Development of Dry and Liquid Duplex Reagent Mix-Based Polymerase Chain Reaction Assays as Novel Tools for the Rapid and Easy Quantification of Bovine Leukemia Virus (BLV) Proviral Loads.

Bovine leukemia virus (BLV) is prevalent worldwide, causing serious problems in the cattle industry. The BLV proviral load (PVL) is a useful index for estimating disease progression and transmission risk. We previously developed a quantitative real-time PCR (qPCR) assay to measure the PVL using the coordination of common motif (CoCoMo) degenerate primers. Here, we constructed a novel duplex BLV-CoCoMo qPCR assay that can amplify two genes simultaneously using a FAM-labeled MGB probe for the BLV LTR gene and a VIC-labeled MGB probe for the BoLA-DRA gene. This liquid duplex assay maintained its original sensitivity and reproducibility in field samples. Furthermore, we developed a dry duplex assay composed of PCR reagents necessary for the optimized liquid duplex assay. We observed a strong positive correlation between the PVLs measured using the dry and liquid duplex assays. Validation analyses showed that the sensitivity of the dry duplex assay was slightly lower than that of the other methods for the detection of a BLV molecular clone, but it showed similar sensitivity to the singleplex assay and slightly higher sensitivity than the liquid duplex assay for the PVL quantification of 82 field samples. Thus, our liquid and dry duplex assays are useful for measuring the BLV PVL in field samples, similar to the original singleplex assay.

Local biosynthesis of corticosterone in rat skeletal muscle.

Adrenal corticosterone plays crucial roles in energy metabolism and immuno-reactivity throughout the body. As we have previously shown that corticosterone biosynthesis in C2C12 myoblasts, we study about corticosterone biosynthesis in rat skeletal muscles. It was found that enzymatic activities producing corticosterone and testosterone except the activity of P450scc in rat skeletal muscle as like as C2C12 cells. The CYP11B mRNA encoding cytochrome P45011ß that mediates 11-deoxycorticosterone hydroxylase activity, producing corticosterone was expressed in skeletal muscles. In immunoblotting analysis, cytochrome P45011ß protein was expressed in rat muscles and whole organs especially higher levels in adrenal and brain. The localizations of corticosterone content and enzymatic activities involved in the production of corticosterone were preferentially observed in gastrocnemius fibers rather than in soleus fibers. The immunohistochemical analysis showed that the fast-twitch or type II muscle fibers positive to antibody against fast myosin heavy chain were preferentially stained with anti-cytochrome P45011ß antibody in the gastrocnemius fiber. In addition, we detected corticosterone biosynthesis from pregnenolone sulfate conjugates in perfusion of the rat hindquarter. Corticosterone is synthesized in rat skeletal muscles and the biosynthesis was localized in the fast-twitch or type II muscle fibers. We speculated that the local synthesized corticosterone might be involved in glucocorticoid-induced muscle atrophy that preferentially occurs in fast muscle fibers, and the initial substrate of the local CORT biosynthesis were supported to be performed from the conjugates such as pregnenolone sulfate circulating in the blood flow.