A lysine rich C-terminal tail is directly involved in the toxicity of CSTX-1, a neurotoxic peptide from the venom of the spider Cupiennius salei.

CSTX-1 (74 amino acids, 8,352.62 Da) is a potent neurotoxin from the venom of Cupiennius salei. With the monoclonal antibody 9H3 against CSTX-1, we identified two similar peptides by Western blot analysis. These two peptides were purified by RP-HPLC: CSTX-2a (61 amino acids, 6865.75 Da) and CSTX-2b (60 amino acids, 6709.57 Da). Using ESI-MS analysis and sequencing we verified that CSTX-2a is a truncated version of CSTX-1. CSTX-2b differs from CSTX-2a by the absence of Arg61. Toxicity of CSTX-1, CSTX-2a, and CSTX-2b to Drosophila melanogaster showed that the absence of the last 13 amino acids of CSTX-1 results in a seven-fold activity loss. CSTX-2b, which lacks Arg61 is 190-fold less toxic. We conclude that the C-terminal part of CSTX-1, especially Arg61, is essential for the expression of toxicity. CSTX-1 is degraded to CSTX-2a and CSTX-2b by proteases that are released from venom gland cells by apocrine secretion.

Immunocytochemical localization and secretion process of the toxin CSTX-1 in the venom gland of the wandering spider Cupiennius salei (Araneae: Ctenidae).

Fluorescein and horseradish peroxidase-labeled monoclonal antibodies were used to localize the predominant toxic peptide CSTX-1 in the venom gland of the spider Cupiennius salei. There was no polarity of CSTX-1 expression in repleted glands, whereas the glands of previously milked spiders showed a decreasing immunofluorescent response from the distal to the proximal portion. Detailed investigation revealed a new structure in the venom-secreting epithelium, which is postulated to be an evolutionary adaptation to increasing gland volume. CSTX-1 was found to be synthesized and stored as a fully active toxin within complex units, composed of long interdigitating cells running perpendicular to the muscular sheath and extending into the central lumen of the gland. These venom-producing units were found in all sectors of the gland, including the transitional region between the main gland and the venom duct. The venom is liberated from the venom-producing units into the glandular lumen following the contraction of the surrounding muscle layer. Free nuclei or other cellular fragments, which would have provided evidence for a holocrine secretion process, were not found in the glandular lumen or in the crude venom obtained by electrical stimulation. The fine regulation of the spider's venom injection process is postulated to be the function of the bulbous ampulla, situated in the anterior third of the venom duct.

Thymidylate synthase inhibitors.

Thymidylate synthase (TS) is a critical enzyme for DNA replication and cell growth because it is the only de novo source of thymine nucleotide precursors for DNA synthesis. TS is the primary target of 5-fluorouracil (5-FU), which has been used for cancer treatment for more than 40 years. However, dissatisfaction with the overall activity of 5-FU against the major cancers, and the recognition that TS still remains an attractive target for anticancer drugs because of its central position in the pathway of DNA synthesis, led to a search for new inhibitors of TS structurally analogous to 5,10-methylenetetrahydrofolate, the second substrate of TS. TS inhibitory antifolates developed to date that are in various stages of clinical evaluation are ZD 1694 and ZD9331 (Astra-Zeneca, London, UK), (Eli Lilly, Indianapolis, IN), LY231514 (BW1843U89 (Glaxo-Wellcome, Research Triangle Park, NC), and AG337 and AG331 (Agouron, La Jolla, CA). Although each of these compounds has TS as its major intracellular site of action, they differ in propensity for polyglutamylation and for transport by the reduced folate carrier. LY231514 also has secondary target enzymes. As a result, each compound is likely to have a different spectrum of antitumor activity and toxicity. This review will summarize the development and properties of this new class of TS inhibitors.

Effects of size, motility and paralysation time of prey on the quantity of venom injected by the hunting spider Cupiennius salei.

Previous experimental studies have shown that neotropical wandering spiders (Cupiennius salei) inject more venom when attacking larger crickets. It has been postulated that this is a consequence of predator-prey interactions during envenomation, which increase in intensity with the size of a given prey species. The present study was designed to test this hypothesis using anaesthetized crickets of different sizes that were moved artificially. Cupiennius salei was found (1) to inject more venom the greater the intensity of the struggling movement of the crickets (prey size kept constant); (2) to inject more venom the longer the duration of the struggling movement of the crickets (prey size and intensity of movement kept constant); and (3) to inject equal amounts into crickets of different size (duration and intensity of movement kept constant). These results indicate that C. salei alters the amount of venom it releases according to the size and motility of its prey. Venom expenditure depends mainly on the extent of the interactions with the prey during the envenomation process, whereas prey size is of minor significance. The regulation of venom injection in concert with behavioural adaptations in response to various types of prey minimizes the energetic cost of venom production, thus increasing the profitability of a given prey item.

Quantifying the venom dose of the spider Cupiennius salei using monoclonal antibodies.

The variation in venom dose with prey size of the neotropical wandering spider Cupieinnius salei was examined experimentally. Monoclonal antibodies were raised against the venom toxins of C. salei. Mab 9H3, recognizing the main toxin CSTX-1, was used to quantify the venom by enzyme-linked immunosorbent assay (ELISA). Crickets (Achta domesticus) in four size classes were randomly offered to sixteen mature female spiders at 14d intervals. The prey items were removed from spiders five minutes after the initial bite and subsequently homogenized for ELISA measurements. The quantity of venom expended was significantly related to the size of prey, ranging from 0.15 microl for the smallest (100 110 mg) to 1.53 microl for the largest (600-660 mg) crickets. Adaptations to prey size were also reflected in capturing behavior. None of the smallest, but almost 50% of the largest crickets were wrapped in silk following the spiders bite. Some other behavioral features may reduce the energetic costs of venom production. In 22% of the smallest crickets no venom was detectable, with the majority showing mechanical damage as a result of fang contact. This indicates. that C. salei does not rely exclusively on its venom when feeding on small prey. Some other aspects such as the site of the bite and the speed of paralyzation and their consequences associated with the amount of venom expended are discussed.