The phosphorylation status and anti-apoptotic activity of Bcl-2 are regulated by ERK and protein phosphatase 2A on the mitochondria

AbstractBcl-2 protein play important roles in the regulation of apoptosis. We previously reported that the phosphorylation of Bcl-2 was augmented by treatment with protein phosphatase 2A (PP2A) inhibitor; however, the kinase responsible for Bcl-2 phosphorylation had not yet been identified. In this study, we identified extracellular-signal-regulated kinase (ERK) as the responsible kinase for the phosphorylation of Bcl-2. We also found that the transmembrane region (TM) deleted form of Bcl-2 (Bcl-2ΔTM), which was unable to localize on the mitochondria was constitutively phosphorylated, whereas wild-type Bcl-2 that localized on the mitochondria, was present in its hypophosphorylated form. The phosphorylation of Bcl-2ΔTM was retarded by treatment with MAP kinase ERK kinase (MEK) inhibitor and PP2A did not bind to Bcl-2ΔTM. These observations suggest that Bcl-2ΔTM is constitutively phosphorylated by ERK, but is not dephosphorylated by PP2A in human tumor cell lines. The phosphorylation of Bcl-2 resulted in a reduction in anti-apoptotic function, implying that dephosphorylation promoted the anti-apoptotic activity of Bcl-2 protein in human tumor cell lines. Thus, the present findings suggest that ERK and PP2A are physiological regulators of Bcl-2 phosphorylation, and these enzymes exert an influence on the anti-apoptotic function of Bcl-2

Genome-Wide Maps of Mononucleosomes and Dinucleosomes Containing Hyperacetylated Histones of Aspergillus fumigatus

It is suggested that histone modifications and/or histone variants influence the nucleosomal DNA length. We sequenced both ends of mononucleosomal and dinucleosomal DNA fragments of the filamentous fungus Aspergillus fumigatus, after treatment with the histone deacetylase inhibitor trichostatin A (TSA). After mapping the DNA fragments to the genome, we identified >7 million mononucleosome positions and >7 million dinucleosome positions. We showed that the distributions of the lengths of the mononucleosomal DNA fragments after 15-min and 30-min treatments with micrococcal nuclease (MNase) showed a single peak at 168 nt and 160 nt, respectively. The distributions of the lengths of the dinucleosomal DNA fragments after 15-min- and 30-min-treatment with MNase showed a single peak at 321 nt and 306 nt, respectively. The nucleosomal DNA fragments obtained from the TSA-treated cells were significantly longer than those obtained from the untreated cells. On the other hand, most of the genes did not undergo any change after treatment. Between the TSA-treated and untreated cells, only 77 genes had ≥2-fold change in expression levels. In addition, our results showed that the locations where mononucleosomes were frequently detected were conserved between the TSA-treated cells and untreated cells in the gene promoters (lower density of the nucleosomes). However, these locations were less conserved in the bodies (higher density of the nucleosomes) of genes with ≥2-fold changes. Our findings indicate that TSA influences the nucleosome positions, especially of the regions with high density of the nucleosomes by elongation of the nucleosomal DNA. However, most of the nucleosome positions are conserved in the gene promoters, even after treatment with TSA, because of the low density of nucleosomes in the gene promoters

Terpendole E, a Kinesin Eg5 Inhibitor, Is a Key Biosynthetic Intermediate of Indole-Diterpenes in the Producing Fungus Chaunopycnis alba

SummaryTerpendole E is the first natural product inhibitor of kinesin Eg5. Because terpendole E production is unstable, we isolated and analyzed the terpendole E biosynthetic gene cluster, which consists of seven genes encoding three P450 monooxygenases (TerP, TerQ, and TerK), an FAD-dependent monooxygenase (TerM), a terpene cyclase (TerB), and two prenyltransferases (TerC and TerF). Gene knockout and feeding experiments revealed that terpendole E is a key intermediate in terpendole biosynthesis and is produced by the action of the key enzyme TerQ from paspaline, a common biosynthetic intermediate of indole-diterpenes. TerP converts terpendole E to a downstream intermediate specific to terpendole biosynthesis and converts paspaline to shunt metabolites. We successfully overproduced terpendole E by disrupting the terP gene. We propose that terpendole E is a key biosynthetic intermediate of terpendoles and related indole-diterpenes

Sulfonamides identified as plant immune-priming compounds in high-throughput chemical screening increase disease resistance in Arabidopsis thaliana

Plant activators are agrochemicals that protect crops from diseases by activating the plant immune system. To isolate lead compounds for use as practical plant activators, we screened two different chemical libraries composed of various bioactive substances by using an established screening procedure that can selectively identify immune-priming compounds. We identified and characterized a group of sulfonamide compounds – sulfameter, sulfamethoxypyridazine, sulfabenzamide, and sulfachloropyridazine – among the various isolated candidate molecules. These sulfonamide compounds enhanced the avirulent Pseudomonas-induced cell death of Arabidopsis suspension cell cultures and increased disease resistance in Arabidopsis plants against both avirulent and virulent strains of the bacterium. These compounds did not prevent the growth of pathogenic bacteria in minimal liquid media at 200 μM. They also did not induce the expression of defense-related genes in Arabidopsis seedlings, at least not at 24 and 48 h after treatment, suggesting that they do not act as salicylic acid analogs. In addition, although sulfonamides are known to be folate biosynthesis inhibitors, the application of folate did not restore the potentiation effects of the sulfonamides on pathogen-induced cell death. Our data suggest that sulfonamides potentiate Arabidopsis disease resistance by their novel chemical properties

Time taken to the maximum increase in the oxygenated hemoglobin level in calf muscle as a predictor of mild and moderate post-thrombotic syndrome

AbstractBackgroundNear-infrared spectroscopy (NIRS) allows continuous noninvasive monitoring of changes in the tissue levels of oxygenated hemoglobin (O2Hb) and deoxygenated hemoglobin (HHb) and can identify the severity of chronic venous diseases. Here we investigated the predictors of post-thrombotic syndrome (PTS) using NIRS in patients with a first episode of deep venous thrombosis (DVT).MethodsThe study enrolled 129 patients with DVT. Risk factors in each patient were assessed at presentation. Venous abnormalities confirmed by ultrasound and parameters derived from NIRS were evaluated at 6 months after DVT. On standing, increases in O2Hb and HHb (ΔO2Hbst and ΔHHbst) and the times taken for each concentration to become maximal (TO2Hbst, and THHbst) were measured. During 10 tiptoe movements, O2Hb showed a continuous decrease (ΔO2Hbex), whereas venous expulsion (ΔHHbEex) and subsequent retention (ΔHHbRex) were observed. The oxygenation index (HbD; HbD = O2Hb − HHb) was also calculated at the end of standing and at the end of 10 tiptoe movements (ΔHbDst and ΔHbDex). Final clinical manifestations were evaluated at 6 years, and PTS was considered to be present if the Villalta score was ≥5.ResultsThirteen patients were excluded and 116 patients were finally included. Of these, 19 (16%) developed PTS. Among various NIRS-derived parameters, TO2Hbst had the highest area under the curve (0.88; 95% confidence interval [CI], 0.80-0.93; P < .01) with the best cutoff value (TO2Hbst ≤48 seconds). On univariate analysis, variables associated with greater risk for development of PTS were stroke (odds ratio [OR], 5.59; 95% CI, 0.74-42.41; P = .06), idiopathic DVT (OR, 4.13; 95% CI, 1.36-12.55; P < .01) and iliofemoral DVT (OR, 4.31; 95% CI, 1.48-12.60; P < .01) at initial presentation, venous occlusion combined with reflux (OR, 4.24; 95% CI, 1.50-12.00; P < .01), and NIRS-derived TO2Hbst ≤48 seconds (OR, 43.03; 95% CI, 9.04-204.81; P < .01) at 6 months. Multivariate logistic regression analysis finally revealed venous occlusion combined with reflux (OR, 4.80; 95% CI, 1.03-22.36; P < .05) and NIRS-derived TO2Hbst ≤48 seconds (OR, 53.73; 95% CI, 8.43-342.41; P < .01) to be independently associated with PTS progression.ConclusionsNIRS-derived TO2Hbst ≤48 seconds is a promising time-course predictor of PTS progression

Cucurbitacin B induces neurogenesis in PC12 cells and protects memory in APP/PS1 mice

Cucurbitacin B (CuB) isolated from Cucumis melo by using a PC12 cell bioassay system exhibited significant nerve growth factor (NGF)-mimic or NGF-enhancing activity in PC12 arid primary neuron cells. It was also demonstrated pro-neurogenesis effects in ICR and APP/PSI mice and improved memory deficit of APP/PSI mice. Its possible mechanism includes significant induction of the phosphorylation of glucocorticoid receptor (GR), protein kinase C (PKC), phospholipase C (PLC) and inhibition of cofilin. ChemProteoBase profiling, binding assay and cellular thermal shift assay (CETSA) were used to determine the target protein. Results revealed that CuB could affect actin dynamics as an actin inhibitor but did not bind with GR. The protein level of cofilin in PC12 cells after treating 0.3 mu M and different temperatures was significantly higher than that of control group. Other neurotrophic signalling pathways, such as TrkA/TrkB, were analysed with specific inhibitors and Western blot. The inhibitors of TrkA, PLC, PKC, Ras, Raf and ERK1/2 significantly decreased the percentage of PC12 cells with neurite outgrowth and shortened the length of neurite outgrowth induced by CuB. CuB significantly induced the phosphorylation of TrkA, ERK and CREB. The phosphorylation of these proteins was obviously decreased by their specific inhibitors. These results suggest that cofilin is a candidate target protein of CuB in PC12 cells and that the GR/PLC/PKC and TrkA/Ras/Raf/ERK signalling pathways play important roles in the neuroprotective effect of CuB.11Ysciescopu