No abstract

We analyzed PCR-amplified carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD) gene fragments from 146 Bombyx mori native strains and found extremely low levels of DNA polymorphism. Two haplotypes were identified, one of which was predominant. CAD haplotype analysis of 42 samples of Japanese B. mandarina revealed four haplotypes. No common haplotype was shared between the two species and at least five base substitutions were detected. This result was suggestive of low levels of gene flow between the two species. The nucleotide diversity (π) scores of the two samples differed markedly: lower π values were estimated for B. mori native strains than Japanese B. mandarina. We further analyzed 12 Chinese B. mandarina derived from seven areas of China, including Taiwan. The results clearly indicated that the π score was ~80-fold greater in Chinese B. mandarina than in B. mori. The extremely low level of DNA polymorphism in B. mori compared to its wild relatives suggested that the CAD gene itself or its tightly linked regions are possible targets for silkworm domestication.

Amyloid beta peptide (Aβ)-related studies require an adequate supply of purified Aβ peptide. However, Aβ peptides are “difficult sequences” to synthesize chemically, and low yields are common due to aggregation during purification. Here, we demonstrate an easier synthesis, deprotection, reduction, cleavage, and purification process for Aβ(1-40) using standard 9-fluorenylmethyloxycarbonyl (Fmoc)-protected amino acids and solid-phase peptide synthesis (SPPS) resin [HMBA (4-hydroxymethyl benzamide) resin] that provides higher yields of Aβ(1-40) than previous standard protocols. Furthermore, purification requires a similar amount of time as conventional purification processes, although the peptide must be cleaved from the resin immediately prior to purification. The method described herein is not limited to the production of Aβ(1-40), and can be used to synthesize other easily-oxidized and aggregating sequences. Our proposed methodology will contribute to various fields using “difficult sequence” peptides, such as pharmaceutical and materials science, as well as research for the diagnosis and treatment of protein/peptide misfolding diseases.

The biological process of skin sensitization depends on the ability of a sensitizer to modify endogenous proteins. A direct peptide reactivity assay (DPRA), based on the biological process of skin sensitization, was developed as an alternative to controversial animal experiments. Although DPRA has been endorsed by industries and is internationally accepted as promising, it has several drawbacks, such as incompatibility with hydrophobic chemicals, inability to perform detailed reaction analysis, and ability to evaluate only single components. Here, we demonstrated that sensitizers and peptide adducts can be easily identified using a mass spectrometry-based solid-phase peptide reaction assay (M-SPRA). We synthesized peptides with a photo-cleavable linker immobilized on resins. We showed the potential of M-SPRA in predicting skin sensitization by measuring the peptide adducts that were selectively eluted from the resin after cleaving the linker post-reaction. M-SPRA provides more detailed information regarding chemical reactivity and accurate assessment of test samples, including mixtures. M-SPRA may be helpful for understanding the binding mechanism of sensitizers (toxicology), which may assist in further refining reactivity assays and aiding in the interpretation of reactivity data.