Synthesis of [2S-2-H]- and [2R-2-H]hexadecanoic acids

[2S-2-²H]- and [2R-2-²H]hexadecanoic acids were synthesized in overall yields of 59–67%. Methyl(2R)-2-hydroxyhexadecanoate, from the acid produced by Hansenula sydowiorum, was converted to the p-toluenesulphonate, reduced to trideutero alcohol with lithium aluminium deuteride and oxidized to [2S-2-²H]hexadecanoic acid. Methyl (2S)-2-chlorohexadecanoate, which was a by-product of tosylation and was also prepared by chlorinatioon of the hydroxy ester with thionyl chloride, on reduction and oxidation as before gave [2R-2-²H]-hexadecanoic acid. Intermediates were fully characterized, isotopic purity was 97% and optical purity was maintained throughout the syntheses. Attempts to reduce the tosyl or chloro groups, only, with sodium borodeuteride gave low yields probably due to preferential reduction of the ester group; 1,2-epoxyhexadecane was obtained from the tosylate and 2-chlorohexadecan-1-ol from the chloro ester.     

不同氧浓度下C2C12细胞的Nrf2抗氧化系统对H2O2刺激的反应*

目的:探讨不同氧浓度下小鼠骨骼肌卫星细胞系(C2C12细胞)对H₂O₂刺激反应的变化及其机制。方法:小鼠骨骼肌卫星细胞系(C2C12细胞),经培养复苏后,将细胞分为7组,每组设8个复孔,各组分别加入浓度为0.1 mmol/L、0.25 mmol/L、0.5 mmol/L、0.75 mmol/L、1 mmol/L、2 mmol/L的H₂O₂,分别作用1 h、2 h后测细胞活力,选择细胞H₂O₂刺激的最佳作用时间和浓度;C2C12细胞分为不同氧浓度组:21% O₂、12% O₂、8% O₂、5% O₂每组设8个复孔,12 h后,H₂O₂作用1 h,收集细胞;检测细胞Nrf2蛋白荧光和蛋白表达量,测定Nrf2和抗氧化酶SOD1、SOD2、CAT、NQO-1、HO-1、GPX-1 的mRNA表达量及细胞ROS水平。结果:选择H₂O₂作用时间相对较短的1 h和浓度0.5 mmol/L作为本实验的H₂O₂刺激条件。与21%O₂组相比,12%O₂组细胞Nrf2蛋白荧光增强,Nrf2 的mRNA和蛋白表达以及抗氧化酶SOD1、SOD2、CAT、NQO-1、HO-1、GPX-1的 mRNA表达均显著增加(P＜0.05或P＜0.01),细胞 ROS水平明显降低(P＜0.01);8%O₂组仅GPX-1 mRNA显著增加(P＜0.05),其他指标变化不大;5%O₂组细胞 Nrf2 mRNA和蛋白表达以及抗氧化酶SOD1、SOD2、NQO-1、GPX-1的 mRNA表达均明显降低(P＜0.05或P＜0.01),细胞 ROS水平则明显升高(P＜0.01)。结论:不同氧浓度下C2C12细胞中Nrf2介导的抗氧化系统对H₂O₂刺激反应不同,12 h的12% O₂浓度可促进C2C12细胞Nrf2的抗氧化作用,而5% O₂浓度的严重低氧则作用相反。     

Evidence for the direct 2beta- and 3beta-hydroxylation of [2H2]GA20-13-O-[6'-2H2]glucoside in seedlings of Phaseolus coccineus

[17-²H₂]GA₂₀-13-O-[6'-²H₂]glucoside was synthesized and applied to seedlings of Phaseolus coccineus L. After incubation for 72 h the conjugate metabolites were purified and shown by LC-ESI-tandem-MS and GC-MS to be [17-²H₂]GA₁-13-O-[6'-²H₂]glucoside and [17-²H₂]GA₂₉-13-O-[6'-²H₂]glucoside. This is the first evidence for the conversion of intact GA-O-glucosides, and represents an additional metabolic pathway of the gibberellin metabolism in P. coccineus L. The results indicate that intact GA-O-glucosides are accepted by 2- and 3-oxidases in the plant.     

PPP2R2A在乳腺癌细胞中结合GFPT2并导致其去磷酸化

PPP2R2A是PP2A磷酸酶的调控亚基之一,以往的研究报道显示,PPP2R2A可促进肿瘤细胞生存和生长。本研究通过串联亲和纯化联合HPLC-Chip-ESI/MS/MS筛选PPP2R2A的相互作用蛋白质,分析结果显示,L-谷氨酰胺-D-果糖-6-磷酸转氨酶1(Glutamine-fructose-6-phosphate transaminase 1,GFPT1)和L-谷氨酰胺-D-果糖-6-磷酸转氨酶2(Glutamine-fructose-6-phosphate transaminase 2,GFPT2)是PPP2R2A可能的结合蛋白。通过免疫荧光共定位、GST Pull-down和免疫共沉淀等方法,进一步确认了PPP2R2A和GFPT1及GFPT2的相互结合。通过shRNA下调PPP2R2A后,GFPT2的磷酸化水平显著增加,但GFPT1的磷酸化水平改变不明显。GFPT2是O-GlcNAC糖基化修饰通路中的一个限速酶,在乳腺癌细胞MDA-MB-231中下调PPP2R2A后,蛋白质O-GlcNAC糖基化修饰水平增加。这些结果表明,PPP2R2A可直接结合GFPT2,并导致其去磷酸化,进而影响细胞内O-GlcNAC糖基化修饰。     

Downregulation of IDH2 exacerbates H2O2-mediated cell death and hypertrophy

Objectives: Reactive oxygen species-mediated cell death contributes to the pathophysiology of cardiovascular disease and myocardial dysfunction. We recently showed that mitochondrial NADP⁺-dependent isocitrate dehydrogenase (IDH2) functions as an antioxidant and anti-apoptotic protein by supplying NADPH to antioxidant systems.

Methods: In the present study, we demonstrated that H₂O₂-induced apoptosis and hypertrophy of H9c2 cardiomyoblasts was markedly exacerbated by small interfering RNA (siRNA) specific for IDH2.

Results: Attenuated IDH2 expression resulted in the modulation of cellular and mitochondrial redox status, mitochondrial function, and cellular oxidative damage. MitoTEMPO, a mitochondria-targeted antioxidant, efficiently suppressed increased caspase-3 activity, increased cell size, and depletion of cellular GSH levels in IDH2 siRNA-transfected cells that were treated with H₂O₂.

Discussion: These results indicated that the disruption of cellular redox balance might be responsible for the enhanced H₂O₂-induced apoptosis and hypertrophy of cultured cardiomyocytes by the attenuated IDH2 expression.     

cAMP potentiates H(2)O(2)-induced ERK1/2 phosphorylation without the requirement for MEK1/2 phosphorylation

In Jurkat T lymphocytes, hydrogen peroxide (H(2)O(2)) potentiates the phosphorylation level of extracellular signal-regulated kinase 1 and 2 (ERK1/2) caused by T cell receptor (TCR) stimulation with anti-CD3 and anti-CD28 or anti-CD3 alone. Submillimolar concentrations of H(2)O(2)-induced phosphorylation of ERK1/2 and MAP/ERK kinase 1 and 2 (MEK1/2) without antigenic stimulation. H(2)O(2) also induced the electrophoretic mobility shift of Lck from 56 to 60 kDa. The MEK inhibitor, PD98059 attenuated ERK1/2 and MEK1/2 phosphorylation, as well as the migration shift of Lck induced by H(2)O(2). The phospholipase C (PLC) inhibitor, U73122, and EGTA reduced the phosphorylation of both ERK1/2 and MEK1/2 induced by H(2)O(2). Interestingly, an increase of intracellular cAMP level with forskolin or 8-(4-chlorophenylthio)-cAMP augmented ERK1/2 phosphorylation by H(2)O(2), while inhibiting MEK1/2 phosphorylation by H(2)O(2). These results demonstrate an alternative pathway that results in augmentation of ERK1/2 phosphorylation without concomitant MEK1/2 phosphorylation in T cells.     

Ash2l-1/Ash2l-2在小鼠胚胎干细胞中的表达特异性及互补效应

H3K4me3是一种重要的表观遗传修饰,主要由MLL(mixed lineage leukemia)甲基转移酶复合体催化,对小鼠胚胎干细胞(mouse embryonic stem cells, mESCs)自我更新能力的维持具有重要作用。ASH2L是MLL复合体中一个重要的核心亚单位,参与调控mESCs中染色质的开放状态。ASH2L在mESCs中有2个异构体：ASH2L-1(80 kDa)和ASH2L-2(65 kDa),且以ASH2L-2的表达为主;而在小鼠胚胎成纤维细胞(mouse embryonic fibroblast, MEF)中,只有ASH2L-1表达。目前,Ash2l-1和Ash2l-2在mESCs中的作用尚不清楚。本文利用CRISPR/Cas9基因组编辑技术,建立了Ash2l-1 ^-/-和Ash2l-2 ^-/-mESCs。通过碱性磷酸酶染色、免疫荧光染色和qRT-PCR发现,Ash2l-1 ^-/-和Ash2l-2 ^-/-mESCs在碱性磷酸酶、多能性调控转录因子(Oct4、Nanog、Sox2和Klf4)的表达与野生型对照无显著差异。通过拟胚体分化实验,发现Ash2l-1 ^-/-mESCs诱导的拟胚体在Snai2(外胚层标记基因)和Gata4(内胚层标记基因)的表达上显著低于野生型mESCs诱导的拟胚体(P<0.01)。通过Western blotting,发现Ash2l-1 ^-/-mESCs中ASH2L-2的表达显著上调(P<0.01),Ash2l-2 ^-/-mESCs中ASH2L-1的表达显著上调(P<0.01),而Ash2l-1 ^-/-和Ash2l-2 ^-/-mESCs中,基因组H3K4me3的表达与野生型对照并无显著差异。这表明Ash2l-1和Ash2l-2之间存在补偿效应。利用JASPAR和KEGG预测分析发现,Ash2l-1和Ash2l-2启动子区分别具有3个和16个潜在的多能性转录因子结合位点,这些转录因子可能介导实现Ash2l-1和Ash2l-2之间的补偿效应。以上结果表明,Ash2l-1和Ash2l-2之间的补偿效应可能参与mESCs多能性的维持和基因组H3K4me3的调控。     

Local Ca2 Rise Near Store Operated Ca2 Channels Inhibits Cell Coupling During Capacitative Ca2 Influx

Using a new fluorescence imaging technique, LAMP, we recently reported that Ca²⁺ influx through store operated Ca²⁺ channels (SOCs) strongly inhibits cell coupling in primary human fibroblasts (HF) expressing Cx43. To understand the mechanism of inhibition, we studied the involvement of cytosolic pH (pH_i) and Ca²⁺([Ca²⁺]_i) in the process by using fluorescence imaging and ion clamping techniques. During the capacitative Ca²⁺ influx, there was a modest decline of pH_i measured by BCECF. Decreasing pH_i below neutral using thioacetate had little effect by itself on cell coupling, and concomitant pH_i drop with thioacetate and bulk [Ca²⁺_i rise with ionomycin was much less effective in inhibiting cell coupling than Ca²⁺ influx. Moreover, clamping pH_i with a weak acid and a weak base during Ca²⁺ influx largely suppressed bulk pH_i drop, yet the inhibition of cell coupling was not affected. In contrast, buffering [Ca²⁺_i with BAPTA, but not EGTA, efficiently prevented cell uncoupling by Ca²⁺ influx. We concluded that local Ca²⁺ elevation subjacent to the plasma membrane is the primary cause for closing Cx43 channels during capacitative Ca²⁺ influx. To assess how Ca²⁺ influx affects junctional coupling mediated by other types of connexins, we applied the LAMP assay to Hela cells expressing Cx26. Capacitative Ca²⁺ influx also caused a strong reduction of cell coupling, suggesting that the inhibitory effect by Ca²⁺ influx may be a more general phenomenon.     

2'-Deoxy-2'-fluoro-beta-D-arabinonucleosides and oligonucleotides (2'F-ANA): synthesis and physicochemical studies

Recently, hybrids of RNA and D-arabinonucleic acids (ANA) as well as the 2'-deoxy-2'-fluoro-D-arabinonucleic acid analog (2'F-ANA) were shown to be substrates of RNase H. This enzyme is believed to be involved in the primary mechanism by which antisense oligonucleotides cause a reduction in target RNA levels in vivo. To gain a better understanding of the properties of arabinose based oligonucleotides, we have prepared a series of 2'F-ANA sequences of homopolymeric (A and T) and mixed base composition (A, T, G and C). UV thermal melting and circular dichroic (CD) studies were used to ascertain the thermodynamic stability and helical conformation of 2'F-ANA/RNA and 2'F-ANA/DNA hybrids. It is shown that 2'F-ANA has enhanced RNA affinity relative to that of DNA and phosphorothioate DNA. The 2'-fluoroarabino modification showed favorable pairing to single-stranded DNA also. This is in sharp contrast to ANA, which forms weak ANA/DNA hybrids at best. According to the measured thermodynamic parameters for duplex formation, the increased stability of hybrids formed by 2'F-ANA (e.g., 2'F-ANA/RNA) appears to originate from conformational pre-organization of the fluorinated sugars and a favorable enthalpy of hybridization. In addition, NMR spectroscopy revealed a five-bond coupling between the 2'F and the base protons (H6/H8) of 2'-deoxy-2'-fluoro-beta-D-arabinonucleosides. This observation is suggestive of a through-space interaction between 2'F and H6/H8 atoms. CD experiments indicate that 2'F-ANA/RNA hybrids adopt an 'A-like' structure and show more resemblance to DNA/RNA hybrids than to the pure RNA/RNA duplex. This feature is believed to be an important factor in the mechanism that allows RNase H to discriminate between 2'F-ANA/RNA (or DNA/RNA) and RNA/RNA duplexes.     

ATP binding site mutagenesis reveals different subunit stoichiometry of functional P2X2/3 and P2X2/6 receptors

The aim of the present experiments was to clarify the subunit stoichiometry of P2X2/3 and P2X2/6 receptors, where the same subunit (P2X2) forms a receptor with two different partners (P2X3 or P2X6). For this purpose, four non-functional Ala mutants of the P2X2, P2X3, and P2X6 subunits were generated by replacing single, homologous amino acids particularly important for agonist binding. Co-expression of these mutants in HEK293 cells to yield the P2X2 WT/P2X3 mutant or P2X2 mutant/P2X3 WT receptors resulted in a selective blockade of agonist responses in the former combination only. In contrast, of the P2X2 WT/P2X6 mutant and P2X2 mutant/P2X6 WT receptors, only the latter combination failed to respond to agonists. The effects of α,β-methylene-ATP and 2-methylthio-ATP were determined by measuring transmembrane currents by the patch clamp technique and intracellular Ca(2+) transients by the Ca(2+)-imaging method. Protein labeling, purification, and PAGE confirmed the assembly and surface trafficking of the investigated WT and WT/mutant combinations in Xenopus laevis oocytes. In conclusion, both electrophysiological and biochemical investigations uniformly indicate that one subunit of P2X2 and two subunits of P2X3 form P2X2/3 heteromeric receptors, whereas two subunits of P2X2 and one subunit of P2X6 constitute P2X2/6 receptors. Further, it was shown that already two binding sites of the three possible ones are sufficient to allow these receptors to react with their agonists.     

Oxygen-dependent H2O2 production by Rubisco

Oxygen and ribulose-1,5-bisphosphate dependent, H(2)O(2) production was observed with several wild type Rubisco enzymes using a sensitive assay. H(2)O(2) and d-glycero-2,3-pentodiulose-1,5-bisphosphate, a known and potent inhibitor of Rubisco activity, are predicted products arising from elimination of H(2)O(2) from a peroxyketone intermediate, specific to oxygenase activity. Parallel assays using varying CO(2) and O(2) concentrations revealed that the partitioning to H(2)O(2) during O(2) consumption by spinach Rubisco was constant at 1/260-1/270. High temperature (38 degrees C), which reduces Rubisco specificity for CO(2) versus O(2), increased the rates of H(2)O(2) production and O(2) consumption, resulting in a small increase in partitioning to H(2)O(2) (1/210). Two Rubiscos with lower specificity than spinach exhibited greater partitioning to H(2)O(2) during catalysis: Chlamydomonas reinhardtii (1/200); and Rhodospirillum rubrum (1/150).     

The inhibitory role of DOC-2/DAB2 in growth factor receptor-mediated signal cascade. DOC-2/DAB2-mediated inhibition of ERK phosphorylation via binding to Grb2

DOC-2/DAB2 (differentially expressed in ovarian carcinoma-2/disabled 2) appears to be a potential tumor suppressor gene with a growth inhibitory effect on several cancer types. Previously, we have shown that DOC-2/DAB2 suppresses protein kinase C-induced AP-1 activation, which is modulated by serine 24 phosphorylation in the N terminus of DOC-2/DAB2. However, the functional impact of the C terminus of DOC-2/DAB2, containing three proline-rich domains, has not been explored. In this study, we examined this functional role in modulating signaling mediated by peptide growth factor receptor tyrosine kinase, particularly because it involves the interaction with Grb2. Using sequence-specific peptides, we found that the second proline-rich domain of DOC-2/DAB2 is the key binding site to Grb2 in the presence of growth factors. Such elevated binding interrupts the binding between SOS and Grb2, which consequently suppresses downstream ERK phosphorylation. Reduced ERK phosphorylation was restored when the binding between DOC-2/DAB2 and Grb2 was interrupted by a specific peptide or by increasing the expression of Grb2. Furthermore, the C terminus of the DOC-2/DAB2 construct can inhibit the AP-1 activity elicited by growth factors. We conclude that DOC-2/DAB2, a potent negative regulator, can suppress ERK activation by interrupting the binding between Grb2 and SOS that is elicited by peptide growth factors. This study further illustrates that DOC-2/DAB2 has multiple effects on the RAS-mediated signal cascades active in cancer cells.     

Mechanisms of H2O2-induced oxidative stress in endothelial cells

Hydrogen peroxide, produced by inflammatory and vascular cells, induces oxidative stress that may contribute to endothelial dysfunction. In smooth muscle cells, H₂O₂ induces production of O₂⁻ by activating NADPH oxidase. However, the mechanisms whereby H₂O₂ induces oxidative stress in endothelial cells are poorly understood. We examined the effects of H₂O₂ on O₂⁻ levels on porcine aortic endothelial cells (PAEC). Treatment with 60 μmol/L H₂O₂ markedly increased intracellular O₂⁻ levels (determined by conversion of dihydroethidium to hydroxyethidium) and produced cytotoxicity (determined by propidium iodide staining) in PAEC. Overexpression of human manganese superoxide dismutase in PAEC reduced O₂⁻ levels and attenuated cytotoxicity resulting from treatment with H₂O₂. L-NAME, an inhibitor of nitric oxide synthase (NOS), and apocynin, an inhibitor of NADPH oxidase, reduced O₂⁻ levels in PAEC treated with H₂O₂, suggesting that both NOS and NADPH oxidase contribute to H₂O₂-induced O₂⁻ in PAEC. Inhibition of NADPH oxidase using apocynin and NOS rescue with L-sepiapterin together reduced O₂⁻ levels in PAEC treated with H₂O₂ to control levels. This suggests interaction-distinct NOS and NADPH oxidase pathways to superoxide. We conclude that H₂O₂ produces oxidative stress in endothelial cells by increasing intracellular O₂⁻ levels through NOS and NADPH oxidase. These findings suggest a complex interaction between H₂O₂ and oxidant-generating enzymes that may contribute to endothelial dysfunction.     

Post-chemiluminescence behaviour of Ni2+, Mg2+, Cd2+ and Zn2+ in the potassium ferricyanide-luminol reaction.

A new chemiluminescence (CL) reaction was observed when Ni2+, Mg2+, Cd2+ or Zn2+ was injected into the reaction mixture after the finish of the CL reaction of alkaline luminol and potassium ferricyanide. This reaction is described as a post-chemiluminescence (PCL) reaction. The possible mechanism for the PCL was proposed based on studies of the CL kinetic characteristic and the CL spectra. The experimental conditions of the CL reactions were optimized and the feasibility of using the reaction to analyse these metal ions was evaluated. The PCL reaction method operates in the ranges: 1 x 10(-7)-8 x 10(-6) g/L Ni2+; 3 x 10(-6)-2 x 10(-4) g/L Mg2+; 8 x 10(-7)-1 x 10(-4) g/L Cd2+; and 2 x 10(-4)-2 x 10(-3) g/L Zn2+, with detection limits of 4 x 10(-8) g/mL, 1 x 10(-6) g/mL, 3 x 10(-7) g/mL, 8 x 10(-5) g/mL, respectively.     

Polymorphism of human liver alcohol dehydrogenase: Identification of ADH2 2-1 and ADH2 2-2 phenotypes in the Japanese by isoelectric focusing

Liver homogenate-supernatants from most Japanese exhibit an atypical pH optimum for ethanol oxidation at pH 8.8 instead of 10.5, the typical pH-activity optimum. It has been proposed that atypical livers contain alcohol dehydrogenase isozymes with ₂ subunits while typical livers contain isozymes with ₁ subunits, both produced by the ADH ₂ gene. Because it is difficult to differentiate the atypical ADH₂ 2-2 phenotype from the ADH₂ 2-1 phenotype by starch gel electrophoresis, an agarose isoelectric focusing procedure was developed that clearly separated the atypical Japanese livers into two groups, A1 and A2. The isozymes in A1 and A2 livers were purified. Type A1 livers contained a single isozyme with an atypical pH-rate profile; it was designated ₂₂. Three isozymes were isolated from A2 livers, two of which corresponded to ₁₁ and ₂₂. A third, absent from the typical and the atypical A1 livers, had an intermediate mobility; it was designated ₂₁. Type A1 livers are, therefore, the homozygous ADH₂ 2-2 phenotype, and type A2 livers, the heterozygous ADH₂ 2-1 phenotype. The ADH₂ 2-2 phenotype was found in 53% of 194 Japanese livers, and the ADH₂ 2-1 phenotype, in 31%. Accordingly, the frequency of ADH ₂ ² was 0.68.This study was supported by U.S. Public Health Service Grant AA 02342.     

Tpl-2/Cot and COX-2 in breast cancer

Background: Breast cancer is the most common cancer in women worldwide and although mortality (129 000/year) stagnates, incidence (370 000/year) is increasing. In addition to histological type, grade, stage, hormonal and c-erbB2 status there is therefore a strong need for new and reliable prognostic and predictive factors. Methods and results: This minireview focuses on two potential prognostic and predictive candidates Tpl2/Cot and COX-2 and summarise information about them. Conclusion: Tumor progression locus 2 (Tpl2/Cot) is a serine/threonine protein kinase belonging to the family of MAP3 kinases. Activated Tpl2/Cot leads to induction of ERK1/2, JNK, NF-kappaB and p38MAPK pathways. The first study on Tpl2/Cot mRNA in breast cancer showed its increase in 40 % of cases of breast cancer but no available data exist on protein expression. Cyclo-oxygenase 2 (COX-2) is inducible by growth and inflammatory factors and contributes to the development of various tumours. Expression of COX-2 in breast cancer varied from 5-100 % in reviewed papers with significantly higher values in poorly differentiated tumours. Tpl2/Cot and COX-2 have their importance in different intracellular pathways and some of these are involved in cancer development. Briefly, the results from recent studies suggest that Tpl2/Cot and COX-2 could be prognostic factors in breast cancer.     

2-(4,5-Dihydroimidazol-2-yl)benzimidazoles as highly selective imidazoline I2/adrenergic alpha2 receptor ligands

2-(4,5-Dihydroimidazol-2-yl)benzimidazoles have been identified as selective imidazoline I2/alpha2-adrenoceptor ligands. 4-Methyl (2) and 4-chloro (4) derivatives display I2 affinity at nanomolar concentration (Ki=4.4 and 17.7 nM, respectively) and high I2/alpha2 selectivity ratio=4226 and 5649, respectively. An evidence has been obtained that pKa value influences considerably the I2/alpha2-selectivity ratio of this class of imidazoline I2 receptor ligands.