Small RNA class transition from siRNA/piRNA to miRNA during pre-implantation mouse development

Recent studies showed that small interfering RNAs (siRNAs) and Piwi-interacting RNA (piRNA) in mammalian germ cells play important roles in retrotransposon silencing and gametogenesis. However, subsequent contribution of those small RNAs to early mammalian development remains poorly understood. We investigated the expression profiles of small RNAs in mouse metaphase II oocytes, 8–16-cell stage embryos, blastocysts and the pluripotent inner cell mass (ICM) using high-throughput pyrosequencing. Here, we show that during pre-implantation development a major small RNA class changes from retrotransposon-derived small RNAs containing siRNAs and piRNAs to zygotically synthesized microRNAs (miRNAs). Some siRNAs and piRNAs are transiently upregulated and directed against specific retrotransposon classes. We also identified miRNAs expression profiles characteristic of the ICM and trophectoderm (TE) cells. Taken together, our current study reveals a major reprogramming of functional small RNAs during early mouse development from oocyte to blastocyst.     

Accumulation of 8-oxo-deoxyguanosine in cardiovascular tissues with the development of hypertension

Accumulation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) in DNA is associated with mutagenesis and cell death. Little attention has been given to the biological significance of 8-oxo-dG accumulation in cardiovascular tissues during the different stage of hypertension and its prevention. We thus investigated the levels and localization of both 8-oxo-dG accumulation and expression of MTH1, which hydrolyzes 8-oxo-dGTP to prevent its incorporation into DNA, in the thoracic aorta prepared from stroke-prone spontaneously hypertensive rats (SHRSP) and age-matched Wister-Kyoto rats (WKY), aged 5-32 weeks. HPLC-MS/MS analysis revealed that the levels of nuclear 8-oxo-dG in the aorta increased significantly in SHRSP, but not WKY, with aging. Immunohistochemical study revealed that both TUNEL reactivity and 8-oxo-dG immunoreactivity were increased in smooth muscle cells (SMC) and endothelial cells (EC) of the aorta with aging, and they exhibited similar distributions in serial sections. The number of 8-oxo-dG and TUNEL positive cells in EC, but not in SMC, was significantly higher in SHRSP than WKY at 32 weeks of age. In contrast, the expression levels of Mth1mRNA and MTH1 protein in the aorta were similarly decreased both in SHRSP and WKY with aging. However, the number of MTH1 expressing EC was remarkably increased in the older SHRSP compared to the younger ones or age-matched WKY. Hypertension significantly increased not only 8-oxo-dG accumulation but also the expression of MTH1 in EC of the aorta during aging. While accumulation of 8-oxo-dG in SMC of the aorta was slightly increased, the expression of MTH1 protein in SMC was rather decreased by hypertension. We thus suggest that MTH1 may protect EC in the aorta from the oxidative damage increased by hypertension.     

Synthesis of closo-Dodecaboryl Lipids and their Liposomal Formation for Boron Neutron Capture Therapy

High accumulation and selective delivery of boron into tumor tissues are the most important requirements to achieve efficient neutron capture therapy of cancers. We focused on liposomal boron delivery system to achieve a large amount of boron delivery to tumor. We succeeded in the synthesis of the double-tailed boron cluster lipids 4a–c and 5a–c, which has a B₁₂H₁₁S-moiety as a hydrophilic function, by S-alkylation of B₁₂H₁₁SH with bromoacetyl and chloroacetocarbamate derivatives of diacylglycerols. Size distribution of liposomes prepared from the boron cluster lipid 4b, dimyristoylphosphatidylcholine, polyethyleneglycol-conjugated distearoylphosphatidylethanolamine, and cholesterol was determined as 100 nm in diameter by an electrophoretic light scattering spectrophotometer. Calcein-encapsulation experiments revealed that these boronated liposomes are stable at 37 °C in fetal bovine serum solution for 24 h.     

Biochemical characterization of the very long-chain fatty acid elongase ELOVL7

Very long-chain fatty acids (VLCFAs) have a variety of physiological functions and are related to numerous disorders. The key step of VLCFA elongation is catalyzed by members of the elongase family, ELOVLs. Mammals have seven ELOVLs (ELOVL1-7), yet none of them has been purified and analyzed. In the presented study we purified ELOVL7 and measured its activity by reconstituting it into proteoliposomes. Purified ELOVL7 exhibited high activity toward acyl-CoAs with C18 carbon chain length. The calculated K(m) values toward C18:3(n-3)-CoA and malonyl-CoA were both in the μM range. We also found that progression of the VLCFA cycle enhances ELOVL7 activity.     

Silymarin attenuated the amyloid β plaque burden and improved behavioral abnormalities in an Alzheimer's disease mouse model

Alzheimer's disease (AD) is characterized by progressive cognitive impairment and the formation of senile plaques. Silymarin, an extract of milk thistle, has long been used as a medicinal herb for liver diseases. Here we report marked suppression of amyloid β-protein (Aβ) fibril formation and neurotoxicity in PC12 cells after silymarin treatment in vitro. In vivo studies had indicated a significant reduction in brain Aβ deposition and improvement in behavioral abnormalities in amyloid precursor protein (APP) transgenic mice that had been preventively treated with a powdered diet containing 0.1% silymarin for 6 months. The silymarin-treated APP mice also showed less anxiety than the vehicle-treated APP mice. These behavioral changes were associated with a decline in Aβ oligomer production induced by silymarin intake. These results suggest that silymarin is a promising agent for the prevention of AD.     

Proton-conductivity assay of plugged and unplugged MotA/B proton channel by cytoplasmic pHluorin expressed in Salmonella

MotA and MotB form the proton-channel complex of the proton-driven bacterial flagellar motor. A plug segment of Escherichia coli MotB suppresses proton leakage through the MotA/B complex when it is not assembled into the motor. Using a ratiometric pH indicator protein, pHluorin, we show that the proton-conductivity of a Salmonella MotA/B complex not incorporated into the motor is two orders of magnitude lower than that of a complex that is incorporated and activated. This leakage is, however, significant enough to change the cytoplasmic pH to a level at which the chemotaxis signal transduction system responds.     

Synthesis and evaluation of a [18F]formyl–Met–Leu–Phe derivative: A positron emission tomography imaging probe for bacterial infections

The tripeptide formyl–Met–Leu–Phe (fMLF) is a prototype of N-formylated chemotactic peptides for neutrophils owing to its ability to bind and activate the G protein-coupled formyl peptide receptor (FPR). Here, we developed an ¹⁸F-labeled fMLF derivative targeting FPR as a positron emission tomography (PET) imaging probe for bacterial infections. The study demonstrates that the fMLF derivative fMLFXYk(FB)k (X?=?Nle) has a high affinity for FPR (Ki?=?0.62?±?0.13?nM). The radiochemical yield and purity of [¹⁸F]fMLFXYk(FB)k were 16% and >96%, respectively. The in vivo biodistribution study showed that [¹⁸F]fMLFXYk(FB)k uptake was higher in the bacterial infected region than in the non-infected region. We observed considerably higher infection-to-muscle ratio of 4.6 at 60?min after [¹⁸F]fMLFXYk(FB)k injection. Furthermore, small-animal PET imaging studies suggested that [¹⁸F]fMLFXYk(FB)k uptake in the bacterial infected region was clearly visualized 60?min after injection.     

Molecular characterization of glycogen synthase 1 and its tissue expression profile with type II hexokinase and muscle-type phosphofructokinase in horses

Muscle glycogen synthase (GYS1) is the rate-limiting enzyme in glycogen synthesis, and its activity is regulated by the phosphorylation states of certain amino acid residues encoded by the GYS1 gene. In the present study, the authors molecularly characterized the full-length equine GYS1 (eGYS1) cDNA and found that it contains a less common polyadenylation signal (AATACA). An amino acid alignment with other mammalian GYS1 showed that the phosphorylation sites in eGYS1 are completely conserved. Genomic DNA analysis revealed that the equine-specific substitutions (Glu 16 Asp and Ala 252 Thr) were completely conserved among six equine species. The tissue expression profiles of eGYS1, equine type II hexokinase (eHKII) and muscle-type phosphofructokinase (ePFKM) were determined by real-time PCR and western blot analysis. The mRNA expression level of eGYS1 was significantly higher in the cervical muscle as compared to other tissues. The cervical muscle and heart tissue samples contained a broad range of eGYS1 protein bands that appeared to reflect multiple phosphorylation states. eHKII was predominately expressed only in the cervical muscle; unlike its expression in other mammals, eHKII was not substantially expressed in the insulin-responsive heart or adipose tissue of horse. The expression level of ePFKM mRNA was significantly higher in the heart than in the cervical muscle, which differs from the PFKM expression pattern of other mammals. These tissue expression profiles are fundamental for the understanding of equine glucose metabolism.