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[Cation-anion selectivity and conductivity of the channels formed by black widow spider whole venom in lipid bilayer].

It is established that BWSV (black widow spider venom) is capable of forming ion channels in lipid bilayer (BPhM). This effect is minimal upon pH value close to 5.0 and is increased either upon oxidation or alkalization of the medium. The amplitudes of the channels are monotonously decreased upon the reduction of pH with simultaneous narrowing of their dispersion. The degree of the change of BPhM conductivity by concentration of BWSV is close to 2 at different pH. Apparently the absorption of the active component of venom on bilayer surface is accompanied by doubling of the degree of its aggregation. Almost ideal cation selectivity of BWSV channels (pH 7.5) is significantly decreased upon the increase in ionic strength. Ionic channel is thought to be a zone of interaction of lipid domains. Possible structure of such a channel is discussed.

Animals↗

Ca2+-dependence of 3H-catecholamines release evoked by black widow spider venom gland extract in rat hypothalamus, occipital cortex and caudate nucleus.

The release of 3H-catecholamines evoked by Black Widow spider venom gland extract (BW-GE) has been studied in the isolated rat hypothalamus (HT), occipital cortex (OC), preloaded with 3H-noradrenaline, and isolated caudate nucleus (CN) preloaded with 3H-dopamine. The BWGE at a concentration of 0.04 gland/ml increased significantly 3H-output in isolated slices of rat HT, OC and CN. This effect was markedly depressed when control calcium concentration in the medium (1.68 mM) was reduced (0-0.56 mM) or enhanced (3 mM), as well as in the presence of an organic Ca2+ antagonist, verapamil (10 microM), or ionophore A 23187 (40 microM), a substance that increases the influx of calcium into the cell. Morphine (up to 0.4 mM) evoked no effect upon 3H-noradrenaline release induced by BWGE. Morphine (10 microM), but not ionophore A 23187 or high Ca2+ (3 mM), reduced 3H-noradrenaline release induced by 20 mM K+. Low Ca2+ and verapamil produced similar effects than those observed for BWGE. Our results demonstrate differences between BWGE and potassium stimuli, and indicate that BWGE releases 3H-catecholamines by a calcium dependent process.

Animals↗

Wandering spider bite--much ado about nothing.

The wandering spider, Palystes natalius, has been regarded as medically important since 1959. Simple experiments recently conducted suggest that serious misinterpretations of an observed result were responsible for this misconception.

Animals↗

Isolation and sequencing of insecticidal peptides from the primitive hunting spider, Plectreurys tristis (Simon).

About 50 peptide toxins were purified from venom of the primitive hunting spider, Plectreurys tristis. Bioassay by injection into larval Heliothis virescens (tobacco budworm) allowed selection of nine toxins for analysis of amino acid sequences. Total sequences were determined for six of the more insecticidal peptides (46-49 amino acids) and three contained free carboxyl-terminal amino acids. These toxins (plectoxins) are paralytic and/or lethal when injected into insect pests such as the lepidopterous larvae H. virescens, Spodoptera exigua (beet armyworm), and Manduca sexta (tobacco hornworm). We expect these plectoxins to be useful in insect control after delivery by a suitable agent such as a recombinant baculovirus.

Amino Acid Sequence↗

Sequence of the cDNA coding for the lethal neurotoxin Tx1 from the Brazilian "armed" spider Phoneutria nigriventer predicts the synthesis and processing of a preprotoxin.

A cDNA library was constructed from the venom glands of the Brazilian "armed" spider, Phoneutria nigriventer, and a clone coding for Tx1, a lethal toxin, was identified and sequenced. The sequence data derived from this cDNA clone combined with the previously determined amino acid sequence predict that Tx1 is initially synthesized as a preprotoxin. Four segments (comprising the signal sequence, a short, 15-amino acid, glutamate-rich sequence, the functional toxin, and 2 glycine residues) can be distinguished. The structure of the preprotoxin and the proposed processing steps required to form the mature Tx1 toxin show similarities with the synthesis and processing of omega-agatoxin IA.

Amino Acid Sequence↗

The release of acetylcholine elicited by extracts of black widow spider glands: studies using rat superior cervical ganglia and inhibitors of electrically stimulated release.

An extract of the venom glands of black widow spiders (BWGE) induces the release of acetylcholine (ACh) from the superior cervical ganglia of rats. The release of ACh follows first-order kinetics, which suggests that the venom either lowers the ganglionic store of ACh, or continually reduces the rate of release. Since ganglionic ACh did not decrease in the presence of BWGE, it is likely that the venom continually reduces the rate of release. The rate constant for BWGE-induced release of ACh is depressed about 45% by treatment of the ganglion with either botulinum toxin or a low Ca++/high Mg++Ringer's solution. The rate constant is depressed about 30% by treatment of the ganglion with 8.3 microng/ml of cytochalasin-B. Since these agents inhibit the release of ACh which is elicited by electrical stimulation of the ganglion, it is suggested that one action of BWGE is the stimulation of some step in the physiological mechanism involved in the release of neurotransmitters. Treatment of the ganglion with tetrodotoxin had no effect on the rate constant for release induced by BWGE. The action of BWGE on the ganglion was not reversible after 10 minutes. When treated with BWGE, ganglia whose stores of transmitter had been labeled by electrical stimulation in the presence of [3H]choline released ACh having uniform specific activity. The data suggest the presence of more than one activity in the extracts.

Acetylcholine↗

Identification and neutralization of biological activities in the venoms of Loxosceles spiders.

The biological activities of the venom of three species of spiders of the genus Loxosceles were studied (L. gaucho, L. laeta and L. intermedia). The dermonecrotic and lethal activities are shared by all three Loxosceles venoms. Only low levels of proteolytic, myotoxic and phospholipase A2 activities were demonstrable even when a large amount of venom was used. No direct hemolytic activity was detected. L. intermedia venom was the most lethal (LD50 0.48 mg/kg), the L. laeta venom was the least lethal (LD50 1.45 mg/kg) whereas L. gaucho venom showed an intermediate value (LD50 0.74 mg/kg). The anti-Loxosceles serum used (anti-arachnidic serum) was able to neutralize the most important activities (i.e., dermonecrotic and lethal activities) of the three venoms. SDS-PAGE and immunoblotting using the anti-arachnidic serum showed that almost all venom antigens were recognized by this antiserum. The possible mechanisms of action of the Loxosceles venom are discussed.

Animals↗

Abdominal-B expression in a spider suggests a general role for Abdominal-B in specifying the genital structure.

We have cloned an Abdominal-B (Abd-B) orthologue from the spider Cupiennius salei and have analysed its expression pattern during embryogenesis. An early expression domain is seen in the posterior part of the embryo, with an initial border in the third opisthosomal segment and later in the fifth opisthosomal segment. During mid-stage of germ band extension, two additional spots of expression appear in the posterior parts of the limb buds on the second opisthosomal segment. These coincide with the position of the future genital opening and Cs-Abd-B remains expressed in these regions until the openings are formed. In view of the fact that Abd-B and its orthologous genes are also required for specifying the genitalia in Drosophila and vertebrates, we suggest that this function may constitute an independent and ancestral role of Abd-B that can be separated from its role in specifying the posterior part of the body region.

Amino Acid Sequence↗

Non-periodic lattice crystals in the hierarchical microstructure of spider (major ampullate) silk.

A commonly adopted model for the microstructure of Nephila clavipes major ampullate silk (MAS) is similar to that used for Bombyx mori (silkworm) silk: a simple composite wherein discrete, essentially perfect crystals are dispersed throughout an amorphous protein matrix. However, inconsistencies arise when researchers using complementary microstructural characterisation techniques attempt to explain their results within that framework. We present here the findings of our parallel studies in x-ray diffraction, electron microscopy, and molecular modeling. These results, combined with other data gleaned from the literature, lead us to propose a revised description of the spider silk microstructure. The new model recognizes that the 70-500 nm sized ordered regions in MAS cannot be constructed from a simple motif of repeating monomers, and develops the concept of non-periodic lattice (NPL) crystals to characterize these structures. The local composition, symmetry, and perfection of order vary over distances that are small compared to the size of an NPL crystal.

Animals↗

Structural studies of spider silk proteins in the fiber.

Although spider silk has been studied for a number of years the structures of the proteins involved have yet to be definitely determined. X-ray diffraction and solid-state nuclear magnetic resonance (NMR) were used to study major ampullate (dragline) silk from Nephila clavipes. The silk was studied in its natural state, in the supercontracted state and in the restretched state following supercontraction. The natural silk structure is dominated by beta-sheets aligned parallel to the fiber axis. Supercontraction is characterized by randomizing of the orientation of the beta-sheet. When the fiber is restretched alignment is regained. However, the same reorientation was observed for wetting of minor ampullate silk which does not supercontract. Thus, the reorientation of beta-sheets alone cannot explain the supercontraction in dragline silk. Cocoon silk showed very little beta-sheet orientation in the natural state and there were no changes upon wetting. NMR and X-ray diffraction data are consistent with the beta-sheets arising from the poly-alanine sequences known to be present in the proteins of major ampullate silk as has been proposed previously.

Amino Acid Sequence↗

Long-term potentiation in the presence of NMDA receptor antagonist arylalkylamine spider toxins.

The role of the NMDA receptor (NMDAR) in long-term potentiation (LTP) is now well established. All potent NMDAR antagonists known to date inhibit the induction of LTP at the Schaffer collateral-CA1 pyramidal cell synapse in rat hippocampus, regardless of their site and mechanism of action. Arylalkylamine toxins are noncompetitive NMDAR antagonists in the mammalian central nervous system (CNS). The synthetic toxins argiotoxin-636 (Arg-636), Joro spider toxin (JSTX-3), alpha-agatoxin-489 and -505 (Agel-489 and Agel-505) and philanthotoxin-433 (delta-PhTX) were found in the present study to have no effect on the induction of LTP in the Schaffer collateral-CA1 pyramidal cell pathway in rat hippocampal slices maintained in vitro. Arylalkylamine toxins represent a class of potent NMDAR antagonists that fail to affect hippocampal LTP, and thus provide novel structural leads for the development of NMDAR antagonists that do not impair cognition.

Animals↗

Studies on oogenesis and oviposition in the brown spider Loxosceles intermedia (Araneae: Sicariidae).

Loxosceles intermedia is a poisonous spider that has a wide distribution in southern Brazil, and constitutes a public health problem. In this study, the ovaries of mature females were examined by light and electron microscopy, and by histochemistry. Oocytes at all stages of development were observed in the same area of the mature ovarian wall, surrounded by a basement membrane and a proteic band. They were supported by a group of pedicular cells, which may function as follicular cells. No follicular cell was seen around the oocyte. Mature oocytes pass through the ovarian epithelial wall and are released into the ovarian lumen, covered by a vitelline membrane. The basement membrane and proteic band remain in the ovarian wall. On its way out, the oocyte is coated by proteins that will form the chorion. The presence of different coats throughout oogenesis, and at the time of egg release, is correlated with conditions that indicate fertilization occurs in the uterus lumen during oviposition.

Animals↗

Orientational order of Australian spider silks as determined by solid-state NMR.

A simple solid-state NMR method was used to study the structure of (13)C- and (15)N-enriched silk from two Australian orb-web spider species, Nephila edulis and Argiope keyserlingi. Carbon-13 and (15)N spectra from alanine- or glycine-labeled oriented dragline silks were acquired with the fiber axis aligned parallel or perpendicular to the magnetic field. The fraction of oriented component was determined from each amino acid, alanine and glycine, using each nucleus independently, and attributed to the ordered crystalline domains in the silk. The relative fraction of ordered alanine was found to be higher than the fraction of ordered glycine, akin to the observation of alanine-rich domains in silk-worm (Bombyx mori) silk. A higher degree of crystallinity was observed in the dragline silk of N. edulis compared with A. keyserlingi, which correlates with the superior mechanical properties of the former.

Animals↗

Upgraded expression of 5S rRNA preludes the production of fibroin by spider glands.

We have developed the large ampullate glands of the orb-web spider Nephila clavipes as a model system in which to study the production of a tissue-specific secretory protein. Through simple manipulations, the glands' fibroin production can be practically abolished and subsequently elicited into high levels of synthesis through a mechanical stimulus applied to the organism. The tissue specific responses evoked by the stimulus can be monitored through time-course studies. The latter have revealed an orchestrated series of tissue and time specific macromolecular syntheses, which optimize the glandular tissues with components of the protein synthesis machinery. This work shows the upgraded accumulation of 5S rRNA in the glands as response to the stimulus within the earliest of the prelude events. Further enquiries on this accumulation must be conducted at the level of differential gene expressions, a chore we have initiated. A DNA fragment containing a single copy 5S rRNA gene has been isolated, cloned, sequenced, and transcribed in a cell-free system. We enclose a discussion on the similarity between the genomic organization of this gene to that of a 5S rRNA gene of Bombyx mori. Our studies have revealed a considerable number of similarities in the silk production strategies of Nephila clavipes and the silkworm Bombyx mori, some of them rather unusual.

Animals↗

Ultrastructural observations of spermatozoa of several tetragnathid spiders with phylogenetic implications (Araneae, Tetragnathidae).

The present study reports on the ultrastructure of spermatozoa and spermatogenesis of 12 tetragnathid spiders (10 Tetragnatha species [T. boydi, T. dearmata, T. extensa, T. montana, T. nigrita, T. obtusa, T. pinicola, T. reimoseri, T. shoshone, T. striata]; Pachygnatha listeri and Metellina segmentata). All species develop typical cleistospermia with a coiled nucleus in the center and a coiled axoneme in the periphery of the cell. Remarkable differences in the sperm ultrastructure of the investigated species comprise the shape of the main sperm cell components (nucleus, acrosomal complex, implantation fossa, and centriolar complex). Within the observed Tetragnatha species, three types of sperms were characterized: T. montana-type, T. boydi-type, and T. striata-type. The highly derivative T. montana-type is characterized by the following remarkable features: an extremely elongated nucleus, shaped like a corkscrew and twisted around the axoneme (before coiling); a deep implantation fossa; a corkscrew-shaped acrosomal vacuole; after the coiling process, the nucleus is coiled five to six times in the center of the spermatozoon and the axoneme is coiled five to six times peripheral to the nucleus. The T. boydi-type hardly differs from the T. montana-type, but is remarkable due to the triangular-shaped nucleus (in cross section). The T. striata-type differs especially by a peculiar acrosomal vacuole with a long, slightly curved process and a short appendix, as well as a nucleus that describes only three loose coils around the axoneme (before coiling). The spermatozoa of Pachygnatha listeri and especially Metellina segmentata differ strikingly from the described Tetragnatha-types and are similar to more primitive araneomorph spermatozoa, such as Hypochilus pococki. The described Tetragnatha-types completely correspond with Okuma's (1988a,b, J Fac Agr Kyushu U 32:165-181, 32:183-213) classification of Tetragnatha species. Furthermore, our results suggest an early derivative systematic position of Pachygnatha within Tetragnathinae and the position of Metellina within the Tetragnathidae.

Acrosome↗

Evolutionary transformation from muscular to hydraulic movements in spider (Arachnida, Araneae) genitalia: a study based on histological serial sections.

The male genitalia of 107 spider species representing 73 families were serially sectioned and studied with an emphasis on muscles moving the genital bulb. As a rule, most non-Entelegynae have two bulbal muscles, most Entelegyne have none, but many exceptions occur. Variation also occurs with regard to origin and attachment of bulbal muscles. There appears to be a trend towards a shift of the origin from proximal (Liphistius, Atypus) to more distal palpal segments (Haplogynae). In most Entelegynae the muscular movement is replaced by hydraulic movement caused by expanding membranes (hematodochae). Hematodochae probably permit increased bulbal rotation and movements of higher complexity. New evidence is presented arguing against Palpimanidae being representatives of Entelegynae. Bulbal glands other than those discharging into the sperm duct (previously known in Amaurobiidae and Dictynidae only) are described in several entelegyne families.

Animals↗

Genome size and endonuclear DNA replication in spiders.

Although genome sizes (C-values) are now available for 115 arachnid species (Gregory and Shorthouse [2003] J Hered 94:285-290), the extent of genome amplification (endonuclear DNA replication or polyploidization) accompanying tissue differentiation in this diverse and abundant class of invertebrates remains unknown. To explore this aspect of arachnid development, samples of hemolymph and other tissues were taken from wild-caught specimens as air-dried smears, stained with the Feulgen reaction for DNA, and assayed using both scanning and image analysis densitometry. Cells from midgut diverticula and Malpighian tubules of Argiope and Lycosa (=Pardosa) often showed giant nuclei with 50-100 pg of DNA per nucleus, reflecting at least four cycles of endonuclear DNA replication when compared to the DNA content of hemocytes or sperm from the same specimen. Nuclei with markedly elevated DNA levels also appeared, but far less frequently, in tissue samples from several other arachnid species (Antrodiaetus, Hypochilus, Latrodectus, Liphistus and Loxosceles), but revealed no correlation with differences in somatic cell (2C) genome sizes. Our data show that several DNA classes of polysomatic nuclei regularly arise during tissue differentiation in some species of spiders and may provide an interesting model system for further study of patterns of tissue-specific variation in DNA endoreduplication during development.

Animals↗