Nested allostery in scorpion hemocyanin (Pandinus imperator)

The oxygen-binding behavior of the 24-meric hemocyanin of the scorpion Pandinus imperator and its dependence on allosteric effectors such as protons can be successfully described by the nesting model; the MWC model is not acceptable. The affinities of the four assumed conformations of the allosteric unit, the 12-meric half-molecule, are not dependent on pH whereas the three allosteric equilibrium constants decrease with decreasing proton concentration. Comparison with the oxygen-binding behavior of the 24-meric tarantula hemocyanin (Eurypelma californicum) reveals that the affinity values for the various conformations seem to be conserved for chelicerata hemocyanin.     

Pandinus imperator scorpion venom blocks voltage-gated potassium channels in GH3 cells

We examined the effects of Pandinus imperator scorpion venom on voltage-gated potassium channels in cultured clonal rat anterior pituitary cells (GH3 cells) using the gigohm-seal voltage-clamp method in the whole-cell configuration. We found that Pandinus venom blocks the voltage-gated potassium channels of GH3 cells in a voltage-dependent and dose-dependent manner. Crude venom in concentrations of 50-500 micrograms/ml produced 50-70% block of potassium currents measured at -20 mV, compared with 25-60% block measured at +50 mV. The venom both decreased the peak potassium current and shifted the voltage dependence of potassium current activation to more positive potentials. Pandinus venom affected potassium channel kinetics by slowing channel opening, speeding deactivation slightly, and increasing inactivation rates. Potassium currents in cells exposed to Pandinus venom did not recover control amplitudes or kinetics even after 20-40 min of washing with venom-free solution. The concentration dependence of crude venom block indicates that the toxins it contains are effective in the nanomolar range of concentrations. The effects of Pandinus venom were mimicked by zinc at concentrations less than or equal to 0.2 mM. Block of potassium current by zinc was voltage dependent and resembled Pandinus venom block, except that block by zinc was rapidly reversible. Since zinc is found in crude Pandinus venom, it could be important in the interaction of the venom with the potassium channel. We conclude that Pandinus venom contains toxins that bind tightly to voltage-dependent potassium channels in GH3 cells. Because of its high affinity for voltage-gated potassium channels and its irreversibility, Pandinus venom may be useful in the isolation, mapping, and characterization of voltage-gated potassium channels.     

Crystallization and preliminary analysis of crystals of the 24-meric hemocyanin of the emperor scorpion (Pandinus imperator)

Hemocyanins are giant oxygen transport proteins found in the hemolymph of several invertebrate phyla. They constitute giant multimeric molecules whose size range up to that of cell organelles such as ribosomes or even small viruses. Oxygen is reversibly bound by hemocyanins at binuclear copper centers. Subunit interactions within the multisubunit hemocyanin complex lead to diverse allosteric effects such as the highest cooperativity for oxygen binding found in nature. Crystal structures of a native hemocyanin oligomer larger than a hexameric substructure have not been published until now. We report for the first time growth and preliminary analysis of crystals of the 24-meric hemocyanin (M_W = 1.8 MDa) of emperor scorpion (Pandinus imperator), which diffract to a resolution of 6.5 Å. The crystals are monoclinc with space group C 1 2 1 and cell dimensions a = 311.61 Å, b = 246.58 Å and c = 251.10 Å (α = 90.00°, β = 90.02°, γ = 90.00°). The asymmetric unit contains one molecule of the 24-meric hemocyanin and the solvent content of the crystals is 56%. A preliminary analysis of the hemocyanin structure reveals that emperor scorpion hemocyanin crystallizes in the same oxygenated conformation, which is also present in solution as previously shown by cryo-EM reconstruction and small angle x-ray scattering experiments.     

Pi7, an orphan peptide from the scorpion Pandinus imperator: a 1H-NMR analysis using a nano-NMR Probe

The three-dimensional solution structure of a novel peptide, Pi7, purified from the venom of the scorpion Pandinus imperator, and for which no specific receptor has been found yet, was determined by two-dimensional homonuclear proton NMR methods from a nanomole amount of compound using a nano-nmr probe. Pandinus imperator peptide 7 does not block voltage-dependent K(+)-channels and does not displace labeled noxiustoxin from rat brain synaptosomal membranes. The toxin has 38 amino acid residues and, similarly to Pi1, is stabilized by four disulfide bridges (Cys6-Cys27, Cys12-Cys32, Cys16-Cys34, and Cys22-Cys37). In addition, the lysine at position 26 crucial for potassium-channel blocking is replaced in Pi7 by an arginine. Tyrosine 34, equivalent to Tyr36 of ChTX is present, but the N-terminal positions 1 and 2 are occupied by two acidic residues Asp and Glu, respectively. The dihedral angles and distance restraints obtained from measured NMR parameters were used in structural calculations in order to determine the conformation of the peptide. The disulfide-bridge topology was established using distance restraints allowing ambiguous partners between S atoms combined with NMR-derived structural information. The structure is organized around a short alpha-helix spanning residues Thr9 to Thr20/Gly21 and a beta-sheet. These two elements of secondary structure are stabilized by two disulfide bridges, Cys12-Cys32 and Cys16-Cys34. The antiparallel beta-sheet is composed of two strands extending from Asn22 to Cys34 with a tight turn at Ile28-Asn29 in contact with the N-terminal fragment Ile4 to Cys6.     

Synthesis and characterization of Pi4, a scorpion toxin from Pandinus imperator that acts on K+ channels.

Pi4 is a 38-residue toxin cross-linked by four disulfide bridges that has been isolated from the venom of the Chactidae scorpion Pandinus imperator. Together with maurotoxin, Pi1, Pi7 and HsTx1, Pi4 belongs to the alpha KTX6 subfamily of short four-disulfide-bridged scorpion toxins acting on K+ channels. Due to its very low abundance in venom, Pi4 was chemically synthesized in order to better characterize its pharmacology and structural properties. An enzyme-based cleavage of synthetic Pi4 (sPi4) indicated half-cystine pairings between Cys6-Cys27, Cys12-32, Cys16-34 and Cys22-37, which denotes a conventional pattern of scorpion toxin reticulation (Pi1/HsTx1 type). In vivo, sPi4 was lethal after intracerebroventricular injection to mice (LD50 of 0.2 microg per mouse). In vitro, addition of sPi4 onto Xenopus laevis oocytes heterologously expressing various voltage-gated K+ channel subtypes showed potent inhibition of currents from rat Kv1.2 (IC50 of 8 pm) and Shaker B (IC50 of 3 nm) channels, whereas no effect was observed on rat Kv1.1 and Kv1.3 channels. The sPi4 was also found to compete with 125I-labeled apamin for binding to small-conductance Ca(2+)-activated K+ (SK) channels from rat brain synaptosomes (IC50 value of 0.5 microm). sPi4 is a high affinity blocker of the Kv1.2 channel. The toxin was docked (BIGGER program) on the Kv channel using the solution structure of sPi4 and a molecular model of the Kv1.2 channel pore region. The model suggests a key role for residues Arg10, Arg19, Lys26 (dyad), Ile28, Lys30, Lys33 and Tyr35 (dyad) in the interaction and the associated blockage of the Kv1.2 channel.     

Chemical synthesis and characterization of Pi1, a scorpion toxin from Pandinus imperator active on K+ channels.

Pi1 is a 35-residue toxin cross-linked by four disulfide bridges that has been isolated from the venom of the chactidae scorpion Pandinus imperator. Due to its very low abundance in the venom, we have chemically synthesized this toxin in order to study its biological activity. Enzyme-based proteolytic cleavage of the synthetic Pi1 (sPi1) demonstrates half-cystine pairings between Cys4-Cys25, Cys10-Cys30, Cys14-Cys32 and Cys20-Cys35, which is in agreement with the disulfide bridge organization initially reported on the natural toxin. In vivo, intracerebroventricular injection of sPi1 in mice produces lethal effects with an LD50 of 0.2 microgram per mouse. In vitro, the application of sPi1 induces drastic inhibition of Shaker B (IC50 of 23 nM) and rat Kv1.2 channels (IC50 of 0.44 nM) heterologously expressed in Xenopus laevis oocytes. No effect was observed on rat Kv1.1 and Kv1.3 currents upon synthetic peptide application. Also, sPi1 is able to compete with 125I-labeled apamin for binding onto rat brain synaptosomes with an IC50 of 55 pM. Overall, these results demonstrate that sPi1 displays a large spectrum of activities by blocking both SK- and Kv1-types of K+ channels; a selectivity reminiscent of that of maurotoxin, another structurally related four disulfide-bridged scorpion toxin that exhibits a different half-cystine pairing pattern.     

Fine structure of the cuticle of the desert scorpion, Hadrurus arizonensis.

The structure of the sclerite and intersegmental cuticle of the opithosoma of the desert scorpion, Hadrurus arizonensis, has been examined by transmission electron microscopy. The sclerite cuticle contains a four-layered epicuticle, a hyaline exocuticle, an inner exocuticle and an endocuticle. The outer part of the hyaline exocuticle and the whole of the inner exocuticle are constructed of helicoidally arranged planes of microfibrils. Within the endocuticle, the overall architecture is not helicoidal as previously assumed, but consists of bundles of microfibrils oriented horizontally and vertically. Microbibrils of the inner exocuticle and the endocutile are seen as simple unstained rods, but those of the hyaline exocuticle are electron dense rods with an unstained central core. The intersegmental cuticle contains a four-layered epicuticle and a procuticle. In detail, its fine structure differs in most respects from that of the sclerite cuticle. Electron microscopy reveals that hyaline exocuticle, previously assumed to be continuous from sclerite to intersegmental membrane, is absent in the latter.     

Imperatoxin A (IpTx(a)) from Pandinus imperator stimulates [(3)H]ryanodine binding to RyR3 channels.

The effect of imperatoxin A (IpTx(a)) on the ryanodine receptor type 3 (RyR3) was studied. IpTx(a) stimulates [(3)H]ryanodine binding to RyR3-containing microsomes, but this effect requires toxin concentrations higher than those required to stimulate RyR1 channels. The effect of IpTx(a) on RyR3 channels was observed at calcium concentrations in the range 0.1 microM to 10 mM. By contrast, RyR2 channels were not significantly affected by IpTx(a) in the same calcium ranges. Single channel current measurements indicated that IpTx(a) induced subconductance state in RyR3 channels that was similar to those observed with RyR1 and RyR2 channels. These results indicate that IpTx(a) is capable of inducing similar subconductance states in all three RyR isoforms, while stimulation of [(3)H]ryanodine binding by this toxin results in isoform-specific responses, with RyR1 being the most sensitive channel, RyR3 displaying an intermediate response and RyR2 the least responsive ones.     

Water uptake through the cuticle of the water scorpion,Nepa cinerea L. (Heteroptera: Nepidae)

The rate of osmotic water uptake through the hydrophilic cuticle of Nepa cinerea has been measured and compared with that found in other water bugs possessing a hydrophobic cuticle.     

Rhythmic changes in the activity of neurosecretory cells of the scorpion, Heterometrus fulvipes (C. Koch)

Changes in the activity of the neurosecretory cells in the cephalothoracic nerve mass (CTNM) of the scorpion, Heterometrus fulvipes (C. Koch) was studied over a 24-h period under natural light-dark cycles. Cytological and cytometric observations revealed that the first 3 groups of type 'A' cells in the sub-oesophageal ganglion of CTNM evidenced ultradian variations in their activity. Two peaks were seen in the activity of these neurosecretory cells, at 12(00) and 00(00). It is inferred that the cells show a biphasic rhythm in the synthesis of two different principles possibly having antagonistic effects on the central nervous system.     

The thermo-photoelectric (TPE) properties of the hornet cuticle: correlation with the morphological structure

The present study investigated thermoelectric phenomena in the cuticle of the Oriental hornet Vespa orientalis (Hymenoptera, Vespinae). This was done in dependence on the pigment extant at various cuticular region, that is, the brown cuticle in which the primary pigment is melanin and embedded within the cuticle, and the yellow stripes in which the yellow pigment is comprised of purines and pteridines that are located in special pockets between the upper part of the cuticle and the basement membrane. The yellow pigment could be separated from the cuticle proper, but the brown pigment was not thus separable. We found that all cuticular regions of the gaster evinced a thermoelectric response, in that with rise in temperature there was a rise in the thermoelectric current, and vice versa. Additionally, the intact hornet displayed a negative photoelectric response in each of its yellow segments, so that upon illumination with UV light, the maximal current dropped by about 40-50%. Measurements taken on individual stripes in the gaster segments revealed that the photoelectric response is elicited only in the yellow stripes. In all the latter the photoelectric response persists but the maximal current level is lower than in the intact whole hornet. If the yellow pigment is detached mechanically or by bacterial incubation, the photoelectric property of the cuticle is abrogated. Likewise the photoelectric property is abrogated upon immersion of the cuticle in alcohol, even though the yellow pigment is still retained. The specific heat of the yellow stripes in the cuticle is about twice as high as that of the same stripes that had been depleted of their yellow pigment, amounting to 1.8-1.9 J/g.K vs. 0.8 J/g.K.     

Fine structure of scorpion trichobothria (Arachnida,Scorpiones)

Summary The fine structure of trichobothria in the scorpions Buthus occitanus (Amoureux, 1789) and Euscorpius carpathicus (Linné, 1767) was investigated by electron microscopy. In both species, cuticular and cellular characteristics are very similar. The articulation of the hair corresponds to that of other arachnid hair sensilla. The receptor endings are excentrically attached to the hair base. They consist of an enveloped S-shaped bundle of seven dendrites in B. occitanus and four in E. carpathicus. Neighbouring outer dendritic segments differ a great deal in diameter and ciliary modification. In B. occitanus, three enveloping cells and several additional secretory cells surround the inner dendritic segments. Structural characteristics are compared to those of other arachnid sensilla and their possible functional significance is discussed.     

Fine structure and development of the cuticle of Intoshia variabili (Orthonectida)

The body of free-swiming mature Intoshia variabili (Orthonectida) is covered by a thin cuticle, 0.3 μm thick. The cuticle is formed at the time when the orthonectid embryos develop in the plasmodium. The process of cuticle formation begins just after the first cilia begin to appear at the surface of the ciliated cells. At first, small extensions of the cell membrane appear at the surface of the cell, more or less parallel to the cell surface. As they develop futher, they stand up, and amorphic material begins to appear between them. The extensions then become microvilli and obtain their final shape, with a small subdistal swelling and a narrower distal part. They are situated very regularly on the surface of the cell. After the microvilli have obtained their final form, material between them begins to get its final structure typical of the adult form. During the period when the mature orthonectid begins to leave the plasmodium and emerge from the host, the regular microvilli begin to disappear, and only small irregular extensions are present under the cuticle on the surface of the cell. During the process of cuticle formation a large amount of smooth endoplasmic reticulum develops in the cells, but once the cuticle is formed it gradually disappears.     

The fine structure and deposition of the larval cuticle of the sheep blowfly (Lucilia Cuprina)

The cuticle of Lucilia is composed of an untanned endocuticle and a complex epicuticle of four layers, superficial layer, outer epicuticle, cuticulin and dense layer. The outer epicuticle and attached epicuticular filaments are resistant to acid hydrolysis. During deposition of the cuticle of each larval instar, the cuticulin and dense layers are formed first, followed by the outer epicuticle, which appears to be laid down by secretions from the epidermis passing through the cuticulin via epicuticular filaments. The outer epicuticle is found in the position normally occupied by the wax layer of other insect species.     

The structure of the cuticle of the shore-crab Carcinus maenas (L.)

The cuticle of Carcinus has been critically examined by light microscopy and found to present features differing from the model proposed by Bouligand (1965), elaborated by Neville and his collaborators (see references), and re-stated by Bouligand (1971). The laminae have the appearance of discrete and separable sheets connected by interlaminar fibres, and between them long unorientated macrofibres pursue a sinuous course and pass from one interlaminar zone to another. Together with the interlaminar connecting fibres the macrofibres appear to account for the plumose appearance of the interlaminar zones.