Mitochondrial polarization in rat hippocampal astrocytes is resistant to cytosolic Ca(2+) loads.

The influence of physiological Ca(2+)-inducing stimuli and agents mimicking ischemic conditions on mitochondrial potential was studied in postnatal (P1) hippocampal astrocytes. Cytosolic Ca(2+) loads with characteristic kinetics of rise and duration, detected by Fura-2, were provoked by extracellular Ca(2+) influx, release from InsP(3)-sensitive intracellular stores, or inhibition of the reloading of endoplasmic reticulum Ca(2+) stores. Inhibitors of mitochondrial respiration caused only moderate release of Ca(2+) from intracellular stores, inducing a rise of less than 60 nM. The maximal Ca(2+) rise was found with InsP(3)-mediated responses (500 nM; via ATP) or with ionophore (4-Br-A23187)-mediated Ca(2+) influx from extracellular medium (770 nM). Remarkably, all these agents causing significant rise of cytosolic Ca(2+), only minimally depolarized the mitochondria. Membrane potential of mitochondria was monitored by Rh123 or TMRE. Depolarization was only found with very high cytosolic Ca(2+) levels (above 60 microM; measured by fura FF). These were achieved with external Ca(2+) influx by ionophore in combination with inhibition of glycolysis. Thus, mitochondria in the astrocytes are obviously not sensitive to moderate cytosolic Ca(2+) loads, irrespective of the source of Ca(2+). Furthermore, isolated rat brain mitochondria display a low sensitivity of respiratory activity to Ca(2+), which is consistent with the data obtained with the astrocytes in vitro. The capacity of isolated mitochondria to build up a potential was gradually reduced at low micromolar Ca(2+) and totally compromised only at Ca(2+) concentrations in the 100 microM range.     

Discovery of a selective inhibitor of oncogenic B-Raf kinase with potent antimelanoma activity

BRAF(V600E) is the most frequent oncogenic protein kinase mutation known. Furthermore, inhibitors targeting "active" protein kinases have demonstrated significant utility in the therapeutic repertoire against cancer. Therefore, we pursued the development of specific kinase inhibitors targeting B-Raf, and the V600E allele in particular. By using a structure-guided discovery approach, a potent and selective inhibitor of active B-Raf has been discovered. PLX4720, a 7-azaindole derivative that inhibits B-Raf(V600E) with an IC(50) of 13 nM, defines a class of kinase inhibitor with marked selectivity in both biochemical and cellular assays. PLX4720 preferentially inhibits the active B-Raf(V600E) kinase compared with a broad spectrum of other kinases, and potent cytotoxic effects are also exclusive to cells bearing the V600E allele. Consistent with the high degree of selectivity, ERK phosphorylation is potently inhibited by PLX4720 in B-Raf(V600E)-bearing tumor cell lines but not in cells lacking oncogenic B-Raf. In melanoma models, PLX4720 induces cell cycle arrest and apoptosis exclusively in B-Raf(V600E)-positive cells. In B-Raf(V600E)-dependent tumor xenograft models, orally dosed PLX4720 causes significant tumor growth delays, including tumor regressions, without evidence of toxicity. The work described here represents the entire discovery process, from initial identification through structural and biological studies in animal models to a promising therapeutic for testing in cancer patients bearing B-Raf(V600E)-driven tumors.     

缺氧血管内皮细胞Rho激酶信号通路活化与通透性的关系

目的 探讨缺氧时血管内皮细胞Rho激酶信号通路活化与通透性的关系. 方法 (1)将人血管内皮细胞株VE细胞接种于6孔板Transwell小室上,按随机数字表法分为对照组(未行缺氧处理)及缺氧1、2、3、6、12h组(分别缺氧处理相应时间),每组5孔.用蛋白质印迹法检测细胞中Rho激酶Ⅰ、Ⅱ及肌球蛋白轻链磷酸酶靶亚单位1(MYPT1)、磷酸化MYPT1( p-MYPT1)、肌球蛋白轻链(MLC)、p-MLC的蛋白表达,计算p-MYPT1/MYPT1、p-MLC/MLC比值.(2)将VE细胞接种于24孔板Transwell小室上,按随机数字表法分为对照组(未行缺氧处理)与缺氧6h组(缺氧处理6h),每组5孔,用荧光分光光度计检测单层细胞通透性.对数据进行单因素方差分析或t检验,多组间两两比较采用Newman-Keuls法. 结果 (1)对照组及缺氧1、2、3、6、12h组细胞Rho激酶Ⅰ蛋白表达量分别为0.63±0.14及0.36±0.08、1.25 ±0.21、1.98±0.16、1.49 ±0.38、0.79±0.24(F=36.52.P ＜0.01),其中缺氧2、3、6h组显著高于对照组(P值均小于0.01).各组细胞Rho激酶Ⅱ蛋白表达量比较,差异具有统计学意义(F =17.84,P＜0.01),其中缺氧2h组为1.33±0.17,显著高于对照组的1.05±0.04(P＜0.01).对照组及缺氧1、2、3、6、12h组细胞p-MYPT1/MYPT1比值分别为0.62±0.13及0.62±0.11、0.65 ±0.10、1.06±0.23、1.37 ±0.16、1.91±0.32(F=37.41,P＜0.01),其中缺氧3、6、12h组显著高于对照组(P值均小于0.01).对照组及缺氧1、2、3、6、12h组细胞p-MLC/MLC比值分别为0.72 ±0.19及0.83 ±0.17、0.91±0.15、1.39 ±0.16、2.02±0.15、0.90±0.25(F=36.92,P＜0.01),其中缺氧3、6h组显著高于对照组(P值均小于0.01).(2)缺氧6h组单层细胞通透性为36.1±8.0,显著高于对照组的9.1±2.1(t=7.30,P＜0.01). 结论 Rho激酶信号通路活化可能参与了缺氧引起的血管内皮通透性增加的发生.     

Effects of Cell Shape Type X Collagen Gene Expression in Hypertrophic Chondrocytes

During endochondral ossification, small flat resting and proliferating chondrocytes mature into large round hypertrophic chondrocytes that synthesize a unique collagen, type X. We have asked whether this change in cell shape during chondrocyte maturation regulates type X collagen gene expression, using immature chick vertebral chondrocytes grown in monolayer or in suspension. The freshly isolated chondrocytes contained no type X collagen RNA, but after 30 days of culture, both attached and suspended cells contained a similar large amount. However, in cells that were grown in monolayer and then resuspended three days before harvest, type X collagen gene expression increased a further 6 fold. These results suggest that the change from a flat to a round shape that occurs during chondrocyte maturation in vivo may be important for maximal expression of the type X collagen gene.     

Prediction of the Repeat Domain Structures and Impact of Parkinsonism‐Associated Variations on Structure and Function of all Functional Domains of Leucine‐Rich Repeat Kinase 2 (LRRK2)

Genetic variations of leucine‐rich repeat kinase 2 (LRRK2) are the major cause of dominantly inherited Parkinson disease (PD). LRRK2 protein contains seven predicted domains: a tandem Ras‐like GTPase (ROC) domain and C‐terminal of Roc (COR) domain, a protein kinase domain, and four repeat domains. PD‐causative variations arise in all domains, suggesting that aberrant functioning of any domain can contribute to neurotoxic mechanisms of LRRK2. Determination of the three‐dimensional structure of LRRK2 is one of the best avenues to decipher its neurotoxic mechanism. However, with the exception of the Roc domain, the three‐dimensional structures of the functional domains of LRRK2 have yet to be determined. Based on the known three‐dimensional structures of repeat domains of other proteins, the tandem Roc–COR domains of the Chlorobium tepidum Rab family protein, and the kinase domain of the Dictyostelium discoideum Roco4 protein, we predicted (1) the motifs essential for protein–protein interactions in all domains, (2) the motifs critical for catalysis and substrate recognition in the tandem Roc–COR and kinase domains, and (3) the effects of some PD‐associated missense variations on the neurotoxic action of LRRK2. Results of our analysis provide a conceptual framework for future investigation into the regulation and the neurotoxic mechanism of LRRK2.     

Functional role of TGF-β receptors during palatal fusion in vitro

Objective

Reported expression patterns for TGF-β receptors (TβR-I, -II, and -III) during palatogenesis suggest that they play essential roles in the mechanisms leading to palatal fusion. The purpose of this study was to compare the functions of the three TβRs during palatal fusion.

Methods

Using organ culture of mouse palatal shelves, expression levels of TβR-I, -II, and -III were suppressed by transfecting the siRNAs siTβR-I, -II, and -III, respectively. Phosphorylation of SMAD2 was examined as an indicator of downstream signalling via each TβR. Linkage between TGF-β signalling and critical events in palatal fusion led to the use of, MMP-13 expression as an outcome measure for the function of the TGF-β receptors.

Results

The siRNA treatment decreased the expression level of each receptor by more than 85%. When treated with either siTβR-I or -II, palatal shelves at E13 + 72 h were not fused, with complete clefting in the anterior and posterior regions. The middle palatal region following treatment with either siTβR-I or -II had fusion from one-half or one-third of the palatal region. Treatment with siTβR-III resulted in a persistent midline seam of medial edge epithelium (MEE) in the anterior region with islands of persistent MEE in the middle and posterior regions of the midline. Treatment with all three siTβRs altered the pattern of SMAD2 phosphorylation. Palatal shelf cultures treated with siTβR-I or -II, but not -III, showed altered MMP-13 expression levels.

Conclusion

The ability to identify and recover MEE and palatal mesenchymal cells during palatal fusion will aid in the evaluation of the different mechanistic events regulated by each TβR during palatogenesis.     

Regulation of inositol 1,3,4-trisphosphate 5/6-kinase (ITPK1) by reversible lysine acetylation

The enzyme inositol 1,3,4-trisphosphate 5/6-kinase (ITPK1) catalyzes the rate-limiting step in the formation of higher phosphorylated forms of inositol in mammalian cells. Because it sits at a key regulatory point in the inositol metabolic pathway, its activity is likely to be regulated. We have previously shown that ITPK1 is phosphorylated, a posttranslational modification used by cells to regulate enzyme activity. We show here that ITPK1 is modified by acetylation of internal lysine residues. The acetylation sites, as determined by mass spectrometry, were found to be lysines 340, 383, and 410, which are all located on the surface of this protein. Overexpression of the acetyltransferases CREB-binding protein or p300 resulted in the acetylation of ITPK1, whereas overexpression of mammalian silent information regulator 2 resulted in the deacetylation of ITPK1. Functionally, ITPK1 acetylation regulates its stability. CREB-binding protein dramatically decreased the half-life of ITPK1. We further found that ITPK1 acetylation down-regulated its enzyme activity. HEK293 cells stably expressing acetylated ITPK1 had reduced levels of the higher phosphorylated forms of inositol, compared with the levels seen in cells expressing unacetylated ITPK1. These results demonstrate that lysine acetylation alters both the stability as well as the activity of ITPK1 in cells.     

Truncating mutations of PPM1D are found in blood DNA samples of lung cancer patients

Background:

PPM1D (WIP1) negatively regulates by dephosphorylation many proteins including p53 tumour suppressor. The truncating mutations (nonsense and frameshift) in exon 6 of PPM1D were found recently in blood cells of patients with breast, ovarian or colorectal cancer. These mutants code for gain-of-function PPM1D with retained phosphatase activity. Their significance in carcinogenesis is unknown.

Methods:

The exon 6 of PPM1D was sequenced in blood DNA of 543 non-small-cell lung cancer patients (NSCLC). The functional significance of selected PPM1D alterations (Arg458X, Lys469Glu) was compared with the wild-type gene and examined by recombinant DNA techniques, immunoblotting and luciferase reporter assays.

Results:

The frameshift mutations were found in five NSCLC patients (5/543; 0.92%), all of them had squamous cell carcinomas (5/328; 1.5%). All patients with the mutations were exposed, before the blood collection, to the DNA damaging agents as a part of chemotherapeutic regimen. Functional tests demonstrated that truncating mutation Arg458X causes enhancement of dephosphorylation activity of PPM1D toward serine 15 of p53, whereas Lys469Glu version is equivalent to the wild-type. Neither version of PPM1D (wild-type, Arg458X, Lys469Glu) significantly modulated the ability of p53 to transactivate promoters of the examined p53-target genes (BAX and MDM2).

Conclusions:

The truncating mutations of PPM1D are present in blood DNA of NSCLC patients at frequency similar to percentage determined for ovarian cancer patients. Our findings raise a question if the detected lesions are a result of chemotherapy.