A new method for measuring cholesterol efflux capacity uses stable isotope-labeled, not radioactive-labeled, cholesterol.

The incidence of cardiovascular events correlates inversely with cholesterol efflux capacity (CEC) more than with HDL-cholesterol level. The measurement of CEC is used to qualify cardiovascular disease risk and is conventionally performed with radioisotope (RI)-labeled cholesterol. Here, we established a CEC measurement technique using stable isotope-labeled cholesterol as an alternative, and we compared the new method with RI and fluorescence (boron dipyrromethene difluoride-cholesterol) methods in cells and in patient serum. We incubated J774 cells labeled with [d7]cholesterol ([d7]C) with patient serum depleted of apoB, and [d7]C extracted from the culture medium was quantified by liquid chromatography/quadrupole time-of-flight mass spectrometry. [d7]C efflux increased with greater apoB-depleted serum concentration and longer incubation time. The assay coefficient of variation (CV) of five consecutive measurements of three sets of samples ranged from 7.3% to 9.5%, and the interassay CV determined by measuring three samples four times ranged from 4.1% to 8.5%, both indicating good precision. We then measured CEC levels of 41 outpatients with serum HDL-cholesterol levels between 36 and 94 mg/dl (mean: 61.7 ± 18.0 mg/dl); in the presence of cAMP, we observed a significant, positive correlation between CEC levels determined with the stable isotope and RI methods that was stronger than the correlation between measurements obtained by the fluorescence and RI methods (r = 0.73, P < 0.0001 vs. r = 0.55, P < 0.001). Therefore, our stable isotope method can be considered useful as a non-RI method and thus deserves evaluation in future clinical studies.

Behavior of ACRBP-deficient mouse sperm in the female reproductive tract.

Gene-knockout mice lacking ACRBP, a proacrosin-binding protein localized in the acrosome of sperm, have been shown to exhibit male subfertility, owing to abnormal formation of the acrosome. In this study, to elucidate the mechanism contributing to the subfertility phenotype, we examined the behavior of ACRBP-deficient mouse sperm in the female reproductive tract. When sperm that had migrated into the uterus and oviduct after mating were counted, the number of ACRBP-deficient sperm was noticeably smaller in the oviduct of mice post mating. However, ACRBP-deficient sperm recovered from the oviduct possessed morphologically normal head shape and retained normal motility. Importantly, ACRBP-deficient sperm displayed a marked reduction in the ability to successfully gain access to unfertilized oocytes. These data suggest that male subfertility of ACRBP-deficient mice may be attributed to incompleteness of the acrosome reaction rather than impairment in sperm migration from the uterus to the oviduct.

Double-stranded RNA-binding protein DRB3 negatively regulates anthocyanin biosynthesis by modulating PAP1 expression in Arabidopsis thaliana.

The model plant Arabidopsis thaliana has five double-stranded RNA-binding proteins (DRB1-DRB5), two of which, DRB1 and DRB4, are well characterized. In contrast, the functions of DRB2, DRB3 and DRB5 have yet to be elucidated. In this study, we tried to uncover their functions using drb mutants and DRB-over-expressed lines. In over-expressed lines of all five DRB genes, the over-expression of DRB2 or DRB3 (DRB2ox or DRB3ox) conferred a downward-curled leaf phenotype, but the expression profiles of ten small RNAs were similar to that of the wild-type (WT) plant. Phenotypes were examined in response to abiotic stresses. Both DRB2ox and DRB3ox plants exhibited salt-tolerance. When these plants were exposed to cold stress, drb2 and drb3 over-accumulated anthocyanin but DRB2ox and DRB3ox did not. Therefore, the over-expression of DRB2 or DRB3 had pleiotropic effects on host plants. Microarray and deep-sequencing analyses indicated that several genes encoding key enzymes for anthocyanin biosynthesis, including chalcone synthase (CHS), dihydroflavonol reductase (DFR) and anthocyanidin synthase (ANS), were down-regulated in DRB3ox plants. When DRB3ox was crossed with the pap1-D line, which is an activation-tagged transgenic line that over-expresses the key transcription factor PAP1 (Production of anthocyanin pigmentation1) for anthocyanin biosynthesis, over-expression of DRB3 suppressed the expression of PAP1, CHS, DFR and ANS genes. DRB3 negatively regulates anthocyanin biosynthesis by modulating the level of PAP1 transcript. Since two different small RNAs regulate PAP1 gene expression, a possible function of DRB3 for small RNA biogenesis is discussed.

Surfing and Swimming of Ejaculated Sperm in the Mouse Oviduct.

To accomplish fertilization in the oviductal ampulla, ejaculated sperm are required to migrate through the female reproductive tract. However, this fundamental process largely remains unknown. In this study, we focused on the role of oviductal smooth muscle (myosalpinx) contractions in the sperm migration. Administration of prifinium bromide, padrin, to mice effectively suppressed myosalpinx contractions, resulting in a decreased rate of fertilization in a dose-dependent manner, and an abrogation of high-speed back-and-forth/shuttling flows of oviductal fluids around the isthmus. Regardless of padrin administration, no shuttling flows were found near the ampulla. In the isthmus, sperm formed a tight assemblage that was synchronized with the shuttling flows. The sperm assemblage was gradually loosened and then completely abolished near the ampulla. No sperm assemblage was formed in the isthmus when padrin was administrated. These results suggest that myosalpinx contractions play important roles in the formation of sperm assemblage in the isthmus, and in the transport of the assemblage to the middle region of the oviduct. It is also suggested that the motility of sperm is essential for the migration of sperm from the middle oviductal region to the ampulla.