Aplydactylonins A-C, three new sesquiterpenes from the Vietnamese sea hare Aplysia dactylomela and their cytotoxicity.

Aplydactylonins A-C (1-3), three new sesquiterpenes, were isolated from the Vietnamese sea hare Aplysia dactylomela. Their structures and absolute configurations were elucidated based on spectroscopic analysis, X-ray crystallography, and density functional theory (DFT) calculations of NMR and ECD data. Compound 2 exhibited cytotoxicity against HepG2, DU145 and A549 cells with respective IC50 values of 4.08 ± 0.63, 38.64 ± 1.04 and 12.33 ± 0.95 µM. In addition, HepG2 cells treated with 5 µM compound 2 for 48 h showed a significant increase in early apoptotic cells (P < 0.05) and increased caspase 3 activity (P < 0.01). Moreover, compound 2 induced sub-G1 phase arrest in HepG2 cells.

Anti-Inflammatory Effects of 5α,8α-Epidioxycholest-6-en-3ß-ol, a Steroidal Endoperoxide Isolated from Aplysia depilans, Based on Bioguided Fractionation and NMR Analysis.

Sea hares of Aplysia genus are recognized as a source of a diverse range of metabolites. 5α,8α-Endoperoxides belong to a group of oxidized sterols commonly found in marine organisms and display several bioactivities, including antimicrobial, anti-tumor, and immunomodulatory properties. Herein we report the isolation of 5α,8α-epidioxycholest-6-en-3ß-ol (EnP(5,8)) from Aplysia depilans Gmelin, based on bioguided fractionation and nuclear magnetic resonance (NMR) analysis, as well as the first disclosure of its anti-inflammatory properties. EnP(5,8) revealed capacity to decrease cellular nitric oxide (NO) levels in RAW 264.7 macrophages treated with lipopolysaccharide (LPS) by downregulation of the Nos2 (inducible nitric oxide synthase, iNOS) gene. Moreover, EnP(5,8) also inhibited the LPS-induced expression of cyclooxygenase-2 (COX-2), interleukin 6 (IL-6), and tumor necrosis factor alpha (TNF-α) at the mRNA and protein levels. Mild selective inhibition of COX-2 enzyme activity was also evidenced. Our findings provide evidence of EnP(5,8) as a potential lead drug molecule for the development of new anti-inflammatory agents.

Nitric oxide and l-arginine regulate feeding in satiated rats.

Nitric Oxide (NO) and its precursor l-arginine were found to inhibit feeding in rats with a low motivation to eat, as they do in Aplysia. In rats that are relatively satiated, treatment with an NO blocker increased feeding, and treatment with an NO donor or with either of 2 doses of l-arginine inhibited feeding. NO and l-arginine modulated several parameters of feeding, such as the total duration of appetitive behaviors, the time spent feeding, the quantity of food eaten and the number of feeding bouts. The inhibitory effect of l-arginine on feeding could not be attributed to changes in locomotion. These data indicate that satiation is partially mediated by increased production of NO. NADPH-Diaphorase histochemical staining, which is specific for tissues actively producing NO, showed significantly greater staining in satiated compared to hungry rats in all 4 hypothalamic nuclei (paraventricular and arcuate nuclei, lateral and ventromedial hypothalamus) that were examined. l-arginine may act as a regulator of feeding by controlling NO production in several hypothalamic nuclei, specifically under condition of a low feeding motivation.

Use of the sea hare (Aplysia fasciata) in marine pollution biomonitoring of harbors and bays.

Our study evaluated heavy metal concentrations in soft tissues of sea hare, Aplysia fasciata, from the Lower Laguna Madre, Texas. Heavy metals in tissues followed Se>As>Pb>Cd. Concentrations ranged As (BDL-28.08), Cd (BDL-5.50), Pb (BDL-12.85) and Se (4.25-93.43ppm). Median As, Cd, Pb, and Se tissue levels exceeded exposure levels. Significant relationships occurred in metal-metal (AsCd, AsPb, CdPb, CdSe, and PbSe), metal-tissue (significant Se uptake by inhalant and exhalant siphons and As in the hepatopancreas), and metal-metal within tissue (AsPb in the hepatopancreas and CdPb in the digestive cecum) analyses (p<0.05). Bioaccumulation factors (BAF) suggested the inhalant siphon, hepatopancreas, and digestive cecum function as macroconcentrators of Cd, hepatopancreas and digestive cecum as macroconcentrators of Pb, and all tissues were deconcentrators for As and Se. As a bioaccumulator of heavy metals, Aplysia was evaluated as a bioindicator of marine pollution in harbors and bays.

Molecular recognition of thiaclopride by Aplysia californica AChBP: new insights from a computational investigation.

The binding of thiaclopride (THI), a neonicotinoid insecticide, with Aplysia californica acetylcholine binding protein (Ac-AChBP), the surrogate of the extracellular domain of insects nicotinic acetylcholine receptors, has been studied with a QM/QM' hybrid methodology using the ONIOM approach (M06-2X/6-311G(d):PM6). The contributions of Ac-AChBP key residues for THI binding are accurately quantified from a structural and energetic point of view. The importance of water mediated hydrogen-bond (H-bond) interactions involving two water molecules and Tyr55 and Ser189 residues in the vicinity of the THI nitrile group, is specially highlighted. A larger stabilization energy is obtained with the THI-Ac-AChBP complex compared to imidacloprid (IMI), the forerunner of neonicotinoid insecticides. Pairwise interaction energy calculations rationalize this result with, in particular, a significantly more important contribution of the pivotal aromatic residues Trp147 and Tyr188 with THI through CH···π/CH···O and π-π stacking interactions, respectively. These trends are confirmed through a complementary non-covalent interaction (NCI) analysis of selected THI-Ac-AChBP amino acid pairs.

Addressing the Genetics of Human Mental Health Disorders in Model Organisms.

Mental health disorders are notoriously difficult to diagnose and treat for a variety of reasons, including genetic heterogeneity, comorbidities, and qualitative diagnostic criteria. Discovery of the molecular pathology underlying these disorders is crucial to the development of quantitative biomarkers and novel therapeutics. In this review, we discuss contributions to the mental health field of different cellular and whole-animal approaches in characterizing psychiatric genetics and molecular pathology. These approaches include mammalian cell and neuronal culture, cerebral organoids, induced pluripotent stem cells, and the whole-animal models of nematodes, flies, mollusks, frogs, mice, and zebrafish, on the last of which we place extra emphasis. Integrative use of these cellular and animal systems in a complementary and informative fashion maximizes the potential contributions to the mental health field as a whole.

Complementary interactions between command-like interneurons that function to activate and specify motor programs.

Motor activity is often initiated by a population of command-like interneurons. Command-like interneurons that reliably drive programs have received the most attention, so little is known about how less reliable command-like interneurons may contribute to program generation. We study two electrically coupled interneurons, cerebral-buccal interneuron-2 (CBI-2) and CBI-11, which activate feeding motor programs in the mollusk Aplysia californica. Earlier work indicated that, in rested preparations, CBI-2, a powerful activator of programs, can trigger ingestive and egestive programs. CBI-2 reliably generated ingestive patterns only when it was repeatedly stimulated. The ability of CBI-2 to trigger motor activity has been attributed to the two program-promoting peptides it contains, FCAP and CP2. Here, we show that CBI-11 differs from CBI-2 in that it contains FCAP but not CP2. Furthermore, it is weak in its ability to drive programs. On its own, CBI-11 is therefore less effective as a program activator. When it is successful, however, CBI-11 is an effective specifier of motor activity; that is, it drives mostly ingestive programs. Importantly, we found that CBI-2 and CBI-11 complement each other's actions. First, prestimulation of CBI-2 enhanced the ability of CBI-11 to drive programs. This effect appears to be partly mediated by CP2. Second, coactivation of CBI-11 with CBI-2 makes CBI-2 programs immediately ingestive. This effect may be mediated by specific actions that CBI-11 exerts on pattern-generating interneurons. Therefore, different classes of command-like neurons in a motor network may make distinct, but potentially complementary, contributions as either activators or specifiers of motor activity.

Cellular, molecular, and epigenetic mechanisms in non-associative conditioning: implications for pain and memory.

Sensitization is a form of non-associative conditioning in which amplification of behavioral responses can occur following presentation of an aversive or noxious stimulus. Understanding the cellular and molecular underpinnings of sensitization has been an overarching theme spanning the field of learning and memory as well as that of pain research. In this review we examine how sensitization, both in the context of learning as well as pain processing, shares evolutionarily conserved behavioral, cellular/synaptic, and epigenetic mechanisms across phyla. First, we characterize the behavioral phenomenon of sensitization both in invertebrates and vertebrates. Particular emphasis is placed on long-term sensitization (LTS) of withdrawal reflexes in Aplysia following aversive stimulation or injury, although additional invertebrate models are also covered. In the context of vertebrates, sensitization of mammalian hyperarousal in a model of post-traumatic stress disorder (PTSD), as well as mammalian models of inflammatory and neuropathic pain is characterized. Second, we investigate the cellular and synaptic mechanisms underlying these behaviors. We focus our discussion on serotonin-mediated long-term facilitation (LTF) and axotomy-mediated long-term hyperexcitability (LTH) in reduced Aplysia systems, as well as mammalian spinal plasticity mechanisms of central sensitization. Third, we explore recent evidence implicating epigenetic mechanisms in learning- and pain-related sensitization. This review illustrates the fundamental and functional overlay of the learning and memory field with the pain field which argues for homologous persistent plasticity mechanisms in response to sensitizing stimuli or injury across phyla.

Cloning analysis of ferritin and the cisplatin-subunit for cancer cell apoptosis in Aplysia juliana hepatopancreas.

Ferritin, an iron storage protein, plays a key role in iron metabolism in vivo. Here, we have cloned an inducible ferritin cDNA with 519 bp within the open reading frame fragment from the hepatopancreas of Aplysia juliana (AJ). The subunit sequence of the ferritin was predicted to be a polypeptide of 172 amino acids with a molecular mass of 19.8291kDa and an isoelectric point of 5.01. The cDNA sequence of hepatopancreas ferritin in AJ was constructed into a pET-32a system for expressing its relative protein efficiently in E. coli strain BL21, under isopropyl-ß-d-thiogalactoside induction. The recombinant ferritin, which was further purified on a Ni-NTA resin column and digested with enterokinase, was detected as a single subunit of approximately 20 kDa mass using both SDS-PAGE and mass spectrometry. The secondary structure and phosphorylation sites of the deduced amino acids were predicted using both ExPASy proteomic tools and the NetPhos 2.0 server, and the subunit space structure of the recombinant AJ ferritin (rAjFer) was built using a molecular operating environment software system. The result of in-gel digestion and identification using MALDI-TOF MS/MS showed that the recombinant protein was AjFer. ICP-MS results indicated that the rAjFer subunit could directly bind to cisplatin[cis-Diaminedichloroplatinum(CDDP)], giving approximately 17.6 CDDP/ferritin subunits and forming a novel CDDP-subunit. This suggests that a nanometer CDDP core-ferritin was constructed, which could be developed as a new anti-cancer drug. The flow cytometry results indicated that CDDP-rAjFer could induce Hela cell apoptosis. Results of the real-time PCR and Western blotting showed that the expression of AjFer mRNA was up-regulated in AJ under Cd(2+) stress. The recombinant AjFer protein should prove to be useful for further study of the structure and function of ferritin in Aplysia.

Virtual screening against acetylcholine binding protein.

The nicotinic acetylcholine receptors (nAChRs) are a member of the ligand-gated ion channel family and play a key role in the transfer of information across neurological networks. The X-ray crystal structure of agonist-bound α(7) acetylcholine binding protein (AChBP) has been recognized as the most appropriate template to model the ligand-binding domain of nAChR for studying the molecular mechanism of the receptor-ligand interactions. Virtual screening of the National Cancer Institute diversity set, a library of 1990 compounds with nonredundant pharmacophore profiles, using AutoDock against AChBPs revealed 51 potential candidates. In vitro radioligand competition assays using [(3)H] epibatidine against the AChBPs from the freshwater snails, Lymnaea stagnalis, and from the marine species, Aplysia californica and the mutant (AcY55W), revealed seven compounds from the list of candidates that had micromolar to nanomolar affinities for the AChBPs. Further investigation on α(7)nAChR expressing in Xenopus oocytes and on the recombinant receptors with fluorescence resonance energy transfer (FRET)-based calcium sensor expressing in HEK cells showed that seven compounds were antagonists of α(7)nAChR, only one compound (NSC34352) demonstrated partial agonistic effect at low dose (10 µM), and two compounds (NSC36369 and NSC34352) were selective antagonists on α(7)nAchR with moderate potency. These hits serve as novel templates/scaffolds for development of more potent and specific in the AChR systems.

A Computational Model of the Temperature-dependent Changes in Firing Patterns in Aplysia Neurons

We performed experiments using Aplysia neurons to identify the mechanism underlying the changes in the firing patterns in response to temperature changes. When the temperature was gradually increased from 11degrees C to 31degrees C the firing patterns changed sequentially from the silent state to beating, doublets, beating-chaos, bursting-chaos, square-wave bursting, and bursting-oscillation patterns. When the temperature was decreased over the same temperature range, these sequential changes in the firing patterns reappeared in reverse order. To simulate this entire range of spiking patterns we modified nonlinear differential equations that Chay and Lee made using temperature-dependent scaling factors. To refine the equations, we also analyzed the spike pattern changes in the presence of potassium channel blockers. Based on the solutions of these equations and potassium channel blocker experiments, we found that, as temperature increases, the maximum value of the potassium channel relaxation time constant, taun(t) increases, but the maximum value of the probabilities of openings for activation of the potassium channels, n(t) decreases. Accordingly, the voltage-dependent potassium current is likely to play a leading role in the temperature-dependent changes in the firing patterns in Aplysia neurons.

VLSI implementation of a template subtraction algorithm for real-time stimulus artifact rejection.

In this paper, we present very-large-scale integrated (VLSI) implementation of a template subtraction algorithm for stimulus artifact rejection (SAR) in real time with applicability to closed-loop neuroprostheses. The SAR algorithm is based upon an infinite impulse response (IIR) temporal filtering technique, which can be efficiently implemented in VLSI with reduced power consumption and silicon area. We demonstrate that initialization of the memory within the system architecture using the first recorded stimulus artifact significantly decreases system response time as compared to the case without memory initialization. Two sets of pre-recorded neural data from an Aplysia californica are used to simulate the functionality of the proposed VLSI architecture in AMS 0.35 microm complementary metal-oxide-semiconductor (CMOS) technology. Depending upon the reproducibility in the shape of stimulus artifacts in vivo, the system eliminates virtually all artifacts in real time and recovers the extracellular neural activity with microW-level power consumption from 1.5 V.

Serotonin stimulation of cAMP-dependent plasticity in Aplysia sensory neurons is mediated by calmodulin-sensitive adenylyl cyclase.

Calmodulin (CaM)-sensitive adenylyl cyclase (AC) in sensory neurons (SNs) in Aplysia has been proposed as a molecular coincidence detector during conditioning. We identified four putative ACs in Aplysia CNS. CaM binds to a sequence in the C1b region of AC-AplA that resembles the CaM-binding sequence in the C1b region of AC1 in mammals. Recombinant AC-AplA was stimulated by Ca(2+)/CaM. AC-AplC is most similar to the Ca(2+)-inhibited AC5 and AC6 in mammals. Recombinant AC-AplC was directly inhibited by Ca(2+), independent of CaM. AC-AplA and AC-AplC are expressed in SNs, whereas AC-AplB and AC-AplD are not. Knockdown of AC-AplA demonstrated that serotonin stimulation of cAMP-dependent plasticity in SNs is predominantly mediated by this CaM-sensitive AC. We propose that the coexpression of a Ca(2+)-inhibited AC in SNs, together with a Ca(2+)/CaM-stimulated AC, would enhance the associative requirement for coincident Ca(2+) influx and serotonin for effective stimulation of cAMP levels and initiation of plasticity mediated by AC-AplA.

Molecular modeling of the alpha9alpha10 nicotinic acetylcholine receptor subtype.

This study reports the comparative molecular modeling, docking and dynamic simulations of human alpha9alpha10 nicotinic acetylcholine receptors complexed with acetylcholine, nicotine and alpha-conotoxin RgIA, using as templates the crystal structures of Aplysia californica and Lymnaea stagnalis acetylcholine binding proteins. The molecular dynamics simulations showed that Arg112 in the complementary alpha10(-) subunit, is a determinant for recognition in the site that binds small ligands. However, Glu195 in the principal alpha9(+), and Asp114 in the complementary alpha10(-) subunit, might confer the potency and selectivity to alpha-conotoxin RgIA when interacting with Arg7 and Arg9 of this ligand.

The Effect of Heat on the Spiking Patterns of the Cells in Aplysia / 의학물리

Fruitful findings have been produced from five out of sixty cells which were obtained from each 63 individual Aplisia caught at the Jeju coast. Spiking patterns of three out of five cells, such as relaxation oscillator, bursting within a short time of the inter-burst interval, chaotic bursting, period doubling sequences, bursting with long trains of action potentials separated by short silent periods, regular repeated beating or elliptic bursting, and silent states had been changed in order as the temperature was lowered to 10 degrees C from 32 degrees C. In the intervals of every about 40 minutes repeated ups and downs of temperature produced similar firing patterns at the allowable temperature ranges. The other two cells showed difference from these. The amplitudes of the action potentials of the two cells will not be highly decreased in 24 hours. Average spike frequencies, the inter-burst interval, peak to peak spike amplitude of action potentials, minimum potential values are compared and analyzed by using the computer programme. The spike frequencies according to temperature show the distribution of bell type, with maximal spike frequencies at intermediate temperatures and minimal ones at either end. The most common pattern consist of high spike frequency during falling and low one during rising temperatures.

Synapse formation and mRNA localization in cultured Aplysia neurons.

mRNA localization and regulated translation provide a means of spatially restricting gene expression within neurons during axon guidance and long-term synaptic plasticity. Here we show that synapse formation specifically alters the localization of the mRNA encoding sensorin, a peptide neurotransmitter with neurotrophin-like properties. In isolated Aplysia sensory neurons, which do not form chemical synapses, sensorin mRNA is diffusely distributed throughout distal neurites. Upon contact with a target motor neuron, sensorin mRNA rapidly concentrates at synapses. This redistribution only occurs in the presence of a target motor neuron and parallels the distribution of sensorin protein. Reduction of sensorin mRNA, but not protein, with dsRNA inhibits synapse formation. Our results indicate that synapse formation can alter mRNA localization within individual neurons. They further suggest that translation of a specific localized mRNA, encoding the neuropeptide sensorin, is required for synapse formation between sensory and motor neurons.

Cloning and biochemical characterization of APIT, a new l-amino acid oxidase from Aplysia punctata.

The purple ink of the sea hare Aplysia punctata contains a 60 kDa protein with tumoricidal activity. This A. punctata ink toxin (APIT) kills tumor cells within 6--8h in an apoptosis independent manner by the production of high amounts of hydrogen peroxide which induce a necrotic form of oxidative stress. Here, we describe the biochemical features of APIT associated with its anti-tumor activity. APIT is a weakly glycosylated FAD-binding L-amino acid oxidase that catalyzes the oxidative deamination of L-lysine and L-arginine and thereby produces hydrogen peroxide (H(2)O(2)), ammonia (NH(4)(+)) and the corresponding alpha-keto acids. The tumoricidal effect is completely abrogated in the absence of the amino acids L-lysine and L-arginine. The enzyme is stable at temperatures from 0 to 50 degrees C. Similar to other FAD-binding enzymes, it is resistant against tryptic digest. Even digest with proteinase K fails to degrade the enzyme. Cloning of the APIT gene and subsequent sequencing revealed a FAD-binding domain followed by a so-called GG-motif, which is typical for L-amino acid oxidases. Strongest homology exists to escapin, aplysianin A precursor, the cyplasins L and S and achacin.

Responses of cerebral GABA-containing CBM neuron to taste stimulation with seaweed extracts in Aplysia kurodai.

Aplysia kurodai distributed along Japan feeds well on Ulva pertusa but rejects Gelidium amansii with distinctive patterned movements of the jaws and radula. On the ventral side of the cerebral M cluster, four cell bodies of higher order neurons that send axons to the buccal ganglia are distributed (CBM neurons). We have previously shown that the dopaminergic CBM1 modulates basic feeding circuits in the buccal ganglia for rejection by firing at higher frequency after application of the aversive taste of seaweed such as Gelidium amansii. In the present experiments immunohistochemical techniques showed that the CBM3 exhibited gamma-aminobutyric acid (GABA)-like immunoreactivity. The CBM3 may be equivalent to the CBI-3 involved in changing the motor programs from rejection to ingestion in Aplysia californica. The responses of the CBM3 to taste stimulation of the lips with seaweed extracts were investigated by the use of calcium imaging. The calcium-sensitive dye, Calcium Green-1, was iontophoretically introduced into a cell body of the CBM3 using a microelectrode. Application of Ulva pertusa or Gelidium amansii extract induced different changes in fluorescence in the CBM3 cell body, indicating that taste of Ulva pertusa initially induced longer-lasting continuous spike responses at slightly higher frequency compared with that of Gelidium amansii. Considering a role of the CBM3 in the pattern selection, these results suggest that elongation of the initial firing response may be a major factor for the CBM3 to switch the buccal motor programs from rejection to ingestion after application of different tastes of seaweeds in Aplysia kurodai.

The appearance of a protein kinase A-regulated splice isoform of slo is associated with the maturation of neurons that control reproductive behavior.

In response to brief synaptic stimulation that activates protein kinase A (PKA), the bag cell neurons of Aplysia trigger the onset of reproductive behaviors by generating a prolonged afterdischarge. In juvenile animals, such afterdischarges are inhibited by a high density of Ca2+ -activated K+ (BK) channels, encoded by the slo gene. An increase in this current also follows an afterdischarge in mature animals, contributing to a subsequent refractory state that limits reproductive behaviors. Using a bag cell cDNA library, we have isolated two alternative transcripts of the slo gene, differing in the presence (slo-a) or absence (slo-b) of a consensus phosphorylation site for PKA. Expression of either isoform in Chinese hamster ovary cells produced Ca2+ - and voltage-dependent channels with macroscopic and unitary properties matching those in bag cell neurons. The isoforms differed, however, in their response to application of the catalytic subunit of PKA, which reduced the open probability of Slo-a, an effect that was reversed by a PKA inhibitor. In contrast, PKA had no effect on Slo-b. By immunocytochemistry, we determined that the PKA-regulated Slo-a subunit is present in adult, but not juvenile, bag cell neurons. Patch clamp recordings from adult and juvenile bag cell neurons confirmed that PKA decreases BK channel activity only in adults. Our findings suggest that a change in the identity of Slo isoforms expressed during development allows mature neurons to generate afterdischarges that are required for reproduction.

Peribysins A-D, potent cell-adhesion inhibitors from a sea hare-derived culture of Periconia species.

Peribysins A-D 1-4, including a new type of furanofuran, have been isolated from a strain of Periconia byssoides originally separated from the sea hare Aplysia kurodai, and their relative stereostructures have been elucidated on the basis of NMR spectral analyses. All these metabolites potently inhibited the adhesion of human-leukemia HL-60 cells to HUVEC. The activity of compound 4, exhibiting the most potent inhibitory activity, was 380 times as potent as herbimycin A (standard).