The gene was cloned and expressed in Pichia pastoris, and the cysteine peptidase was successfully expressed, secreted, and purified using affinity chromatography with a yield of approximately 10 mg/L. A polyclonal antibody produced against cysteine peptidase from X. citri subsp citri fused with HIS tag ((HIS)CPXAC) recognized the purified recombinant cysteine peptidase (HIS)CPXAC, confirming the correct production of this protein
in P. pastoris. The same antibody detected the protein in the culture supernatant of Xac grown in pathogenicity-inducing medium. Kinetic analysis revealed that (HIS)CPXAC hydrolyzed the carbobenzoxy-Leu-Arg-7-amido-4-methylcoumarin Akt inhibition substrate with a catalytic efficiency (k(cat)/K-m) of 47 mu M-1.s(-1). The purified (HIS)CPXAC displayed maximal catalytic activity at pH 5.5 and 30 degrees C. The recombinant enzyme was inhibited by the specific cysteine peptidase inhibitor E-64, as well as by the recombinant cysteine peptidase inhibitors CaneCPI-1, CaneCPI-2, CaneCPI-3, and CaneCPI-4, with K-i values of 1.214, 84.64, 0.09, 0.09, and 0.012 nM, respectively. Finally, the N-terminal sequencing of the purified protein enabled the identification of
the first 5 amino acid residues (AVHGM) immediately after the putative signal peptide, thereby enabling the identification of the cleavage point and corroborating previous studies that have identified this sequence GANT61 in a secreted protein from Xanthomonas spp.”
“PURPOSE: To study the impact on the subjective depth of field of 4th-order spherical aberration and its combination with 6th-order spherical aberration find more and analyze the accuracy of image-quality metrics in predicting the impact.
Laboratoire Aime Cotton, Centre National de la Recherche Scientifique, Universite Paris-Sud, Orsay, France.
DESIGN: Case series.
METHODS: Subjective depth of field was defined as the range of defocus at which the target (3 high-contrast letters at 20/50) was perceived acceptable. Depth of field was measured using 0.18 diopter (D) steps in young subjects with the addition of the following spherical aberration values: +/- 0.3 mu m and +/- 0.6 mu m 4th-order spherical aberration with 3.0 mm and 6.0 mm pupils and +/- 0.3 mu m 4th-order spherical aberration with +/- 0.1 mu m 6th-order spherical aberration for 6.0 mm pupils.
RESULTS: The addition of 0.3 and +/- 0.6 mu m 4th-order spherical aberration increased depth of field by 30% and 45%, respectively. The combination of 4th-order spherical aberration and 6th-order spherical aberration of opposite signs increased depth of field more than 4th-order spherical aberration alone (ie, 63%), while the combination of 4th-order spherical aberration and 6th-order spherical aberration of the same signs did not (ie, 24%). Whereas the midpoint of the depth of field could be predicted by image-quality metrics, none was found a good predictor of objectionable depth of field.