Isolation and identification of the toxic peptides from Lophyrotoma zonalis (Pergidae) sawfly larvae.

The broad-leaved paper bark tree Melaleuca quinquenervia (Cav) (Myrtaceae) was introduced into Florida (USA) early in this century it has proliferated to such an extent that urgent measures are now required to control it. The sawfly Lophyrotoma zonalis (Pergidae) has been introduced as a possible biological control agent due to its ability to defoliate M. quinquenervia. Because toxic D-amino acid- containing peptides have been isolated from some sawfly species, L. zonalis larvae were processed using the previously reported method for the recovery of these compounds. The toxins lophyrotomin (as the free C-terminal acid) and a mixture of pergidin and Val (4)-pergidin were isolated at 0.36 and 0.43% yield of the dried larvae, respectively. Both compounds when dosed intraperitoneally to C57/Bl6 male mice were hepatotoxic with lowest lethal doses of 8 and 32 mg/kg, respectively. The pathology of the liver was different for each compound, with the lophyrotomin free acid causing a periportal haemorrhagic necrosis and the pergidin causing a periacinar coagulative necrosis.

Unique toxic peptides isolated from sawfly larvae in three continents.

D-Amino acid containing peptides have been found to be responsible for sawfly larvae poisoning in many parts of the world. These compounds, unique in the animal kingdom, were isolated from three different species of sawfly indigenous to Australia, Denmark and South America. The octapeptide, lophyrotomin, is the major toxin in the Australian and Danish species and is present in small amounts in the South American sawfly. Pergidin, the main toxin in the South American sawfly, is a heptapeptide containing a phosphoseryl residue. This, as far as we are aware, is the first example of such a peptide to be isolated from an animal source. Small amounts of pergidin have been found in the other two species. All available evidence suggests that both peptides are biosynthesised 'de novo' possibly as a protective device, however it cannot be excluded that microorganisms may be responsible. These compounds are stable to enzymatic breakdown because of their configuration and their strong chemical bonding and lipophilic character provide a potential for residues to remain in the host animal and cause significant changes.

Structural investigation of resin glycosides from Ipomoea lonchophylla.

A fraction from Ipomoea lonchophylla, which was toxic to mice, contained an inseparable mixture of resin glycosides with differing numbers of C5 ester groups on the hexasaccharide chain. After alkaline hydrolysis of the esters, the structure of the major component (1) was elucidated using high-field NMR spectroscopy, mass spectrometry, chemical studies, and comparison with known resin glycosides. Compound 1 was identified as 3,11-dihydroxytetradecanoic acid 11-O-beta-quinovopyranosyl-(1-->2)-beta-glucopyranosyl-(1-->3)- [alpha-rhamnopyranosyl- (1-->4)]-quinovopyranosyl-(1-->2)-beta-glucopyranosyl-(1-->2)-beta -fucopyranoside.

Isolation and characterisation of urushiol components from the Australian native cashew (Semecarpus australiensis).

Native cashew (Semecarpus australiensis) is a well-known food source for aboriginal people of northeastern Queensland and the Northern Territory. It is also well known that contact with the seeds at a certain stage of growth can cause sever dermatitis in susceptible individuals. To prepare the fruits for eating, they are commonly treated by leaching for 2-7 days with water followed by heating in bark, and this treatment apparently produces an edible cashew nut. Recently, attempts have been made to use this valuable source of food by a commercial company. It was necessary to identify the active principle(s) in the seeds to determine the most effective way of rendering the seed suitable for human consumption without altering the flavour. By using solvent extraction and silica-gel chromatography, a fraction containing one major urushiol was obtained. Its structure was confirmed by comparison of its NMR and mass spectral (MS) data with that previously reported. This compound, which is found in high yield, is also found in poison ivy (Toxiodenron radicans) and is responsible for dermatitis in susceptible individuals. A method of removing the active principle from the seed has been suggested.

Isolation and identification of a compound from avocado (Persea americana) leaves which causes necrosis of the acinar epithelium of the lactating mammary gland and the myocardium.

It is well known that when lactating livestock eat avocado (Persea americana) leaves they may develop non-infectious mastitis and agalactia. This is associated with extensive coagulation necrosis of the secretory acinar epithelium and interstitial oedema, congestion, and haemorrhage. Similar lesions have been produced in mammary glands of lactating mice fed a diet containing a small percentage of freeze-dried avocado leaf. Tests using these animals have been used to isolate the active principle, termed "persin," from avocado leaves. The purified persin was examined using IR, NMR, and UV spectroscopy and mass spectrometry, and identified as (Z,Z)-1-(acetyloxy)-2-hydroxy-12,15-heneicosadien-4-one. Persin has previously been isolated from avocado leaves and shown to have antifungal properties and to be toxic to silkworms. Our tests have shown that persin at the dose rate of 60-100 mg/kg has the same effect on mammary glands in lactating mice as leaves from avocado. Enantioselective syntheses of the R and the S isomers of persin and related derivatives were carried out. These compounds were tested for activity required to induce widespread lactating mammary gland necrosis in mice, and only the R isomer was found active. At doses of persin above 100 mg/kg necrosis of myocardial fibres may occur and hydrothorax may be present in severely affected animals. The mechanism of action of persin on both the mammary gland and the myocardium remain to be resolved.

Isolation of a compound from Eupatorium adenophorum (Spreng.) [Ageratina adenophora (Spreng.)] causing hepatotoxicity in mice.

Regular ingestion of Eupatorium adenophorum [Ageratina adenophora (Spreng.)] or Crofton weed causes chronic pulmonary disease in horses mainly in Australia, New Zealand, and the Himalayas. The disease is characterized by pulmonary interstitial fibrosis, emphysema, alveolar epithelisation and reduced tolerance to exercise. Horses apparently are the only animals affected and there are numerous reports of farms losing all their horses. The disorder was produced experimentally in horse feeding trials, and it was shown that characteristic lesions occurred in the lungs. In studies with laboratory animals, mice were shown to be suitable test animals, but in this species lesions occur in the liver rather than the lungs. The hepatic injury in these animals is characterized by multiple areas of focal necrosis of the parenchyma associated with degeneration and loss of the epithelium lining the small bile ducts. The active principle 9-oxo-10,11 dehydroagerophorone responsible for these lesions in mice has been isolated from E. adenophorum. Although the compound has been shown to exhibit toxicity to larvae of invertebrate species, no mammalian toxicity studies have been previously reported involving the isolated toxin. The mechanism of the toxic effect of the compound as well as its possible relevance to the respiratory disease in the horse remain to be investigated.

Isolation and structure determination of terminalin A toxic condensed tannin from Terminalia oblongata.

Terminalia oblongata (yellow wood) is a small deciduous tree growing over an area of central Queensland that supports a large proportion of this state's cattle population. Cattle and sheep that consume yellow wood leaves are poisoned and die. Severe losses of these animals can occur, and this problem is considered the main cause of economic loss to the cattle industry in the area apart from drought. A new toxic condensed tannin, terminalin was isolated from Terminalia oblongata. Its structure was deduced following NMR, IR, UV, MS analyses and in the knowledge that these data show good correlations to those obtained from the related punicalagin molecule which is present in the plant. Terminalin has a high toxicity (20 mg/kg) to white Quackenbush male mice and produces a vascular renal necrosis with slight liver necrosis, unlike punicalagin, which produces liver lesions but not kidney lesions. Similar results were obtained with sheep. A most interesting aspect is that there are two different specific toxins in the plant.

Identification of the octapeptide lophyrotomin in the European birch sawfly (Arge pullata).

Larvae of the European birch sawfly Arge pullata were shown to contain lophyrotomin, an octapeptide liver toxin containing four D-amino acids. Lophyrotomin was previously isolated from Lophyrotoma interrupta sawfly larvae in Australia.

Cell-free synthesis of the depsipeptide beauvericin.

The enzymatic formation of the cyclodepsipeptide beauvericin was demonstrated in cell-free extracts from Beauveria bassiana. In analogy to the enniatin synthetase system formation of beauvericin is strictly dependent on the presence of the constituent amino and hydroxy acid, S-adenosylmethionine, and ATP/Mg2+. Synthesizing activity could be enriched about 12-fold by fractional ammonium sulfate precipitation. Besides the enniatin synthetase system this represents another example of the cell-free synthesis of a depsipeptide from eucaryotic origin.

Lophyrotomin, a new toxic octapeptide from the larvae of sawfly, Lophyrotoma interrupta.

Lophyrotomin, a new toxic octapeptide, has been isolated from sawfly (Lophyrotoma interrupta) larvae using solvent extraction, dialysis, adsorption on polyamide, ion exchange chromatography on DEAE cellulose and silica gel G chromatography. On the basis of evidence from chemical methods, pmr and mass spectrometry, a partial structure is proposed. Lophyrotomin has an approximate intraperitoneal LD50 of 2 mg/kg for mice.