Analysis of a Secreted Aspartic Peptidase Disruption Mutant of Glomerella cingulata

Peptidases have been implicated in the pathogenicity of fungi that cause disease in plants. Expression of the secreted aspartic peptidase gene (gcsap), of a Glomerella cingulata isolate pathogenic on apples, is induced during appressorium formation. To determine whether the secreted aspartic peptidase (GcSAP) is essential to pathogenicity, gcsap was disrupted using a vector containing a 637 bp fragment of genomic DNA that encodes the sequence spanning the two active site aspartic acid (Asp) residues. To ensure that the truncated gcsap gene products could not have residual peptidase activity the codons for the active site residues Asp¹¹² and Asp²⁹⁷ were both mutated to histidine residues. Both PCR and Southern analysis confirmed disruption of gcsap. Neither gcsap mRNA nor GcSAP activity was detected in the disruption mutant. Pathogenicity tests on fruit from three apple cultivars showed that GcSAP was not required for pathogenicity. The disruption mutant grew on medium containing protein as the sole source of nitrogen because G. cingulata secretes a previously undetected peptidase(s). A serine peptidase that had a pH optimum between pH 7.0 and 8.0 and a K _m of 0.25 mM for the synthetic substrate succinyl-Ala–Ala–Pro–Phe-p-nitroanilide was identified.     

In this issue – June 2011

Computer model predictions and field observations of anthelmintic resistance in sheep · Dangers of off‐label use of barium selenate · Elbow luxation in dogs and cats · Prognosis of joint infections in adult horses · Omentalisation for mediastinal abscess in a dog · Adenoviruses in lizards     

Assessment of corneal thickness,intraocular pressure,optical corneal diameter,and axial globe dimensions in Miniature Horses

OBJECTIVE: To determine corneal thickness, intraocular pressure (IOP), and horizontal and vertical corneal diameter (HCD and VCD) and to obtain axial measurements of the anterior chamber depth (ACD), crystalline lens thickness (CLT), vitreous chamber depth (VtCD), and axial globe length (AGL) in eyes of Miniature Horses. ANIMALS: 41 healthy Miniature Horses. PROCEDURE: Ocular component measurements were obtained via ultrasonic pachymetry, applanation tonometry, ultrasound, and by use of a Jameson caliper. RESULTS: Mean IOP and corneal thickness for all eyes were 26.0 mm Hg and 785.6 microm, respectively. There was no correlation of age with IOP or corneal thickness and no difference in these variables between right and left eyes or between females and males. Mean HCD andVCD were 25.8 and 19.4 mm, respectively; although there were no differences between sexes or between right and left eyes, there was positive correlation of optical corneal diameters with increasing age. Mean ACD, CLT, VtCD, and AGL were smaller in Miniature Horses (5.6, 10.0, 18.1, and 33.7 mm, respectively), compared with values for full-sized horses; there was no difference in these variables between sexes or between right and left eyes in Miniature Horses, but they were correlated with increasing age. CONCLUSIONS AND CLINICAL RELEVANCE: In Miniature Horses, corneal thickness and IOP are similar to values reported for full-sized horses and do not increase with advancing age. Vertical corneal diameter, HCD, and AGL increase until 5, 7, and 2 years of age, respectively.