ABSTRACT
In free-living and parasitic nematodes, the methylation of phosphoethanolamine to phosphocholine provides a key metabolite to sustain phospholipid biosynthesis for growth and development. Because the phosphoethanolamine methyltransferases (PMT) of nematodes are essential for normal growth and development, these enzymes are potential targets of inhibitor design. The pine wilt nematode (Bursaphelenchus xylophilus) causes extensive damage to trees used for lumber and paper in Asia. As a first step toward testing BxPMT1 as a potential nematicide target, we determined the 2.05 Å resolution x-ray crystal structure of the enzyme as a dead-end complex with phosphoethanolamine and S-adenosylhomocysteine. The three-dimensional structure of BxPMT1 served as a template for site-directed mutagenesis to probe the contribution of active site residues to catalysis and phosphoethanolamine binding using steady-state kinetic analysis. Biochemical analysis of the mutants identifies key residues on the ß1d-α6 loop (W123F, M126I, and Y127F) and ß1e-α7 loop (S155A, S160A, H170A, T178V, and Y180F) that form the phosphobase binding site and suggest that Tyr127 facilitates the methylation reaction in BxPMT1.
Subject(s)
Ethanolamines/chemistry , Helminth Proteins/chemistry , Methyltransferases/chemistry , Nematoda/enzymology , Pinus/parasitology , Plant Diseases/parasitology , Amino Acid Sequence , Animals , Binding Sites , Cloning, Molecular , Crystallography, X-Ray , Escherichia coli/genetics , Escherichia coli/metabolism , Ethanolamines/metabolism , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Helminth Proteins/genetics , Helminth Proteins/metabolism , Kinetics , Methyltransferases/genetics , Methyltransferases/metabolism , Models, Molecular , Nematoda/genetics , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Substrate Specificity , ThermodynamicsABSTRACT
PURPOSE: To determine diagnostic yield of renal biopsies performed in patients referred for image-guided tumor ablation (IGTA) and the frequency with which biopsy results would have obviated the need for subsequent ablation. MATERIALS AND METHODS: Retrospective review of an internal ablation database of a single institution revealed 401 consecutive percutaneous renal mass IGTAs performed from April 2000 to April 2015. Of 401 ablations, 32 were excluded, yielding 369 ablation events in 342 patients, which represented the study cohort. Patients were subdivided into groups according to whether or not biopsy was performed. Lesions were categorized according to size, malignancy/benignity, and pathology. RESULTS: IGTA was performed with biopsy for 317/369 (85.9%) and without biopsy for 52/369 (14.1%) lesions. Overall diagnostic yield for percutaneous biopsy was 94.3% (299/317). Based on biopsy results, 82.6% (262/317) were classified as malignant or suspicious, 9.5% (30/317) were classified as likely benign, and 2.2% (7/317) were classified as definitively benign. Only definitively benign lesions were designated as obviating the need for IGTA. IGTA was supported by biopsy results in the remaining 97.8% (310/317), including renal cell carcinomas, oncocytic neoplasms, metastases, and nondiagnostic biopsy results. CONCLUSIONS: Biopsy of renal masses with suspicious imaging features rarely (2.2%) obviated the need for IGTA. For patients who have undergone counseling and have elected to forgo active surveillance and surgical options, biopsy can safely be performed concomitantly with ablation.
