Molecular cloning and sequencing of cDNAs encoding insecticidal peptides from the primitive hunting spider, Plectreurys tristis (Simon).

Plectreurys tristis cephalothorax mRNA was isolated and amplified by PCR using degenerate primers corresponding to reverse translated mature Plt-VI toxin. An oligonucleotide corresponding to a portion of the amplified product was then used to screen a P. tristis cDNA library. The cDNAs from 10 positive clones were sequenced. Eight of these cDNAs corresponded to Plt-VI toxin, one to Plt-XI toxin, and one was very similar to Plt-VIII toxin, with the exception of a single amino acid substitution. Analysis of these cDNAs indicated that these toxins are initially synthesized as prepro-forms which undergo signal cleavage followed by additional processing at both their N- and C-termini to produce the mature products.

Characterization of the precursor for Manduca sexta diuretic hormone Mas-DH.

We have isolated a cDNA clone encoding a precursor form of the diuretic hormone from the tobacco hornworm Manduca sexta (Mas-DH). Translation of the cDNA revealed a 138-amino acid precursor consisting of the Mas-DH amino acid sequence bounded by dibasic amino acid processing sites, a putative signal sequence, and additional peptide sequence on either side of the Mas-DH coding sequence. The region of the precursor upstream of the mature Mas-DH sequence shows limited (28%) homology to the cryptic region of the ovine corticotropin-releasing factor precursor. The Mas-DH RNA is 1.5-1.6 kilobases long; it is present in both the heads and bodies of adult and larval insects. In prewandering fifth stadium larvae, Mas-DH mRNA is expressed in brain, nerve cord, gut, and Malpighian tubules, but not in the fat body. There is a single genomic copy of the Mas-DH gene; the message is multiply spliced.

Purification and characterization of two classes of neurotoxins from the funnel web spider, Agelenopsis aperta.

Two classes of paralytic toxins were isolated from the venom of Agelenopsis aperta and their chemical and larvicidal properties characterized. Five acylpolyamine toxins (alpha-agatoxins) of molecular masses 452, 488, 489, 504, and 505 Da produce immediate but reversible paralysis in Manduca sexta following injection. Six insecticidal peptides (mu-agatoxins) produce a gradual but irreversible paralysis. The complete amino acid sequences (36-38 residues) of the mu-agatoxins are presented. These peptides contain eight half-cystines and are quite similar in sequence. At least four of these toxins are amidated at the carboxyl terminus. The secondary structure of one of these toxins (mu-Aga V) was investigated.

Structures and biological activities of three synaptic antagonists from orb weaver spider venom.

The venom of Argiope aurantia, an orb weaver spider, contains a mixture of low molecular weight "argiotoxins", which block neuromuscular transmission in insects. Complete structure elucidation of three argiotoxins reveals common features; a hydrophilic, basic domain of arginine, a polyamine and asparagine is connected to an aromatic moiety contributed either by 4-hydroxyindole-3-acetic acid or 2,4-dihydroxyphenylacetic acid. Structural assignments of two argiotoxins are verified by chemical synthesis. The argiotoxins cause reversible paralysis when injected into insects and this is correlated with a stimulus-dependent inhibition of skeletal neuromuscular transmission at submicromolar concentrations.

Intestinal degradation and absorption of the glycosidic sweeteners stevioside and rebaudioside A.

Contrary to prior indications, the glycosidic sweeteners stevioside and rebaudioside A are degraded to the diterpenoid aglycone steviol by rat intestinal microflora in vitro. Additional studies with steviol-17-[14C] show almost total absorption from the rat lower bowel following intracecal administration.

Intestinal absorption of polymeric derivatives of the food dyes sunset yellow and tartrazine in rats.

1. Absorption and metabolism of 14C-labelled sunset yellow (FD & C Yellow No. 6), tartrazine (FD & C Yellow No. 5) and high molecular weight polymeric derivatives of the two azo dyes were compared in rats. 2. A trace to 1.5 percent of unchanged monomeric dyes was excreted in urine and bile during the first 24 h after dosing. No unchanged dye was absorbed after administration of the polymeric derivatives. 3. In animals dosed with sunset yellow and its polymer derivative, absorption of the azo-bound cleavage product 1-amino-2-naphthol-6-sulphonic acid was 8.5 and 6.9 percent, respectively, while absorption of the cleavage product sulphanilic acid was 37.4 and 0 percent, respectively. 4. In animals dosed with tartrazine and its polymer derivative, absorption of the cleavage product aminopyrazolone and its metabolites was 4.0 and 4.6 percent, respectively. 5. Azo bond cleavage did not appear to be decreased in the polymer derivatives. However, the sulphanilic acid moiety of both dyes remained attached to the polymer backbone, resulting in a 95 percent decrease in sulphanilic acid absorption with polymeric tartrazine. 6. Decreased absorption of unchanged dyes and certain metabolites with the stable, non-absorbed polymeric derivatives may be significant in developing non-sensitizing substitutes for these two commonly used food colourants.

Long term bile collection in the rat.

Bile was collected from the rat continuously, over an extended period, using an externalized cannula brought through an intrascapular incision. The cannula was protected by a 30-cm, 16-gauge stainless steel tube attached to the skin through the incision. The rat was housed in a 10.2 x 10.2 x 12.7 cm cage at a lever higher than the collection vial to aid in drainage of bile. A hole in the vial cap permitted the cannula to turn freely as the rat moved about the cage. This method allowed bile to be collected conveniently over long periods of time with minimal restraint of the animal.