Combinatorial mutagenesis to restrict amino acid usage in an enzyme to a reduced set

We developed an effective strategy to restrict the amino acid usage in a relatively large protein to a reduced set with conservation of its in vivo function. The 213-residue Escherichia coli orotate phosphoribosyltransferase was subjected to 22 cycles of segment-wise combinatorial mutagenesis followed by 6 cycles of site-directed random mutagenesis, both coupled with a growth-related phenotype selection. The enzyme eventually tolerated 73 amino acid substitutions: In the final variant, 9 amino acid types (A, D, G, L, P, R, T, V, and Y) occupied 188 positions (88%), and none of 7 amino acid types (C, H, I, M, N, Q, and W) appeared. Therefore, the catalytic function associated with a relatively large protein may be achieved with a subset of the 20 amino acid. The converged sequence also implies simpler constituents for proteins in the early stage of evolution.     

Second cancer during long-term survival after resection of biliary tract carcinoma

Curative surgery for biliary tract malignancy has improved the prognosis of patients; however, during long-term follow up after extensive surgery, four of our patients (two with gallbladder carcinoma and two with bile duct carcinoma) developed a second primary cancer (one each in the duodenum, skin, descending colon, and lung). Regular examination of the upper gastrointestinal tract, colorectum, and lungs, and testing for tumor markers (carcinoembryonic antigen and CA19-9) were performed as follow-up studies. As a result, the second cancers were all found at a relatively early stage and all four patients are still alive 6–14 years after the initial operation. A review of the annual autopsy reports over the last 5 years in Japan showed that the incidence of second cancer was 14.8% in patients with gallbladder carcinoma and 13.5% in those with bile duct carcinoma. Furthermore, the rate of second gastric and colorectal carcinoma was significantly higher in patients with primary bile duct cancer than in those with primary gallbladder cancer. In conclusion, the incidence of a second cancer after resection of biliary tract malignancy is more than 10%, but this second cancer can be detected relatively easily and treated at an early stage during the course of regular long-term follow up for the first cancer.     

Mitochondrial reactive oxygen species suppress humoral immune response through reduction of CD19 expression in B cells in mice

Reactive oxygen species (ROS) are implicated in the modulation of diverse processes including immune responses. To evaluate the effects of metabolic ROS produced by mitochondria on B‐cell function and development, we created transgenic (Tg) mice expressing a phosphorylation‐defective mutant of succinate dehydrogenase A in B cells (bSDHA^Y215F). Splenic B cells in male, but not female, bSDHA^Y215F mice produced three times more ROS than those in the control mice, and had decreased production of IgM, IgG₁, and IgG₃, and affinity maturation of IgG₁ against T‐cell‐dependent antigens. Following immunization, the male bSDHA^Y215F mice further displayed suppressed germinal center (GC) formation, and proliferation of GC B cells. Signaling analysis revealed defects in the intrinsic BCR responses, such as activation of Lyn, Btk, and PLCγ2, thus resulting in reduced intracellular Ca²⁺ mobilization. Notably, the expression levels of B‐cell co‐receptor CD19 and its interaction with Lyn after BCR ligation were significantly reduced in B cells from male bSDHA^Y215F mice. These results suggest that mitochondrial ROS suppress humoral immune responses through reduction of CD19 expression and resultant BCR signaling in B cells. Therefore, B‐cell immunity may be more labile to oxidative stress in male mice than in female mice.