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Granulosa cells play important roles in the regulation of ovarian functions. Phospholipase C is crucial in several signalling pathways and could participate in the molecular mechanisms of cell proliferation, differentiation and ageing. The objective of this study was to identify the effects of phospholipase C on the steroidogenesis of oestradiol and progesterone in porcine granulosa cells cultured in vitro. Inhibitor U73122 or activator m-3M3FBS of phospholipase C was added to the in vitro medium of porcine granulosa cells, respectively. The secretion of oestradiol decreased after 2 hr, 8 hr, 12 hr, 24 hr and 48 hr of treatment with 500 nM U73122 (p < .05) and decreased after 2 hr of treatment in the 500 nM m-3M3FBS addition group (p < .05). The secretion of progesterone increased after 4 hr of treatment with 500 nM U73122 (p < .05) and increased after 2 hr and 8 hr of treatment in the 500 nM m-3M3FBS addition group (p < .05). The ratio of oestradiol to progesterone decreased at each time point, except 8 hr after the addition of 500 nM U73122 (p < .05). The ratio of oestradiol to progesterone decreased after 2 hr (p < .05) of treatment with 500 nM m-3M3FBS. In genes that regulate the synthesis of oestradiol or progesterone, the mRNA expression of CYP11A1 was markedly increased (p < .05), and the mRNA expression of other genes did not change significantly in the U73122 treatment group, while the addition of m-3M3FBS did not change those genes significantly despite the contrary trend. Our results demonstrated that phospholipase C can be a potential target to stimulate the secretion of oestradiol and suppress progesterone secretion in porcine granulosa cells cultured in vitro, which shed light on a novel biological function of phospholipase C in porcine granulosa cells.

Coenzyme A (CoA) thioesters (acyl-CoAs) are essential intermediates of metabolism. Inborn errors of acyl-CoA metabolism include a large fraction of the classical organic acidemias. These conditions can involve liver, muscle, heart and brain, and can be fatal. These conditions are increasingly detected by newborn screening. There is a renewed interest in CoA metabolism and in developing effective new treatments. Here, we review theories of the pathophysiology in relation to mitochondrial CoA sequestration, toxicity and redistribution (CASTOR).

Phospholipase C (PLC) can participate in cell proliferation, differentiation and aging. However, whether it has a function in apoptosis in porcine primary granulosa cells is largely uncertain. The objective of this study was to examine the effects of PLC on apoptosis of porcine primary granulosa cells cultured in vitro. The mRNA expression of BAK, BAX and CASP3, were upregulated in the cells treated with U73122 (the PLC inhibitor). The abundance of BCL2 mRNA, was upregulated, while BAX and CASP3 mRNA expression was decreased after treatment with m-3M3FBS (the PLC activator). Both the early and late apoptosis rate were maximized with 0.5 μM U73122 for 4 h. The rate of early apoptosis was the highest at 4 h and the rate of late apoptosis was the highest at 12 h in the m-3M3FBS group. The protein abundance of PLCβ1, protein kinase C β (PKCβ), calmodulin-dependent protein kinaseII α (CAMKIIα) and calcineurinA (CalnA) were decreased by U73122, and CAMKIIα protein abundance was increased by m-3M3FBS. The mRNA expression of several downstream genes (CDC42, NFATc1, and NFκB) was upregulated by PLC. Our results demonstrated that apoptosis can be inhibited by altering PLC signaling in porcine primary granulosa cells cultured in vitro, and several calcium−sensitive targets and several downstream genes might take part in the processes.

Parkinson's disease (PD) is characterized by the loss of dopaminergic neurons in the substantia nigra and striatum. Aging is the most important risk factor of PD. Ferroptosis is an iron-dependent form of cell death associated with PD. However, it is not clear whether ferroptosis accelerates PD by promoting cellular senescence. This study investigated the mechanism of 1-methyl-4-phenylpyridinium (MPP+) -induced PC12 cells injury. We found that MPP+ induced cell senescence with increased β-galactosidase activity and the expression of p53, p21 and p16 activation in cells. In addition, MPP+ treatment showed smaller mitochondria and increased membrane density, downregulation of ferritin heavy chain 1 expression and upregulation of acyl-CoA synthetase long chain family member 4 expression, and enhanced levels of oxidative stress, which were important characteristics of ferroptosis. Ferrostatin-1 (Fer-1), a ferroptosis inhibitor, was tested to eliminate MPP+-induced cell senescence. Fer-1 downregulated the expression of p53 and upregulated the expression of solute carrier family 7 member 11 (SLC7A11) and glutathione peroxidase-4 (GPX4) in MPP+-induced ferroptosis. Inhibition of p53 eliminated cell senescence by upregulation the expression of of SLC7A11 and GPX4. Thus, these results suggest that MPP+ induces senescence in PC12 cells via the p53/ SLC7A11/ GPX4 signaling pathway in the ferroptosis regulation mechanism.

Melanoma is characterized by high malignancy and early onset of metastasis. Epithelial-to-mesenchymal transition (EMT) is an early event during tumor metastasis. Tumor cells that develop EMT can escape apoptosis, but they are vulnerable to ferroptosis inducers. Gambogenic acid (GNA), a xanthone found in Gamboge, has cytotoxic effects in highly invasive melanoma cells. This study investigated the anti-melanoma effect and mechanism of action of GNA in TGF-β1-induced EMT melanoma cells. We found that GNA significantly inhibited the invasion, migration and EMT in melanoma cells, and these cells exhibited small mitochondrial wrinkling (an important feature of ferroptosis). An iron chelator, but not an apoptosis inhibitor or a necrosis inhibitor, abolished the inhibitory effects of GNA on proliferation, invasion and migration of TGF-β1-stimulated melanoma cells. GNA upregulated the expression of p53, solute carrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4) in the model cells, contributing to the mechanisms underlying GNA-induced ferroptosis. Collectively, our findings suggest that GNA induces ferroptosis in TGF-β1-stimulated melanoma cells via the p53/SLC7A11/GPX4 signaling pathway.

Alcohol consumption causes liver steatosis in humans. Metabolic disorders of lipids are one of the factors that cause liver steatosis in hepatocytes. Hepatic Niemann‑Pick C1‑like 1 (NPC1L1) regulates lipid homeostasis in mammals. The relationship between NPC1L1 and autophagy in those with a history of alcohol abuse is unclear. The present study aimed to investigate the function of NPC1L1 in the activation of hepatic autophagy in a mouse model with a human (h)NPC1L1 transgene under alcohol feeding conditions. The mice expressing hNPC1L1 (Ad‑L1) or controls (Ad‑null) were created by retro‑orbital adenovirus injection. The Ad‑L1 and Ad‑null mice were fed with alcohol or a non‑alcoholic diet to mimic chronic alcohol consumption in humans. Hepatic autophagy was demonstrated in isolated primary hepatocytes by monitoring autophagic vacuoles under fluorescence microscopy, and by western blotting for autophagic makers. Isolated hepatocytes from the livers of Ad‑L1 mice were treated with different doses of ezetimibe to study the restoration of autophagy. Chronic alcohol feeding caused liver injury and steatosis, shown by significantly higher levels of plasma alanine transaminase and aspartate transaminase activity, and by hematoxylin and eosin staining in Ad‑L1 and Ad‑null mice. Compared to Ad‑null control mice, the microtubule‑associated proteins 1A/1B light chain 3 (LC3) particles in the isolated hepatocytes of Ad‑L1 mice were decreased, both under alcohol and non‑alcoholic feeding. The ratio of LC3II/LC3I was significantly decreased, and the level of p62/sequestosome‑1 protein was significantly increased in Ad‑L1 mice compared with Ad‑null mice after alcohol feeding. Levels of LC3II protein were statistically increased in hepatocytes isolated from Ad‑L1 mice with ezetimibe treatment. The increase in LC3II expression was dose dependent. Within the tested range, it reached its highest level at 40 µM. The livers of Ad‑L1 mice represent a more human‑like state for the study of hepatic autophagy. Hepatic expression of human NPC1L1 resulted in an inhibition of autophagy; it may contribute to alcoholic fatty liver disease in humans.

The last decade saw major advances in understanding the metabolism of Coenzyme A (CoA) thioesters (acyl-CoAs) and related inborn errors (CoA metabolic diseases, CAMDs). For diagnosis, acylcarnitines and organic acids, both derived from acyl-CoAs, are excellent markers of most CAMDs. Clinically, each CAMD is unique but strikingly, three main patterns emerge: first, systemic decompensations with combinations of acidosis, ketosis, hypoglycemia, hyperammonemia and fatty liver; second, neurological episodes, particularly acute "stroke-like" episodes, often involving the basal ganglia but sometimes cerebral cortex, brainstem or optic nerves and third, especially in CAMDs of long chain fatty acyl-CoA metabolism, lipid myopathy, cardiomyopathy and arrhythmia. Some patients develop signs from more than one category. The pathophysiology of CAMDs is not precisely understood. Available data suggest that signs may result from CoA sequestration, toxicity and redistribution (CASTOR) in the mitochondrial matrix has been suggested to play a role. This predicts that most CAMDs cause deficiency of CoA, limiting mitochondrial energy production, and that toxic effects from the abnormal accumulation of acyl-CoAs and from extramitochondrial functions of acetyl-CoA may also contribute. Recent progress includes the following. (1) Direct measurements of tissue acyl-CoAs in mammalian models of CAMDs have been related to clinical features. (2) Inborn errors of CoA biosynthesis were shown to cause clinical changes similar to those of inborn errors of acyl-CoA degradation. (3) CoA levels in cells can be influenced pharmacologically. (4) Roles for acetyl-CoA are increasingly identified in all cell compartments. (5) Nonenzymatic acyl-CoA-mediated acylation of intracellular proteins occurs in mammalian tissues and is increased in CAMDs.

Background and aims Niemann-Pick C1-like1 (NPC1L1), a crucial cholesterol absorption receptor expressed in human intestine and liver. But in mouse it is only expressed in intestine. Previous studies elucidated that expression of human NPC1L1 in mouse liver led to increase of plasma cholesterol due to activation of absorption from bile. However, hepatic NPC1L1 function was not elucidated in LDL receptor deficient mouse (LDLR−/−) in which LDL was a main lipoprotein as in human. Methods and results L1-Tg/LDLR−/− mouse was created by crossing liver-specific NPC1L1 transgenic mouse (L1-Tg) with LDLR−/−. L1-Tg/LDLR−/− mice developed hyperlipidemia when fed with atherogenic diet (AD) containing 0.2% cholesterol for 21days. Compared with control mice, biliary cholesterol level in L1-Tg/LDLR−/− mice was significantly lower, plasma cholesterol level was significantly higher in L1-Tg/LDLR−/− mice under both chow diet and AD feeding. New finding in this study is augmentations of plasma TAG L1-Tg/LDLR−/− mice fed with AD. Results were shown that very low density lipoprotein (VLDL) secretion was elevated in L1-Tg/LDLR−/− mice after AD fed. The increase of VLDL secretion was further confirmed by higher expression of hepatic triacylglycerol hydrolase (TGH) and microsomal triglyceride transfer protein (MTP). Conclusion L1-Tg/LDLR−/− mouse is a humanized model to study cholesterol absorption and transportation. The results obtained from L1-Tg/LDLR−/− mouse indicated no feedback mechanism to inhibit NPC1L1 function in liver and hepatic expression of NPC1L1 correlated with VLDL secretion in hypercholesterolemia state.

The asymmetrical crystal [Na(15C5)2][Na(15C5)]2(AsMo12O40)]·CH3CN (1) was synthesized. Interestingly, the huge α-keggin ion AsMo12 is distorted by introduction of CH3CN molecules and adopts a chiral assembly with space group P4. In contrast, the analogous compound AsW12 co-crystallizes with trace H2O molecules. The hydrogen atoms of H2O are positionally disordered with 50 % occupation to correlate with AsW12 in space group P4/m. Solid and solution CD spectra indicate that the crystals of 1 are in R configuration. This leads to the conclusion that molybdenum has greater affinity toward organic CH3CN, whereas tungsten has greater affinity to inorganic H2O. Additionally, AsMo12 shows greater deformability than AsW12. It is suggested that POMo shows a greater potential as chiral material than POW because of the flexibility of Mo. As a highlight, CH3CN can function as a general chiral inducer for the innovation of other chiral crystals, if fixed along one dimension.

Fudosteine [(-)-(R)-2-amino-3-(3-hydroxypropylthio) propionic acid] is a synthetic derivative of l-cysteine as a novel expectorant. To facilitate pharmacokinetics studies of fudosteine in man, a sensitive and specific LC–MS–MS method for the quantitative detection of fudosteine in human plasma was developed and validated. The method involved the addition of fosfomycin as internal standards, protein-precipitation, HPLC separation, and quantification by MS/MS system using negative electrospray ionization in the multiple reaction monitoring mode. The precursor → product ion transitions were monitored at 177.6 → 90.9 and 136.8 → 78.9 for fudosteine and the IS, respectively. The lower limit of quantitation (LLOQ) was 5 ng mL−1. The calibration curves for fudosteine was linear over a concentration range of 0.005–10 µg mL−1. The intra- and inter-day analyses of QC samples at 0.01, 1, 10 µg mL−1 indicated good precision %RSD between 92.00 and 101.02%. The fudosteine was stable in human plasma stored at room temperature for at least 12 h, 4 °C for at least 12 h, −20 °C for at least 20 days, and at least three freeze–thaw cycles procedures. This accurate and highly specific assay provides a useful method for evaluating the pharmacokinetics of fudosteine in human.

The role of calcium in mediating resistance to several auxinic herbicides (i.e., 2,4-dichlorophenoxyacetic acid, [4-chloro-2-methylphenoxy] acetic acid, (±)-2-(4-chloro-2-methylphenoxy) propanoic acid [mecoprop], 3,6-dichloro-2-methoxy-benzoic acid [dicamba], or 4-amino-3, 5,6-trichloro-2-pyridinecarboxylic acid [picloram]) was investigated by modulating calcium dynamics of a susceptible (S) and resistant (R) biotype of wild mustard. The inhibitory effects of the auxinic herbicides on root length of the S seedlings were significantly reduced upon pretreatment with calcium in the presence of the calcium ionophore A23187. Conversely, the addition of verapamil, a calcium channel blocker, to the R seedlings increased their sensitivity to the auxinic herbicides. Valinomycin, a potassium channel ionophore, did not ameliorate the effect of the auxinic herbicides on both biotypes of wild mustard, thus indicating that the observed effects were specific for calcium. These results demonstrate that calcium p...

5-tert-Butyl-carbamoyloxy-3-(3-trifluoromethyl) phenyl-4-thiazolidinone (compound 1), a representative of a novel class of thiazolidinone herbicides, shows potential for the pre-emergence control of grasses and small-seeded broad-leaved weeds in crops such as soyabean. This compound powerfully inhibited the growth and lateral branching of the roots of agar-grown seedlings of susceptible species but had no apparent effect on the growth of Escherichia coli, Aspergillus nidulans, Fusarium culmorum or of insects cells in liquid culture. It inhibited the growth of plant (Daucus carota and Zea mays) cells in liquid culture and this inhibition was not reversed by addition to the medium of mixtures of either amino acids or of nucleosides. It did not inhibit fatty acid biosynthesis, respiration, the biosynthesis of sterols, or the biosynthesis of protein in the systems examined herein. By contrast, it induced potent (IC50 c.50 nM) and rapid inhibition of the incorporation of [3H]glucose into the acid-insoluble cell-wall fraction of roots. Thus, compound 1 exerts its herbicidal effect, directly or indirectly, through inhibition of the biosynthesis of cellulose and cellulose-like polysaccharides, in a manner similar to isoxaben and dichlobenil. Mutant lines of Arabidopsis thaliana selected for resistance to isoxaben were cross-resistant to compound 1. Consistent with the selective herbicidal activity observed, the compound was a potent inhibitor of [3H]glucose incorporation into the polysaccharide of seedling roots of Zea mays and of Setaria viridis but only relatively weakly active on Glycine max and Ipomoea hederacea. Given that compound 1 appeared to be metabolised only slowly and that [3H]glucose incorporation experiments were conducted within a total period of less than 90 min, it seems probable that, as in the case of the acetyl CoA carboxylase-inhibitor graminicides, the observed selectivity is determined by species-dependent differences at the (in this case unknown) molecular target site for the herbicide. © 1998 Society of Chemical Industry