Sea Anemone Bunodosoma Caissarum Research Articles

Multiomic Approach for Bioprospection: Investigation of Toxins and Peptides of Brazilian Sea Anemone Bunodosoma caissarum.

Sea anemones are sessile invertebrates of the phylum Cnidaria and their survival and evolutive success are highly related to the ability to produce and quickly inoculate venom, with the presence of potent toxins. In this study, a multi-omics approach was applied to characterize the protein composition of the tentacles and mucus of Bunodosoma caissarum, a species of sea anemone from the Brazilian coast. The tentacles transcriptome resulted in 23,444 annotated genes, of which 1% showed similarity with toxins or proteins related to toxin activity. In the proteome analysis, 430 polypeptides were consistently identified: 316 of them were more abundant in the tentacles while 114 were enriched in the mucus. Tentacle proteins were mostly enzymes, followed by DNA- and RNA-associated proteins, while in the mucus most proteins were toxins. In addition, peptidomics allowed the identification of large and small fragments of mature toxins, neuropeptides, and intracellular peptides. In conclusion, integrated omics identified previously unknown or uncharacterized genes in addition to 23 toxin-like proteins of therapeutic potential, improving the understanding of tentacle and mucus composition of sea anemones.

Determination and speciation of cadmium in microcosms with Bunodosoma caissarum and Perna perna using isotopically enriched 116Cd

The study of the uptake and distribution of elements in marine environments is of great interest for understanding their pathways and accumulation. Here, we investigated in laboratory experiments the accumulation behavior of Cd in the sea anemone Bunodosoma caissarum and the mussel Perna perna. Specimens were incubated with isotopically enriched 116Cd in aquariums. Cd concentrations in the seawater and in the tissues of B. caissarum and P. perna were followed by inductively coupled plasma-mass spectrometry (ICP-MS) by means of isotope dilution analysis. Bioconcentration factors for B. caissarum and P. perna exposed to 0.9μg·L−1 of 116Cd were determined to be 80.5 and 850, respectively. P. perna specimens exposed to 4.5μg·L−1 of 116Cd reached 530. Cytosolic proteins associated with Cd from the tissues were extracted and further analyzed by size-exclusion chromatography coupled to ICP-MS. Cd accumulation could be detected in both organisms ranging from high-molecular to low-molecular species.

Assessing mercury contamination in a tropical coastal system using the mussel Perna perna and the sea anemone Bunodosoma caissarum.

Total mercury concentrations in the mussel Perna perna and the sea anemone Bunodosoma caissarum were determined to assess Hg contamination in Guanabara Bay, Rio de Janeiro, Brazil, and an adjacent sea area. Concentrations in the tissues of these species were compared. Average total mercury concentrations ranged from 3.54 to 21.01μgkg-1 (wet wt.) in P. perna and from 4.51 to 23.19μgkg-1 (wet wt.) for B. caissarum. Concentrations varied according to the sampling stations. Distribution of concentrations for both species was similar along the sampling stations, and a significant correlation was observed. Results suggest that B. caissarum could be a suitable biomonitor species for mercury contamination in the study area and could be used as a complementary species for monitoring studies. Further research is, however, needed to assess how environmental conditions and other variables affect Hg concentrations in B. caissarum.

Mercury distribution, methylation and volatilization in microcosms with and without the sea anemone Bunodosoma caissarum

Mercury (Hg) has a complex biogeochemical cycle in aquatic environments. Its most toxic form, methylmercury (MeHg), is produced by microorganisms. This study investigated how the sea anemone Bunodosoma caissarum affects Hg distribution, methylation and volatilization in laboratory model systems. 203Hg was added to microcosms and its distribution in seawater, specimens and air was periodically measured by gamma spectrometry. MeHg was measured by liquid scintillation. After the uptake period, specimens had a bioconcentration factor of 70 and in microcosms with and without B. caissarum, respectively 0.05% and 0.32% of the initial spike was found as MeHg. After depuration, MeHg in specimens ranged from 0.2% to 2.4% of total Hg. Microcosms with B. caissarum had higher Hg volatilization (58%) than controls (17%), possibly due to Hg2+ reduction mediated by microorganisms associated with its tissues and mucus secretions. Marine organisms and their associated microbiota may play a role in Hg and MeHg cycling.

Purification and characterization of the biological effects of phospholipase A 2 from sea anemone Bunodosoma caissarum

Sea anemones contain a variety of biologically active substances. Bunodosoma caissarum is a sea anemone from the Cnidaria phylum, found only in Brazilian coastal waters. The aim of the present work was to study the biological effects of PLA 2 isolated from the sea anemone B. caissarum on the isolated perfused kidney, the arteriolar mesenteric bed and on insulin secretion. Specimens of B. caissarum were collected from the São Vicente Channel on the southern coast of the State of São Paulo, Brazil. Reverse phase HPLC analysis of the crude extract of B. caissarum detected three PLA 2 proteins (named BcPLA 21, BcPLA 22 and BcPLA 23) found to be active in B. caissarum extracts. MALDI-TOF mass spectrometry of BcPLA 21 showed one main peak at 14.7 kDa. The N-terminal amino acid sequence of BcPLA 21 showed high amino acid sequence identity with PLA 2 group III protein isolated from the Mexican lizard (PA23 HELSU, HELSU, PA22 HELSU) and with the honey bee Apis mellifera (PLA 2 and 1POC_A). In addition, BcPLA 21 also showed significant overall homology to bee PLA 2. The enzymatic activity induced by native BcPLA 21 (20 μg/well) was reduced by chemical treatment with p-bromophenacyl bromide ( p-BPB) and with morin. BcPLA 21 strongly induced insulin secretion in presence of high glucose concentration. In isolated kidney, the PLA 2 from B. caissarum increased the perfusion pressure, renal vascular resistance, urinary flow, glomerular filtration rate, and sodium, potassium and chloride levels of excretion. BcPLA 21, however, did not increase the perfusion pressure on the mesenteric vascular bed. In conclusion, PLA 2, a group III phospholipase isolated from the sea anemone B. caissarum, exerted effects on renal function and induced insulin secretion in conditions of high glucose concentration.

BcIV, a new paralyzing peptide obtained from the venom of the sea anemone Bunodosoma caissarum. A comparison with the Na + channel toxin BcIII

Sea anemones produce a wide variety of biologically active compounds, such as the proteinaceous neurotoxins and cytolysins. Herein we report a new peptide, purified to homogeneity from the neurotoxic fraction of B. caissarum venom, by using gel filtration followed by rp-HPLC, naming it as BcIV. BcIV is a 41 amino acid peptide (molecular mass of 4669 amu) possessing 6 cysteines covalently linked by three disulfide bonds. This toxin has 45 and 48% of identity when compared to APETx1 and APETx2 from Anthopleura elegantissima, respectively, and 42% of identity with Am-II and BDS-I and-II obtained from Antheopsis maculata and Anemonia sulcata, respectively. This neurotoxin presents only a weak-paralyzing action (minimal Lethal Dose close to 2000 μg/kg) in swimming crabs Callinectes danae. This appears to be a different effect to that caused by the type 1 sea anemone toxin BcIII that is lethal to the same animals at lower doses (LD 50 = 219 μg/kg). Circular dichroism spectra of BcIII and BcIV show a high content of β-strand secondary structure in both peptides, very similar to type 1 sodium channel toxins from various sea anemones, and to APETx1 and APETx2 from A. elegantissima, a HERG channel modulator and an ASIC3 inhibitor, respectively. Interestingly, BcIII and BcIV have similar effects on the action potential of the crab leg nerves, suggesting the same target in this tissue. As BcIII was previously reported as a Na + channel effector and BcIV is inactive over Na + currents of mammalian GH3 cells, we propose a species-specific action for this new molecule. A molecular model of BcIV was constructed using the structure of the APETx1 as template and putative key residues are discussed.

Caissarolysin I (Bcs I), a new hemolytic toxin from the Brazilian sea anemone Bunodosoma caissarum: Purification and biological characterization

Two cationic proteins, C1 and C3, were purified to homogeneity from the hemolytic fraction of the venom of Bunodosoma caissarum sea anemone. The purification processes employed gel filtration followed by ion exchange chromatography, being the purity and molecular mass confirmed by SDS-PAGE and mass spectrometry. Protein C1 represented the second major peak of the hemolytic fraction and was previously believed to be a cytolysin belonging to a new class of hemolysins. The C1 protein has a molecular mass of 15495 Da and was assayed for hemolysis, PLA 2 activity and acute toxicity in crabs and mice, showing no activity in these assays. It has an amino terminal with no similarity to all known hemolysins and, therefore, should not be considered a toxin, being its function completely unknown. The protein C3 (19757 Da), that also lacks PLA 2 activity, was recognized by antiserum against Eqt II and presented high hemolytic activity to human erythrocytes (ED 50 of 0.270 μg/ml), being named Caissarolysin I (Bcs I). Its activity was inhibited by pre-incubation with sphingomyelin (SM) and also when in presence of erythrocytes pre-treated with the SMase P2, a phospholipase D from the brown spider Loxosceles intermedia, indicating that SM is the main target of Bcs I. Caissarolysin I is the first hemolysin purified from a sea anemone belonging to the genus Bunodosoma and belongs to the Actinoporin family of sea anemone hemolysins.

The Sea Anemone Toxin Bc2 Induces Continuous or Transient Exocytosis, in the Presence of Sustained Levels of High Cytosolic Ca2+ in Chromaffin Cells

We have isolated and characterized a new excitatory toxin from the venom of the sea anemone Bunodosoma caissarum, named Bc2. We investigated the mechanism of action of the toxin on Ca(2+)-regulated exocytosis in single bovine adrenal chromaffin cells, monitoring simultaneously fura-2 fluorescence measurements and electrochemical recordings using a carbon fiber microelectrode. Bc2 induced quantal release of catecholamines in a calcium-dependent manner. This release was associated with a sustained rise in cytosolic Ca(2+) and displayed two different patterns of response: a continuous discharge of prolonged duration that changed to a transient burst as the toxin concentration (or incubation time) increased. Continuous secretion was dependent on the activity of native voltage-dependent Ca(2+) channels and showed a pattern similar to that of alpha-latrotoxin; however, its kinetics adjusted better to that of continuous cell depolarization with high K(+) concentration. In contrast, transient secretion was independent of Ca(2+) entry through native voltage-dependent Ca(2+) channels and showed inhibition of late vesicle fusion that was accompanied by "freezing" of F-actin disassembly. These new features make Bc2 a promising new tool for studying the machinery of neurotransmitter release.

Synaptosomal glutamate release induced by the fraction Bc2 from the venom of the sea anemone Bunodosoma caissarum

The effect of a fraction (Bc2) from the venom of the sea anemone Bunodosoma caissarum on [3H]glutamate release from rat cortical synaptosomes was investigated. Bc2 (2-20 microg/ml) provoked massive glutamate release without causing synaptosome disruption. Glutamate release stimulated by Bc2 was independent of extracellular Ca2+ and of voltage-sensitive Na+ channels, and it was completely abolished by the addition of sphingomyelin. No definitive evidence about the mechanism underlying the stimulatory effect of Bc2 is available as yet. However, a direct interaction with the exocytotic machinery cannot be ruled out.

Characterization of peptides in sea anemone venom collected by a novel procedure

Peptide neurotoxins were isolated from the venom obtained by electrical stimulation of the sea anemone Bunodosoma caissarum. This technique allows almost pure venom to be collected, and the animals to survive. Three neurotoxins (assayed on crustacean nerves) were isolated by gel filtration and reversed-phase high performance liquid chromatography. Hemolysins were also detected in the venom. The amino acid sequence of a major neurotoxin BcIII was determined. BcIII has 48 amino acid residues with six half-cystine residues. This sequence has homology with the type 1 long sea anemone neurotoxins. Two minor toxins (BcI and II) have similar amino acid composition and amino-terminal sequences to BcIII.

Purines. LVII. Regioselective Alkylation of N6,9-Disubstituted 8-Oxoadenines: Syntheses of the Sea Anemone Purine Caissarone and Some Positional Isomers and Analogues.

The first total synthesis of caissarone hydrochloride (1), a constituent of the sea anemone Bunodosoma caissarum, has been accomplished via a two-step route starting from N6, 9-dimethyl-8-oxoadenine (3), which is obtainable from 9-methyladenine (5) through a four-step route. The key step in the synthesis is the regioselective methylation of 3 at N(3), which has been designed on the basis of a methylation study of N6-benzyl-9-methyl-8-oxoadenine (11). Some positional isomers (19, 24, and 31) and analogues (8, 26, and 32) of 1 have also been synthesized. A 1H-NMR spectroscopic study has suggested that the free base (23) of caissarone is capable of forming a hetero-base pair (such as 41) with 2', 3', 5'-tri-O-acetylguanosine (40) in Me2SO-d6.

Increase of mammalian intestinal motility by the iminopurine caissarone isolated from the sea anemone Bunodosoma caissarum

The effects of caissarone (C 8H 11N 5O) on guinea-pig ileum and rat duodenum preparations are reported. Caissarone evoked, at first, a small response, as compared with that of applied acetylcholine. This effect was blocked by atropine, hexamethonium and slightly reduced by tetrodotoxin. The main caissarone effect seems to be on the myenteric plexus as it increased spontaneous activity, tone and peristalsis; these effects being counteracted by tetrodotoxin, hexamethonium and atropine. Extracellular electrophysiological recording of the action potentials of myenteric plexus neurons showed an increase in frequency in the presence of caissarone. Caissarone potentiated the longitudinal muscle tension responses elicited by transmural electric stimulation in both control conditions, and when tension was reduced by pretreatment with tetrodotoxin or adenosine triphosphate. Caissarone also potentiated the longitudinal muscle tension responses to applied acetylcholine and this effect was reduced, but not abolished, by tetrodotoxin. Caissarone may have an indirect excitatory action on nicotinic receptors at the ganglia or may act as a reversible antagonist of purine transmitters released by neurons of the myenteric plexus involved in the physiological inhibition of intestinal motility.

Anomalies in sea-urchin egg development induced by a novel purine isolated from the sea-anemone Bunodosoma caissarum

Caissarone (mol. wt 229.5; melting point 285–290°C) is a novel purine isolated and purified from the sea-anemone Bunodosoma caissarum. The purine inhibits the detachment of the vitelline layer from the sea-urchin egg plasma membrane after fertilization and this effect leads to polyspermy. Various abnormalities were detected at various embryonic stages, from multipolar egg division through unequal cleavages and exogastrulation up to teratogenic effects on the sea-urchin larvae (echinopluteus).

X-Ray molecular structure of caissarone, a novel purine derivative from the sea anemone Bunodosoma caissarum Correa 1964

A new quaternary purine derivative isolated from the sea anemone Bunodosoma caissarum has been identified, by spectroscopic measurements and X-ray diffraction analysis, to be 3,9-dimethyl-6-methylimino-8-oxo-3,6,8,9-tetrahydropurine (caissarone) and isolated as the hydrochloride. The X-ray structure indicates that the exocyclic nitrogen is apparently the most electron-rich centre and that the molecule is planar.

Absence of cholinesterase activity in body wall homogenates from the sea anemone Bunodosoma caissarum Corrêa

1. 1. Attempts to detect cholinesterase activity in body wall homogenates of the sea anemone Bunodosoma caissarum were made using gasometric and colorimetric techniques. 2. 2. No cholinesterase activity was found in the homogenates using either of these techniques. 3. 3. Control experiments for cholinesterase activity were performed using crude homogenates from the polychaete Eurythoë complanata and the marine fish Bathygobius soporator. 4. 4. An evolutionary line of acetylcholine-meditated transmission at the junction level is discussed.

Action of crude and fractioned homogenates of the midgut gland of the sea hare Aplysia brasiliana rang, 1828 on some cholinoceptive structures

1. Crude and fractioned homogenates of the midgut gland of Aplysia brasiliana were tested on the heart of the toad Bufo ictericus ictericus and cholinoceptive structures of some invertebrates. 2. In the in situ heart of the toad the fractions, with increasing concentrations, first decreased both the frequency and the amplitude and then stopped the organ in diastole. 3. On recovery from the effect of the fractions, the toad heart could not be stopped by vagus stimulation. This result is admitted as due to the urocanylcholine present in the fractions. 4. The application of acetylcholine on an isolated toad heart previously treated by the active fractions was also ineffective. Atropine blocked the action of the active fractions in the isolated heart. 5. The tonus and the frequency of beating of the isolated heart of A. brasiliana are enhanced by the active fractions. This action can be blocked by atropine but not by B.O.L. The quiescent isolated heart of this gastropod resumes its beating on the application of the active fractions. 6. In the isolated heart of the bivalve Perna perna the active fractions also rise the amplitude of the beating. 7. Crude homogenates and the active fractions of the midgut gland strongly contract the protractor muscle of the sea urchin lantern and the longitudinal muscle of the sea cucumber. In both cases this action can be blocked by atropine. 8. Column muscle preparations of the sea anemone Bunodosoma caissarum are also sensitive to the active fractions. 9. The present results confirm and extend to A. brasiliana the existence of cholinergic principles in the midgut gland, probably acetylcholine and urocanylcholine, as found by Blankenship et al. (1975) in another sea here.