Sea Anemone Anemonia Sulcata Research Articles

AsKC11, a Kunitz Peptide from Anemonia sulcata, Is a Novel Activator of G Protein-Coupled Inward-Rectifier Potassium Channels.

(1) Background: G protein-coupled inward-rectifier potassium (GIRK) channels, especially neuronal GIRK1/2 channels, have been the focus of intense research interest for developing drugs against brain diseases. In this context, venom peptides that selectively activate GIRK channels can be seen as a new source for drug development. Here, we report on the identification and electrophysiological characterization of a novel activator of GIRK1/2 channels, AsKC11, found in the venom of the sea anemone Anemonia sulcata. (2) Methods: AsKC11 was purified from the sea anemone venom by reverse-phase chromatography and the sequence was identified by mass spectrometry. Using the two-electrode voltage-clamp technique, the activity of AsKC11 on GIRK1/2 channels was studied and its selectivity for other potassium channels was investigated. (3) Results: AsKC11, a Kunitz peptide found in the venom of A. sulcata, is the first peptide shown to directly activate neuronal GIRK1/2 channels independent from Gi/o protein activity, without affecting the inward-rectifier potassium channel (IRK1) and with only a minor effect on KV1.6 channels. Thus, AsKC11 is a novel activator of GIRK channels resulting in larger K+ currents because of an increased chord conductance. (4) Conclusions: These discoveries provide new insights into a novel class of GIRK activators.

Engineering of a monomeric fluorescent protein AsGFP499 and its applications in a dual translocation and transcription assay

The tetrameric green fluorescent protein AsGFP(499) from the sea anemone Anemonia sulcata was converted into a dimeric and monomeric protein by site-directed mutagenesis. The protein was engineered without prior knowledge of its crystal structure based on a sequence alignment of multiple proteins belonging to the GFP-family. Crucial residues for oligomerisation of AsGFP(499) were predicted and selected for mutation. By introduction of a single site mutation (S103K) the A/B subunit was disrupted whereas two substitutions were necessary to separate the A/C subunit (T159K/F173E). This method can be applied as a general predictive method for designing monomeric proteins from multimeric fluorescent proteins. The maturation temperature was optimised to 37 degrees C by a combination of Site-directed and random mutagenesis. In cell-based assays, the NFATc1A (nuclear factor of activated T-cells, subtype 1, isoform A)-AsGFP(499) chimera formed massive cytoplasmic aggregates in HeLa cells, which prevented the shuttling of NFATc1A into the nucleus and consequentially its transcriptional activity. In contrast, the cells expressing the NFATc1A in fusion with our engineered dimeric and monomeric fluorescent mutants were homogeneously distributed throughout the cytoplasm, making these stable cell lines functional in both translocation and transcriptonal assays. This new dual cellular assay will allow the screening and discovery of new drugs that target NFAT cellular processes.

Modulation of Kv3 Subfamily Potassium Currents by the Sea Anemone Toxin BDS: Significance for CNS and Biophysical Studies

Kv3 potassium channels, with their ultra-rapid gating and high activation threshold, are essential for high-frequency firing in many CNS neurons. Significantly, the Kv3.4 subunit has been implicated in the major CNS disorders Parkinson's and Alzheimer's diseases, and it is claimed that selectively targeting this subunit will have therapeutic utility. Previous work suggested that BDS toxins ("blood depressing substance," from the sea anemone Anemonia sulcata) were specific blockers for rapidly inactivating Kv3.4 channels, and consequently these toxins are increasingly used as diagnostic agents for Kv3.4 subunits in central neurons. However, precisely how selective are these toxins for this important CNS protein? We show that BDS is not selective for Kv3.4 but markedly inhibits current through Kv3.1 and Kv3.2 channels. Inhibition comes about not by "pore block" but by striking modification of Kv3 gating kinetics and voltage dependence. Activation and inactivation kinetics are slowed by BDS-I and BDS-II, and V(1/2) for activation is shifted to more positive voltages. Alanine substitution mutagenesis around the S3b and S4 segments of Kv3.2 reveals that BDS acts via voltage-sensing domains, and, consistent with this, ON gating currents from nonconducting Kv3.2 are markedly inhibited. The altered kinetics and gating properties, combined with lack of subunit selectivity with Kv3 subunits, seriously affects the usefulness of BDS toxins in CNS studies. Furthermore, our results do not easily fit with the voltage sensor "paddle" structure proposed recently for Kv channels. Our data will be informative for experiments designed to dissect out the roles of Kv3 subunits in CNS function and dysfunction.

Chromophore Environment Provides Clue to “Kindling Fluorescent Protein” Riddle

asCP, the unique green fluorescent protein-like nonfluorescent chromoprotein from the sea anemone Anemonia sulcata, becomes fluorescent ("kindles") upon green light irradiation, with maximum emission at 595 nm. The kindled protein then relaxes to a nonfluorescent state or can be "quenched" instantly by blue light irradiation. In this work, we used asCP mutants to investigate the mechanism underlying kindling. Using site-directed mutagenesis we showed that amino acids spatially surrounding Tyr(66) in the chromophore are crucial for kindling. We propose a model of the kindling mechanism, in which the key event is chromophore turning or cis-trans isomerization. Using site-directed mutagenesis we also managed to transfer the kindling property to the two other coral chromoproteins. Remarkably, most kindling mutants were capable of both reversible and irreversible kindling. Also, we obtained novel variants that kindled upon blue light irradiation. The diversity of photoactivated fluorescent proteins that can be developed by site-directed mutagenesis is promising for biotechnological needs.

Cracks in the beta-can: fluorescent proteins from Anemonia sulcata (Anthozoa, Actinaria).

We characterize two green fluorescent proteins (GFPs), an orange fluorescent protein, and a nonfluorescent red protein isolated from the sea anemone Anemonia sulcata. The orange fluorescent protein and the red protein seem to represent two different states of the same protein. Furthermore, we describe the cloning of a GFP and a nonfluorescent red protein. Both proteins are homologous to the GFP from Aequorea victoria. The red protein is significantly smaller than other GFP homologues, and the formation of a closed GFP-like beta-can is not possible. Nevertheless, the primary structure of the red protein carries all features necessary for orange fluorescence. We discuss a type of beta-can that could be formed in a multimerization process.

Natural Animal Coloration Can Be Determined by a Nonfluorescent Green Fluorescent Protein Homolog

It is generally accepted that the colors displayed by living organisms are determined by low molecular weight pigments or chromoproteins that require a prosthetic group. The exception to this rule is green fluorescent protein (GFP) from Aequorea victoria that forms a fluorophore by self-catalyzed protein backbone modification. Here we found a naturally nonfluorescent homolog of GFP to determine strong purple coloration of tentacles in the sea anemone Anemonia sulcata. Under certain conditions, this novel chromoprotein produces a trace amount of red fluorescence (emission lambda(max) = 595 nm). The fluorescence demonstrates unique behavior: its intensity increases in the presence of green light but is inhibited by blue light. The quantum yield of fluorescence can be enhanced dramatically by single amino acid replacement, which probably restores the ancestral fluorescent state of the protein. Other fluorescent variants of the novel protein have emission peaks that are red-shifted up to 610 nm. They demonstrate that long wavelength fluorescence is attainable in GFP-like fluorescent proteins.

An all-electron calculation of an antihypertensive protein with the Gaussian-based density functional method

An all-electron calculation of the antihypertensive and antiviral protein BDS-I from the sea anemone Anemonia sulcata was carried out using the density functional method based on Gaussian-type orbitals with workstations. Our computer program ProteinDF coded by the object-oriented language C++, was originally designed for all-electron calculations of proteins and controlled in parallel with high efficiency. The numbers of residues, atoms, orbitals and auxiliary functions of the calculated protein were 43, 629, 3615 and 6545, respectively. This Letter reports timing data of the computation and some calculated electronic properties. Our program will open the door for advanced studies of larger proteins.

Effect of Deltamethrin Treatment on Lepidopteran Larvae Infected with Baculoviruses Expressing Insect-Selective Toxins μ-Aga-IV, As II, or Sh 1

Abstract Modification of sodium channels by pyrethroids is promoted by channel opening. The insect-specific neurotoxins μ-Aga-IV from the spider Agelenopsis aperta, As II from the sea anemone Anemonia sulcata, and Sh I from the sea anemone Stichadactyla helianthus all increase sodium channel opening and so we tested each of them in combination with deltamethrin for synergistic insecticidal activity. We found that As II increased the insecticidal activity of deltamethrin against the blowfly Lucilia sericata. We also examined the effect of deltamethrin on the infectivity and virulence of Autographa californica nuclear polyhedrosis virus (AcMNPV) and recombinant toxin-expressing viruses in both dose-dependent and time-mortality studies. The recombinant viruses expressed chimeric synthetic genes for μ-Aga-IV, As II, or Sh I (genes mag4, sat2, and ssh1, respectively). When Trichoplusia ni larvae were infected with a LC 20 of the recombinant viruses and treated with a LC 20 of deltamethrin in diet incorporation bioassays, the doses were strictly additive. A cooperative decrease in mortality rate was observed in droplet feeding bioassays when a low dose (LC 20 ) of deltamethrin and high doses of toxin-expressing viruses were fed to larvae but this effect was host-dependent. In T. ni, only wild-type AcMNPV and a recombinant expressing mag4 under very late viral promoter control exhibited reduced times in controlling host feeding and mortality in the presence of deltamethrin. For Heliothis virescens, larvae infected with sat2 -expressing viruses displayed shorter feeding times in the presence of deltamethrin than those infected with virus alone but only at very high virus doses. H. virescens larvae infected with wild-type AcMNPV and a mag4 -expressing recombinant had shorter feeding times and also shorter effective and lethal times in the presence of deltamethrin. Our results indicate that coapplication of this pyrethroid will confer little or no advantage in field applications of recombinant baculovirus pesticides and that application of pyrethroids will probably have no adverse affect on recombinant virus efficacy unless it deters larval feeding and virus acquisition.

Effects of Simultaneous Expression of Two Sodium Channel Toxin Genes on the Properties of Baculoviruses as Biopesticides

Abstract Previously, we have described the properties of recombinant baculoviruses expressing three chimeric genes, mag4, sat2, and ssh1, that encode secretable insect selective sodium channel toxins, μ-Aga-IV from the spider Agelenopsis aperta, As II from the sea anemone Anemonia sulcata, and Sh I from the sea anemone Stichadactyla helianthus, respectively. We now show that μ-Aga-IV and As II act at distinct sites on voltage-sensitive sodium channels of insects and synergistically promote channel opening. We also show that these toxins have synergistic insecticidal activity against the blowfly Lucilia sericata and the fall armyworm Spodoptera frugiperda. To determine if toxin synergy also occurs in the context of virus replication, we inserted the chimeric toxin genes into nonessential sites of the Autographa californica nuclear polyhedrosis virus (AcMNPV) genome under the control of either a modified promoter, P synXIV , or an insect derived promoter, P hsp70 . Comparative analyses showed that viruses expressing toxin genes under the control of the P hsp70 promoter were more effective as biopesticides than under the control of P synXIV . Two toxins, μ-Aga-IV and As II, exerted the most potent effects in S. frugiperda and Trichoplusia ni larvae, respectively. A virus simultaneously expressing two P hsp70 -promoted toxin genes, mag4 and sat2, exhibited properties similar to the two viruses expressing each of the toxin genes individually except that larval feeding time (FT 50 ) was reduced an additional 10%, indicating a small advantage to coproducing synergistic toxins.

A simple biochemical method in the search for bioactive polypeptides in a sea anemone ( Anemonia sulcata)

The sea anemone Anemonia sulcata is a well-known natural source of supply of biologically active polypeptides. So far, five toxins, ATX I, II, III, IV and AS V, several polyvalent protease inhibitors, an elastase inhibitor, two blood pressure-depressive polypeptides and very recently peptides that inhibit competitively the binding of 125I-dendrotoxin to rat brain membranes and block the voltage-sensitive K + channels, have been isolated from it. The sea anemone toxins (especially toxin II of A. sulcata, ATX II) are very important tools in neurophysiological and pharmacological research, and their structure-function relationship has been investigated. Because of the great scientific value of the sea anemone toxins a simplification of their purification procedure was elaborated.

Kalicludines and kaliseptine: Two different classes of sea anemone toxins for voltage-sensitive K + channels

New peptides have been isolated from the sea anemone Anemonia sulcata which inhibit competitively the binding of 125I-dendrotoxin I (a classical ligand for K+ channel) to rat brain membranes and behave as blockers of voltage-sensitive K+ channels. Sea anemone kalicludines are 58-59-amino acid peptides cross-linked with three disulfide bridges. They are structurally homologous both to dendrotoxins which are snake venom toxins and to the basic pancreatic trypsin inhibitor (Kunitz inhibitor) and have the unique property of expressing both the function of dendrotoxins in blocking voltage-sensitive K+ channels and the function of the Kunitz inhibitor in inhibiting trypsin. Kaliseptine is another structural class of peptide comprising 36 amino acids with no sequence homology with kalicludines or with dendrotoxins. In spite of this structural difference, it binds to the same receptor site as dendrotoxin and kalicludines and is as efficient as a K+ channel inhibitor as the most potent kalicludine.

The sea anemone Anemonia sulcata is a rich source of biologically active polypeptides

The sea anemone Anemonia sulcata is a rich source of biologically active polypeptides

Kalicludines and Kaliseptine: TWO DIFFERENT CLASSES OF SEA ANEMONE TOXINS FOR VOLTAGE-SENSITIVE K+ CHANNELS

New peptides have been isolated from the sea anemone Anemonia sulcata which inhibit competitively the binding of 125I-dendrotoxin I (a classical ligand for K+ channel) to rat brain membranes and behave as blockers of voltage-sensitive K+ channels. Sea anemone kalicludines are 58-59-amino acid peptides cross-linked with three disulfide bridges. They are structurally homologous both to dendrotoxins which are snake venom toxins and to the basic pancreatic trypsin inhibitor (Kunitz inhibitor) and have the unique property of expressing both the function of dendrotoxins in blocking voltage-sensitive K+ channels and the function of the Kunitz inhibitor in inhibiting trypsin. Kaliseptine is another structural class of peptide comprising 36 amino acids with no sequence homology with kalicludines or with dendrotoxins. In spite of this structural difference, it binds to the same receptor site as dendrotoxin and kalicludines and is as efficient as a K+ channel inhibitor as the most potent kalicludine.

Three-dimensional structure in solution of neurotoxin III from the sea anemone Anemonia sulcata.

The three-dimensional structure in aqueous solution of the 27-residue polypeptide neurotoxin Anemonia sulcata toxin III (ATX III) has been determined from 1H NMR data. As ATX III self-associates in the millimolar concentration range, causing a marked concentration dependence for the chemical shifts of several residues [Norton, R. S., Cross, K., Braach-Maksvytis, V., & Wachter, E. (1993) Biochem. J. 293, 545-551], it was necessary to record NOESY spectra over a range of concentrations in order to eliminate any intermolecular interactions from the NOE restraint set. The pairings of the six half-cystine residues were also unknown and had to be determined (as 3-17, 4-11, and 6-22) from preliminary structure calculations performed using both upper bound distance restraints from NOESY data and a substantial number of lower bound restraints inferred from the absence of NOESY cross-peaks. Final structures were determined, using the program X-PLOR, from interproton distance restraints inferred from NOEs, backbone and side chain dihedral angle restraints from spin-spin coupling measurements, and a smaller number of lower bound restraints. Stereospecific assignments for 11 beta-methylene pairs were also included. The final set of 28 structures had an average pairwise RMS difference of 1.32 A over the backbone heavy atoms (N, C alpha, and C) and 2.18 A over all heavy atoms. For the well-defined region encompassing residues 3-22, the corresponding values were 0.62 and 1.28 A, respectively. ATX III adopts a compact structure containing four reverse turns (a distorted type I beta-turn at residues 6-9, a type I beta-turn at residues 8-11, and inverse gamma-turns at residues 12-14 and 15-17) and two other chain reversals, but no regular alpha-helix or beta-sheet. Several of the residues most affected by aggregation are located on the surface of the molecule, forming a hydrophobic patch which may constitute part of the sodium channel binding surface. Possible relationships between the structure of ATX III and those of other sea anemone toxins that interact with the same site on the voltage-gated sodium channel are considered.

1H-n.m.r. study of the solution properties and secondary structure of neurotoxin III from the sea anemone Anemonia sulcata.

The solution properties, secondary structure and global fold of the 27-residue polypeptide neurotoxin III (ATX III), from the sea anemone Anemonia sulcata, have been investigated using high-resolution 1H-n.m.r. spectroscopy. Studies of the concentration dependence of the n.m.r. spectrum indicate that the molecule self-associates in the millimolar concentration range useable for n.m.r. analysis, the association being less pronounced at acidic pH values. The dependence on pH of association implies that electrostatic interactions play a role in this process, while the significant concentration-dependent shifts of the aromatic resonances of Tyr-7 and Trp-13 indicate that hydrophobic interactions also contribute. Individual pKa values have been determined for most ionizable groups in the molecule. Sequence-specific resonance assignments were obtained for all protons using a range of two-dimensional homonuclear-correlated and nuclear-Overhauser-effect (nOe) spectra. The secondary structure of the polypeptide was identified from sequential (i, i+1) and medium-range (i, i+2/3/4) nOe connectivities, NH to C alpha H coupling constants, C alpha H chemical shifts, and the location of slowly exchanging backbone-amide protons. ATX III contains no regular alpha-helix or beta-sheet, consisting instead of a network of reverse turns. nOe connectivities between half-cystine residues are consistent with the disulphide pairings 3-17, 4-11 and 6-22. ATX III has a well-defined structure and appears to lack the disordered loop which, in the longer sea anemone toxins (46-49 residues), may be part of the receptor-binding surface.

A proton nuclear magnetic resonance study of the antihypertensive and antiviral protein BDS-I from the sea anemone Anemonia sulcata: sequential and stereospecific resonance assignment and secondary structure

The sequential resonance assignment of the 1H NMR spectrum of the antihypertensive and antiviral protein BDS-I from the sea anemone Anemonia sulcata is presented. This is carried out with two-dimensional NMR techniques to identify through-bond and through-space (less than 5 A) connectivities. Added spectral complexity arises from the fact that the sample is an approximately 1:1 mixture of two BDS-I isoproteins, (Leu-18)-BDS-I and (Phe-18)-BDS-I. Complete assignments, however, are obtained, largely due to the increased resolution and sensitivity afforded at 600 MHz. In addition, the stereospecific assignment of a large number of beta-methylene protons is achieved from an analysis of the pattern of 3J alpha beta coupling constants and the relative magnitudes of intraresidue NOEs involving the NH, C alpha H, and C beta H protons. Regular secondary structure elements are deduced from a qualitative interpretation of the nuclear Overhauser enhancement, 3JHN alpha coupling constant, and amide NH exchange data. A triple-stranded antiparallel beta-sheet is found to be related to that found in partially homologous sea anemone polypeptide toxins.

Determination of the three-dimensional solution structure of the antihypertensive and antiviral protein BDS-I from the sea anemone Anemonia sulcata: a study using nuclear magnetic resonance and hybrid distance geometry-dynamical simulated annealing

The three-dimensional solution structure of the antihypertensive and antiviral protein BDS-I from the sea anemone Anemonia sulcata has been determined on the basis of 489 interproton and 24 hydrogen-bonding distance restraints supplemented by 23 phi backbone and 21 chi 1 side-chain torsion angle restraints derived from nuclear magnetic resonance (NMR) measurements. A total of 42 structures is calculated by a hybrid metric matrix distance geometry-dynamical simulated annealing approach. Both the backbone and side-chain atom positions are well defined. The average atomic rms difference between the 42 individual SA structures and the mean structure obtained by averaging their coordinates is 0.67 +/- 0.12 A for the backbone atoms and 0.90 +/- 0.17 A for all atoms. The core of the protein is formed by a triple-stranded antiparallel beta-sheet composed of residues 14-16 (strand 1), 30-34 (strand 2), and 37-41 (strand 3) with an additional mini-antiparallel beta-sheet at the N-terminus (residues 6-9). The first and second strands of the triple-stranded antiparallel beta-sheet are connected by a long exposed loop (residues 17-30). A number of side-chain interactions are discussed in light of the structure.

Three-dimensional structure of the neurotoxin ATX Ia from Anemonia sulcata in aqueous solution determined by nuclear magnetic resonance spectroscopy.

With the aid of 1H nuclear magnetic resonance (NMR) spectroscopy, the three-dimensional structure in aqueous solution was determined for ATX Ia, which is a 46 residue polypeptide neurotoxin of the sea anemone Anemonia sulcata. The input for the structure calculations consisted of 263 distance constraints from nuclear Overhauser effects (NOE) and 76 vicinal coupling constants. For the structure calculation several new or ammended programs were used in a revised strategy consisting of five successive computational steps. First, the program HABAS was used for a complete search of all backbone and chi 1 conformations that are compatible with the intraresidual and sequential NMR constraints. Second, using the program DISMAN, we extended this approach to pentapeptides by extensive sampling of all conformations that are consistent with the local and medium-range NMR constraints. Both steps resulted in the definition of additional dihedral angle constraints and in stereospecific assignments for a number of beta-methylene groups. In the next two steps DISMAN was used to obtain a group of eight conformers that contain no significant residual violations of the NMR constraints or van der Waals contacts. Finally, these structures were subjected to restrained energy refinement with a modified version of the molecular mechanics module of AMBER, which in addition to the energy force field includes potentials for the NOE distance constraints and the dihedral angle constraints. The average of the pairwise minimal RMS distances between the resulting refined conformers calculated for the well defined molecular core, which contains the backbone atoms of 35 residues and 20 interior side chains, is 1.5 +/- 0.3 A. This core is formed by a four-stranded beta-sheet connected by two well-defined loops, and there is an additional flexible loop consisting of the eleven residues 8-18. The core of the protein is stabilized by three disulfide bridges, which are surrounded by hydrophobic residues and shielded on one side by hydrophilic residues.

MALE-FEMALE ASSOCIATION IN THE SPIDER CRAB INACHUS PHALANGIUM: THE INFLUENCE OF FEMALE REPRODUCTIVE STAGE AND SIZE

The distribution of Inachus phalangium (Decapoda: Majidae) on the sea anemone Anemonia sulcata was investigated from 1982 to 1984 in the coastal region of Banyuls-sur-Mer (southern France). Most adult crabs (64.5%) were found alone on an anemone, but groups of 2, 3, and 4 adults sharing an anemone were decreasingly frequent. Spider crabs were randomly distributed on the anemones; 79.5% of the occupied anemones held only one crab. In cases of multiple occupation of a given anemone, (1) heterosexual pairs were more frequent than could be expected for random association, and homosexual pairs less frequent, (2) reproductive males were paired more frequently than expected with females ready to spawn, and (3) adult males pairing with females ready to spawn were larger than solitary males or those males with females not yet ready to spawn. In multiparous females 27% of the variance in the number of eggs per brood was explained by body size; egg number was positively correlated with body size but varied strongly within the same body-size class. Assortative pairing for body size in pairs was not found, probably because any fitness advantage accruing to males in choosing large females is offset by the costs of longer seeking time and the loss of mating opportunities with smaller females.

The covalent structure of the elastase inhibitor from Anemonia sulcata--a "non-classical" Kazal-type protein.

The amino-acid sequence of the proteinase inhibitor specific for elastases from the sea anemone Anemonia sulcata was determined from performic-acid oxidized inhibitor and from three cyanogen bromide fragments of reduced and carboxymethylated inhibitor. The molecule consists of a single polypeptide chain formed from 48 amino-acid residues and is stabilized by three intramolecular disulfide bridges. After cyanogen bromide cleavage of the native protein at methionines 10 and 28 followed by chymotryptic cleavage two fragments each containing a single disulfide bridge were isolated. These indicated the location of three intramolecular disulfide linkages between Cys4 and Cys34 (part of A-loop), Cys8 and Cys27 (B-loop) and Cys16 and Cys48 (C-loop). The sequential homology and the disulfide pattern identified the elastase inhibitor as a Kazal-type inhibitor in which, however, not only the CysI-CysII segment is rather short but interestingly the Cys4-Cys34 disulfide anchoring point (i.e. CysI-CysV) in the C-loop is shifted by one turn in the alpha-helical segment towards the C-terminus. Thus, the elastase inhibitor is a non-classical Kazal-type inhibitor with respect to the positioning of the half-cystines. The inhibitor molecule was modelled based on the known three-dimensional structure of the silver pheasant ovomucoid third domain. The shortened amino-terminal segment was arranged in such a manner to allow disulfide bridge formation between the first cysteine Cys4 and the replaced Cys34 under maintenance of a suitable binding loop conformation. The characteristic ovomucoid scaffold consisting of a central alpha-helix, an adjacent three-stranded beta-sheet and the proteinase-binding loop cross-connected through disulfide bridges CysI-CysV and CysIII-CysVI was conserved.