Highly specific unnatural base pair systems as a third base pair for PCR amplification

Toward the expansion of the genetic alphabet of DNA, we present highly efficient unnatural base pair systems as an artificial third base pair for PCR. Hydrophobic unnatural base pair systems between 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds) and 2-nitro-4-propynylpyrrole (Px) were fine-tuned for efficient PCR, by assessing the amplification efficiency and fidelity using different polymerases and template sequence contexts and modified Px bases. Then, we found that some modifications of the Px base reduced the misincorporation rate of the unnatural base substrates opposite the natural bases in templates without reducing the Ds-Px pairing selectivity. Under optimized conditions using Deep Vent DNA polymerase, the misincorporation rate was extremely low (0.005%/bp/replication), which is close to that of the natural base mispairings by the polymerase. DNA fragments with different sequence contexts were amplified ～10(10)-fold by 40 cycles of PCR, and the selectivity of the Ds-Px pairing was >99.9%/replication, except for 99.77%/replication for unfavorable purine-Ds-purine motifs. Furthermore, >97% of the Ds-Px pair in DNA survived in the 10(28)-fold amplified products after 100-cycle PCR (10 cycles repeated 10 times). This highly specific Ds-Px pair system provides a framework for new biotechnology.     

Synthesis and binding properties of new selective ligands for the nucleobase opposite the AP site

DNA is continuously damaged by endogenous and exogenous factors such as oxidative stress or DNA alkylating agents. These damaged nucleobases are removed by DNA N-glycosylase and form apurinic/apyrimidinic sites (AP sites) as intermediates in the base excision repair (BER) pathway. AP sites are also representative DNA damages formed by spontaneous hydrolysis. The AP sites block DNA polymerase and a mismatch nucleobase is inserted opposite the AP sites by polymerization to cause acute toxicities and mutations. Thus, AP site specific compounds have attracted much attention for therapeutic and diagnostic purposes. In this study, we have developed nucleobase-polyamine conjugates as the AP site binding ligand by expecting that the nucleobase part would play a role in the specific recognition of the nucleobase opposite the AP site by the Watson-Crick base pair formation and that the polyamine part should contribute to the access of the ligand to the AP site by a non-specific interaction to the DNA phosphate backbone. The nucleobase conjugated with 3,3'-diaminodipropylamine (A-ligand, G-ligand, C-ligand, T-ligand and U-ligand) showed a specific stabilization of the duplex containing the AP site depending on the complementary combination with the nucleobase opposite the AP site; that is A-ligand to T, G-ligand to C, C-ligand to G, T- and U-ligand to A. The thermodynamic binding parameters clearly indicated that the specific stabilization is due to specific binding of the ligands to the complementary AP site. These results have suggested that the complementary base pairs of the Watson-Crick type are formed at the AP site.     

AMP-activated protein kinase regulates PDGF-BB-stimulated interleukin-6 synthesis in osteoblasts: involvement of mitogen-activated protein kinases

AimWe have previously reported that platelet-derived growth factor (PDGF)-BB stimulates synthesis of interleukin-6 (IL-6), a potent bone resorptive agent, in osteoblast-like MC3T3-E1 cells, and that the activation of p44/p42 mitogen-activated protein (MAP) kinase, p38MAP kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) is implicated in the IL-6 synthesis. In the present study,we investigated the involvement of AMP-activated protein kinase (AMPK), a regulator of energy metabolism, in the PDGF-BB-stimulated IL-6 synthesis in MC3T3-E1 cells.Main methodsThe levels of IL-6 were measured by ELISA. The phosphorylation of each protein kinases was analyzed by Western blotting. The mRNA levels of IL-6 were determined by real-time RT-PCR.Key findingsPDGF-BB time-dependently induced the phosphorylation of AMPK. Compound C, an inhibitor of AMPK, which reduced PDGF-BB-induced acetyl-CoA carboxylase phosphorylation, dose-dependently suppressed the PDGF-BB-stimulated IL-6 release. In addition, the PDGF-BB-stimulated IL-6 release in human osteoblasts was also inhibited by compound C. The mRNA expression of IL-6 induced by PDGF-BB was markedly reduced by compound C. The PDGF-BB-induced phosphorylation of p44/p42 MAP kinase, p38 MAP kinase and SAPK/JNK was inhibited by compound C.SignificanceThese results strongly suggest that AMPK positively regulates PDGF-BB-stimulated IL-6 synthesis via the MAP kinases in osteoblasts.     

Development of a repeated-dose liver micronucleus assay using adult rats: an investigation of diethylnitrosamine and 2,4-diaminotoluene

Various liver micronucleus assay methods, such as those involving partial hepatectomy, treatment with mitogens, and the use of juvenile animals, have been developed. These assays have been proven to be of high sensitivity and specificity to predict hepatocarcinogenicity of compounds that cannot be detected by bone marrow micronucleus assays. On the contrary, the existing assays have only been evaluated for their use in detecting micronucleus induction in the settings of relatively short-term cell proliferation. However, the integration of in vivo genotoxicity endpoints into routine toxicity studies is increasingly desired from the viewpoint of animal welfare to reduce the number of animals used. In the present study, the rodent hepatocarcinogens diethylnitrosamine (DEN) and 2,4-diaminotoluene (2,4-DAT) were repeatedly administered orally to male Crl:CD (SD) rats (6 weeks old at the beginning of administration) for 5, 14, and 28 days, and changes in the frequency of hepatocytes with micronuclei in liver tissues that had undergone no artificial treatment to accelerate cell proliferation were evaluated. At the same time, a new method of hepatocyte isolation involving the treatment of a portion of the liver with collagenase in a centrifuge tube, without the use of in situ perfusion, was established. The induction of micronucleated hepatocytes was achieved after the repeated administration of DEN for 5 days or longer and of 2,4-DAT for 14 days or longer. Micronucleus frequencies were increased depending on the number of administrations, indicating that micronucleated hepatocytes had possibly remained for a long period of time and accumulated additively. It therefore appears that even in adult rat liver with low mitotic activity, a repeated-dose of a chemical substance for 14 days or longer enables the detection of micronucleus induction. In addition, the establishment of a method to isolate hepatocytes without perfusion using only a part of the liver enables the integration of liver micronucleus assays into general toxicity studies.     

Prevention of Disuse Muscle Atrophy by Dietary Ingestion of 8-Prenylnaringenin in Denervated Mice

Flavonoids have attracted considerable attention in relation to their effects upon health. 8-Prenylnaringenin (8-PN) is found in the common hop (Humulus lupulus) and assumed to be responsible for the health impact of beer consumption. We wanted to clarify the effects of prenylation on the physiological functions of dietary flavonoids by comparing the effects of 8-PN with that of intact naringenin in the prevention of disuse muscle atrophy using a model of denervation in mice. Consumption of 8-PN (but not naringenin) prevented loss of weight in the gastrocnemius muscle further supported by the lack of induction of the protein content of a key ubiquitin ligase involved in muscle atrophy, atrogin-1, and by the activation of Akt phosphorylation. 8-PN content in the gastrocnemius muscle was tenfold higher than that of naringenin. These results suggested that, compared with naringenin, 8-PN was effectively concentrated into skeletal muscle to exert its preventive effects upon disuse muscle atrophy. It is likely that prenylation generates novel functions for 8-PN by enhancing its accumulation into muscle tissue through dietary intake.     

Regulatory Roles of Cytokinins and Cytokinin Signaling in Response to Potassium Deficiency in Arabidopsis

Potassium (K) is an important plant macronutrient that has various functions throughout the whole plant over its entire life span. Cytokinins (CKs) are known to regulate macronutrient homeostasis by controlling the expression of nitrate, phosphate and sulfate transporters. Although several studies have described how CKs signal deficiencies for some macronutrients, the roles of CKs in K signaling are poorly understood. CK content has been shown to decrease under K-starved conditions. Specifically, a CK-deficient mutant was more tolerant to low K than wild-type; however, a plant with an overaccumulation of CKs was more sensitive to low K. These results suggest that K deprivation alters CK metabolism, leading to a decrease in CK content. To investigate this phenomenon further, several Arabidopsis lines, including a CK-deficient mutant and CK receptor mutants, were analyzed in low K conditions using molecular, genetic and biochemical approaches. ROS accumulation and root hair growth in low K were also influenced by CKs. CK receptor mutants lost the responsiveness to K-deficient signaling, including ROS accumulation and root hair growth, but the CK-deficient mutant accumulated more ROS and exhibited up-regulated expression of HAK5, which is a high-affinity K uptake transporter gene that is rapidly induced by low K stress in ROS- and ethylene-dependent manner in response to low K. From these results, we conclude that a reduction in CK levels subsequently allows fast and effective stimulation of low K-induced ROS accumulation, root hair growth and HAK5 expression, leading to plant adaptation to low K conditions.