A Genetic Analysis of Lysosomal Enzyme Activities in Brahman Cattle

Analyses of variance and co variance were carried out on the activities of three lysosomal enzymes in mononuclear blood cells from Brahman cattle. These were hexosaminidase (HEX), beta-D-galacto-sidase (GAL) and acid alpha-glucosidase (GLU) which had been measured in blood mononuclear cells from 1752 cattle from 6 herds in a Pompe's disease control programme. Herd of origin and date of bleeding significantly affected the level of activity of all enzymes. In addition, HEX and GAL were affected by age and HEX by the sex of the animal bled. Estimates of heritability from sire variances were 0.29:t 0.09 for HEX, 0.31 :t 0.09 for GAL and 0.44:t 0.09 for GLU. Genetic correlations between all enzymes were positive. The data indicate the existence of a major gene causing Pompe's disease and responsible for 16% of the genetic variation in GLU. One standard deviation of selection differential for high GLU should almost eliminate Pompe's disease from the population. The effi-ciency of selection would be aided by estimating the breeding value for GLU using measurements of HEX and GLU and taking account of an animal's sex, age, date of bleeding and herd of origin.

Validation of a Competitive Enzyme-Linked Immunosorbent Assay for Detection of Babesia bigemina Antibodies in Cattle

A competitive enzyme-linked immunosorbent assay (cELISA) based on a broadly conserved, species-specific, B-cell epitope within the C terminus of Babesia bigemina rhoptry-associated protein 1a was validated for international use. Receiver operating characteristic analysis revealed 16% inhibition as the threshold for a negative result, with an associated specificity of 98.3% and sensitivity of 94.7%. Increasing the threshold to 21% increased the specificity to 100% but modestly decreased the sensitivity to 87.2%. By using 21% inhibition, the positive predictive values ranged from 90.7% (10% prevalence) to 100% (95% prevalence) and the negative predictive values ranged from 97.0% (10% prevalence) to 48.2% (95% prevalence). The assay was able to detect serum antibody as early as 7 days after intravenous inoculation. The cELISA was distributed to five different laboratories along with a reference set of 100 defined bovine serum samples, including known positive, known negative, and field samples. The pairwise concordance among the five laboratories ranged from 100% to 97%, and all kappa values were above 0.8, indicating a high degree of reliability. Overall, the cELISA appears to have the attributes necessary for international application.

An asparaginyl endopeptidase mediates in vivo protein backbone cyclization

Proteases can catalyze both peptide bond cleavage and formation, yet the hydrolysis reaction dominates in nature. This presents an interesting challenge for the biosynthesis of backbone cyclized (circular) proteins, which are encoded as part of precursor proteins and require post-translational peptide bond formation to reach their mature form. The largest family of circular proteins are the plant-produced cyclotides; extremely stable proteins with applications as bioengineering scaffolds. Little is known about the mechanism by which they are cyclized in vivo but a highly conserved Asn (occasionally Asp) residue at the C terminus of the cyclotide domain suggests that an enzyme with specificity for Asn (asparaginyl endopeptidase; AEP) is involved in the process. Nicotiana benthamiana does not endogenously produce circular proteins but when cDNA encoding the precursor of the cyclotide kalata B1 was transiently expressed in the plants they produced the cyclotide, together with linear forms not commonly observed in cyclotide-containing plants. Observation of these species over time showed that in vivo asparaginyl bond hydrolysis is necessary for cyclization. When AEP activity was suppressed, either by decreasing AEP gene expression or using a specific inhibitor, the amount of cyclic cyclotide in the plants was reduced compared with controls and was accompanied by the accumulation of extended linear species. These results suggest that an AEP is responsible for catalyzing both peptide bond cleavage and ligation of cyclotides in a single processing event.

A free-enzyme catalyst for the bioremediation of environmental atrazine contamination.

Herbicide contamination from agriculture is a major issue worldwide, and has been identified as a threat to freshwater and marine environments in the Great Barrier Reef World Heritage Area in Australia. The triazine herbicides are of particular concern because of potential adverse effects, both on photosynthetic organisms and upon vertebrate development. To date a number of bioremediation strategies have been proposed for triazine herbicides, but are unlikely to be implemented due to their reliance upon the release of genetically modified organisms. We propose an alternative strategy using a free-enzyme bioremediant, which is unconstrained by the issues surrounding the use of live organisms. Here we report an initial field trial with an enzyme-based product, demonstrating that the technology is technically capable of remediating water bodies contaminated with the most common triazine herbicide, atrazine.

A core metabolic enzyme mediates resistance to phosphine gas

Phosphine is a small redox-active gas that is used to protect global grain reserves, which are threatened by the emergence of phosphine resistance in pest insects. We find that polymorphisms responsible for genetic resistance cluster around the redox-active catalytic disulfide or the dimerization interface of dihydrolipoamide dehydrogenase (DLD) in insects (Rhyzopertha dominica and Tribolium castaneum) and nematodes (Caenorhabditis elegans). DLD is a core metabolic enzyme representing a new class of resistance factor for a redox-active metabolic toxin. It participates in four key steps of core metabolism, and metabolite profiles indicate that phosphine exposure in mutant and wild-type animals affects these steps differently. Mutation of DLD in C. elegans increases arsenite sensitivity. This specific vulnerability may be exploited to control phosphine-resistant insects and safeguard food security.

Genetic conservation of Phosphine Resistance in the Rice Weevil Sitophilus oryzae (L.)

High levels of resistance to phosphine in the rice weevil Sitophilus oryzae have been detected in Asian countries including China and Vietnam, however there is limited knowledge of the genetic mechanism of resistance in these strains. We find that the genetic basis of strong phosphine resistance is conserved between strains of S. oryzae from China, Vietnam and Australia. Each of four strongly resistant strains has an identical amino acid variant in the encoded dihydrolipoamide dehydrogenase (DLD) enzyme that was previously identified as a resistance factor in Rhyzopertha dominica and Tribolium castaneum. The unique amino acid substitution, Asparagine > Threonine (N505T) of all strongly resistant S. oryzae corresponds to the position of an Asparagine > Histidine variant (N506H) that was previously reported in strongly resistant R. dominica. Progeny (F16 and F18) from two independent crosses showed absolute linkage of N505T to the strong resistance phenotype, indicating that if N505T was not itself the resistance variant that it resided within 1 or 2 genes of the resistance factor. Non-complementation between the strains confirmed the shared genetic basis of strong resistance, which was supported by the very similar level of resistance between the strains, with LC50 values ranging from 0.20 to 0.36 mgL-1 for a 48 hour exposure at 25°C. Thus, the mechanism of high level resistance to phosphine is strongly conserved between R. dominica, T. castaneum and S. oryzae. A fitness cost associated with strongly resistant allele was observed in segregating populations in the absence of selection.