stonikacidin A ( 1 ) was proposed. The alkaloid 1 inhibited the growth of Staphylococcus aureus and Escherichia coli test strains, as well as affected the formation of S. aureus and E. coli biofilms. Compound 1 inhibited the activity of sortase A. Molecular docking data showed that stonikacidin A ( 1 ) can bind with sortase A due to the interactions between its bromine atoms and some amino acid residues of the enzyme.

Three new monosulfated polyhydroxysteroid glycosides, spiculiferosides A ( 1 ), B ( 2 ), and C ( 3 ), along with new related unsulfated monoglycoside, spiculiferoside D ( 4 ), were isolated from an ethanolic extract of the starfish Henricia leviuscula spiculifera collected in the Sea of Okhotsk. Compounds 1 - 3 contain two carbohydrate moieties, one of which is attached to C-3 of the steroid tetracyclic core, whereas another is located at C-24 of the side chain of aglycon. Two glycosides ( 2 , 3 ) are biosides, and one glycoside ( 1 ), unlike them, includes three monosaccharide residues. Such type triosides are a rare group of polar steroids of sea stars. In addition, the 5-substituted 3-OSO 3 -α-L-Araf unit was found in steroid glycosides from starfish for the first time. Cell viability analysis showed that 1 - 3 (at concentrations up to 100 μM) had negligible cytotoxicity against human embryonic kidney HEK293, melanoma SK-MEL-28, breast cancer MDA-MB-231, and colorectal carcinoma HCT 116 cells. These compounds significantly inhibited proliferation and colony formation in HCT 116 cells at non-toxic concentrations, with compound 3 having the greatest effect. Compound 3 exerted anti-proliferative effects on HCT 116 cells through the induction of dose-dependent cell cycle arrest at the G2/M phase, regulation of expression of cell cycle proteins CDK2, CDK4, cyclin D1, p21, and inhibition of phosphorylation of protein kinases c-Raf, MEK1/2, ERK1/2 of the MAPK/ERK1/2 pathway.

The lipopolysaccharides of Herbaspirillum lusitanum P6-12 T (HlP6-12 T ) and H. frisingense GSF30 T (HfGSF30 T ) was isolated by phenol-water extraction from bacterial cells and was characterized using chemical analysis and SDS-PAGE. It was shown that these bacteria produce LPSs that differ in their physicochemical properties and macromolecular organization. In this paper, the lipid A structure of the HlP6-12 T LPS, was characterized through chemical analyses and matrix-assisted laser desorption ionization (MALDI) mass spectrometry. To prove the effect of the size of micelles on their bioavailability, we examined the activity of both LPSs toward the morphology of wheat seedlings. Analysis of the HlP6-12 T and HfGSF30 T genomes showed no significant differences between the operons that encode proteins involved in the biosynthesis of the lipids A and core oligosaccharides. The difference may be due to the composition of the O-antigen operon. HfGSF30 T has two copies of the rfb operon, with the main one divided into two fragments. In contrast, the HlP6-12 T genome contains only a single rfb-containing operon, and the other O-antigen operons are not comparable at all. The integrity of O-antigen-related genes may also affect LPS variability of. Specifically, we have observed a hairpin structure in the middle of the O-antigen glycosyltransferase gene, which led to the division of the gene into two fragments, resulting in incorrect protein synthesis and potential abnormalities in O-antigen production., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The effect of Kamchatka crab hepatopancreas containing three collagenolytic isoenzymes Collagenase KK and proteinases of Streptomyces lavendulae on metabolic activity and cell death were carried out on in vitro models. It was shown that changes in the protein structure under the influence of Collagenase KK occurred earlier than under the effect of bacterial proteinases. At the same time, activity of Collagenase KK was significantly higher than that of bacterial proteinases (p<0.01). Both preparations had a pronounced time- and dose-dependent effects on metabolic activity of cells. Collagenase KK had low cytotoxic effect, and cells mainly died by apoptosis. Thus, hepatopancreas collagenase has a high activity and proapoptotic effect on cells and can be used in low concentrations for enzymatic disaggregation of tissues., (© 2024. Springer Science+Business Media, LLC, part of Springer Nature.)

Six previously unknown triterpene glycosides, pacificusosides L-Q ( 1 - 6 ), and two previously known triterpene glycosides, cucumariosides B 1 ( 7 ) and A 5 ( 8 ), were isolated from an alcoholic extract of Pacific sun star, Solaster pacificus . The structures of 1 - 6 were determined using 1D and 2D NMR, ESIMS, and chemical modifications. Compound 1 is a rare type of triterpene glycoside with non-holostane aglycon, having a linear trisaccharide carbohydrate chain. Pacificusosides M-P ( 2 - 5 ) have new structures containing a Δ 8(9) -3,16,18-trihydroxy tetracyclic triterpene moiety. This tetracyclic fragment in sea star or sea cucumber triterpene glycosides was described for the first time. All the compounds under study exhibit low or moderate cytotoxic activity against colorectal carcinoma HCT 116 cells, and breast cancer MDA-MB-231 cells were assessed by MTS assay. Compound 2 effectively suppresses the colony formation of cancer cells at a non-toxic concentration, using the soft-agar assay. A scratch assay has shown a significant anti-invasive potential of compound 2 against HCT 116 cells, but not against MDA-MB-231 cells.

Four new mono- and trisulfated triterpene penta- and tetraosides, djakonoviosides C 1 ( 1 ), D 1 ( 2 ), E 1 ( 3 ), and F 1 ( 4 ) were isolated from the Far Eastern sea cucumber Cucumaria djakonovi (Cucumariidae, Dendrochirotida), along with six known glycosides found earlier in other Cucumaria species. The structures of unreported compounds were established on the basis of extensive analysis of 1D and 2D NMR spectra as well as by HR-ESI-MS data. The set of compounds contains six different types of carbohydrate chains including two new ones. Thus, djakonovioside C 1 ( 1 ) is characterized by xylose as the second residue, that was a branchpoint in the pentasaccharide chain. Meanwhile, only quinovose and rarely glucose have been found earlier in pentasaccharide chains branched at C-2 of the second sugar unit. Djakonovioside E 1 ( 3 ) is characterized by a tetrasaccharide trisulfated chain, with glucose as the second residue. So, in the series of isolated glycosides, three types of sugars in the second position were presented: the most common, quinovose-in six compounds; glucose-in three substances; and the rare xylose-in one glycoside. The set of aglycones was composed of holostane- and non-holostane-type polycyclic systems; the latter comprised normal and reduced side chains. Noticeably, isokoreoside A ( 9 ), isolated from C. djakonovi , was a single glycoside having a 9(11)-double bond, indicating two oxidosqualenecyclases are operating in the process of the biosynthesis of aglycones. Some of the glycosides from C. djakonovi , which were characterized by pentasaccharide branched chains containing one to three sulfate groups, are chemotaxonomic features of the representatives of the genus Cucumaria . The assortment of sugar parts of Cucumaria's glycosides was broadened with previously undescribed penta- and tetrasaccharide moieties. The metabolic network of sugar parts and aglycones is constructed based on biogenetic relationships. The cytotoxic action of compounds 1 - 10 , isolated from C. djakonovi , against human breast cancer cell lines was investigated along with the hemolytic activity. Erythrocytes were, as usual, more sensitive to the membranolytic action of the glycosides than cancer cells. The triple-negative breast cancer MDA-MB-231 cell line was more vulnerable to the action of glycosides in comparison with the other tested cancer cells, while the MCF-7 cell line was less susceptible to cytotoxic action. Djakonovioside E 1 ( 3 ) demonstrated selective action against ER-positive MCF-7 and triple-negative MDA-MB-231 cell lines, while the toxic effect in relation to normal mammary epithelial cells (MCF-10A) was absent. Cucumarioside A 2 -5 ( 6 ) inhibited the formation and growth of colonies of cancer cells to 44% and tumor cell migration to 85% of the control. Quantitative structure-activity relationships (QSAR) were calculated on the basis of the correlational analysis of the physicochemical properties and structural features of the glycosidic molecules and their membranolytic activity. QSAR revealed the extremely complex nature of such relationships, but these calculations correlated well with the observed SAR.

Investigation of the Vietnamese marine sponge Rhabdastrella globostellata led to the isolation of two new polar isomalabaricanes: rhabdastrellosides A ( 1 ) and B ( 2 ). Their structures and stereochemistry were elucidated with the application of 1D and 2D NMR, HRESIMS, and HRESIMS/MS methods, as well as chemical modifications and GC-MS analysis. Metabolites 1 and 2 are the first isomalabaricanes with non-oxidized cyclopentane ring in the tricyclic core system. Moreover, having a 3-O-disaccharide moiety in their structures, they increase a very rare group of isomalabaricane glycosides. We report here a weak cytotoxicity of 1 and 2 toward human neuroblastoma SH-SY5Y cells and normal rat H9c2 cardiomyocytes, as well as the cytoprotective activity of rhabdastrelloside B ( 2 ) at 1 µM evaluated using CoCl 2 -treated SH-SY5Y and H9c2 cells.

Due to the increasing prevalence of fungal diseases caused by fungi of the genus Candida and the development of pathogen resistance to available drugs, the need to find new effective antifungal agents has increased. Azole antifungals, which are inhibitors of sterol-14α-demethylase or CYP51, have been widely used in the treatment of fungal infections over the past two decades. Of special interest is the study of C. krusei CYP51, since this fungus exhibit resistance not only to azoles, but also to other antifungal drugs and there is no available information about the ligand-binding properties of CYP51 of this pathogen. We expressed recombinant C. krusei CYP51 in E. coli cells and obtained a highly purified protein. Application of the method of spectrophotometric titration allowed us to study the interaction of C. krusei CYP51 with various ligands. In the present work, the interaction of C. krusei CYP51 with azole inhibitors, and natural and synthesized steroid derivatives was evaluated. The obtained data indicate that the resistance of C. krusei to azoles is not due to the structural features of CYP51 of this microorganism, but rather to another mechanism. Promising ligands that demonstrated sufficiently strong binding in the micromolar range to C. krusei CYP51 were identified, including compounds 99 (Kd = 1.02 ± 0.14 µM) and Ch-4 (Kd = 6.95 ± 0.80 µM). The revealed structural features of the interaction of ligands with the active site of C. krusei CYP51 can be taken into account in the further development of new selective modulators of the activity of this enzyme.

Antibodies recognizing RBD and the S-protein have been previously demonstrated to be formed in humans after SARS-CoV-2 infection and vaccination with the Sputnik V adenovirus vaccine. These antibodies were found to be active when hydrolyzing FITC-labeled oligopeptides corresponding to linear epitopes of the S-protein. The thin-layer chromatography method allows the relative accumulation of the reaction product to be estimated but cannot identify hydrolysis sites. This study used the MALDI-TOF MS method to establish oligopeptide hydrolysis sites. Using the MALDI-TOF MS method in combination with the analysis of known hydrolysis sites characteristic of canonical proteases allowed us to establish the unique hydrolysis sites inherent only to catalytically active antibodies. We have discovered two 12-mer oligopeptides to have six hydrolysis sites equally distributed throughout the oligopeptide. The other three oligopeptides were found to have two to three closely spaced hydrolysis sites. In contrast to trypsin and chymotrypsin proteases, the catalytically active antibodies of COVID-19 patients have their peptide bond hydrolyzed mainly after proline, threonine, glycine, or serine residues. Here, we propose a new high-throughput experimental method for analyzing the proteolytic activity of natural antibodies produced in viral pathology.

Extracellular vesicles (EVs), carriers of molecular signals, are considered a critical link in maintaining homeostasis in mammals. Currently, there is growing interest in studying the role of EVs, including exosomes (subpopulation of EVs), in animals of other evolutionary levels, including marine invertebrates. We have studied the possibility of obtaining appropriate preparations of EVs from whole-body extract of holothuria Eupentacta fraudatrix using a standard combination of centrifugation and ultracentrifugation. However, the preparations were heavily polluted, which did not allow us to conclude that they contained vesicles. Subsequent purification by FLX gel filtration significantly reduced the pollution but did not increase vesicle concentration to a necessary level. To detect EVs presence in the body of holothurians, we used transmission electron microscopy of ultrathin sections. Late endosomes, producing the exosomes, were found in the cells of the coelom epithelium covering the gonad, digestive tube and respiratory tree, as well as in the parenchyma cells of these organs. The study of purified homogenates of these organs revealed vesicles (30-100 nm) morphologically corresponding to exosomes. Thus, we can say for sure that holothurian cells produce EVs including exosomes, which can be isolated from homogenates of visceral organs.

Seven new monosulfated triterpene glycosides, djakonoviosides A ( 1 ), A 1 ( 2 ), A 2 ( 3 ), and B 1 -B 4 ( 4 - 7 ), along with three known glycosides found earlier in the other Cucumaria species, namely okhotoside A 1 -1, cucumarioside A 0 -1, and frondoside D, have been isolated from the far eastern sea cucumber Cucumaria djakonovi (Cucumariidae, Dendrochirotida). The structures were established on the basis of extensive analysis of 1D and 2D NMR spectra and confirmed by HR-ESI-MS data. The compounds of groups A and B differ from each other in their carbohydrate chains, namely monosulfated tetrasaccharide chains are inherent to group A and pentasaccharide chains with one sulfate group, branched by C-2 Qui2, are characteristic of group B. The aglycones of djakonoviosides A 2 ( 3 ), B 2 ( 5 ), and B 4 ( 7 ) are characterized by a unique structural feature, a 23,16-hemiketal fragment found first in the sea cucumbers' glycosides. The biosynthetic pathway of its formation is discussed. The set of aglycones of C. djakonovi glycosides was species specific because of the presence of new aglycones. At the same time, the finding in C. djakonovi of the known glycosides isolated earlier from the other species of Cucumaria , as well as the set of carbohydrate chains characteristic of the glycosides of all investigated representatives of the genus Cucumaria , demonstrated the significance of these glycosides as chemotaxonomic markers. The membranolytic actions of compounds 1 - 7 and known glycosides okhotoside A 1 -1, cucumarioside A 0 -1, and frondoside D, isolated from C. djakonovi against human cell lines, including erythrocytes and breast cancer cells (MCF-7, T-47D, and triple negative MDA-MB-231), as well as leukemia HL-60 and the embryonic kidney HEK-293 cell line, have been studied. Okhotoside A 1 -1 was the most active compound from the series because of the presence of a tetrasaccharide linear chain and holostane aglycone with a 7(8)-double bond and 16 β - O -acetoxy group, cucumarioside A 0 -1, having the same aglycone, was slightly less active because of the presence of branching xylose residue at C-2 Qui2. Generally, the activity of the djakonoviosides of group A was higher than that of the djakonoviosides of group B containing the same aglycones, indicating the significance of a linear chain containing four monosaccharide residues for the demonstration of membranolytic action by the glycosides. All the compounds containing hemiketal fragments, djakonovioside A 2 ( 3 ), B 2 ( 5 ), and B 4 ( 7 ), were almost inactive. The most aggressive triple-negative MDA-MB-231 breast cancer cell line was the most sensitive to the glycosides action when compared with the other cancer cells. Okhotoside A 1 -1 and cucumarioside A 0 -1 demonstrated promising effects against MDA-MB-231 cells, significantly inhibiting the migration, as well as the formation and growth, of colonies.

Sea cucumber triterpene glycosides are a class of secondary metabolites that possess distinctive chemical structures and exhibit a variety of biological and pharmacological activities. The application of MS-based approaches for the study of triterpene glycosides allows rapid evaluation of the structural diversity of metabolites in complex mixtures. However, the identification of the detected triterpene glycosides can be challenging. The objective of this study is to establish the first spectral library containing the mass spectra of sea cucumber triterpene glycosides using ultraperformance liquid chromatography-quadrupole time-of-flight mass spectrometry. The library contains the electrospray ionization tandem mass spectra and retention times of 191 triterpene glycosides previously isolated from 15 sea cucumber species and one starfish at the Laboratory of the Chemistry of Marine Natural Products of the G.B. Elyakov Pacific Institute of Bioorganic Chemistry. In addition, the chromatographic behavior and some structure-related neutral losses in tandem MS are discussed. The obtained data will accelerate the accurate dereplication of known triterpene glycosides and the annotation of novel compounds, as we demonstrated by the processing of LC-MS/MS data of Eupentacta fraudatrix extract.

Histones play vital roles in chromatin function and gene transcription; however, they are very harmful in the intercellular space because they stimulate systemic inflammatory and toxic responses. Myelin basic protein (MBP) is the major protein of the axon myelin-proteolipid sheath. Antibodies-abzymes with various catalytic activities are specific features of some autoimmune diseases. IgGs against individual histones (H2A, H1, H2B, H3, and H4) and MBP were isolated from the blood of experimental-autoimmune-encephalomyelitis-prone C57BL/6 mice by several affinity chromatographies. These Abs-abzymes corresponded to various stages of EAE development: spontaneous EAE, MOG, and DNA-histones accelerated the onset, acute, and remission stages. IgGs-abzymes against MBP and five individual histones showed unusual polyreactivity in the complex formation and enzymatic cross-reactivity in the specific hydrolysis of the H2A histone. All the IgGs of 3-month-old mice (zero time) against MBP and individual histones demonstrated from 4 to 35 different H2A hydrolysis sites. The spontaneous development of EAE over 60 days led to a significant change in the type and number of H2A histone hydrolysis sites by IgGs against five histones and MBP. Mice treatment with MOG and the DNA-histone complex changed the type and number of H2A hydrolysis sites compared to zero time. The minimum number (4) of different H2A hydrolysis sites was found for IgGs against H2A (zero time), while the maximum (35) for anti-H2B IgGs (60 days after mice treatment with DNA-histone complex). Overall, it was first demonstrated that at different stages of EAE evolution, IgGs-abzymes against individual histones and MBP could significantly differ in the number and type of specific sites of H2A hydrolysis. The possible reasons for the catalytic cross-reactivity and great differences in the number and type of histone H2A cleavage sites were analyzed.

In our research on sphingolipids from marine invertebrates, a mixture of phytoceramides was isolated from the sponge Monanchora clathrata (Western Australia). Total ceramide, ceramide molecular species (obtained by RP-HPLC, high-performance liquid chromatography on reversed-phase column) and their sphingoid/fatty acid components were analyzed by NMR (nuclear magnetic resonance) spectroscopy and mass spectrometry. Sixteen new ( 1b , 3a , 3c , 3d , 3f , 3g , 5c , 5d , 5f , 5g , 6b - g ) and twelve known ( 2b , 2e , 2f , 3b , 3e , 4a - c , 4e , 4f , 5b , 5e ) compounds were shown to contain phytosphingosine-type backbones i -t17:0 ( 1 ), n -t17:0 ( 2 ), i -t18:0 ( 3 ), n -t18:0 ( 4 ), i -t19:0 ( 5 ), or ai -t19:0 ( 6 ), N -acylated with saturated (2 R )-2-hydroxy C 21 ( a ), C 22 ( b ), C 23 ( c ), i- C 23 ( d ), C 24 ( e ), C 25 ( f ), or C 26 ( g ) acids. The used combination of the instrumental and chemical methods permitted the more detailed investigation of the sponge ceramides than previously reported. It was found that the cytotoxic effect of crambescidin 359 (alkaloid from M. clathrata ) and cisplatin decreased after pre-incubation of MDA-MB-231 and HL-60 cells with the investigated phytoceramides. In an in vitro paraquat model of Parkinson's disease, the phytoceramides decreased the neurodegenerative effect and ROS (reactive oxygen species) formation induced by paraquat in neuroblastoma cells. In general, the preliminary treatment (for 24 or 48 h) of the cells with the phytoceramides of M. clathrata was necessary for their cytoprotective functions, otherwise the additive damaging effect of these sphingolipids and cytotoxic compounds (crambescidin 359, cisplatin or paraquat) was observed.

Purinergic P2X7 receptors (P2X7) have now been proven to play an important role and represent an important therapeutic target in many pathological conditions including neurodegeneration. Here, we investigated the impact of peptides on purinergic signaling in Neuro-2a cells through the P2X7 subtype in in vitro models. We have found that a number of recombinant peptides, analogs of sea anemone Kunitz-type peptides, are able to influence the action of high concentrations of ATP and thereby reduce the toxic effects of ATP. The influx of calcium, as well as the fluorescent dye YO-PRO-1, was significantly suppressed by the studied peptides. Immunofluorescence experiments confirmed that the peptides reduce the P2X7 expression level in neuronal Neuro-2a cells. Two selected active peptides, HCRG1 and HCGS1.10, were found to specifically interact with the extracellular domain of P2X7 and formed stable complexes with the receptor in surface plasmon resonance experiments. The molecular docking approach allowed us to establish the putative binding sites of the most active HCRG1 peptide on the extracellular domain of the P2X7 homotrimer and propose a mechanism for regulating its function. Thus, our work demonstrates the ability of the Kunitz-type peptides to prevent neuronal death by affecting signaling through the P2X7 receptor.

Three new tetrasulfated triterpene glycosides, chilensosides E ( 1 ), F ( 2 ), and G ( 3 ), have been isolated from the Far-Eastern sea cucumber Paracaudina chilensis (Caudinidae, Molpadida). The structures were established based on extensive analysis of 1D and 2D NMR spectra and confirmed by HR-ESI-MS data. The compounds differ in their carbohydrate chains, namely in the number of monosaccharide residues (five or six) and in the positions of sulfate groups. Chilensosides E ( 1 ) and F ( 2 ) are tetrasulfated pentaosides with the position of one of the sulfate groups at C-3 Glc3, and chilensoside G ( 3 ) is a tetrasulfated hexaoside. The biogenetic analysis of the glycosides of P. chilensis has revealed that the structures form a network due to the attachment of sulfate groups to almost all possible positions. The upper semi-chain is sulfated earlier in the biosynthetic process than the lower one. Noticeably, the presence of a sulfate group at C-3 Glc3-a terminal monosaccharide residue in the bottom semi-chain of compounds 1 and 2 -excludes the possibility of this sugar chain's further elongation. Presumably, the processes of glycosylation and sulfation are concurrent biosynthetic stages. They can be shifted in time in relation to each other, which is a characteristic feature of the mosaic type of biosynthesis. The hemolytic action of compounds 1 - 3 against human erythrocytes and cytotoxic activities against five human cancer cell lines were tested. The compounds showed moderate hemolytic activity but were inactive against cancer cells, probably because of their structural peculiarities, such as the combination of positions of four sulfate groups.

Exosomes are nanovesicles with a 40-150 nm diameter and are essential for communication between cells. Literature data suggest that exosomes obtained from different sources (cell cultures, blood plasma, urea, saliva, tears, spinal fluid, milk) using a series of centrifugations and ultracentrifugations contain hundreds and thousands of different protein and nucleic acid molecules. However, most of these proteins are not an intrinsic part of exosomes; instead, they co-isolate with exosomes. Using consecutive ultracentrifugation, gel filtration, and affinity chromatography on anti-CD9- and anti-CD63-Sepharoses, we isolated highly purified vesicle preparations from 18 horse milk samples. Gel filtration of the initial preparations allowed us to remove co-isolating proteins and their complexes and to obtain highly purified vesicles morphologically corresponding to exosomes. Using affinity chromatography on anti-CD9- and anti-CD63-Sepharoses, we obtained extra-purified CD9 + and CD63 + exosomes, which simultaneously contain these two tetraspanins, while the CD81 tetraspanin was presented in a minor quantity. SDS-PAGE and MALDI analysis detected several major proteins with molecular masses over 10 kDa: CD9, CD63, CD81, lactadherin, actin, butyrophilin, lactoferrin, and xanthine dehydrogenase. Analysis of extracts by trifluoroacetic acid revealed dozens of peptides with molecular masses in the range of 0.8 to 8.5 kDa. Data on the uneven distribution of tetraspanins on the surface of horse milk exosomes and the presence of peptides open new questions about the biogenesis of these extracellular vesicles.

Two new guanidine alkaloids, batzelladines O ( 1 ) and P ( 2 ), were isolated from the deep-water marine sponge Monanchora pulchra. The structures of these metabolites were determined by NMR spectroscopy, mass spectrometry, and ECD. The isolated compounds exhibited cytotoxic activity in human prostate cancer cells PC3, PC3-DR, and 22Rv1 at low micromolar concentrations and inhibited colony formation and survival of the cancer cells. Batzelladines O ( 1 ) and P ( 2 ) induced apoptosis, which was detected by Western blotting as caspase-3 and PARP cleavage. Additionally, induction of pro-survival autophagy indicated as upregulation of LC3B-II and suppression of mTOR was observed in the treated cells. In line with this, the combination with autophagy inhibitor 3-methyladenine synergistically increased the cytotoxic activity of batzelladines O ( 1 ) and P ( 2 ). Both compounds were equally active in docetaxel-sensitive and docetaxel-resistant prostate cancer cells, despite exhibiting a slight p-glycoprotein substrate-like activity. In combination with docetaxel, an additive effect was observed. In conclusion, the isolated new guanidine alkaloids are promising drug candidates for the treatment of taxane-resistant prostate cancer.

The structural characterization of lipopolysaccharides has critical implications for some biomedical applications, and marine bacteria are an inimitable source of new glyco-structures potentially usable in medicinal chemistry. On the other hand, lipopolysaccharides of marine Gram-negative bacteria present certain structural features that can help the understanding of the adaptation processes. The deep-sea marine Gram-negative bacterium Idiomarina zobellii KMM 231 T , isolated from a seawater sample taken at a depth of 4000 m, represents an engaging microorganism to investigate in terms of its cell wall components. Here, we report the structural study of the R-type lipopolysaccharide isolated from I. zobellii KMM 231 T that was achieved through a multidisciplinary approach comprising chemical analyses, NMR spectroscopy, and MALDI mass spectrometry. The lipooligosaccharide turned out to be characterized by a novel and unique pentasaccharide skeleton containing a very short mono-phosphorylated core region and comprising terminal neuraminic acid. The lipid A was revealed to be composed of a classical disaccharide backbone decorated by two phosphate groups and acylated by i13:0(3-OH) in amide linkage, i11:0 (3-OH) as primary ester-linked fatty acids, and i11:0 as a secondary acyl chain.

Histones have a specific key role in the remodeling of chromatin and gene transcription. In the blood, free histones are damage-connected proteins. Myelin basic protein (MBP) is the major component of the myelin-proteolipid sheath of axons. Antibodies possessing enzymatic activities (abzymes, ABZs) are the specific features of several autoimmune pathologies. IgGs against five histones, MBP, and DNA were obtained from the sera of multiple sclerosis (MS) patients using several affinity chromatographies. The sites of H3 histone splitting by Abs against five individual histones, MBP, and DNA were revealed by MALDI mass spectrometry. It was shown that the number of H3 splitting sites by IgGs against five various histones is different (number of sites): H3 (11), H1 (14), H2A (11), H4 (17), MBP (22), and DNA (29). IgGs against five different histones hydrolyze H3 at different sites, and only a few them coincide. The main reason for the enzymatic cross-reactivity of Abs against H3 and four other histones, as well as MBP, might be the high level of these proteins' homology. The effective hydrolysis of the H3 histone at 29 sites with IgGs against DNA can be explained by the formation of chimeric abzymes against hybrid antigenic determinants formed by different histones and MBP at the junction of these protein sequences with DNA. The active centers of such abzymes contain structural elements of canonical DNases and proteases. Since free histones are pernicious proteins, antibodies-ABZs against five histones, MBP, and DNA could have a negative role in the pathogenesis of MS and probably other various autoimmune diseases.

The nicotinic acetylcholine receptors (nAChRs) are prototypical ligand-gated ion channels, provide cholinergic signaling, and are modulated by various venom toxins and drugs in addition to neurotransmitters. Here, four APETx-like toxins, including two new toxins, named Hmg 1b-2 Met ox and Hmg 1b-5, were isolated from the sea anemone Heteractis magnifica and characterized as novel nAChR ligands and acid-sensing ion channel (ASIC) modulators. All peptides competed with radiolabeled α-bungarotoxin for binding to Torpedo californica muscle-type and human α7 nAChRs. Hmg 1b-2 potentiated acetylcholine-elicited current in human α7 receptors expressed in Xenopus laevis oocytes. Moreover, the multigene family coding APETx-like peptides library from H. magnifica was described and in silico surface electrostatic potentials of novel peptides were analyzed. To explain the 100% identity of some peptide isoforms between H. magnifica and H. crispa , 18S rRNA, COI, and ITS analysis were performed. It has been shown that the sea anemones previously identified by morphology as H. crispa belong to the species H. magnifica .

The indicators of spermatogenesis and the state of LPO and antioxidant protection in men with pathozoospermia after COVID-19 were assessed before and after treatment an antioxidant complex. Blood plasma served as the material for biochemical studies. In the examined patients, the parameters of spermatogenesis, as well as blood concentration of LPO components (diene conjugates and TBA-reactive substances) were analyzed. The total antioxidant activity of the blood was determined as an indicator characterizing the total activity of LPO inhibitors and determining its buffer capacity. In patients recovered from COVID-19, an increase in spermatogenesis disorders and shifts towards the predominance of prooxidant factors were observed. After a course (1 month) of antioxidant complex, patients showed increased sperm motility, decreased leukocyte count in the ejaculate, and restored balance in the prooxidant-antioxidant system towards antioxidant components. The effectiveness of correction of post-COVID disorders largely depends on the degree of damage to the structure and function of cell membranes caused by oxidative stress. The use of the antioxidant complex is a promising option, because it reduces the level of LPO, enhances antioxidant protection of the body, and also normalizes some parameters of spermatogenesis., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Histones play vital roles in chromatin functioning and gene transcription, but in intercellular space, they are harmful due to stimulating systemic inflammatory and toxic responses. Myelin basic protein (MBP) is the most important protein of the axon myelin-proteolipid sheath. Antibodies-abzymes with different catalytic activities are critical and specific features of some autoimmune diseases. Five IgG preparations against histones (H4, H1, H2A, H2B, and H3) and against MBP corresponding to different spontaneous, MOG (myelin oligodendrocyte glycoprotein of mice), and DNA-histones that accelerated onset, acute, and remission stages of experimental autoimmune encephalomyelitis (EAE; model of human multiple sclerosis) development were obtained from EAE-prone C57BL/6 mice by several affinity chromatographies. IgG-abzymes against five histones and MBP possess unusual polyreactivity in complexation and catalytic cross-reactivity in the hydrolysis of histone H4. IgGs against five histones and MBP corresponding to 3 month-old mice (zero time) in comparison with Abs corresponding to spontaneous development of EAE during 60 days differ in type and number of H4 sites for hydrolysis. Immunization of mice with MOG and DNA-histones complex results in an acceleration of EAE development associated with an increase in the activity of antibodies in H4 hydrolysis. Twenty days after mouse immunization with MOG or DNA-histones complex, the IgGs hydrolyze H4 at other additional sites compared to zero time. The maximum number of different sites of H4 hydrolysis was revealed for IgGs against five histones and MBP at 60 days after immunization of mice with MOG and DNA-histones. Overall, it first showed that at different stages of EAE development, abzymes could significantly differ in specific sites of H4 hydrolysis.

The bacterium Streptomyces sp. KMM 9044 from a sample of marine sediment collected in the northwestern part of the Sea of Japan produces highly chlorinated depsiheptapeptides streptocinnamides A ( 1 ) and B ( 2 ), representatives of a new structural group of antibiotics. The structures of 1 and 2 were determined using nuclear magnetic resonance and mass spectrometry studies and confirmed by a series of chemical transformations. Streptocinnamide A potently inhibits Micrococcus sp. KMM 1467, Arthrobacter sp. ATCC 21022, and Mycobacterium smegmatis MC2 155.

Only some human organs, including the liver, are capable of very weak self-regeneration. Some marine echinoderms are very useful for studying the self-regeneration processes of organs and tissues. For example, sea cucumbers Eupentacta fraudatrix (holothurians) demonstrate complete restoration of all organs and the body within several weeks after their division into two parts. Therefore, these cucumbers are a prospective model for studying the general mechanisms of self-regeneration. However, there is no data available yet concerning biomolecules of holothurians, which can stimulate the processes of organ and whole-body regeneration. Investigation of these restoration mechanisms is very important for modern medicine and biology because it can help to understand which hormones, nucleic acids, proteins, enzymes, or complexes play an essential role in self-regeneration. It is possible that stable, polyfunctional, high-molecular-weight protein complexes play an essential role in these processes. It has recently been shown that sea cucumbers Eupentacta fraudatrix contain a very stable multiprotein complex of about 2000 kDa. The first analysis of possible enzymatic activities of a stable protein complex was carried out in this work, revealing that the complex possesses several protease and DNase activities. The complex metalloprotease is activated by several metal ions (Zn2+ > Mn2+ > Mg2+). The relative contribution of metalloproteases (~63.4%), serine-like protease (~30.5%), and thiol protease (~6.1%) to the total protease activity of the complex was estimated. Metal-independent proteases of the complex hydrolyze proteins at trypsin-specific sites (after Lys and Arg). The complex contains both metal-dependent and metal-independent DNases. Mg2+, Mn2+, and Co2+ ions were found to strongly increase the DNase activity of the complex.

Five new triterpene di-, tri- and tetrasulfated hexaosides (chitonoidosides I ( 1 ), J ( 2 ), K ( 3 ), K 1 ( 4 ) and L ( 5 )) were isolated from the Far-Eastern sea cucumber Psolus chitonoides , collected near Bering Island (Commander Islands) from a depth of 100-150 m. The structural variability of the glycosides concerned both the aglycones (with 7(8)- or 9(11)-double bonds) and carbohydrate chains differing from each other by the third sugar residue (Xyl or sulfated by C-6 Glc) and/or by the fourth-terminal in the bottom semi-chain-residue (Glc or sulfated by C-6 MeGlc) as well as by the positions of a sulfate group at C-4 or C-6 in the sixth-terminal in the upper semi-chain-residue (MeGlc). Hemolytic activities of these compounds 1 - 5 against human erythrocytes as well as cytotoxicity against human cancer cell lines, HeLa, DLD-1 and HL-60, were studied. The hexaosides, chitonoidosides K ( 3 ) and L ( 5 ) with four sulfate groups, were the most active against tumor cells in all the tests. Noticeably, the sulfate group at C-4 of MeGlc6 did not decrease the membranolytic effect of 5 as compared with 3 , having the sulfate group at C-6 of MeGlc6. Erythrocytes were, as usual, more sensitive to the action of the studied glycosides than cancer cells, although the sensitivity of leukemia promyeloblast HL-60 cells was higher than that of other tumor cells. The glycosides 1 and 2 demonstrated some weaker action in relation to DLD-1 cells than against other tumor cell lines. Chitonoidoside K 1 ( 4 ) with a hydroxyl at C 25 of the aglycone was not active in all the tests. The metabolic network formed by the carbohydrate chains of all the glycosides isolated from P. chitonoides as well as the aglycones biosynthetic transformations during their biosynthesis are discussed and illustrated with schemes.

Today, marine natural products are considered one of the main sources of compounds for drug development. Starfish and sea cucumbers are potential sources of natural products of pharmaceutical interest. Among their metabolites, polar steroids, triterpene glycosides, and polar lipids have attracted a great deal of attention; however, studying these compounds by conventional methods is challenging. The application of modern MS-based approaches can help to obtain valuable information about such compounds. This review provides an up-to-date overview of MS-based applications for starfish and sea cucumber bioactive compounds analysis. While describing most characteristic features of MS-based approaches in the context of starfish and sea cucumber metabolites, including sample preparation and MS analysis steps, the present paper mainly focuses on the application of MS-based metabolic profiling of polar steroid compounds, triterpene glycosides, and lipids. The application of MS in metabolomics studies is also outlined.

Sea stars or starfish (class Asteroidea) and holothurians or sea cucumbers (class Holothuroidea), belonging to the phylum Echinodermata (echinoderms), are characterized by different sets of glycosidic metabolites: the steroid type in starfish and the triterpene type in holothurians. However, herein we report the isolation of eight new triterpene glycosides, pacificusosides D−K (1−3, 5−9) along with the known cucumarioside D (4), from the alcoholic extract of the Far Eastern starfish Solaster pacificus. The isolated new compounds are closely related to the metabolites of sea cucumbers, and their structures of 1−3 and 5−9 were determined by extensive NMR and ESIMS techniques. Compounds 2, 5, and 8 have a new type of tetrasaccharide chain with a terminal non-methylated monosaccharide unit. Compounds 3, 6, and 9 contain another new type of tetrasaccharide chain, having 6-O-SO3-Glc as one of the sugar units. The cytotoxic activity of 1−9 against non-cancerous mouse epidermal cells JB6 Cl41 and human melanoma cell lines SK-MEL-2, SK-MEL-28, and RPMI-7951 was determined by MTS assay. Compounds 1, 3, 4, 6, and 9 showed potent cytotoxicity against these cell lines, but the cancer selectivity (SI > 9) was observed only against the SK-MEL-2 cell line. Compounds 1, 3, 4, 6, and 9 at the non-toxic concentration of 0.1 μM significantly inhibited neoplastic cell transformation of JB6 Cl41 cells induced by chemical carcinogens (EGF, TPA) or ionizing radiation (X-rays and UVB). Moreover, compounds 1 and 4 at the non-toxic concentration of 0.1 µM possessed the highest inhibiting activity on colony formation among the investigated compounds and decreased the colonies number of SK-MEL-2 cells by 64% and 70%, respectively. Thus, triterpene glycosides 1 and 4 can be considered as prospective cancer-preventive and anticancer-compound leaders.

Metal ions and other elements play many different critical roles in all biological processes. They can be especially important in high concentrations for the functioning of organisms living in seawater. It is important to understand how much the concentrations of different trace elements in such organisms can be higher than in seawater. Some marine organisms capable of rapid recovery after different injuries are fascinating in this regard. Sea cucumbers Eupentacta fraudatrix can completely restore all organs and the whole body within several weeks after their division into two parts. Here, for the first time, a comparison of the content of different elements in seawater, sea cucumber, and its very stable multiprotein complex (2000 kDa) was performed using two-jet plasma atomic emission spectrometry. Among the 18 elements we found in sea cucumbers, seawater contained only six elements in detectable amounts, and their content decreased in the following order: Mg > Ca > B > Sr ≈ Si > Cr (0.13−930 µg/g of seawater). The content of these elements in sea cucumbers was higher compared with seawater (-fold): Ca (714) > Sr (459) > Cr (75) > Si (42)> B (12) > Mg (6.9). Only four of them had a higher concentration in the protein complex than in seawater (-fold): Si (120.0) > Cr (31.5) > Ca (9.1) > Sr (8.8). The contents of Mg and B were lower in the protein complex than in seawater. The content of elements additionally found in sea cucumbers decreased in the order (µg/g of powder) of P (1100) > Fe (47) > Mn (26) > Ba (15) > Zn (13) > Al (9.3) > Mo (2.8) > Cu (1.4) > Cd (0.3), and in the protein complex, in the order of P (290) > Zn (51) > Fe (23) > Al (14) ≈ Ni (13) > Cu (7.5) > Ba (2.5) ≈ Co (2.0) ≈ Mn (1.6) > Cd (0.7) >Ag (0.2). Thus, sea cucumbers accumulate various elements, including those contained in very low concentrations in seawater. The possible biological roles of these elements are discussed here.

Mother's milk provides newborns with various nutrients (e.g., enzymes, proteins, peptides, hormones, antibodies) that help babies grow and protect them from bacterial and viral infections. The functions of many components of breast milk can be very different from their corresponding functions in body fluids of healthy adults. Catalytic antibodies (abzymes) that hydrolyze peptides, proteins, DNA, RNA, and oligosaccharides were detected not only in human milk, but also in the blood sera of autoimmune patients. However, abzymes with unexpected synthetic activities (lipids, oligosaccharides, and protein kinase activities) were revealed in milk that were not found in the blood of autoimmune patients. The nutrition of infants with fresh milk has a very specific role; newborns are well protected by antibodies of mother's milk (passive immunity). Protease abzymes were found in the blood sera of autoimmune patients, whereas healthy humans usually do not contain such autoantibodies. Here, we present the first evidence that the milk of healthy mothers contains secretory (s)IgA that effectively hydrolyze 5 histones (e.g., H1, H2A, H2B, H3, and H4) and myelin basic protein (MBP). Several rigid criteria were applied to show that protease activity is an intrinsic property of sIgA. Milk abzymes against 5 histones cannot hydrolyze different control proteins except histones and MBP, whereas autoantibodies against MBP split this protein and 5 histones. Antibodies against histones and MBP exhibit complexation polyreactivity as well as specific and unusual catalytic cross-reactivity. With some exceptions, the specific sites of hydrolysis of H1, H2A, and H2B by sIgA against histones do not coincide with the sites of hydrolysis by abzymes against MBP. On the whole, fresh human milk is a very specific source of many of the most unusual antibodies and abzymes., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Four new triterpene disulfated glycosides, chitonoidosides E 1 ( 1 ), F ( 2 ), G ( 3 ), and H ( 4 ), were isolated from the Far-Eastern sea cucumber Psolus chitonoides and collected near Bering Island (Commander Islands) at depths of 100-150 m. Among them there are two hexaosides ( 1 and 3 ), differing from each other by the terminal (sixth) sugar residue, one pentaoside ( 4 ) and one tetraoside ( 2 ), characterized by a glycoside architecture of oligosaccharide chains with shortened bottom semi-chains, which is uncommon for sea cucumbers. Some additional distinctive structural features inherent in 1 - 4 were also found: the aglycone of a recently discovered new type, with 18(20)-ether bond and lacking a lactone in chitonoidoside G ( 3 ), glycoside 3- O -methylxylose residue in chitonoidoside E 1 ( 1 ), which is rarely detected in sea cucumbers, and sulfated by uncommon position 4 terminal 3- O -methylglucose in chitonoidosides F ( 2 ) and H ( 4 ). The hemolytic activities of compounds 1 - 4 and chitonoidoside E against human erythrocytes and their cytotoxic action against the human cancer cell lines, adenocarcinoma HeLa, colorectal adenocarcinoma DLD-1, and monocytes THP-1, were studied. The glycoside with hexasaccharide chains ( 1 , 3 and chitonoidoside E) were the most active against erythrocytes. A similar tendency was observed for the cytotoxicity against adenocarcinoma HeLa cells, but the demonstrated effects were moderate. The monocyte THP-1 cell line and erythrocytes were comparably sensitive to the action of the glycosides, but the activity of chitonoidosides E and E 1 ( 1 ) significantly differed from that of 3 in relation to THP-1 cells. A tetraoside with a shortened bottom semi-chain, chitonoidoside F ( 2 ), displayed the weakest membranolytic effect in the series.

Oceanalin B ( 1 ), an α,ω-bipolar natural product belonging to a rare family of sphingoid tetrahydoisoquinoline β-glycosides, was isolated from the EtOH extract of the lyophilized marine sponge Oceanapia sp. as the second member of the series after oceanalin A ( 2 ) from the same animal. The compounds are of particular interest due to their biogenetically unexpected structures as well as their biological activities. The structure and absolute stereochemistry of 1 as a α,ω-bifunctionalized sphingoid tetrahydroisoquinoline β-glycoside was elucidated using NMR, CD and MS spectral analysis and chemical degradation. Oceanalin B exhibited in vitro antifungal activity against Candida glabrata with a MIC of 25 μg/mL.

In contrast to many human organs, only the human liver can self-regenerate, to some degree. Some marine echinoderms are convenient objects for studying the processes of regenerations of organs and tissues. For example, sea cucumbers Eupentacta fraudatrix can completely restore within several weeks, the internal organs and the whole body after their division into two or three parts. Therefore, these cucumbers are a very convenient model for studying the general mechanisms of regeneration. However, there is no literature data yet on which biomolecules of these cucumbers can stimulate the regeneration of organs and the whole-body processes. Studying the mechanisms of restoration is very important for modern biology and medicine, since it can help researchers to understand which proteins, enzymes, hormones, or possible complexes can play an essential role in regeneration. This work is the first to analyze the possible content of very stable protein complexes in sea cucumbers Eupentacta fraudatrix . It has been shown that their organisms contain a very stable multiprotein complex of about 2000 kDa. This complex contains 15 proteins with molecular masses (MMs) >10 kDa and 21 small proteins and peptides with MMs 2.0-8.6 kDa. It is effectively destroyed only in the presence of 3.0 M MgCl 2 and, to a lesser extent, 3.0 M NaCl, while the best dissociation occurs in the presence of 8.0 M urea + 0.1 M EDTA. Our data indicate that forming a very stable proteins complex occurs due to the combination of bridges formed by metal ions, electrostatic contacts, and hydrogen bonds.

Six new triterpene tetra-, penta- and hexaosides, chitonoidosides A ( 1 ), A 1 ( 2 ), B ( 3 ), C ( 4 ), D ( 5 ), and E ( 6 ), containing one or two sulfate groups, have been isolated from the Far-Eastern sea cucumber Psolus chitonoides , collected near Bering Island (Commander Islands) from the depth of 100-150 m. Three of the isolated compounds ( 1 , 3 and 6 ) are characterized by the unusual aglycone of new type having 18(20)-ether bond and lacking a lactone in contrast with wide spread holostane derivatives. Another unexpected finding is 3- O -methylxylose residue as a terminal unit in the carbohydrate chains of chitonoidosides B ( 3 ), C ( 4 ), and E ( 6 ), which has never been found before in the glycosides from holothurians belonging to the Psolidae family. Moreover, this monosaccharide is sulfated in the compound 4 into unprecedented 3- O -methylxylose 4- O -sulfate residue. Chitonoidoside C ( 4 ) is characterized by tetrasaccharide moiety lacking a part of the bottom semi-chain, but having disaccharide fragment attached to C-4 of Xyl1. Such architecture is not common in sea cucumber glycosides. Cytotoxic activities of the compounds 1 - 5 against mouse and human erythrocytes and human cancer cell lines: adenocarcinoma HeLa, colorectal adenocarcinoma DLD-1, and leukemia promyeloblast HL-60 cells were studied. The cytotoxic effect of chitonoidoside d ( 5 ) was the most significant in this series due to the presence of pentasaccharide disulfated sugar chain in combination with holostane aglycone. Surprisingly, the glycosides 1 and 3 , comprising the new aglycone without γ-lactone, demonstrated similar activity to the known compounds with holostane aglycones. Chitonoidoside C ( 4 ) was less cytotoxic due to the different architecture of the carbohydrate chain compared to the other glycosides and probably due to the presence of a sulfate group at C-4 in 3- O -MeXyl4.

Histones play a key role in chromatin remodeling and gene transcription. Further, free histones in the blood act as damage-associated molecules. Administration of histones to animals results in systemic inflammatory and toxic effects. Myelin basic protein is the principal constituent element of the myelin-proteolipid sheath of axons. Abzymes (antibodies with catalytic activities) are the original features of some autoimmune diseases. In this study, electrophoretically homogeneous IgGs against H1, H2A, H2B, H3, and H4 histones and myelin basic protein (MBP) were isolated from the blood sera of multiple sclerosis (MS) patients by several affinity chromatographies. Using MALDI mass spectrometry, the sites of H1 histone cleavage by IgGs against H1, H2A, H2B, H3, H4, and MBP were determined. It was shown that IgGs against H1 split H1 at 12 sites, while the number of cleavage sites by abzymes against other histones was lower: H2A (9), H2B (7), H3 (3), and H4 (3). The minimum rate of H1 hydrolysis was observed for antibodies against H3 and H4. A high rate of hydrolysis and the maximum number of H1 hydrolysis sites (17) were found for antibodies against MBP. Only a few sites of H1 hydrolysis by anti-H1 antibodies coincided with those for IgGs against H2A, H2B, H3, H4, and MBP. Thus, the polyreactivity of complexation and the enzymatic cross-activity of antibodies against H1, four other histones, and MBP have first been shown. Since histones act as damage molecules, abzymes against histones and MBP can play a negative role in the pathogenesis of MS and probably other different diseases as well.

In the milk of healthy women, antibodies were found with different catalytic activities (abzymes), which are absent in the sera of other healthy people. Moreover, it was previously shown that DNase antibodies-abzymes of patients with autoimmune diseases are cytotoxic to cancer cells. In this work, it was first shown that IgG and secretory IgA (sIgA) do not possess embryotoxicity; they practically do not affect the development of fertilized eggs of sea urchins but demonstrate sperm toxicity. After addition to the eggs of sperm preincubated with IgG and sIgA, the number of unfertilized eggs was increased, in the case of sIgA 1.6-fold higher than that for IgG. The suppression of the growth of MCF-7 breast cancer cells by sIgA was 2.2 times more effective than with IgG antibodies. The relative enzymatic activity of milk sIgA was higher than IgG (-fold): 1.9 (DNase), 4.6 (amylase), 1.7 (peroxidase), 1.3 (protease), 3.7 [hydrolysis of poly(C)], 3.3 [hydrolysis of poly(U)], and 1.7 (oxidation of 3,3'-diaminobenzidine). One of the possible reasons for the observed difference between sIgA and IgG could be that all 6 catalytic activities of sIgA were, on average, 2.6 times higher than that for IgG. Correlation coefficients between all the relative 6 enzymatic activities of IgG and sIgA and their toxicity to sea urchin sperm and to cancer cells were calculated. Maximum correlation coefficients were observed for DNase (+0.71), protease (+0.64) activities for sIgA, as well as protease (+0.59) and RNase (+0.77) of IgG with their toxicity toward sperm. The correlation coefficients were also high between peroxidase activity (+0.85) of sIgA and poly(U) hydrolysis by IgG (+0.58) with their suppression of tumor cell growth. It has been suggested that the catalytic activities of abzymes may be important in the manifestation of their sperm toxicity and inhibition of cancer cell growth., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).)

Nine new mono-, di-, and trisulfated triterpene penta- and hexaosides, kurilosides A 3 ( 1 ), D 1 ( 2 ), G ( 3 ), H ( 4 ), I ( 5 ), I 1 ( 6 ), J ( 7 ), K ( 8 ), and K 1 ( 9 ) and two desulfated derivatives, DS-kuriloside L ( 10 ), having a trisaccharide branched chain, and DS-kuriloside M ( 11 ), having hexa- nor -lanostane aglycone with a 7(8)-double bond, have been isolated from the Far-Eastern deep-water sea cucumber Thyonidium (=Duasmodactyla) kurilensis (Levin) and their structures were elucidated based on 2D NMR spectroscopy and HR-ESI mass-spectrometry. Five earlier unknown carbohydrate chains and two aglycones (having a 16 β ,(20S)-dihydroxy-fragment and a 16 β -acetoxy,(20S)-hydroxy fragment) were found in these glycosides. All the glycosides 1 - 9 have a sulfate group at C-6 Glc, attached to C-4 Xyl1, while the positions of the other sulfate groups vary in different groups of kurilosides. The analysis of the structural features of the aglycones and the carbohydrate chains of all the glycosides of T. kurilensis showed their biogenetic relationships. Cytotoxic activities of the compounds 1 - 9 against mouse neuroblastoma Neuro 2a, normal epithelial JB-6 cells, and erythrocytes were studied. The highest cytotoxicity in the series was demonstrated by trisulfated hexaoside kuriloside H ( 4 ), having acetoxy-groups at C(16) and C(20), the latter one obviously compensated the absence of a side chain, essential for the membranolytic action of the glycosides. Kuriloside I 1 ( 6 ), differing from 4 in the lacking of a terminal glucose residue in the bottom semi-chain, was slightly less active. The compounds 1 - 3 , 5 , and 8 did not demonstrate cytotoxic activity due to the presence of hydroxyl groups in their aglycones.

Three new triterpene glycosides, pacificusosides A-C ( 1 - 3 ), and three previously known triterpene glycosides, cucumariosides C 1 ( 4 ), C 2 ( 5 ), and A 10 ( 6 ), were isolated from the alcoholic extract of the Far Eastern starfish Solaster pacificus . The structures of 1 - 3 were elucidated by extensive NMR and ESIMS techniques and chemical transformations. Compound 1 has a novel, unique structure, containing an aldehyde group of side chains in its triterpene aglycon. This structural fragment has not previously been found in the sea cucumber triterpene glycosides or starfish steroidal glycosides. Probably, pacificusoside A ( 1 ) is a product of the metabolism of the glycoside obtained through dietary means from a sea cucumber in the starfish. Another two new triterpene glycosides ( 2 , 3 ) have closely related characteristics to sea cucumber glycosides. The cytotoxicity of compounds 1 - 6 was tested against human embryonic kidney HEK 293 cells, colorectal carcinoma HT-29 cells, melanoma RPMI-7951 cells, and breast cancer MDA-MB-231 cells using MTS assay. Compounds 4 - 6 revealed the highest cytotoxic activity against the tested cell lines, while the other investigated compounds had moderate or slight cytotoxicity. The cytotoxic effects of 2 - 6 were reduced by cholesterol like the similar effects of the previously investigated individual triterpene glycosides. Compounds 3 , 4 , and 5 almost completely suppressed the colony formation of the HT-29, RPMI-7951, and MDA-MB-231 cells at a nontoxic concentration of 0.5 µM.

Histones play important roles in chromatin functioning and gene transcription, but in the intercellular space, they are harmful since they stimulate systemic inflammatory and toxic responses. Electrophoretically homogeneous IgGs against myelin basic protein (MBP), as well as H3 and H4 histones, were isolated from sera of HIV-infected patients. In contrast to known classical proteases, these IgGs split exclusively only histones and MBP but no other control proteins. Among 13 sites of hydrolysis of H3 by IgGs against H3 and 14 sites for anti-MBP IgGs, only two sites of the hydrolysis were the same. Between seven cleavage sites of H4 with IgGs against H4 and 9 sites of this histone hydrolysis by antibodies against MBP, only three sites were the same. The sites of hydrolysis of H3 (and H4) with abzymes against these histones and against MBP were different, but several expended protein clusters containing hydrolysis sites are partially overlapped. The existence of enzymatic cross-reactivity of abzymes against H3 and H4 and MBP represents a great menace to humans since due to cell apoptosis, histones constantly occur in human blood. They can hydrolyze MBP of the myelin sheath of axons and play a negative role in the pathogenesis of HIV-infected patients.

The structural elucidation of primary and secondary peroxidation products, formed from complex lipids, is a challenge in lipid analysis. In the present study, rare minor oxidized cerebrosides, isolated from the extract of a far eastern deep-sea glass sponge, Aulosaccus sp., were analyzed as constituents of a multi-component RP-HPLC (high-performance liquid chromatography on reversed-phase column) fraction using NMR (nuclear magnetic resonance) spectroscopy, mass spectrometry, GC (gas chromatography), and chemical transformations (including hydrogenation or derivatization with dimethyl disulfide before hydrolysis). Eighteen previously unknown β-D-glucopyranosyl-(1→1)-ceramides ( 1a - a // , 1b - b // , 2a - a // , 2b - b // , 3c - c // , 3d - d // ) were shown to contain phytosphingosine-type backbones (2 S ,3 S ,4 R ,11 Z )-2-aminoeicos-11-ene-1,3,4-triol (in 1 ), (2 S ,3 S ,4 R ,13 Z )-2-aminoeicos-13-ene-1,3,4-triol (in 2 ), and (13 S *,14 R *)-2-amino-13,14-methylene-eicosane-1,3,4-triol (in 3 ). These backbones were N -acylated with straight-chain monoenoic (2 R )-2-hydroxy acids that had allylic hydroperoxy/hydroxy/keto groups on C-17 / in the 15 / E -23:1 chain ( a - a // ), C-16 / in the 17 / E -23:1 ( b - b // ) and 14 / E -22:1 ( c - c // ) chains, and C-15 / in the 16 / E -22:1 chain ( d - d // ). Utilizing complementary instrumental and chemical methods allowed for the first detailed structural analysis of a complex mixture of glycosphingolipids, containing allylically oxygenated monoenoic acyl chains.

Six new monosulfated triterpene tetra-, penta- and hexaosides, namely, the kurilosides A 1 ( 1 ), A 2 ( 2 ), C 1 ( 3 ), D ( 4 ), E ( 5 ) and F ( 6 ), as well as the known earlier kuriloside A ( 7 ), having unusual non-holostane aglycones without lactone, have been isolated from the sea cucumber Thyonidium (= Duasmodactyla) kurilensis (Levin) (Cucumariidae, Dendrochirotida) , collected in the Sea of Okhotsk near Onekotan Island from a depth of 100 m. Structures of the glycosides were established by 2D NMR spectroscopy and HR-ESI mass spectrometry. Kurilosides of the groups A and E contain carbohydrate moieties with a rare architecture (a pentasaccharide branched by C(4) Xyl1), differing from each other in the second monosaccharide residue (quinovose or glucose, correspondingly); kurilosides of the group C are characterized by a unique tetrasaccharide branched by a C(4) Xyl1 sugar chain; and kurilosides of the groups D and F are hexaosides differing from each other in the presence of an O -methyl group in the fourth (terminal) sugar unit. All these glycosides contain a sulfate group at C-6 of the glucose residue attached to C-4 Xyl1 and the non-holostane aglycones have a 9(11) double bond and lack γ-lactone. The cytotoxic activities of compounds 1 - 7 against mouse neuroblastoma Neuro 2a, normal epithelial JB-6 cells and erythrocytes were studied. Kuriloside A 1 ( 1 ) was the most active compound in the series, demonstrating strong cytotoxicity against the erythrocytes and JB-6 cells and a moderate effect against Neuro 2a cells.

Herpes simplex virus type 1 (HSV-1) is one of the most prevalent pathogens worldwide requiring the search for new candidates for the creation of antiherpetic drugs. The ability of sea urchin spinochromes-echinochrome A (EchA) and its aminated analogues, echinamines A (EamA) and B (EamB)-to inhibit different stages of HSV-1 infection in Vero cells and to reduce the virus-induced production of reactive oxygen species (ROS) was studied. We found that spinochromes exhibited maximum antiviral activity when HSV-1 was pretreated with these compounds, which indicated the direct effect of spinochromes on HSV-1 particles. EamB and EamA both showed the highest virucidal activity by inhibiting the HSV-1 plaque formation, with a selectivity index (SI) of 80.6 and 50.3, respectively, and a reduction in HSV-1 attachment to cells (SI of 8.5 and 5.8, respectively). EamA and EamB considerably suppressed the early induction of ROS due to the virus infection. The ability of the tested compounds to directly bind to the surface glycoprotein, gD, of HSV-1 was established in silico. The dock score of EchA, EamA, and EamB was -4.75, -5.09, and -5.19 kcal/mol, respectively, which correlated with the SI of the virucidal action of these compounds and explained their ability to suppress the attachment and penetration of the virus into the cells.

A new neurotoxin RTX-VI that modulates the voltage-gated sodium channels (Na V ) was isolated from the ethanolic extract of the sea anemone Heteractis crispa. Its amino acid sequence was determined using the combination of Edman degradation and tandem mass spectrometry. RTX-VI turned out to be an unusual natural analogue of the previously described sea anemone toxin RTX-III. The RTX-VI molecule consists of two disulfide-linked peptide chains and is devoid of Arg13, which is important for the selectivity and affinity of such peptides for the Na V channels. Electrophysiological screening of RTV-VI on Na V channel subtypes showed its selective interaction with the central nervous system (Na V 1.2, Na V 1.6) and insect (BgNa V 1, VdNa V 1) sodium channels.

Echinochrome A (Ech A, 1 ) is one of the main pigments of several sea urchin species and is registered in the Russian pharmacopeia as an active drug substance (Histochrome ® ), used in the fields of cardiology and ophthalmology. In this study, Ech A degradation products formed during oxidation by O 2 in air-equilibrated aqueous solutions were identified, isolated, and structurally characterized. An HPLC method coupled with diode-array detection (DAD) and mass spectrometry (MS) was developed and validated to monitor the Ech A degradation process and identify the appearing compounds. Five primary oxidation products were detected and their structures were proposed on the basis of high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) as 7-ethyl-2,2,3,3,5,7,8-heptahydroxy-2,3-dihydro-1,4-naphthoquinone ( 2 ), 6-ethyl-5,7,8-trihydroxy-1,2,3,4-tetrahydronaphthalene-1,2,3,4-tetraone ( 3 ), 2,3-epoxy-7-ethyl-2,3-dihydro-2,3,5,6,8-pentahydroxy-1,4-naphthoquinone ( 4 ), 2,3,4,5,7-pentahydroxy-6-ethylinden-1-one ( 5 ), and 2,2,4,5,7-pentahydroxy-6-ethylindane-1,3-dione ( 6 ). Three novel oxidation products were isolated, and NMR and HR-ESI-MS methods were used to establish their structures as 4-ethyl-3,5,6-trihydroxy-2-oxalobenzoic acid ( 7 ), 4-ethyl-2-formyl-3,5,6-trihydroxybenzoic acid ( 8 ), and 4-ethyl-2,3,5-trihydroxybenzoic acid ( 9 ). The known compound 3-ethyl-2,5-dihydroxy-1,4-benzoquinone ( 10 ) was isolated along with products 7 - 9 . Compound 7 turned out to be unstable; its anhydro derivative 11 was obtained in two crystal forms, the structure of which was elucidated using X-ray crystallography as 7-ethyl-5,6-dihydroxy-2,3-dioxo-2,3-dihydrobenzofuran-4-carboxylic acid and named echinolactone. The chemical mechanism of Ech A oxidative degradation is proposed. The in silico toxicity of Ech A and its degradation products 2 and 7 - 10 were predicted using the ProTox-II webserver. The predicted median lethal dose (LD 50 ) value for product 2 was 221 mg/kg, and, for products 7 - 10 , it appeared to be much lower (≥2000 mg/kg). For Ech A, the predicted toxicity and mutagenicity differed from our experimental data.

Seven new polyoxygenated steroids belonging to a new structural group of sponge steroids, gracilosulfates A-G ( 1 - 7 ), possessing 3 β - O -sulfonato, 5 β, 6 β epoxy (or 5(6)-dehydro), and 4 β ,23-dihydroxy substitution patterns as a common structural motif, were isolated from the marine sponge Haliclona gracilis. Their structures were determined by NMR and MS methods. The compounds 1, 2, 4, 6 , and 7 inhibited the expression of prostate-specific antigen (PSA) in 22Rv1 tumor cells.