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J P Purcell

Publications and source records attributed to J P Purcell.

10 recordsLinked to original sources
PubMed2001

Expression and chloroplast targeting of cholesterol oxidase in transgenic tobacco plants.

Cholesterol oxidase represents a novel type of insecticidal protein with potent activity against the cotton boll weevil (Anthonomus grandis grandis Boheman). We transformed tobacco (Nicotiana tabacum) plants with the cholesterol oxidase choM gene and expressed cytosolic and chloroplast-targeted versions of the ChoM protein. Transgenic leaf tissues expressing cholesterol oxidase exerted insecticidal activity against boll weevil larvae. Our results indicate that cholesterol oxidase can metabolize phytosterols in vivo when produced cytosolically or when targeted to chloroplasts. The transgenic plants exhibiting cytosolic expression accumulated low levels of saturated sterols known as stanols, and displayed severe developmental aberrations. In contrast, the transgenic plants expressing chloroplast-targeted cholesterol oxidase maintained a greater accumulation of stanols, and appeared phenotypically and developmentally normal. These results are discussed within the context of plant sterol distribution and metabolism.

Actinomyces↗
PubMed1994

Cloning of an insecticidal cholesterol oxidase gene and its expression in bacteria and in plant protoplasts.

We cloned and sequenced structural gene choM, which encodes an insecticidally active cholesterol oxidase in Streptomyces sp. strain A19249. The primary translation product was predicted to be a 547-amino-acid protein whose first 43 amino acids constitute a secretory signal peptide. Expression of the gene with the signal sequence in Escherichia coli resulted in production of a protein that had enzymatic and insecticidal properties which were indistinguishable from those of the cholesterol oxidase secreted by Streptomyces sp. strain A19249. Expression of the gene with or without the signal sequence in tobacco protoplasts resulted in production of an enzymatically active cholesterol oxidase.

Amino Acid Sequence↗
PubMed1993

Cholesterol oxidase: a potent insecticidal protein active against boll weevil larvae.

The discovery of proteins that control insects is critical for the continued growth of the agricultural biotechnology industry. A highly efficacious protein that killed boll weevil (Anthonomus grandis grandis Boheman) larvae was discovered in Streptomyces culture filtrates. The protein was identified as cholesterol oxidase (E.C. 1.1.3.6). Purified cholesterol oxidase was active against boll weevil larvae at a concentration (LC50 = 20.9 micrograms/ml) comparable to the bioactivity of Bacillus thuringiensis proteins against other insect pests. Histological studies demonstrated that cholesterol oxidase lysed the boll weevil midgut epithelium, suggesting that this is the primary mechanism of lethality.

Amino Acid Sequence↗
PubMed1990

Biochemical characterization of cuticle polypeptides from the infective larvae of Haemonchus contortus.

1. Ecdysis of infective Haemonchus contortus larvae is effected by the enzymatic degradation of a specialized region of the second molt cuticle containing a biochemically unique polypeptide (mol. wt = 160,000). 2. The 160,000 mol. wt polypeptide and related polypeptides are synthesized at approximately 6 days of larval development. Antigenically similar polypeptides occur in other ruminant trichostrongyles. 3. Cuticle polypeptides digested during ecdysis differ from second molt cuticle collagens in amino acid composition and collagenase sensitivity. However, some antigenic homology between the 160,000 mol. wt polypeptide and cuticle collagens suggests structurally similar regions.

Aging↗
PubMed1990

Scanning electron microscopy of the sheathed infective larva and parasitic third-stage larva of Haemonchus contortus (Nematoda: Trichostrongyloidea).

Scanning electron microscopy was used to describe the infective and parasitic third-stage larvae of Haemonchus contortus, the large stomach worm of ruminants. Infective larvae are ensheathed in the cuticle of the second stage, so the descriptions are of the second- and third-stage cuticles. Both larval stages had an inner circle of 6 labial papillae, an outer circle of 6 labial papillae and 4 somatic papillae, and lateral amphidial pits. Infective larvae (cuticle of the second stage) had the 6 internal labial papillae on prominent bluntly rectangular lappets in a star-shaped arrangement around a large triradiate mouth, small triangular or round amphidial pits, flattened ribbonlike lateral alae, and phasmidial apertures opening on the ventral surface of the lateral alae. Parasitic third-stage larvae had the 6 internal labial papillae on small elevations without lappets around a small mouth; large, oval amphidial pits; ribbonlike lateral alae for most of their length, but with the anterior 30-40 microns of the alae cordlike; and phasmidial apertures on the body cuticle ventral to the lateral alae.

Animals↗
PubMed1989

Purification of a 44 kilodalton protease which mediates the ecdysis of infective Haemonchus contortus larvae.

We have characterized and purified a parasite protease which mediates the ecdysis of Haemonchus contortus, and at least several other ruminant trichostrongyles. The protease, with an apparent approximate molecular weight of 44,000, is a zinc metalloprotein which hydrolyzes several large protein substrates in in vitro assays. In vivo and in biological assays on isolated second molt cuticles this protease hydrolyzes a specific circular region of the second stage cuticle which results in removal of a cuticular cap, providing a rapid and synchronous method for the escape of infective larvae during the transition from free-living to parasitic environments.

Animals↗
PubMed1989

Light and scanning electron microscopy of the ecdysis of Haemonchus contortus infective larvae.

During the second ecdysis of ruminant trichostrongyles, a region of the second molt cuticle is digested by a 44-kDa Zn-metalloprotease. We have examined this digestion process by light and scanning electron microscopy (SEM). The substrate region of the cuticle appeared, during the ecdysis process, as an indented ring at the 20th cuticular annulus coincident with the anterior terminus of the lateral alae. Continued digestion of the cuticle resulted in holes in the ring region that expanded until they became continuous and separation occurred between the anterior and posterior portions of the cuticle. Mechanical movements of the L3 forced aside the cuticle cap that generally remained attached on one side to the posterior portion as the larva escaped from the sheath. The site of secretion of the 44-kDa ecdysing enzyme causing cuticle digestion was not clear from morphological observations; however, existing evidence strongly points to the release of enzyme from the esophageal (pharyngeal) glands through the mouth.

Animals↗