ABSTRACT
Structure-activity relationships of diazinoyl nicotinic insecticides (diazinoyl isomers and 5- or 6-substituted pyrazin-2-oyl analogues) are considered in terms of affinity to the insect nicotinic acetylcholine receptor (nAChR) and insecticidal activity against the imidacloprid-resistant brown planthopper. Among the test compounds, 3-(6-chloropyridin-3-ylmethyl)-2-(pyrazinoyl)iminothiazoline shows the highest potency in nAChR affinity and insecticidal activity. Aplysia californica acetylcholine binding protein (AChBP) mutants (Y55W + Q57R and Y55W + Q57T) are utilized to compare molecular recognition of nicotinic insecticides with diverse pharmacophores. N-nitro- or N-cyanoimine imidacloprid or acetamiprid, respectively, exhibits a high affinity to these AChBP mutants at a similar potency level. Intriguingly, the pyrazin-2-oyl analogue has a higher affinity to AChBP Y55W + Q57R than that to Y55W + Q57T, thereby indicating that pyrazine nitrogen atoms contact Arg57 guanidinium and Trp55 indole NH. Furthermore, nicotine prefers AChBP Y55W + Q57T over Y55W + Q57R, conceivably suggesting that the protonated nicotine is repulsed by Arg57 guanidinium, consistent with its inferior potency to insect nAChR.
Subject(s)
Hemiptera , Insect Proteins , Insecticides , Neonicotinoids , Receptors, Nicotinic , Animals , Insecticides/chemistry , Insecticides/pharmacology , Receptors, Nicotinic/metabolism , Receptors, Nicotinic/chemistry , Receptors, Nicotinic/genetics , Hemiptera/chemistry , Hemiptera/genetics , Hemiptera/drug effects , Hemiptera/metabolism , Structure-Activity Relationship , Insect Proteins/metabolism , Insect Proteins/genetics , Insect Proteins/chemistry , Neonicotinoids/chemistry , Neonicotinoids/pharmacology , Neonicotinoids/metabolism , Nitro Compounds/chemistry , Nitro Compounds/pharmacology , Nitro Compounds/metabolism , Aplysia/chemistry , Aplysia/metabolism , Aplysia/genetics , Nicotine/chemistry , Nicotine/metabolism , Nicotine/analogs & derivatives , Nicotine/pharmacologyABSTRACT
A d-galacturonic acid-specific lectin, named AcL, was purified from the sea hare Aplysia californica by galactose-agarose affinity chromatography. AcL has a molecular mass of 27.5 kDa determined by MALDI-TOF mass spectrometry. This lectin shows a good affinity for d-galacturonic acid and a lower affinity for galactosides: raffinose, melibiose, α and ß-lactose, and d-galactose. We determined the amino acid sequence of AcL by trypsin digestion and subsequent peptide analysis by mass spectrometry, resulting in a 238 amino acid protein with a theoretical molecular mass of 26.4 kDa. The difference between the theoretical and experimental values can be attributed to post-translational modifications. Thiol-disulfide quantification discerned five disulfide bonds and three free cysteines. The structure of Acl is mainly comprised of beta sheets, determined by circular dichroism, and predicted with AlphaFold. Theoretical models depict three nearly identical tandem domains consisting of two beta sheets each. From docking analysis, we identified AcL glycan-binding sites as multiple conserved motifs in each domain. Furthermore, phylogenetic analysis based on its structure and sequence showed that AcL and its closest homologues (GalULs) form a clear monophyletic group, distinct from other glycan-binding proteins with a jelly-roll fold: lectins of types F and H. GalULs possess four conserved sequence regions that distinguish them and are either ligand-binding motifs or stabilizing network hubs. We suggest that this new family should be referred to as GalUL or D-type, following the traditional naming of lectins; D standing for depilans, the epithet for the species (Aplysia depilans) from which a lectin of this family was first isolated and described.
Subject(s)
Aplysia , Hares , Animals , Aplysia/chemistry , Aplysia/metabolism , Hares/metabolism , Galectins/chemistry , Phylogeny , Galactose/metabolism , Polysaccharides/metabolismABSTRACT
New bromoditerpenes having an α-methylene carbonyl structure, azuriaplysins A (1) and B (2), were isolated from the sea hare Aplysia kurodai. Their relative stereostructures were determined based on one- and two-dimensional NMR spectroscopic analysis. In addition, the absolute stereostructures were determined by the total synthesis of both enantiomers of azuriaplysins A (1) and B (2), the key points of which were bromocyclization of farnesol and optical resolution of a key intermediate. Azuriaplysin B (2) and its enantiomer exhibited moderate cytotoxicity against HeLa S3 cells.
Subject(s)
Aplysia , Hares , Animals , Aplysia/chemistry , Magnetic Resonance Spectroscopy , StereoisomerismABSTRACT
BACKGROUND: Amyloids are ordered, insoluble protein aggregates, characterized by a cross-ß sheet quaternary structure in which molecules in a ß-strand conformation are stacked along the filament axis via intermolecular interactions. While amyloids are typically associated with pathological conditions, functional amyloids have also been identified and are present in a wide variety of organisms ranging from bacteria to humans. The cytoplasmic polyadenylation element-binding (CPEB) prion-like protein is an mRNA-binding translation regulator, whose neuronal isoforms undergo activity-dependent aggregation, a process that has emerged as a plausible biochemical substrate for memory maintenance. CPEB aggregation is driven by prion-like domains (PLD) that are divergent in sequence across species, and it remains unknown whether such divergent PLDs follow a similar aggregating assembly pathway. Here, we describe the amyloid-like features of the neuronal Aplysia CPEB (ApCPEB) PLD and compare them to those of the Drosophila ortholog, Orb2 PLD. RESULTS: Using in vitro single-molecule and bulk biophysical methods, we find transient oligomers and mature amyloid-like filaments that suggest similarities in the late stages of the assembly pathway for both ApCPEB and Orb2 PLDs. However, while prior to aggregation the Orb2 PLD monomer remains mainly as a random coil in solution, ApCPEB PLD adopts a diversity of conformations comprising α-helical structures that evolve to coiled-coil species, indicating structural differences at the beginning of their amyloid assembly pathways. CONCLUSION: Our results indicate that divergent PLDs of CPEB proteins from different species retain the ability to form a generic amyloid-like fold through different assembly mechanisms.
Subject(s)
Amyloid/metabolism , Aplysia/metabolism , Prions/metabolism , Animals , Aplysia/chemistry , Polyadenylation , Prions/chemistryABSTRACT
RNA modifications are emerging as critical players in the spatiotemporal regulation of gene expression. Although liquid chromatography-tandem mass spectrometry (LC-MS/MS) enables the simultaneous quantification of numerous enzymatically modified RNAs in a biological sample, conventional RNA extraction and enzymatic digestion protocols that are employed prior to analysis have precluded the application of this technique for small-volume samples. In this study, a biphasic liquid microjunction (LMJ) extraction system using coaxial capillaries that direct and aspirate extraction solvents onto a â¼350 µm diameter sample spot was developed and applied for the extraction of RNA from individual cell clusters in the central nervous system of the marine mollusk Aplysia californica. To maximize RNA recoveries, optimized extraction solvents consisting of 10% methanol and chloroform were evaluated under dynamic and static extraction conditions. An MS-compatible RNA digestion buffer was developed to minimize the number of sample-transfer steps and facilitate the direct enzymatic digestion of extracted RNA within the sample collection tube. Compared to RNA extraction using a conventional phenol-chloroform method, the LMJ-based method provided a 3-fold greater coverage of the neuronal epitranscriptome for similar amounts of tissues and also produced mRNA of sufficient purity for reverse transcription polymerase chain reaction amplification. Using this approach, the expression of RNA-modifying enzymes in a given neuronal cell cluster can be characterized and simultaneously correlated with the LC-MS/MS analysis of RNA modifications within the same subset of neurons.
Subject(s)
Aplysia/chemistry , Neurons/chemistry , RNA/isolation & purification , Animals , Chromatography, Liquid , Neurons/metabolism , RNA/chemistry , RNA/metabolism , Tandem Mass SpectrometryABSTRACT
Eisenia hydrolysis-enhancing protein (EHEP), which is a novel protein that has been identified in Aplysia kurodai, protects ß-glucosidases from phlorotannin inhibition to facilitate the production of glucose from the laminarin abundant in brown algae. Hence, EHEP has attracted attention for its potential applications in producing biofuel from brown algae. In this study, EHEP was purified from the natural digestive fluid of A. kurodai and was crystallized using the sitting-drop vapor-diffusion method. Native and SAD (single-wavelength anomalous diffraction) data sets were successfully collected at resolutions of 1.20 and 2.48â Å using wavelengths of 1.0 and 2.1â Å, respectively, from crystals obtained in initial screening. The crystals belonged to space group P212121 and contained one EHEP molecule in the asymmetric unit. All 20 S-atom sites in EHEP were located and the phases were determined by the SAD method using the S atoms in the natural protein as anomalous scatterers (native-SAD). After phase improvement, interpretable electron densities were obtained and 58% of the model was automatically built.
Subject(s)
Aplysia/chemistry , Crystallization/methods , Proteins/chemistry , Animals , Aplysia/enzymology , Aplysia/genetics , Aplysia/metabolism , Crystallography, X-Ray , Hydrolysis , Mass Spectrometry , Models, Molecular , Protein Conformation , Protein Domains/genetics , Proteins/isolation & purificationABSTRACT
d-Amino acid-containing peptides (DAACPs) make up a class of post-translationally modified peptides in animals that play important roles as cell-to-cell signaling molecules. Despite the functional importance of l- to d-residue isomerization, little is known about its prevalence, mostly due to difficulties associated with detecting differences in peptide stereochemistry. Prior efforts to discover DAACPs have been largely focused on pursuing peptides based on homology to known DAACPs or DAACP-encoding precursors. Here, we used a combination of enzymatic screening, mass spectrometry, and chromatographic analysis to identify novel DAACPs in the central nervous system (CNS) of Aplysia californica. We identified five new DAACPs from the pleurin precursor and three DAACPs from previously uncharacterized proteins. In addition, two peptides from the pleurin precursor, Plrn2 and Plrn3, exist as DAACPs with the d-residue found at position 2 or 3. These differentially modified forms of Plrn2 and Plrn3 are located in specific regions of the animal's CNS. Plrn2 and Plrn3 appear to be the first animal DAACPs in which the d-residue is found at more than one position, and this suggests that l- to d-residue isomerization may be a more variable/dynamic modification than previously thought. Overall, this study demonstrates the utility of nontargeted DAACP discovery approaches for identifying new DAACPs and demonstrates that isomerization is prevalent throughout the CNS of A. californica.
Subject(s)
Amino Acids/chemistry , Aplysia/chemistry , Neuropeptides/chemistry , Peptide Fragments/chemistry , Protein Processing, Post-Translational/genetics , Amino Acid Sequence , Animals , Aplysia/genetics , Central Nervous System/chemistry , Chromatography, High Pressure Liquid , Proteomics , Stereoisomerism , Tandem Mass SpectrometryABSTRACT
The lipid and fatty acid compositions of two species of gastropods, Aplysia kurodai and Aplysia juliana (collected from shallow sea water), were examined to assess their lipid profiles, health benefits, and the trophic relationships between herbivorous gastropods and their diets. The primary polyunsaturated fatty acids (PUFAs) found in the neutral lipids of all gastropod organs consisted of four shorter chain n-3 PUFAs: linolenic acid (LN, 18:3n-3), icosatetraenoic acid (ITA, 20:4n-3), icosapentaenoic acid (EPA, 20:5n3), and docosapentaenoic acid (DPA, 22:5n-3). The PUFAs found in polar lipids were various n-3 and n-6 PUFAs: arachidonic acid (ARA, 20:4n-6), adrenic acid (docosatetraenoic acid, DTA, 22:4n-6), icosapentaenoic acid (EPA, 20:5n-3), and docosapentaenoic acid (DPA, 22:5n-3) in addition to trace levels of docosahexaenoic acid (DHA, 22:6n-3). Various n-3 and n-6 PUFAs (18:2n-6, 20:2n-6, 18:3n-6, 20:3n-6, 18:3n-3, 18:4n-3, 20:3n-3, n-3 ITA, and 22:3n-6,9,15) comprised the biosynthetic profiles of A. kurodai and A. juliana. Both Aplysia species have traditionally been eaten as local foods in Japan, and the high levels of n-3 (EPA and n-3 DPA) and n-6 (ARA and DTA) PUFAs indicate that they are a healthful addition to a human's diet.
Subject(s)
Aplysia/chemistry , Fatty Acids, Unsaturated/analysis , Animals , Fatty Acids, Unsaturated/isolation & purification , Fatty Acids, Unsaturated/metabolismABSTRACT
Structural features and binding properties of sulfoxaflor (SFX) with Ac-AChBP, the surrogate of the insect nAChR ligand binding domain (LBD), are reported herein using various complementary molecular modeling approaches (QM, molecular docking, molecular dynamics, and QM/QM'). The different SFX stereoisomers show distinct behaviors in terms of binding and interactions with Ac-AChBP. Molecular docking and Molecular Dynamics (MD) simulations highlight the specific intermolecular contacts involved in the binding of the different SFX isomers and the relative contribution of the SFX functional groups. QM/QM' calculations provide further insights and a significant refinement of the geometric and energetic contributions of the various residues leading to a preference for the SS and RR stereoisomers. Notable differences in terms of binding interactions are pointed out for the four stereoisomers. The results point out the induced fit of the Ac-AChBP binding site according to the SFX stereoisomer. In this process, the water molecules-mediated contacts play a key role, their energetic contribution being among the most important for the various stereoisomers. In all cases, the interaction with Trp147 is the major binding component, through CH···π and π···π interactions. This study provides a rationale for the binding of SFX to insect nAChR, in particular with respect to the new class of sulfoximine-based insect nAChR competitive modulators, and points out the requirements of various levels of theory for an accurate description of ligand-receptor interactions.
Subject(s)
Aplysia/metabolism , Insecticides/metabolism , Pyridines/metabolism , Receptors, Cholinergic/metabolism , Sulfur Compounds/metabolism , Animals , Aplysia/chemistry , Aplysia/drug effects , Binding Sites , Insecticides/chemistry , Molecular Docking Simulation , Molecular Dynamics Simulation , Protein Binding , Pyridines/chemistry , Receptors, Cholinergic/chemistry , Sulfur Compounds/chemistry , ThermodynamicsABSTRACT
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.
Subject(s)
Anti-Inflammatory Agents/chemistry , Anti-Inflammatory Agents/pharmacology , Aplysia/chemistry , Cholesterol Esters/chemistry , Cholesterol Esters/pharmacology , Ergosterol/analogs & derivatives , Macrophages/drug effects , Adjuvants, Immunologic/pharmacology , Animals , Anti-Inflammatory Agents/isolation & purification , Chemical Fractionation , Cholesterol Esters/isolation & purification , Down-Regulation/drug effects , Enzyme Activation/drug effects , Ergosterol/chemistry , Ergosterol/isolation & purification , Ergosterol/pharmacology , Lipopolysaccharides/pharmacology , Magnetic Resonance Spectroscopy , Mice , Nitric Oxide Synthase Type II/genetics , RAW 264.7 CellsABSTRACT
Many marine animals use chemicals to defend themselves and their eggs from predators. Beyond their ecologically relevant functions, these chemicals may also have properties that make them beneficial for humans, including biomedical and industrial applications. For example, some chemical defenses are also powerful antimicrobial or antitumor agents with relevance to human health and disease. One such chemical defense, escapin, an l-amino acid oxidase in the defensive ink of the sea hare Aplysia californica, and related proteins have been investigated for their biomedical properties. This review details our current understanding of escapin's antimicrobial activity, including the array of molecules generated by escapin's oxidation of its major substrates, l-lysine and l-arginine, and mechanisms underlying these molecules' bactericidal and bacteriostatic effects on planktonic cells and the prevention of formation and removal of bacterial biofilms. Models of escapin's effects are presented, and future directions are proposed.
Subject(s)
Anti-Bacterial Agents/chemistry , Aplysia/enzymology , L-Amino Acid Oxidase/chemistry , Animals , Anti-Bacterial Agents/pharmacology , Aplysia/chemistry , Bacteria/drug effects , Biofilms/drug effects , L-Amino Acid Oxidase/pharmacologyABSTRACT
The profile of essential and non-essential elements was traced in the edible sea hares Aplysia depilans Gmelin, Aplysia fasciata Poiret and Aplysia punctata Cuvier. Manganese (Mn), iron (Fe), zinc (Zn), copper (Cu) and selenium (Se) were identified as the major essential elements. Risk assessment evidenced that the levels of cadmium (Cd) and lead (Pb) did not exceed the maximum limit value established by the European Regulation, the contents of chromium (Cr), nickel (Ni) and arsenic (As) being also below the levels established by the FDA guide. A correlation between trace elements levels and desaturation-elongation indexes of fatty acids was found. While Cd, Se and molybdenum (Mo) seem to promote the desaturation-elongation process involved on the production of C20:4n-6c, Ni, Cr and Fe may potentiate the conversion of C18:3n-3c to C20:5n-3c. Furthermore, cobalt (Co), Ni and Cu appear to decrease Δ9 desaturation index. Besides the suggested biosynthetic switch modulated by trace elements, the nutritional value of the species is further strengthened.
Subject(s)
Aplysia/chemistry , Fatty Acids/metabolism , Trace Elements/analysis , Trace Elements/pharmacology , Acetyltransferases/drug effects , Animals , Fatty Acid Desaturases/drug effects , Fatty Acid Elongases , Nutrition AssessmentABSTRACT
Characterization of endogenous neuropeptides produced from post-translational proteolytic processing of precursor proteins is a demanding task. A variety of complex prohormone processing steps generate molecular diversity from neuropeptide prohormones, making in silico neuropeptide discovery difficult. In addition, the wide range of endogenous peptide concentrations as well as significant peptide complexity further challenge the structural characterization of neuropeptides. Liquid chromatography-mass spectrometry (MS), performed in conjunction with bioinformatics, allows for high-throughput characterization of peptides. Mass analyzers and molecular dissociation techniques render specific characteristics to the acquired data and thus, influence the analysis of the MS data using bioinformatic algorithms for follow-up peptide identification. Here we evaluated the efficacy of several distinct peptidomic workflows using two mass spectrometers, the Thermo Orbitrap Fusion Tribrid and Bruker Impact HD UHR-QqTOF, for confident peptide discovery and characterization. We compared the results in several categories, including the numbers of identified peptides, full-length mature neuropeptides among all identifications, and precursor proteins mapped by the identified peptides. We also characterized the peptide false discovery rate (FDR) based on the occurrence of amidation, a known post-translational modification (PTM) that has been shown to require the presence of a C-terminal glycine. Thus, amidation events without a preceding glycine were considered false-positive amidation assignments. We compared the FDR calculated by the search engine used here to the minimum FDR estimated via false amidation assignments. The search engine severely underestimated the rate of false PTM assignments among the identified peptides, regardless of the specific MS platform used.
Subject(s)
Ganglia/chemistry , High-Throughput Screening Assays , Mass Spectrometry/methods , Neuropeptides/isolation & purification , Protein Processing, Post-Translational , Algorithms , Amides/chemistry , Amides/metabolism , Animals , Aplysia/chemistry , Aplysia/physiology , Chromatography, Liquid , Computational Biology , False Positive Reactions , Glycine/chemistry , Glycine/metabolism , Mass Spectrometry/instrumentation , Neuropeptides/chemistry , ProteolysisABSTRACT
A better understanding of neuromodulation in a behavioral system requires identification of active modulatory transmitters. Here, we used identifiable neurons in a neurobiological model system, the mollusc Aplysia, to study neuropeptides, a diverse class of neuromodulators. We took advantage of two types of feeding neurons, B48 and B1/B2, in the Aplysia buccal ganglion that might contain different neuropeptides. We performed a representational difference analysis (RDA) by subtraction of mRNAs in B48 versus mRNAs in B1/B2. The RDA identified an unusually long (2025 amino acids) peptide precursor encoding Aplysia leucokinin-like peptides (ALKs; e.g. ALK-1 and ALK-2). Northern blot analysis revealed that, compared with other ganglia (e.g. the pedal-pleural ganglion), ALK mRNA is predominantly present in the buccal ganglion, which controls feeding behavior. We then used in situ hybridization and immunohistochemistry to localize ALKs to specific neurons, including B48. MALDI-TOF MS on single buccal neurons revealed expression of 40 ALK precursor-derived peptides. Among these, ALK-1 and ALK-2 are active in the feeding network; they shortened the radula protraction phase of feeding motor programs triggered by a command-like neuron. We also found that this effect may be mediated by the ALK-stimulated enhancement of activity of an interneuron, which has previously been shown to terminate protraction. We conclude that our multipronged approach is effective for determining the structure and defining the diverse functions of leucokinin-like peptides. Notably, the ALK precursor is the first verified nonarthropod precursor for leucokinin-like peptides with a novel, marked modulatory effect on a specific parameter (protraction duration) of feeding motor programs.
Subject(s)
Aplysia/physiology , Ganglia, Invertebrate/physiology , Neuropeptides/metabolism , Animals , Aplysia/chemistry , Aplysia/cytology , Aplysia/genetics , Feeding Behavior , Ganglia, Invertebrate/chemistry , Ganglia, Invertebrate/metabolism , Neurons/chemistry , Neurons/cytology , Neurons/metabolism , Neuropeptides/analysis , Neuropeptides/genetics , Protein Processing, Post-Translational , RNA, Messenger/analysis , RNA, Messenger/genetics , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
Venom peptide toxins such as conotoxins play a critical role in the characterization of nicotinic acetylcholine receptor (nAChR) structure and function and have potential as nervous system therapeutics as well. However, the lack of solved structures of conotoxins bound to nAChRs and the large size of these peptides are barriers to their computational docking and design. We addressed these challenges in the context of the α4ß2 nAChR, a widespread ligand-gated ion channel in the brain and a target for nicotine addiction therapy, and the 19-residue conotoxin α-GID that antagonizes it. We developed a docking algorithm, ToxDock, which used ensemble-docking and extensive conformational sampling to dock α-GID and its analogs to an α4ß2 nAChR homology model. Experimental testing demonstrated that a virtual screen with ToxDock correctly identified three bioactive α-GID mutants (α-GID[A10V], α-GID[V13I], and α-GID[V13Y]) and one inactive variant (α-GID[A10Q]). Two mutants, α-GID[A10V] and α-GID[V13Y], had substantially reduced potency at the human α7 nAChR relative to α-GID, a desirable feature for α-GID analogs. The general usefulness of the docking algorithm was highlighted by redocking of peptide toxins to two ion channels and a binding protein in which the peptide toxins successfully reverted back to near-native crystallographic poses after being perturbed. Our results demonstrate that ToxDock can overcome two fundamental challenges of docking large toxin peptides to ion channel homology models, as exemplified by the α-GID:α4ß2 nAChR complex, and is extendable to other toxin peptides and ion channels. ToxDock is freely available at rosie.rosettacommons.org/tox_dock.
Subject(s)
Algorithms , Aplysia/chemistry , Conotoxins/chemistry , Molecular Docking Simulation/methods , Nicotinic Antagonists/chemistry , Receptors, Nicotinic/chemistry , Animals , HumansABSTRACT
Diffusion functional magnetic resonance imaging (DfMRI) has been proposed as a method for functional neuroimaging studies, as an alternative to blood oxygenation level dependent (BOLD)-fMRI. DfMRI is thought to more directly reflect neural activation, but its exact mechanism remains unclear. It has been hypothesized that the water apparent diffusion coefficient (ADC) decrease observed upon neural activation results from swelling of neurons or neuron parts. To elucidate the origin of the DfMRI response at cellular level we performed diffusion MR microscopy at 17.2 T in Aplysia californica buccal ganglia and compared the water ADCs at cellular and ganglia levels before and after neuronal activation induced by perfusion with a solution containing dopamine. Neural cell swelling, evidenced from optical microscopy imaging, resulted in an intracellular ADC increase and an ADC decrease at ganglia level. Furthermore, the intracellular ADC increase was found to have a significant positive correlation with the increase in cell size. Our results strongly support the hypothesis that the ADC decrease observed with DfMRI upon neuronal activation at tissue level reflects activation-induced neural cell swelling.
Subject(s)
Aplysia/chemistry , Neuroimaging/methods , Neurons/cytology , Water/chemistry , Animals , Diffusion , Diffusion Magnetic Resonance Imaging , Oxygen/bloodABSTRACT
A new lectin from Aplysia dactylomela eggs (ADEL) was isolated by affinity chromatography on HCl-activated Sepharose™ media. Hemagglutination caused by ADEL was inhibited by several galactosides, mainly galacturonic acid (Ka = 6.05 × 106 M-1). The primary structure of ADEL consists of 217 residues, including 11 half-cystines involved in five intrachain and one interchain disulfide bond, resulting in a molecular mass of 57,228 ± 2 Da, as determined by matrix-assisted laser desorption/ionization time of flight mass spectrometry. ADEL showed high similarity with lectins isolated from Aplysia eggs, but not with other known lectins, indicating that these lectins could be grouped into a new family of animal lectins. Three glycosylation sites were found in its polypeptide backbone. Data from peptide-N-glycosidase F digestion and MS suggest that all oligosaccharides attached to ADEL are high in mannose. The secondary structure of ADEL is predominantly ß-sheet, and its tertiary structure is sensitive to the presence of ligands, as observed by CD. A 3D structure model of ADEL was created and shows two domains connected by a short loop. Domain A is composed of a flat three-stranded and a curved five-stranded ß-sheet, while domain B presents a flat three-stranded and a curved four-stranded ß-sheet. Molecular docking revealed favorable binding energies for interactions between lectin and galacturonic acid, lactose, galactosamine, and galactose. Moreover, ADEL was able to agglutinate and inhibit biofilm formation of Staphylococcus aureus, suggesting that this lectin may be a potential alternative to conventional use of antimicrobial agents in the treatment of infections caused by Staphylococcal biofilms.
Subject(s)
Anti-Bacterial Agents/chemistry , Aplysia/chemistry , Biofilms/drug effects , Lectins/chemistry , Staphylococcus aureus/drug effects , Zygote/chemistry , Amino Acid Sequence , Animals , Anti-Bacterial Agents/isolation & purification , Anti-Bacterial Agents/metabolism , Anti-Bacterial Agents/pharmacology , Aplysia/genetics , Aplysia/metabolism , Biofilms/growth & development , Escherichia coli/genetics , Escherichia coli/metabolism , Galactosides/pharmacology , Gene Expression , Hemagglutination Inhibition Tests , Hexuronic Acids/pharmacology , Lectins/genetics , Lectins/isolation & purification , Lectins/pharmacology , Molecular Docking Simulation , Protein Domains , Protein Structure, Secondary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/pharmacology , Sequence Alignment , Staphylococcus aureus/growth & developmentABSTRACT
We describe a reflection imaging system that consists of a plasmonic crystal, a common laboratory microscope, and band-pass filters for use in the quantitative imaging and in situ monitoring of live cells and their substrate interactions. Surface plasmon resonance (SPR) provides a highly sensitive method to monitor changes in physicochemical properties occurring at metal-dielectric interfaces. Polyelectrolyte thin films deposited using the layer-by-layer (LBL) self-assembly method provide a reference system for calibrating the reflection contrast changes that occur when the polyelectrolyte film thickness changes and provide insight into the optical responses that originate from the multiple plasmonic features supported by this imaging system. Finite-difference time-domain (FDTD) simulations of the optical responses measured experimentally from the polyelectrolyte reference system are used to provide a calibration of the optical system for subsequent use in quantitative studies investigating live cell dynamics in cultures supported on a plasmonic crystal substrate. Live Aplysia californica pedal ganglion neurons cultured in artificial seawater were used as a model system through which to explore the utility of this plasmonic imaging technique. Here, the morphology of cellular peripheral structures â²80 nm in thickness were quantitatively analyzed, and the dynamics of their trypsin-induced surface detachment were visualized. These results illustrate the capacities of this system for use in investigations of the dynamics of ultrathin cellular structures within complex bioanalytical environments.
Subject(s)
Aplysia/chemistry , Animals , Nanostructures , Neurons , Optical Devices , Surface Plasmon ResonanceABSTRACT
The Ca2+-activated K+ channel, Slo1, has an unusually large conductance and contains a voltage sensor and multiple chemical sensors. Dual activation by membrane voltage and Ca2+ renders Slo1 central to processes that couple electrical signalling to Ca2+-mediated events such as muscle contraction and neuronal excitability. Here we present the cryo-electron microscopy structure of a full-length Slo1 channel from Aplysia californica in the presence of Ca2+ and Mg2+ at a resolution of 3.5 Å. The channel adopts an open conformation. Its voltage-sensor domain adopts a non-domain-swapped attachment to the pore and contacts the cytoplasmic Ca2+-binding domain from a neighbouring subunit. Unique structural features of the Slo1 voltage sensor suggest that it undergoes different conformational changes than other known voltage sensors. The structure reveals the molecular details of three distinct divalent cation-binding sites identified through electrophysiological studies of mutant Slo1 channels.
Subject(s)
Aplysia/ultrastructure , Cryoelectron Microscopy , Large-Conductance Calcium-Activated Potassium Channels/ultrastructure , Allosteric Regulation/drug effects , Amino Acid Sequence , Animals , Aplysia/chemistry , Aplysia/genetics , Binding Sites/drug effects , Calcium/chemistry , Calcium/pharmacology , Cations, Divalent/metabolism , Cytoplasm/metabolism , Electrophysiological Phenomena , Large-Conductance Calcium-Activated Potassium Channels/chemistry , Large-Conductance Calcium-Activated Potassium Channels/genetics , Lipid Bilayers/chemistry , Lipid Bilayers/metabolism , Magnesium/chemistry , Magnesium/pharmacology , Models, Molecular , Protein Domains/drug effects , Protein Subunits/chemistry , Protein Subunits/metabolismABSTRACT
The precise control of an ion channel gate by environmental stimuli is crucial for the fulfilment of its biological role. The gate in Slo1 K+ channels is regulated by two separate stimuli, intracellular Ca2+ concentration and membrane voltage. Slo1 is thus central to understanding the relationship between intracellular Ca2+ and membrane excitability. Here we present the Slo1 structure from Aplysia californica in the absence of Ca2+ and compare it with the Ca2+-bound channel. We show that Ca2+ binding at two unique binding sites per subunit stabilizes an expanded conformation of the Ca2+ sensor gating ring. These conformational changes are propagated from the gating ring to the pore through covalent linkers and through protein interfaces formed between the gating ring and the voltage sensors. The gating ring and the voltage sensors are directly connected through these interfaces, which allow membrane voltage to regulate gating of the pore by influencing the Ca2+ sensors.