Isolation and identification of four major impurities in capreomycin sulfate.

Capreomycin has good clinical utility to treat multidrug resistant tuberculosis, but it is only used as a second line drug due to its adverse reactions. Literature has demonstrated that the toxicity of capreomycin product is significantly influenced by the impurities in it. Unfortunately, so far, no one impurity in capreomycin has ever been isolated and definitely identified due to its extremely strong basic character and high polarity. An ion-pair method reported in literature can provide separation of capreomycin and its impurities, but it is hard to be used for the preparative purpose. In this study, this ion-pair method was further improved to detect more impurities in capreomycin sulfate substance. Besides the four main components (IA, IB, IIA and IIB), four impurities (impurity A-D) with their contents much higher than the identification threshold were observed. Furthermore, a two dimensional (2D) LC quadrupole-time of flight (Q-TOF) MS method was established to realize high resolution MS analysis of these impurities. For the purpose of preparative isolation, a hydrophilic interaction chromatography (HILIC) method was established. The four main components were well isolated, but unfortunately, the four impurities were co-eluted with each other or with IB by the HILIC method. Fortunately, the degradation experiments revealed that IA and IB could yield clean impurity A and B respectively in acidic medium, and can yield clean impurity D and C respectively in alkaline medium. Therefore, IA and IB were first isolated by the preparative HILIC method, then pure IA and IB underwent acid degradation and base degradation separately and followed by re-isolation by the HILIC method to obtain pure impurity A-D respectively. Based on Q-TOF MS and NMR analysis, the structures (including absolute configuration) of the four isolated impurities were definitely identified.

Characterization of a late expression gene of Bombyx mori nucleopolyhedrovirus.

Bombyx mori nucleopolyhedrovirus (BmNPV) ORF5 (Bm5) is a gene present in many lepidopteran nucleopolyhedroviruses (NPVs), but its function is unknown. In this study, Bm5 was characterized. The transcript of Bm5 was detected 12-72 h post infection (p.i.). Polyclonal antiserum raised to a His-BM5 fusion protein recognized BM5 in infected cell lysates from 24 to 72 h p.i., suggesting that Bm5 is a late gene. Immunofluorescence analysis by confocal microscopy showed that the BM5 protein is localized primarily in the cytoplasm. Localization of BM5 in budded virion (BV) and occlusion-derived virion (ODV) by Western analyses demonstrated that BM5 is not a structural protein associated with BV or ODV.

Bombyx mori nucleopolyhedrovirus ORF94, a novel late protein is identified to be a component of ODV structural protein.

Orf94 (Bm94) of Bombyx mori nucleopolyhedrovirus (BmNPV) potentially encodes 424-amino acids with a predicted molecular weight of 49.4 kDa, but its function remains unknown. Blast search results revealed that Bm94 homologues exist in 10 completely sequenced Lepidopteron NPVs with identities ranging from 95 to 37%. Results of our recent study showed that Bm94 was transcribed from 12 to 72 h and the corresponding protein was detected from 24 to 72 h post-infection. Furthermore, Western blot analysis revealed that Bm94 was present in occlusion-derived virus (ODV) and in total protein from BmNPV-infected BmN cells, but not in budded virus. Immunofluorescence analysis revealed that the protein located primarily in the cytoplasm and was also present in the nucleus in the later infection. In conclusion, these results together indicated that Bm94 was a late gene, which distributed both in the cytoplasm and in the nucleus, and was identified to be a component of BmNPV ODV.