During development of a novel detection method for the UDP-glucuronosyl transferase 1A1 (UGT1A1)*28, the fluorescence intensity of a dye conjugated to cytosine (C) at the end of a DNA strand decreased upon hybridization with guanine (G). This phenomenon is referred to as photoinduced electron transfer (PeT). Using this phenomenon, we devised a method for the naked-eye detection of UGT1A1*28 (thymine-adenine (TA)-repeat polymorphism). Fluorescently labeled single-stranded DNA (ssDNA) oligonucleotides (probes) were designed and hybridized with complementary strand DNAs (target DNAs). Base pair formation at the blunt end between fluorescently labeled C (probe side) and G (target side), induced dramatic fluorescence quenching. Additionally, when the labeled-CG pair formed near the TA-repeat sequence, different TA-repeat numbers were discriminated. However, obtaining enough target DNA for this probe by typical polymerase chain reaction (PCR) was difficult. To enable the practical use of the probe, producing sufficient target DNA remains problematic.
Attention deficit/ hyperactivity disorder (AD/HD) is a mild developmental disorder . We reported that stroke-prone spontaneously hypertensive rat/Ezo (SHRSP/Ezo) had high validity as an AD/HD animal model, because of its behavioral phenotype. Recently, we revealed the NMDA receptor dysfunction on excitatory synapse in the prefrontal cortex (PFC) of SHRSP/Ezo. D-serine, an endogenous co-ligand for NMDA receptor, is one of new drug targets for the treatment of several psychiatric disorders. D-serine is biosynthesized from the optical conversion of L-to D-by serine racemase (SR), and metabolized by D-amino acid oxidase (DAAO). Here, we evaluated the serine kinetics in the PFC of SHRSP/Ezo and assessed the effect of DAAO inhibitor on AD/HD-like behaviors of SHRSP/Ezo. At 6 weeks old, SHRSP/Ezo and its genetic control, WKY/Ezo were anesthetized and immediately decapitated. The PFC tissue was trimmed on ice for analysis of SR and DAAO expression by western blotting and measurement of serine concentration by HPLC-ECD system. Moreover, we performed local injection of DAAO inhibitor (5 microgram/side) into the PFC and assessed AD/HD-like behaviors using Y-maze test. DAAO in the PFC of SHRSP/Ezo was significantly higher expression compared with WKY/Ezo, although SR was not significant. Correspondingly, D-/L-ratio of serine in the PFC of SHRSP/Ezo was lower than that of WKY/Ezo. Furthermore, local injection of DAAO inhibitor into the PFC improved inattention and hyperactivity of SHRSP/Ezo. These results suggest that DAAO inhibitor can be a possible candidate for treatment of AD/HD.
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