Transcriptomic and proteomic analyses reveal the first occurrence of diverse toxin groups in Millepora alcicornis.

Millepora alcicornis is a reef-forming cnidarian widely distributed in the Mexican Caribbean. Millepora species or "fire corals" inflict a painful stinging reaction in humans when touched. Even though hundreds of organic and polypeptide toxins have been characterized from sea anemones and jellyfish, there are few reports regarding the diversity of toxins synthesized by fire corals. Here, based on transcriptomic analysis of M. alcicornis, several predicted proteins that show amino acid sequence similarity to toxins were identified, including neurotoxins, metalloproteases, hemostasis-impairing toxins, serin proteases, cysteine-rich venom proteins, phospholipases, complement system-impairing toxins, phosphodiesterases, pore-forming toxins, and L-aminoacid oxidases. The soluble nematocyst proteome of this organism was shown to induce hemolytic, proteolytic, and phospholipase A2 effects by gel zymography. Protein bands or spots on 1D- and 2D-PAGE gels corresponding to zones of hemolytic and enzymatic activities were excised, subjected to in-gel digestion with trypsin, and analyzed by mass spectrometry. These proteins exhibited sequence homology to PLA2s, metalloproteinases, pore-forming toxins, and neurotoxins, such as actitoxins and CrTX-A. The complex array of venom-related transcripts that were identified in M. alcicornis, some of which are first reported in "fire corals", provide novel insight into the structural richness of Cnidarian toxins and their distribution among species. SIGNIFICANCE: Marine organisms are a promising source of bioactive compounds with valuable contributions in diverse fields such as human health, pharmaceuticals, and industrial application. Currently, not much attention has been paid to the study of fire corals, which possess a variety of molecules that exhibit diverse toxic effects and therefore have great pharmaceutical and biotechnological potential. The isolation and identification of novel marine-derived toxins by classical approaches are time-consuming and have low yields. Thus, next-generation strategies, like base-'omics technologies, are essential for the high-throughput characterization of venom compounds such as those synthesized by fire corals. This study moves the field forward because it provides new insights regarding the first occurrence of diverse toxin groups in Millepora alcicornis. The findings presented here will contribute to the current understanding of the mechanisms of action of Millepora toxins. This research also reveals important information related to the potential role of toxins in the defense and capture of prey mechanisms and for designing appropriate treatments for fire coral envenomation. Moreover, due to the lack of information on the taxonomic identification of Millepora, the insights presented here can advise the taxonomic classification of the species of this genus.

Vasodilation Elicited by Isoxsuprine, Identified by High-Throughput Virtual Screening of Compound Libraries, Involves Activation of the NO/cGMP and H2S/KATP Pathways and Blockade of α1-Adrenoceptors and Calcium Channels.

Recently, our research group demonstrated that uvaol and ursolic acid increase NO and H2S production in aortic tissue. Molecular docking studies showed that both compounds bind with high affinity to endothelial NO synthase (eNOS) and cystathionine gamma-lyase (CSE). The aim of this study was to identify hits with high binding affinity for the triterpene binding-allosteric sites of eNOS and CSE and to evaluate their vasodilator effect. Additionally, the mechanism of action of the most potent compound was explored. A high-throughput virtual screening (HTVS) of 107,373 compounds, obtained from four ZINC database libraries, was performed employing the crystallographic structures of eNOS and CSE. Among the nine top-scoring ligands, isoxsuprine showed the most potent vasodilator effect. Pharmacological evaluation, employing the rat aorta model, indicated that the vasodilation produced by this compound involved activation of the NO/cGMP and H2S/KATP signaling pathways and blockade of α1-adrenoceptors and L-type voltage-dependent Ca2+ channels. Incubation of aorta homogenates in the presence of isoxsuprine caused 2-fold greater levels of H2S, which supported our preliminary in silico data. This study provides evidence to propose that the vasodilator effect of isoxsuprine involves various mechanisms, which highlights its potential to treat a wide variety of cardiovascular diseases.

Affinin (Spilanthol), Isolated from Heliopsis longipes, Induces Vasodilation via Activation of Gasotransmitters and Prostacyclin Signaling Pathways.

Heliopsis longipes roots have been widely used in Mexican traditional medicine to relieve pain, mainly, toothaches. Previous studies have shown that affinin, the major alkamide of these roots, induces potent antinociceptive and anti-inflammatory activities. However, the effect of H. longipes root extracts and affinin on the cardiovascular system have not been investigated so far. In the present study, we demonstrated that the dichloromethane and ethanolic extracts of H. longipes roots, and affinin, isolated from these roots, produce a concentration-dependent vasodilation of rat aorta. Affinin-induced vasorelaxation was partly dependent on the presence of endothelium and was significantly blocked in the presence of inhibitors of NO, H2S, and CO synthesis (NG-nitro-l-arginine methyl ester (l-NAME), dl-propargylglycine (PAG), and chromium mesoporphyrin (CrMP), respectively); K⁺ channel blockers (glibenclamide (Gli) and tetraethyl ammonium (TEA)), and guanylate cyclase and cyclooxygenase inhibitors (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and indomethacin (INDO), respectively). Our results demonstrate, for the first time, that affinin induces vasodilation by mechanisms that involve gasotransmitters, and prostacyclin signaling pathways. These findings indicate that this natural alkamide has therapeutic potential in the treatment of cardiovascular diseases.

Role of Nitric Oxide and Hydrogen Sulfide in the Vasodilator Effect of Ursolic Acid and Uvaol from Black Cherry Prunus serotina Fruits.

The present research aimed to isolate the non-polar secondary metabolites that produce the vasodilator effects induced by the dichloromethane extract of Prunus serotina (P. serotina) fruits and to determine whether the NO/cGMP and the H2S/KATP channel pathways are involved in their mechanism of action. A bioactivity-directed fractionation of the dichloromethane extract of P. serotina fruits led to the isolation of ursolic acid and uvaol as the main non-polar vasodilator compounds. These compounds showed significant relaxant effect on rat aortic rings in an endothelium- and concentration-dependent manner, which was inhibited by NG-nitro-L-arginine methyl ester (L-NAME), DL-propargylglycine (PAG) and glibenclamide (Gli). Additionally, both triterpenes increased NO and H2S production in aortic tissue. Molecular docking studies showed that ursolic acid and uvaol are able to bind to endothelial NOS and CSE with high affinity for residues that form the oligomeric interface of both enzymes. These results suggest that the vasodilator effect produced by ursolic acid and uvaol contained in P. serotina fruits, involves activation of the NO/cGMP and H2S/KATP channel pathways, possibly through direct activation of NOS and CSE.

Composition and biological activities of the aqueous extracts of three scleractinian corals from the Mexican Caribbean: Pseudodiploria strigosa, Porites astreoides and Siderastrea siderea

Background: Scleractinian corals (stony corals) are the most abundant reef-forming cnidarians found in coral reefs throughout the world. Despite their abundance and ecological importance, information about the diversity of their toxins and their biological activities is very scarce. In this study, the chemical composition and the biological activities of the aqueous extracts of Pseudodiploria strigosa, Porites astreoides and Siderastrea siderea, three scleractinian corals from the Mexican Caribbean, have been assessed for the first time. Methods: Toxicity of the extracts was assessed in crickets; the presence of cytolysins was detected by the hemolysis assay; the vasoconstrictor activity was determined by the isolated rat aortic ring assay; the nociceptive activity was evaluated by the formalin test. The presence of phospholipases A2 (PLA2), serine proteases, and hyaluronidases was determined by enzymatic methods. Low-molecular-weight fractions were obtained by gel filtration chromatography and ultrafiltration. Results: Extracts from the three species were toxic to crickets, induced hemolysis in human and rat erythrocytes, produced vasoconstriction on isolated rat aortic rings, and presented phospholipase A2 and serine-protease activity. Despite the fact that these corals are not considered to be harmless to humans, the extracts generated significant nociceptive responses. The matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry analysis of the low-molecular-weight fractions revealed the presence of peptides within a mass range of 3000 to 6000 Da. These fractions were toxic to crickets and two of them induced a transitory vasoconstrictor effect on isolated rat aortic rings. Conclusion: This study suggests that scleractinian corals produce low-molecular-weight peptides that are lethal to crickets and induce vasoconstriction.(AU)

Composition and biological activities of the aqueous extracts of three scleractinian corals from the Mexican Caribbean: Pseudodiploria strigosa, Porites astreoides and Siderastrea siderea

Scleractinian corals (stony corals) are the most abundant reef-forming cnidarians found in coral reefs throughout the world. Despite their abundance and ecological importance, information about the diversity of their toxins and their biological activities is very scarce. In this study, the chemical composition and the biological activities of the aqueous extracts of Pseudodiploria strigosa, Porites astreoides and Siderastrea siderea, three scleractinian corals from the Mexican Caribbean, have been assessed for the first time. Methods: Toxicity of the extracts was assessed in crickets; the presence of cytolysins was detected by the hemolysis assay; the vasoconstrictor activity was determined by the isolated rat aortic ring assay; the nociceptive activity was evaluated by the formalin test. The presence of phospholipases A2 (PLA2), serine proteases, and hyaluronidases was determined by enzymatic methods. Low-molecular-weight fractions were obtained by gel filtration chromatography and ultrafiltration. Results: Extracts from the three species were toxic to crickets, induced hemolysis in human and rat erythrocytes, produced vasoconstriction on isolated rat aortic rings, and presented phospholipase A2 and serine-protease activity. Despite the fact that these corals are not considered to be harmless to humans, the extracts generated significant nociceptive responses. The matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry analysis of the low-molecular-weight fractions revealed the presence of peptides within a mass range of 3000 to 6000 Da. These fractions were toxic to crickets and two of them induced a transitory vasoconstrictor effect on isolated rat aortic rings. Conclusion: This study suggests that scleractinian corals produce low-molecular-weight peptides that are lethal to crickets and induce vasoconstriction.

Cytolytic and systemic toxic effects induced by the aqueous extract of the fire coral Millepora alcicornis collected in the Mexican Caribbean and detection of two types of cytolisins

Background Millepora alcicornis is a branching hydrocoral common throughout the Caribbean Sea. Like other members of this genus, this species is capable of inducing skin eruptions and blisters with severe pain after contact. In the present study, we investigated the toxicity of theM. alcicornis aqueous extract on several animal models. Considering that some cnidarian hemolysins have been associated to local tissue damage, since they also induce lysis of other cell types, we also made a partial characterization of the hemolytic activity of M. alcicornis aqueous extract. This information is important for understanding the defense mechanisms of the "fire corals".Methods The effects of pH, temperature, and some divalent cations on the hemolytic activity of the extract were assayed, followed by a zymogram analysis to detect the cytolysins and determine their approximate molecular weight. The toxicity of the aqueous extract was assayed in mice, by intravenous administration, and histopathological changes on several tissues were analyzed by light microscopy. The toxicity of the extract was also tested inArtemia salina nauplii, and the damages caused on the crustaceans were analyzed by transmission and scanning electron microscopy.Results The hemolytic activity of the hydrocoral extract was enhanced in the presence of Ca 2+ (≥2 mM), Mg 2+ (≥6 mM), and Ba2+ (≥0.1 mM); however, it was reduced in the presence of Cu2+(≥0.1 mM), Zn 2+ (≥6 mM), and EDTA (≥0.34 mM). Differences in the pH did not affect the hemolytic activity, but it was temperature-sensitive, since preincubation at ≥ 50 °C sharply reduced hemolysis. The zymogram showed the presence of two types of hemolysins: ~ 28-30 kDa proteins with phospholipase A 2 activity and ~ 200 kDa proteins that do not elicit enzymatic activity. The aqueous extract of this cnidarian was lethal to mice (LD 50 = 17 μg protein/g), and induced kidney, liver, and lung damages. Under denaturing conditions, the aqueous extract completely lost its toxic and hemolytic activities.Conclusions The results showed that the M. alcicornis aqueous extract contains two types of thermolabile hemolysins: proteins of approximately 28-30 kDa with PLA 2 activity, while the others are larger proteins of approximately 200 kDa, which do not possess PLA 2activity. Those thermolabile cytolysins, which are stable to pH changes and whose activity is calcium dependent, are capable of inducing damage in lung, kidney and liver tissues, resulting in a slow death of mice. M. alcicorniscytolysins also provoke tissue dissociation inArtemia salina nauplii that might be attributed to pore forming mechanisms.(AU)

Cytolytic and systemic toxic effects induced by the aqueous extract of the fire coral Millepora alcicornis collected in the Mexican Caribbean and detection of two types of cytolisins

Background Millepora alcicornis is a branching hydrocoral common throughout the Caribbean Sea. Like other members of this genus, this species is capable of inducing skin eruptions and blisters with severe pain after contact. In the present study, we investigated the toxicity of theM. alcicornis aqueous extract on several animal models. Considering that some cnidarian hemolysins have been associated to local tissue damage, since they also induce lysis of other cell types, we also made a partial characterization of the hemolytic activity of M. alcicornis aqueous extract. This information is important for understanding the defense mechanisms of the fire corals.Methods The effects of pH, temperature, and some divalent cations on the hemolytic activity of the extract were assayed, followed by a zymogram analysis to detect the cytolysins and determine their approximate molecular weight. The toxicity of the aqueous extract was assayed in mice, by intravenous administration, and histopathological changes on several tissues were analyzed by light microscopy. The toxicity of the extract was also tested inArtemia salina nauplii, and the damages caused on the crustaceans were analyzed by transmission and scanning electron microscopy.Results The hemolytic activity of the hydrocoral extract was enhanced in the presence of Ca 2+ (2 mM), Mg 2+ (6 mM), and Ba2+ (0.1 mM); however, it was reduced in the presence of Cu2+(0.1 mM), Zn 2+ (6 mM), and EDTA (0.34 mM). Differences in the pH did not affect the hemolytic activity, but it was temperature-sensitive, since preincubation at 50 °C sharply reduced hemolysis. The zymogram showed the presence of two types of hemolysins: ~ 2830 kDa proteins with phospholipase A 2 activity and ~ 200 kDa proteins that do not elicit enzymatic activity. The aqueous extract of this cnidarian was lethal to mice (LD 50 = 17 g protein/g), and induced kidney, liver, and lung damages. Under denaturing conditions, the aqueous extract completely lost its toxic and hemolytic activities.Conclusions The results showed that the M. alcicornis aqueous extract contains two types of thermolabile hemolysins: proteins of approximately 2830 kDa with PLA 2 activity, while the others are larger proteins of approximately 200 kDa, which do not possess PLA 2activity. Those thermolabile cytolysins, which are stable to pH changes and whose activity is calcium dependent, are capable of inducing damage in lung, kidney and liver tissues, resulting in a slow death of mice. M. alcicorniscytolysins also provoke tissue dissociation inArtemia salina nauplii that might be attributed to pore forming mechanisms.

Nutraceutical value of black cherry Prunus serotina Ehrh. fruits: antioxidant and antihypertensive properties.

In Mexico black cherry (Prunus serotina Ehrh.) fruits are consumed fresh, dried or prepared in jam. Considering the evidence that has linked intake of fruits and vegetables rich in polyphenols to cardiovascular risk reduction, the aim of this study was to characterize the phenolic profile of black cherry fruits and to determine their antioxidant, vasorelaxant and antihypertensive effects. The proximate composition and mineral contents of these fruits were also assessed. Black cherry fruits possess a high content of phenolic compounds and display a significant antioxidant capacity. High-performance liquid chromatography/mass spectrometric analysis indicated that hyperoside, anthocyanins and chlorogenic acid were the main phenolic compounds found in these fruits. The black cherry aqueous extract elicited a concentration-dependent relaxation of aortic rings and induced a significant reduction on systolic blood pressure in L-NAME induced hypertensive rats after four weeks of treatment. Proximate analysis showed that black cherry fruits have high sugar, protein, and potassium contents. The results derived from this study indicate that black cherry fruits contain phenolic compounds which elicit significant antioxidant and antihypertensive effects. These findings suggest that these fruits might be considered as functional foods useful for the prevention and treatment of cardiovascular diseases.