Flux balance analysis predicts Warburg-like effects of mouse hepatocyte deficient in miR-122a.

The liver is a vital organ involving in various major metabolic functions in human body. MicroRNA-122 (miR-122) plays an important role in the regulation of liver metabolism, but its intrinsic physiological functions require further clarification. This study integrated the genome-scale metabolic model of hepatocytes and mouse experimental data with germline deletion of Mir122a (Mir122a-/-) to infer Warburg-like effects. Elevated expression of MiR-122a target genes in Mir122a-/-mice, especially those encoding for metabolic enzymes, was applied to analyze the flux distributions of the genome-scale metabolic model in normal and deficient states. By definition of the similarity ratio, we compared the flux fold change of the genome-scale metabolic model computational results and metabolomic profiling data measured through a liquid-chromatography with mass spectrometer, respectively, for hepatocytes of 2-month-old mice in normal and deficient states. The Ddc gene demonstrated the highest similarity ratio of 95% to the biological hypothesis of the Warburg effect, and similarity of 75% to the experimental observation. We also used 2, 6, and 11 months of mir-122 knockout mice liver cell to examined the expression pattern of DDC in the knockout mice livers to show upregulated profiles of DDC from the data. Furthermore, through a bioinformatics (LINCS program) prediction, BTK inhibitors and withaferin A could downregulate DDC expression, suggesting that such drugs could potentially alter the early events of metabolomics of liver cancer cells.

Self-compatibility of 'Zaoguan' (Pyrus bretschneideri Rehd.) is associated with style-part mutations.

The pear cultivar 'Zaoguan' (S(4)S(34)) is the a self-compatible descendant of 'Yali' (S(21)S(34)) × 'Qingyun'(S(4)S(9)). Two self-incompatible cultivars 'Xinya' and 'Yaqing', also S-genotyped as S(4)S(34) for the S-RNase gene, were used as controls. Field pollination data revealed that 'Zaoguan' displayed SC, whereas 'Xinya' and 'Yaqing' showed self-incompatibility (SI) upon self-pollination. Reciprocal pollinations between the varieties showed that most of the 'Zaoguan' flowers pollinated with 'Xinya' or 'Yaqing' pollen set fruits but that few of the 'Xinya' or 'Yaqing' flowers set fruit when pollinated with 'Zaoguan' pollen. The pollen performance was monitored with fluorescence microscopy, and we observed that 'Zaoguan' accepted self-pollen as well as 'Xinya' or 'Yaqing' pollen, whereas 'Xinya' or 'Yaqing' rejected self-pollen and 'Zaoguan' pollen. The S(34)-RNase but not the S(4)-RNase could be detected in all selfed progeny of 'Zaoguan'. Comparisons of the 2D-PAGE profiles of the stylar extracts from the three cultivars showed that the S(4)-RNase protein expressed normally, but the S(34)-RNase of 'Zaoguan' was not found. Thus, we concluded that the stylar S(34) products were defective in 'Zaoguan' and that the S (4)-allele functioned normally. The nucleotide sequences of the S(4)- and S(34)-RNase of 'Zaoguan' showed no differences from those of 'Xinya' or 'Yaqing', and they transcribed normally. These results indicate that SC in 'Zaoguan' was due to the loss of the S(34)-RNase caused by unknown post-transcriptional factors.

Reciprocal regulation of Ca²+-activated outward K+ channels of Pyrus pyrifolia pollen by heme and carbon monoxide.

• The regulation of plant potassium (K+) channels has been extensively studied in various systems. However, the mechanism of their regulation in the pollen tube is unclear. • In this study, the effects of heme and carbon monoxide (CO) on the outward K+ (K+(out)) channel in pear (Pyrus pyrifolia) pollen tube protoplasts were characterized using a patch-clamp technique. • Heme (1 µM) decreased the probability of K+(out) channel opening without affecting the unitary conductance, but this inhibition disappeared when heme was co-applied with 10 µM intracellular free Ca²+. Conversely, exposure to heme in the presence of NADPH increased channel activity. However, with tin protoporphyrin IX treatment, which inhibits hemeoxygenase activity, the inhibition of the K+(out) channel by heme occurred even in the presence of NADPH. CO, a product of heme catabolism by hemeoxygenase, activates the K+(out) channel in pollen tube protoplasts in a dose-dependent manner. The current induced by CO was inhibited by the K+ channel inhibitor tetraethylammonium. • These data indicate a role of heme and CO in reciprocal regulation of the K+(out) channel in pear pollen tubes.

Self-compatibility of 'Katy' apricot (Prunus armeniaca L.) is associated with pollen-part mutations.

Apricot (Prunus armeniaca L.) cultivars originated in China display a typical S-RNase-based gametophytic self-incompatibility (GSI). 'Katy', a natural self-compatible cultivar belonging to the European ecotype group, was used as a useful material for breeding new cultivars with high frequency of self-compatibility by hybridizing with Chinese native cultivars. In this work, the pollen-S genes (S-haplotype-specific F-box gene, or SFB gene) of 'Katy' were first identified as SFB1 and SFB (8), and the S-genotype was determined as S1 S8. Genetic analysis of 'Katy' progenies under controlled pollination revealed that the stylar S1-RNase and S8-RNase have a normal function in rejecting wild-type pollen with the same S-haplotype, while the pollen grains carrying either the SFB1 or the SFB8 gene are both able to overcome the incompatibility barrier. However, the observed segregation ratios of the S-genotype did not fit the expected ratios under the assumption that the pollen-part mutations are linked to the S-locus. Moreover, alterations in the SFB1 and SFB8 genes and pollen-S duplications were not detected. These results indicated that the breakdown of SI in 'Katy' occurred in pollen, and other factors not linked to the S-locus, which caused a loss of pollen S-activity. These findings support a hypothesis that modifying factors other than the S-locus are required for GSI in apricot.

S-RNase disrupts tip-localized reactive oxygen species and induces nuclear DNA degradation in incompatible pollen tubes of Pyrus pyrifolia.

Pear (Pyrus pyrifolia L.) has an S-RNase-based gametophytic self-incompatibility (SI) mechanism, and S-RNase has also been implicated in the rejection of self-pollen and genetically identical pollen. However, RNA degradation might be only the beginning of the SI response, not the end. Recent in vitro studies suggest that S-RNase triggers mitochondrial alteration and DNA degradation in the incompatible pollen tube of Pyrus pyrifolia, and it seems that a relationship exists between self S-RNase, actin depolymerization and DNA degradation. To further uncover the SI response in pear, the relationship between self S-RNase and tip-localized reactive oxygen species (ROS) was evaluated. Our results show that S-RNase specifically disrupted tip-localized ROS of incompatible pollen tubes via arrest of ROS formation in mitochondria and cell walls. The mitochondrial ROS disruption was related to mitochondrial alteration, whereas cell wall ROS disruption was related to a decrease in NADPH. Tip-localized ROS disruption not only decreased the Ca(2+) current and depolymerized the actin cytoskeleton, but it also induced nuclear DNA degradation. These results indicate that tip-localized ROS disruption occurs in Pyrus pyrifolia SI. Importantly, we demonstrated nuclear DNA degradation in the incompatible pollen tube after pollination in vivo. This result validates our in vitro system in vivo.

S-RNase triggers mitochondrial alteration and DNA degradation in the incompatible pollen tube of Pyrus pyrifolia in vitro.

Pear (Pyrus pyrifolia L.) has a S-RNase-based gametophytic self-incompatibility (SI) mechanism, and S-RNase has also been implicated in the rejection of self-pollen and genetically identical pollen. No studies, however, have examined the extent of organelle alterations during the SI response in Pyrus pyrifolia. Consequently, this study focused on the alterations to mitochondria and nuclear DNA in incompatible pollen tubes of the pear. Methylthiazolyldiphenyl-tetrazolium bromide was used to evaluate the viability of pollen tubes under S-RNase challenge. The results showed that the viability of the control and compatible pollen tubes decreased slightly, but that of the incompatible pollen and pollen tubes began to decline at 30 min. The mitochondrial membrane potential (Delta psi(mit)) was also tested with rhodamine 123 30 min after SI challenge, and was shown to have collapsed in the incompatible pollen tubes after exposure to S-RNase. Western blotting 2 h after SI challenge confirmed that the Delta psi(mit) collapse induced leakage of cytochrome c into the cytosol. Swollen mitochondria were detected by transmission electron microscopy as early as 1 h after SI challenge and the degradation of nuclear DNA was observed by both 4,6-diamidino-2-phenylindole and transferase-mediated dUTP nick-end labeling. These diagnostic features of programmed cell death (PCD) suggested that PCD may specifically occur in incompatible pollen tubes.