Glomerulonephritis Caused by Bartonella spp. Infective Endocarditis: The Difficulty and Importance of Differentiation from Anti-neutrophil Cytoplasmic Antibody-related Rapidly Progressive Glomerulonephritis.

A 65-year-old man with valvular disorder presented to his physician because of widespread purpura in both lower extremities. Blood tests showed elevated serum creatinine levels and proteinase 3-anti-neutrophil cytoplasmic antibody (ANCA) with hematuria, suggesting ANCA-related rapidly progressive glomerulonephritis (RPGN). Although multiple blood cultures were negative, transthoracic echocardiography revealed warts in the valves, and a renal biopsy also showed findings of glomerular infiltration by mononuclear leukocytes and C3 deposition in the glomeruli, suggesting infection-related glomerulonephritis. Later, Bartonella antibody turned positive. Antimicrobial treatment improved the purpura and renal function without any recurrence. ANCA-positive RPGN requires the exclusion of infective endocarditis, especially that induced by Bartonella spp.

A simple system for expression of proteins containing 3-azidotyrosine at a pre-determined site in Escherichia coli.

We developed an efficient method for introduction of 3-azidotyrosine (N(3)-Y) into proteins in Escherichia coli cells. We constructed a plasmid that is adaptable for the constitutive expression of both Methanosarcina acetivorans tyrosyl-tRNA synthetase (TyrRS) and tRNA(()(CUA)), and made an orthogonal tRNA((CUA)) that is recognized as a substrate only by the archaeal TyrRS. Random mutations were introduced into M. acetivorans TyrRS around the tyrosine binding pocket, and a TyrRS mutant recognizing N(3)-Y was selected. We then expressed rat calmodulin (CaM) containing N(3)-Y, using the CaM gene with an amber codon at position 80. Mass analyses confirmed production of CaM containing N(3)-Y, but a significant amount of CaM containing 3-aminotyrosine was also detected. To more efficiently express CaM containing N(3)-Y, we added an arabinose-inducible gene for the mutant TyrRS to the plasmid carrying the mutant TyrRS/tRNA(()(CUA)) gene. Although the yields of full-length CaM increased ~3-fold, the ratio of N(3)-Y introduction was not significantly improved. Following screening for a suitable host cell, we found that CaM expressed in E. coli SHuffle (K-12) had 97% N(3)-Y at the pre-determined site. Finally, we obtained up to 2 mg of CaM containing N(3)-Y per 100 ml of culture media, sufficient for use in various proteomics experiments, including photo-crosslinking.