Comparative whole-genome and proteomics analyses of the next seed bank and the original master seed bank of MucoRice-CTB 51A line, a rice-based oral cholera vaccine.

BACKGROUND: We have previously developed a rice-based oral vaccine against cholera diarrhea, MucoRice-CTB. Using Agrobacterium-mediated co-transformation, we produced the selection marker-free MucoRice-CTB line 51A, which has three copies of the cholera toxin B subunit (CTB) gene and two copies of an RNAi cassette inserted into the rice genome. We determined the sequence and location of the transgenes on rice chromosomes 3 and 12. The expression of alpha-amylase/trypsin inhibitor, a major allergen protein in rice, is lower in this line than in wild-type rice. Line 51A was self-pollinated for five generations to fix the transgenes, and the seeds of the sixth generation produced by T5 plants were defined as the master seed bank (MSB). T6 plants were grown from part of the MSB seeds and were self-pollinated to produce T7 seeds (next seed bank; NSB). NSB was examined and its whole genome and proteome were compared with those of MSB. RESULTS: We re-sequenced the transgenes of NSB and MSB and confirmed the positions of the three CTB genes inserted into chromosomes 3 and 12. The DNA sequences of the transgenes were identical between NSB and MSB. Using whole-genome sequencing, we compared the genome sequences of three NSB with three MSB samples, and evaluated the effects of SNPs and genomic structural variants by clustering. No functionally important mutations (SNPs, translocations, deletions, or inversions of genic regions on chromosomes) between NSB and MSB samples were detected. Analysis of salt-soluble proteins from NSB and MSB samples by shot-gun MS/MS detected no considerable differences in protein abundance. No difference in the expression pattern of storage proteins and CTB in mature seeds of NSB and MSB was detected by immuno-fluorescence microscopy. CONCLUSIONS: All analyses revealed no considerable differences between NSB and MSB samples. Therefore, NSB can be used to replace MSB in the near future.

Variability in cimetidine absorption and plasma double peaks following oral administration in the fasted state in humans: correlation with antral gastric motility.

The role of gastrointestinal motility and pH in determining cimetidine bioavailability as well as double peaks in plasma profiles following oral administration, in the quiescent or active phase of antral motility, to humans in the fasted state was examined. Plasma cimetidine-time curves did not show the presence of double peaks in any subject following intravenous administration. The incidence of double peaks was 73% following oral administration and was independent of antral migrating motility complex phase. Further, it was found that oral administration of cimetidine in the quiescent phase resulted in significantly higher bioavailability and in other pharmacokinetic parameters compared to that obtained following administration in the active phase. Excellent linearity in plots of motility peaks vs. plasma peaks with slopes close to unity were evident for both quiescent (r(2)=0.93) and active phase (r(2)=0.97) administration. A total of 14 peaks out of 22 (10 subjects, 64%) and 20 out of 27 peaks (11 subjects, 74%), were accounted for in quiescent and active phase oral administration, respectively. The proximal occurrence of plasma peaks to antral motility peaks typical of phase III contractions strongly implies that motility patterns may be responsible for secondary maxima following oral cimetidine administration in the fasted state.