Diacylglycerol kinase inhibitor R59022-induced autophagy and apoptosis in the neuronal cell line NG108-15

Phosphatidic acid (PA) is a lipid second messenger and is believed to be involved in cell proliferation and survival. PA is mainly produced by phospholipase D (PLD) and diacylglycerol kinase (DGK). Elevated PLD activity is believed to suppress apoptosis via activation of the mammalian target of rapamycin (mTOR). On the other hand, DGK inhibition has been demonstrated to induce apoptosis, but it is unclear whether DGK can regulate mTOR. Here, we investigated whether DGK inhibition can induce apoptosis and autophagy in neuronal cells, since mTOR is a key mediator of autophagy and the simultaneous activation of apoptosis and autophagy has been detected. A DGK inhibitor, R59022 induced autophagy and apoptosis without serum in NG108-15 cells. Autophagy preceded apoptosis, and apoptosis inhibition did not affect R59022-induced autophagy. R59022-induced autophagy was inhibited by exogenous PA, and protein kinase C activation and increases in intracellular Ca²⁺ levels, which are assumed to be caused by diacylglycerol accumulation, did not appear to be involved in R59022-induced autophagy. We also investigated the effects of R59022 on mTOR signaling pathway, and found that the pathway was not inhibited by R59022. These results imply that DGK plays an important role in cell survival via mTOR-independent mechanism.     

Differences in sex expression and mating systems in three pollination morphs of Cimicifuga simplex

Cimicifuga simplex (Ranunculaceae) has three genetically distinct pollination morphs. Here, we report that each of the three pollination morphs of C. simplex differs from the others with regard to sex expression and mating system: morph I consists mostly of ramets with hermaphroditic flowers and ramets with only female flowers, morph II consists of ramets with hermaphroditic flowers and ramets with hermaphroditic and male flowers, and morph III consists mostly of ramets with hermaphroditic flowers. A microsatellite analysis of seed DNA showed that morph III has a high self-fertilization rate. Flowering season and flower visitor assemblages, which also differ among the three morphs, may influence the evolution and maintenance of the differences in sex expression and mating systems in the morphs.     

MPN estimation of qPCR target sequence recoveries from whole cell calibrator samples

DNA extracts from enumerated target organism cells (calibrator samples) have been used for estimating Enterococcus cell equivalent densities in surface waters by a comparative cycle threshold (Ct) qPCR analysis method. To compare surface water Enterococcus density estimates from different studies by this approach, either a consistent source of calibrator cells must be used or the estimates must account for any differences in target sequence recoveries from different sources of calibrator cells. In this report we describe two methods for estimating target sequence recoveries from whole cell calibrator samples based on qPCR analyses of their serially diluted DNA extracts and most probable number (MPN) calculation. The first method employed a traditional MPN calculation approach. The second method employed a Bayesian hierarchical statistical modeling approach and a Monte Carlo Markov Chain (MCMC) simulation method to account for the uncertainty in these estimates associated with different individual samples of the cell preparations, different dilutions of the DNA extracts and different qPCR analytical runs. The two methods were applied to estimate mean target sequence recoveries per cell from two different lots of a commercially available source of enumerated Enterococcus cell preparations. The mean target sequence recovery estimates (and standard errors) per cell from Lot A and B cell preparations by the Bayesian method were 22.73 (3.4) and 11.76 (2.4), respectively, when the data were adjusted for potential false positive results. Means were similar for the traditional MPN approach which cannot comparably assess uncertainty in the estimates. Cell numbers and estimates of recoverable target sequences in calibrator samples prepared from the two cell sources were also used to estimate cell equivalent and target sequence quantities recovered from surface water samples in a comparative Ct method. Our results illustrate the utility of the Bayesian method in accounting for uncertainty, the high degree of precision attainable by the MPN approach and the need to account for the differences in target sequence recoveries from different calibrator sample cell sources when they are used in the comparative Ct method.     

Structural requirements of flavonoids for the adipogenesis of 3T3-L1 cells

To search for a new class of antidiabetic compounds, effects of 44 flavonoids on the adipogenesis of 3T3-L1 cells were examined. Among them, 3,4',7-trimethylkaempferol, tetramethylkaempferol, and pentamethylquercetin concentration-dependently enhanced the accumulation of triglyceride, a marker of adipogenesis. With regard to structural requirements of flavonoids for the activity, it was fond that: (1) most flavonoids having hydroxy groups lacked the effect; (2) flavonols with methoxy groups showed stronger effects particularly those with a methoxy group at the 3-position; and (3) a methoxy group of flavonols at the B ring was also important. 3,4',7-Trimethylkaempferol, tetramethylkaempferol, and pentamethylquercetin significantly increased the amount of adiponectin released into the medium and the uptake of 2-deoxyglucose into the cells. Furthermore, tetramethylkaempferol and pentamethylquercetin also increased mRNA levels of adiponectin, glucose transporter 4 (GLUT4), and fatty acid-binding protein (aP2). Both compounds also increased the mRNA levels of peroxisome proliferator-activated receptor (PPAR)γ2 and CCAAT/enhancer-binding protein (C/EBP)α, β, and/or δ, although, different from troglitazone, they did not activate PPARγ directly in a nuclear receptor cofactor assay.     

In vitro evaluation of the behaviour of human polymorphonuclear neutrophils in direct contact with chitosan-based membranes

Several novel biodegradable materials have been proposed for wound healing applications in the past few years. Taking into consideration the biocompatibility of chitosan-based biomaterials, and that they promote adequate cell adhesion, this work aims at investigating the effect of chitosan-based membranes, over the activation of human polymorphonuclear neutrophils (PMNs). The recruitment and activation of polymorphonuclear neutrophils (PMNs) reflects a primary reaction to foreign bodies. Activation of neutrophils results in the production of reactive oxygen species (ROS) such as O(2)(-) and HO(-) and the release of hydrolytic enzymes which are determinant factors in the inflammatory process, playing an essential role in the healing mechanisms. PMNs isolated from human peripheral blood of healthy volunteers were cultured in the presence of chitosan or chitosan/soy newly developed membranes. The effect of the biomaterials on the activation of PMNs was assessed by the quantification of lysozyme and ROS. The results showed that PMNs, in the presence of the chitosan-based membranes secrete similar lysozyme amounts, as compared to controls (PMNs without materials) and also showed that the materials do not stimulate the production of either O(2)(-) or HO(-). Moreover, PMNs incubated with the biomaterials when stimulated with phorbol 12-myristate 13-acetate (PMA) or formyl-methionyl-leucyl-phenylalanine (fMLP) showed a chemiluminescence profile with a slightly lower intensity, to that observed for positive controls (cells without materials and stimulated with PMA), which reflects the maintenance of their stimulation capacity. Our data suggests that the new biomaterials studied herein do not elicit activation of PMNs, as assessed by the low lysozyme activity and by the minor detection of ROS by chemiluminescence. These findings reinforce previous statements supporting the suitability of chitosan-based materials for wound healing applications.     

Elevated cardiac tissue level of aldosterone and mineralocorticoid receptor in diastolic heart failure: Beneficial effects of mineralocorticoid receptor blocker

Cardiac aldosterone levels have not been evaluated in diastolic heart failure (DHF), and its roles in this type of heart failure remain unclear. This study aimed to detect cardiac aldosterone by use of a liquid chromatographic-mass spectrometric method and to assess the effects of mineralocorticoid receptor blockade on hypertensive DHF. Dahl salt-sensitive rats fed 8% NaCl diet from 7 wk (hypertensive DHF model) were divided at 13 wk into three groups: those treated with subdepressor doses of eplerenone (12.5 or 40 mg x kg(-1) x day(-1)) and an untreated group. Dahl salt-sensitive rats fed 0.3% NaCl diet served as controls. Cardiac aldosterone was detected in the DHF rats but not in the control rats, with increased ventricular levels of mineralocorticoid receptor. Cardiac levels of 11-deoxycorticosterone, corticosterone, and 11-dehydrocorticosterone were not different between the control and DHF rats, but the tissue level of corticosterone that has an affinity to mineralocorticoid receptor was 1,000 times as high as that of aldosterone. Aldosterone synthase activity and CYP11B2 mRNA were undetectable in the ventricular tissue of the DHF rats. Administration of eplerenone attenuated ventricular hypertrophy, ventricular fibrosis, myocardial stiffening, and relaxation abnormality, leading to the prevention of overt DHF. In summary, the myocardial aldosterone level increased in the DHF rats. However, its value was extremely low compared with corticosterone, and no evidence for enhancement of intrinsic myocardial aldosterone production was found. The upregulation of mineralocorticoid receptor may play a central role in the pathogenesis of DHF, and blockade of mineralocorticoid receptor is likely an effective therapeutic regimen of DHF.     

AMSH, an ESCRT-III associated enzyme, deubiquitinates cargo on MVB/late endosomes

The appropriate sorting of vesicular cargo, including cell-surface proteins, is critical for many cellular functions. Ubiquitinated cargo is targeted to endosomes and digested by lysosomal enzymes. We previously identified AMSH, a deubiquitination enzyme (DUB), to be involved in vesicular transport. Here, we purified an AMSH-binding protein, CHMP3, which is an ESCRT-III subunit. ESCRT-III functions on maturing endosomes, indicating AMSH might also play a role in MVB/late endosomes. Expression of an AMSH mutant lacking CHMP3-binding ability resulted in aberrant endosomes with accumulations of ubiquitinated cargo. Nevertheless, CHMP3-binding capability was not essential for AMSH's in vitro DUB activity or its endosomal localization, suggesting that, in vivo, the deubiquitination of endosomal cargo is CHMP3-dependent. Ubiquitinated cargo also accumulated on endosomes when catalytically inactive AMSH was expressed or AMSH was depleted. These results suggest that both the DUB activity of AMSH and its CHMP3-binding ability are required to clear ubiquitinated cargo from endosomes.