Some Properties of Neutral Proteolytic System in Rabbit Skeletal Muscle

The properties of the neutral proteolytic activity concentrated in a fraction (F–1) separated from rabbit muscle homogenate were examined by measuring the effects of various reagents and metal ions, the time course of the proteolysis and Ca-stability. The obtained results have indicated that F–1 contains two types of neutral protease active on proteins, tentatively named Protease I and II, The former, which is activated by Ca²⁺ and Ca-labile, shows an explosive production of Cu-Folin phenol reagent positive materials at the early stage of incubation. The latter, which is Ca-stable, shows a large production of ninhydrin positive materials throughout the incubation time. The proteolysis by F–1 was similar to the autolysis of muscle homogenate in all the properties examined. Therefore, Proteases I and II were assumed to be main enzymes responsible for the muscle proteolysis at the neutral pH region. As there has been no factor denying their functioning in living muscle, it is probable that Proteases I and II take important parts in the muscle catabolism.     

New microbiota found in sputum from patients with community-acquired pneumonia

Community-acquired pneumonia （CAP） is a major concern in hospitals and the bacterial community of which has not been systemically discussed yet. Sputum from patients in the acute stages is a kind of accessible sample reflecting its fea- tures. In our study, we analyzed 45 sputum samples from 45 patients with CAP. Eighteen sputum samples from healthy people were chosen as the controls. Pyrosequencing of the 16s rDNA V3 hypervariable regions of aH the bacteria con- tained in the sputum was used as a culture-independent method to disclose the community constitution. Also, our published data for hospital-acquired pneumonia （HAP） in sputum was used for comparison. By pyrosequencing, 〉90,000 DNA reads were detected. After being analyzed by tools in the Ribosomal Database Project, the reads were clas- sified into five main phyla and 〉100 genera. At the phyla level, the reads＇ distribution of CAP is similar to that of healthy people and at genera level, the occurrence of each genus possesses their feature in three categories. Genera such as Streptococcus and Neisseria showed stability in their percentages, indicating that such genera are rarely affected by exogenous bacteria or antibiotics. The role of other genera such as Moraxella and Rothia in CAP should be emphasized. According to our analysis, the bacterial communities of CAP are with slight change when compared with those of healthy people, but have a large gap between HAP. Meanwhile, Rothia might be an important endogenous pneumonia-causing factor.     

Identification and characterization of a -1 reading frameshift in the heavy chain constant region of an IgG1 recombinant monoclonal antibody produced in CHO cells

Frameshifts lead to complete alteration of the intended amino acid sequences, and therefore may affect the biological activities of protein therapeutics and pose potential immunogenicity risks. We report here the identification and characterization of a novel -1 frameshift variant in a recombinant IgG1 therapeutic monoclonal antibody (mAb) produced in Chinese hamster ovary cells during the cell line selection studies. The variant was initially observed as an atypical post-monomer fragment peak in size exclusion chromatography. Characterization of the fragment peak using intact and reduced liquid chromatography-mass spectrometry (LC-MS) analyses determined that the fragment consisted of a normal light chain disulfide-linked to an aberrant 26 kDa fragment that could not be assigned to any HC fragment even after considering common modifications. Further analysis using LC-MS/MS peptide mapping revealed that the aberrant fragment contained the expected HC amino acid sequence (1-232) followed by a 20-mer novel sequence corresponding to expression of heavy chain DNA sequence in the -1 reading frame. Examination of the DNA sequence around the frameshift initiation site revealed that a mononucleotide repeat GGGGGG located in the IgG1 HC constant region was most likely the structural root cause of the frameshift. Rapid identification of the frameshift allowed us to avoid use of a problematic cell line containing the frameshift as the production cell line. The frameshift reported here may be observed in other mAb products and the hypothesis-driven analytical approaches employed here may be valuable for rapid identification and characterization of frameshift variants in other recombinant proteins.