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PubMed2006

Synthesis and characterization of multiblock copolymers based on spider dragline silk proteins.

Spider dragline silk with its superlative tensile properties provides an ideal system to study the relationship between morphology and mechanical properties of a structural protein. Accordingly, we synthesized two hybrid multiblock copolymers by condensing poly(alanine) [(Ala)(5)] blocks of the structural proteins (spidroin MaSp1 and MaSp2) of spider dragline silk with different oligomers of isoprene (2200 and 5000 Da) having reactive end groups. The synthetic multiblock polymer displayed similar secondary structure to that of natural spidroin, the peptide segment forming a beta-sheet structure. These multiblock polymers showed a significant solubility in the component solvents. Moreover, the copolymer which contains the short polyisoprene segment would aggregate into a micellar-like structure, as observed by TEM.

Animals↗
PubMed2001

Self-assembly of beta-sheets into nanostructures by poly(alanine) segments incorporated in multiblock copolymers inspired by spider silk.

Selective replacement of the amorphous peptide domain of a spider silk with poly(ethylene glycol) gave N. clavipes silk-inspired polymers having similar solid-state structures and very good mechanical properties. The tendency of poly(alanine) having appropriate chain length to form beta-sheets and the facility with which the beta-sheets self-assemble have been retained in the polymers. Solid-state (13)C NMR, solid-state FTIR, X-ray diffraction, and AFM studies showed that the polymers formed predominantly antiparallel beta-sheets that self-assembled into discrete nanostructures. The longer the peptide segment was, the greater was the tendency to self-assemble into antiparallel beta-sheet aggregates. AFM revealed that the morphology of the polymers was a microphase-separated architecture that contained irregularly shaped 100-200 nm poly(alanine) nanodomains interspersed within the PEG phase. The results suggest that the poly(alanine) domain influences the solid-state properties of spider silk through beta-sheet self-assembly into temporary cross-links. The results further demonstrate that by selectively replacing certain segments of a naturally occurring biopolymer with a judiciously selected nonnative segment while, at the same time, retaining other segments known to be critical for the essential properties of the native biopolymer, a synthetic polymer with similar properties and function can be obtained.

Amino Acid Sequence↗
PubMed2000

Effect of brown spider venom on basement membrane structures.

Loxoscelism or necrotic arachnidism are terms used to describe lesions and reactions induced by bites (envenomation) from spiders of the genus Loxosceles. Envenomation has been reported to provoke dermonecrosis and haemorrhage at the bite site and haemolysis, disseminated intravascular coagulation and renal failure. The purpose of this work was to study the effect of the venom of the brown spider Loxosceles intermedia on basement membrane structures and on its major constituent molecules. Light microscopy observations showed that L. intermedia venom obtained through electric shock, which reproduces two major signals of Loxoscelism in the laboratory, exhibits activity toward basement membrane structures in mouse Engelbreth-Holm-Swarm (EHS) sarcoma. Basement degradation was seen by a reduced periodic acid-Schiff (PAS) and alcian blue staining as well as by a reduced immunostaining for laminin when compared to control experiments. Electron microscopy studies confirmed the above results, showing the action of the venom on EHS-basement membranes and demonstrating that these tissue structures are susceptible to the venom. Using purified components of the basement membrane, we determined through SDS-PAGE and agarose gel that the venom is not active toward laminin or type IV collagen, but is capable of cleaving entactin and endothelial heparan sulphate proteoglycan. In addition, when EHS tissue was incubated with venom we detected a release of laminin into the supernatant, corroborating the occurrence of some basement membrane disruption. The venom-degrading effect on entactin was blocked by 1, 10-phenanthroline, but not by other protease inhibitors such as PMSF, NEM or pepstatin-A. By using light microscopy associated with PAS staining we were able to identify that 1,10-phenanthroline also inhibits EHS-basement membrane disruption evoked by venom, corroborating that a metalloprotease of venom is involved in these effects. Degradation of these extracellular matrix molecules and the observed susceptibility of the basement membrane could lead to loss of vessel and glomerular integrity, resulting in haemorrhage and renal problems after envenomation.

Animals↗
PubMed2001

Wolf spiders show graded antipredator behavior in the presence of chemical cues from different sized predators.

The wolf spider, Pardosa milvina, displays effective antipredator behavior (reduced activity) in the presence of silk and excreta cues from adults of another cooccurring wolf spider, Hogna helluo. However, Pardosa and Hogna engage in size-structured intraguild predation, where Pardosa may be either the prey or predator of Hogna. We tested the ability of adult female Pardosa to vary antipredator responses toward kairomones produced by Hogna that vary in size. Hogna were maintained on filter paper for 24 hr. We then presented the paper to adult female Pardosa simultaneously paired with a blank sheet of paper. One treatment had two sheets of blank paper to serve as a control. The Hogna stimulus treatments were as follows (N = 15/treatment): (1) 1 Hogna half the mass of Pardosa; (2) 1 Hogna of equal mass of a Pardosa; (3) 1 adult Hogna, 30 times the mass of Pardosa; and (4) 8 Hogna each 0.25 the mass of Pardosa. Pardosa decreased activity in the presence of kairomones from Hogna of equal or larger size, but showed no change in activity in the presence of a blank control or from a single Hogna smaller than itself. Pardosa showed a reduction in activity in the presence of cues from eight small Hogna. Pardosa avoided substrates with adult Hogna cues, but showed no avoidance response to any other treatment. These results suggest that Pardosa is showing graded antipredator behavior relative to the quantity of predator kairomones present rather than directly discriminating among the different sizes of the predator.

Animals↗
PubMed2002

Structure-activity relationships of pyrrolizidine alkaloids in insect chemical defense against the orb-weaving spider Nephila clavipes.

Pyrrolizidine alkaloids (PAs) are known to protect Arctiidae moths and Danainae and Ithomiinae butterflies against the orb-weaving spider Nephila clavipes (Araneae, Araneidae, Tetragnathinae), which liberates adults of these insects unharmed from its web. We tested against this spider the role of eight PAs and one derived structure [an 89:11 mixture of the 12-membered macrocyclic diester free base integerrimine and senecionine and the respective N-oxide; two hydrolysis products from this mixture (the necine base retronecine, its respective N-oxide, and a mixture of integerriminic and senecionic necic acids); the 12-membered macrocyclic senkirkine; the 9-O-monoester free base senecioylretronecine and its respective N-oxide; and the 9-O-monoester free base callimorphine (a PA biosynthesized only by insects from retronecine)]. The mixture integerrimine-senecionine N-oxide seems to be more active than the respective free base [LibD50 (liberation dose 50) = 0.042 and 0.153 microg/dry weight of prey, respectively], but the difference in activity between the N-oxide and free base of the 9-O-monoester senecioylretronecine was slight (LibD50 = 0.167 and 0.104, respectively). Senkirkine, an otonecine base PA that does not form N-oxide and is not found in insects, was the less active, showing the highest LibD50 (0.354). The difference in antipredator activity between N-oxides and free bases from macrocyclic diesters and monoesters may be correlated with physicochemical properties of these molecules in interaction with the Nephila receptors. For the active structures, there was a significant correlation between dosage and antipredator activity. Both forms of retronecine and a necic acid mixture were inactive, supporting the hypothesis that PAs biosynthesized by insects from retronecine were originally produced and stored in order to optimize chemical defense. Comparison of dose/activity data with reported amounts of PAs in butterflies suggested that, in general, PA-specialist insects are protected against predation by Nephila.

Animals↗
PubMed2003

Determination of the torsion angles of alanine and glycine residues of model compounds of spider silk (AGG)(10) using solid-state NMR methods.

Spiders synthesize several kinds of silk fibers. In the primary structure of spider silk, one of the major ampullate (dragline, frame) silks, spidroin 1, and flagelliform silk (core fibers of adhesive spiral), there are common repeated X-Gly-Gly (X = Ala, Leu, Pro, Tyr, Glu, and Arg) sequences, which are considered to be related to the elastic character of these fibers. In this paper, two dimensional spin diffusion solid-state NMR under off magic angle spinning (OMAS), (13)C chemical shift contour plots, and Rotational Echo DOuble Resonance (REDOR) were applied to determine the torsion angles of one Ala and two kinds of Gly residues in the Ala-Gly-Gly sequence of (13)C=O isotope-labeled (Ala-Gly-Gly)(10). The torsion angles were determined to be (phi, psi) = (-90 degrees, 150 degrees ) within an experimental error of +/-10 degrees for each residue. This conformation is characterized as 3(1) helix which is in agreement with the structure proposed from the X-ray powder diffraction pattern of poly(Ala-Gly-Gly). The 3(1) helix of (Ala-Gly-Gly)(10) does not change by formic acid treatment although (Ala-Gly)(15) easily changes from the silk I conformation (the structure of Bombyx mori silk fibroin before spinning in the solid state) to silk II conformation (the structure of the silk fiber after spinning) by such treatment. Thus, the 3(1) helix conformation of (Ala-Gly-Gly)(10) is considered very stable. Furthermore, the torsion angles of the 16th Leu residue of (Leu-Gly-Gly)(10) were also determined as (phi, psi) = (-90 degrees, 150 degrees ) and this peptide is also considered to take 3(1) helix conformation.

Alanine↗
PubMed2004

Chemical signaling in a wolf spider: a test of ethospecies discrimination.

Chemical signals from female wolf spiders that elicit exploratory behavior and courtship in males are often assumed to be species-specific, but males of some species court in response to silk cues deposited by closely related heterospecific females. Such is the case with the wolf spiders Schizocosa ocreata and S. rovneri, ethospecies reproductively isolated on the basis of differences in behavioral mechanisms during courtship. We explored whether male S. ocreata and S. rovneri reciprocally discriminate species-specific chemical or mechanical cues associated with female silk by using male behavioral response as an assay. Males were exposed to stimulus treatment categories including silk, washed silk, silk extract, and appropriate controls within conspecific or heterospecific female stimulus categories. Male S. ocreata and S. rovneri did not discriminate between conspecific or heterospecific female stimuli, and courtship intensity was greatest on untreated silk. There were no differences in latency to begin courtship or in rates of courtship behaviors attributed to species origin of silk. However, silk treatment (washed silk, extract) had a significant effect on display and exploratory behaviors (e.g., chemoexplore) in both species. Methanol extraction of female silk successfully removed or inactivated a component necessary to elicit active courtship, but extraction did not significantly reduce exploratory behavior, suggesting that a separate compound may be responsible for releasing this behavior. Together, these experiments support the characterization of S. ocreata and S. rovneri as ethospecies reproductively isolated only by female discrimination of species-specific male courtship, and indicate that chemical, but not mechanical cues associated with silk are critical for eliciting male courtship in both species.

Animal Communication↗
PubMed2001

Implicit associations for fear-relevant stimuli among individuals with snake and spider fears.

This study investigated an implicit measure of cognitive processing, the Implicit Association Test (IAT; A. G. Greenwald, D. E. McGhee, & J. L. K. Schwartz, 1998), as a measure of fear-related automatic associations. Sixty-seven students with snake or spider fears completed 4 IAT tasks in which they classified pictures of snakes and spiders along with descriptive words indicating valence, fear, danger, or disgust. Results indicated that all 4 tasks discriminated between fear groups in terms of their implicit associations, and fear-specific effects were significant even after controlling for the impact of valence evaluation. Findings are discussed in terms of applications of the IAT methodology to examine cognitive processing and schemata in anxiety and potential uses for assessing anxiety disorders.

Adult↗
PubMed2002

Predator and prey views of spider camouflage.

Crab-spiders (Thomisus onustus) positioned for hunting on flowers disguise themselves by assuming the same colour as the flower, a strategy that is assumed to fool both bird predators and insect prey. But although this mimicry is obvious to the human observer, it has never been examined with respect to different visual systems. Here we show that when female crab-spiders mimic different flower species, they are simultaneously cryptic in the colour-vision systems of both bird predators and hymenopteran prey.

Animals↗
PubMed2002

Fibre science: supercontraction stress in wet spider dragline.

Unrestrained spider dragline 'super-contracts' when it is wetted, causing its length to shrink by about half and its diameter to almost double. Here we measure the supercontraction stresses generated upon initial exposure of spider dragline to moisture and find that they are transient, as well as being greater than previously estimated. Our findings cast doubt on suggestions that supercontraction may help to maintain tension in wet webs and could limit the potential load-bearing applications of silk and its analogues.

Animals↗
PubMed1977

The origin of the muscle fasciculation caused by funnel-web spider venom.

The origin of the fasciculation of skeletal muscle produced by funnel-web spider venom (FSV) has been examined in mouse phrenic nerve hemi-diaphragm preparations, FSV from male spiders at concentrations greater than 10(-6) g/ ml invariably produced muscle fasciculation which could be prevented by d-tubocurarine (14micron), tetrodotoxin (0.3 micron) or by increasing the external magnesium concentration or calcium concentration. Diphenyl hydantoin (3-6 X 10(-5) M) was able to reduce these fasciculations in some experiments. In curarized preparations, multiple end plate potentials (EPPs) in response to single stimuli and bursts of spontaneous EPPs were seen in the presence of FSV (10(-5) g/ml). Extracellular recordings from phrenic nerves in the presence of FSV (10(-5) g/ml) revealed additional components in compound action potentials elicited by single stimuli, and "spontaneous" electrical activity was observed in unstimulated nerves. This spontaneous activity was abolished by raising the divalent cation concentration in the bathing solution. These results suggest that a primary site of action of FSV is the surface membrane of nerve fibres and that muscle fasciculation arises as a consequence of spontaneous action potentials produced by the venom in motor nerves.

Action Potentials↗
PubMed2005

Relationships between supercontraction and mechanical properties of spider silk.

Typical spider dragline silk tends to outperform other natural fibres and most man-made filaments. However, even small changes in spinning conditions can have large effects on the mechanical properties of a silk fibre as well as on its water uptake. Absorbed water leads to significant shrinkage in an unrestrained dragline fibre and reversibly converts the material into a rubber. This process is known as supercontraction and may be a functional adaptation for the silk's role in the spider's web. Supercontraction is thought to be controlled by specific motifs in the silk proteins and to be induced by the entropy-driven recoiling of molecular chains. In analogy, in man-made fibres thermal shrinkage induces changes in mechanical properties attributable to the entropy-driven disorientation of 'unfrozen' molecular chains (as in polyethylene terephthalate) or the 'broken' intermolecular hydrogen bonds (as in nylons). Here we show for Nephila major-ampullate silk how in a biological fibre the spinning conditions affect the interplay between shrinkage and mechanical characteristics. This interaction reveals design principles linking the exceptional properties of silk to its molecular orientation.

Animals↗
PubMed2006

Comparing the rheology of native spider and silkworm spinning dope.

Silk production has evolved to be energetically efficient and functionally optimized, yielding a material that can outperform most industrial fibres, particularly in toughness. Spider silk has hitherto defied all attempts at reproduction, despite advances in our understanding of the molecular mechanisms behind its superb mechanical properties. Spun fibres, natural and man-made, rely on the extrusion process to facilitate molecular orientation and bonding. Hence a full understanding of the flow characteristics of native spinning feedstock (dope) will be essential to translate natural spinning to artificial silk production. Here we show remarkable similarity between the rheologies for native spider-dragline and silkworm-cocoon silk, despite their independent evolution and substantial differences in protein structure. Surprisingly, both dopes behave like typical polymer melts. This observation opens the door to using polymer theory to clarify our general understanding of natural silks, despite the many specializations found in different animal species.

Animals↗
PubMed2000

Neurotoxic activity of venom from the Australian eastern mouse spider (Missulena bradleyi) involves modulation of sodium channel gating.

Mouse spiders represent a potential cause of serious envenomation in humans. This study examined the activity of Missulena bradleyi venom in several in vitro preparations. Whilst female M. bradleyi venom at doses up to 0.05 microl ml(-1) failed to alter twitch or resting tension in all preparations used, male venom (0.02 and 0.05 microl ml(-1)) produced potent effects on transmitter release in both smooth and skeletal neuromuscular preparations. In the mouse phrenic nerve diaphragm preparation, male M. bradleyi venom (0.02 microl ml(-1)) caused rapid fasciculations and an increase in indirectly evoked twitches. Male venom (0.02 and 0.05 microl ml(-1)) also caused a large contracture and rapid decrease in indirectly evoked twitches in the chick biventer cervicis muscle, however had no effect on responses to exogenous ACh (1 mM) or potassium chloride (40 mM). In the chick preparation, contractile responses to male M. bradleyi venom (0.05 microl ml(-1)) were attenuated by (+)-tubocurarine (100 microM) and by tetrodotoxin (TTX, 1 microM). Both actions of male M. bradleyi venom were blocked by Atrax robustus antivenom (2 units ml(-1)). In the unstimulated rat vas deferens, male venom (0.05 microl ml(-1)) caused contractions which were inhibited by a combination of prazosin (0.3 microM) and P(2X)-receptor desensitization (with alpha,beta-methylene ATP 10 microM). In the rat stimulated vas deferens, male venom (0.05 microl ml(-1)) augmented indirectly evoked twitches. Male venom (0.1 microl ml(-1)) causes a slowing of inactivation of TTX-sensitive sodium currents in acutely dissociated rat dorsal root ganglion neurons. These results suggest that venom from male M. bradleyi contains a potent neurotoxin which facilitates neurotransmitter release by modifying TTX-sensitive sodium channel gating. This action is similar to that of the delta-atracotoxins from Australian funnel-web spiders.

Acetylcholine↗
PubMed2001

Novel mechanism of blocking axonal Na(+) channels by three macrocyclic polyamine analogues and two spider toxins.

1. The mechanism of Na(+) channel block by three macrocyclic polyamine derivatives and two spider toxins was studied with voltage clamp and internal perfusion method in squid axons. 2. All these chemicals specifically block Na(+) channels in the open state only from the internal surface, and do not affect K(+) channels. 3. The blocking effect is enhanced as the depolarizing pulse becomes larger. Blocked channels are unable to shift to the inactivated state. 4. In the case of cyclam and guanidyl-side armed cyclam (G-cyclam), quick release of these chemicals from the binding sites is proven by the increase in the tail current and prolongation of the time course of the off gating current. On the other hand, in the presence of N-4 and the spider toxins, their detachment was delayed significantly. 5. Molecular requirements for the block of Na(+) channels by these molecules are the presence of positive charge and hydrophobicity.

Algorithms↗
PubMed2003

Phoneutria nigriventer spider venom activates 5-HT4 receptors in rat-isolated vagus nerve.

1. The venom of Phoneutria nigriventer spider (PNV) causes intense pain and inflammation following an attack. We have investigated the involvement of capsaicin-sensitive nerve fibres by utilizing an in vitro nerve preparation. Extracellular DC potential recordings were made from the rat-isolated vagus nerve, a preparation that is rich in capsaicin-sensitive, that is, nociceptive, C-fibres. 2. PNV (1-10 microg ml(-1)), capsaicin (0.03-0.3 microM) or 5-hydroxytriptamine (5-HT) (0.3-3 microM) induced dose-dependent depolarizations of vagus nerve fibres. Depolarizing responses to capsaicin were blocked by ruthenium red (RR, 10 microM), but responses to PNV were not. Depolarizing responses to PNV or veratridine (50 microM) were inhibited by tetrodotoxin (TTX, 10 microM), but those to capsaicin were not. This suggests that capsaicin and PNV depolarize the nerve fibres by distinct mechanisms. 3. Depolarization in response to 5-HT (3 microM) was reduced by the 5-HT(3) receptor antagonists Y25130 (0.5 micro M) and tropisetron (10 nM) or, to a lesser extent, by the 5-HT(4) receptor antagonist RS39604 (1 or 10 microM). Depolarizing responses to PNV were not affected significantly by Y25130 or tropisetron, but were blocked by RS39604. 4. These data show that 5-HT(4) receptors play a significant role in the activation of nociceptive sensory nerve fibres by PNV and suggest that this is of importance in the development of the pain and inflammation associated with bites from the P. nigriventer spider.

Action Potentials↗
PubMed2001

Agrobacterium tumefaciens-mediated transformation of rice with the spider insecticidal gene conferring resistance to leaffolder and striped stem borer.

Immature embryos of rice varieties "Xiushuill" and "Chunjiang 11" precultured for 4d were infected and transformed by Agrobacterium tumefaciens strain EHA101/pExT7 (containing the spider insecticidal gene). The resistant calli were transferred onto the differentiation medium and plants were regenerated. The transformation frequency reached 56% approximately 72% measured as numbers of Geneticin (G418)-resistant calli produced and 36% approximately 60% measured as numbers of transgenic plants regenerated, respectively. PCR and Southern blot analysis of transgenic plants confirmed that the T-DNA had been integrated into the rice genome. Insect bioassays using T1 transgenic plants indicated that the mortality of the leaffolder (Cnaphalocrasis medinalis) after 7d of leaf feeding reached 38% approximately 61% and the corrected mortality of the striped stem borer (Chilo suppressalis) after 7d of leaf feeding reached 16% approximately 75%. The insect bioassay results demonstrated that the transgenic plants expressing the spider insecticidal protein conferred enhanced resistance to these pests.

Agrobacterium tumefaciens↗
PubMed1988

Amino acid sequence of versutoxin, a lethal neurotoxin from the venom of the funnel-web spider Atrax versutus.

The complete amino acid sequence of versutoxin, a lethal neurotoxic polypeptide isolated from the venom of male and female funnel-web spiders of the species Atrax versutus, was determined. Sequencing was performed in a gas-phase protein sequencer by automated Edman degradation of the S-carboxymethylated toxin and fragments of it produced by reaction with CNBr. Versutoxin consisted of a single chain of 42 amino acid residues. It was found to have a high proportion of basic residues and of cystine. The primary structure showed marked homology with that of robustoxin, a novel neurotoxin recently isolated from the venom of another funnel-web-spider species, Atrax robustus.

Amino Acid Sequence↗