Biosynthesis,isolation, and NMR analysis of leukotriene A epoxides: substrate chirality as a determinant of the cis or trans epoxide configuration

Leukotriene (LT)A₄ and closely related allylic epoxides are pivotal intermediates in lipoxygenase (LOX) pathways to bioactive lipid mediators that include the leukotrienes, lipoxins, eoxins, resolvins, and protectins. Although the structure and stereochemistry of the 5-LOX product LTA₄ is established through comparison to synthetic standards, this is the exception, and none of these highly unstable epoxides has been analyzed in detail from enzymatic synthesis. Understanding of the mechanistic basis of the cis or trans epoxide configuration is also limited. To address these issues, we developed methods involving biphasic reaction conditions for the LOX-catalyzed synthesis of LTA epoxides in quantities sufficient for NMR analysis. As proof of concept, human 15-LOX-1 was shown to convert 15S-hydroperoxy-eicosatetraenoic acid (15S-HPETE) to the LTA analog 14S,15S-trans-epoxy-eicosa-5Z,8Z,10E,12E-tetraenoate, confirming the proposed structure of eoxin A₄. Using this methodology we then showed that recombinant Arabidopsis AtLOX1, an arachidonate 5-LOX, converts 5S-HPETE to the trans epoxide LTA₄ and converts 5R-HPETE to the cis epoxide 5-epi-LTA₄, establishing substrate chirality as a determinant of the cis or trans epoxide configuration. The results are reconciled with a mechanism based on a dual role of the LOX nonheme iron in LTA epoxide biosynthesis, providing a rational basis for understanding the stereochemistry of LTA epoxide intermediates in LOX-catalyzed transformations.     

The Metastasis Suppressor,N-myc Downregulated Gene 1 (NDRG1), Is a Prognostic Biomarker for Human Colorectal Cancer

Metastasis remains to be one of the most prevalent causes leading to poor long-term survival of colorectal cancer (CRC) patients. The clinical significances of tumor metastatic suppressor, N-myc downregulated gene 1 (NDRG1), have been inconsistently reported in a variety of cancerous diseases. In this study with 240 CRC clinical specimens, we showed that NDRG1 expression was significantly decreased in most of CRC tissues compared to the paired non-tumor counterparts. Statistical analysis revealed a significant inverse correlation of NDRG1 expression with tumor stage, differentiation status and metastasis. Compared with NDRG1-negative group, NDRG1-positve group had better disease-free/overall survival (p = 0.000) over 5 years’ follow-up. Furthermore, NDRG1 was considered to be an independent prognostic factor for overall survival (p = 0.001) and recurrence (p = 0.003). Our study concludes that NDRG1 is a novel favorable predictor for the prognosis in CRC patients.     

Identification of HSP90 as Potential Biomarker of Biliary Atresia Using Two-Dimensional Electrophoresis and Mass Spectrometry

Biliary atresia (BA) is a devastating cholestatic liver disease targeting infants. Current diagnosis depends on surgical exploration of the biliary tree. The aim of the present study was to identify potential biomarkers for the diagnosis of biliary atresia (BA). Two-dimensional electrophoresis was utilized for the identification of proteins that were differentially expressed in liver biopsies of 20 BA patients and 12 infants with non-BA neonatal cholestasis (NC) as controls. Using mass spectrometry, we identified 15 proteins with expressions significantly altered. Out of the 15 proteins identified, heat shock protein (HSP) 90 was the most significantly altered and was down-regulated in BA samples compared to NC samples using immunoblotting analysis. Our findings suggest that HSP90 might be a potential biomarker for the diagnosis of BA and may be used for monitoring further development and therapy for BA. This study demonstrated that a comprehensive strategy of proteomic identification combined with further validation should be adopted in biomarker discovery.     

Applying Reversible Mutations of Nodulation and Nitrogen-Fixation Genes to Study Social Cheating in Rhizobium etli-Legume Interaction

Mutualisms are common in nature, though these symbioses can be quite permeable to cheaters in situations where one individual parasitizes the other by discontinuing cooperation yet still exploits the benefits of the partnership. In the Rhizobium-legume system, there are two separate contexts, namely nodulation and nitrogen fixation processes, by which resident Rhizobium individuals can benefit by cheating. Here, we constructed reversible and irreversible mutations in key nodulation and nitrogen-fixation pathways of Rhizobium etli and compared their interaction with plant hosts Phaseolus vulgaris to that of wild type. We show that R. etli reversible mutants deficient in nodulation factor production are capable of intra-specific cheating, wherein mutants exploit other Rhizobium individuals capable of producing these factors. Similarly, we show that R. etli mutants are also capable of cheating inter-specifically, colonizing the host legume yet contributing nothing to the partnership in terms of nitrogen fixation. Our findings indicate that cheating is possible in both of these frameworks, seemingly without damaging the stability of the mutualism itself. These results may potentially help explain observations suggesting that legume plants are commonly infected by multiple bacterial lineages during the nodulation process.     

Hepatic Parenchymal Changes following Transcatheter Embolization and Chemoembolization in a Rabbit Tumor Model

Objective

To compare the effects of transcatheter arterial chemoembolization (TACE) with transcatheter arterial embolization (TAE) on liver function, hepatic damage, and hepatic fibrogenesis in a rabbit tumor model.

Materials and Methods

Thirty-nine New Zealand white rabbits implanted with VX2 tumors in the left liver lobes were randomly divided into three groups: TAE, TACE, and control group. In the TAE group (n = 15), polyvinyl alcohol particles (PVAs) were used for left hepatic artery embolization. In the TACE group (n = 15), the tumors were treated with left hepatic arterial infusions of a suspension of 10-hydroxycamptothecin and lipiodol, followed by embolization with PVAs. In the control group (n = 9), the animals received sham treatment with distilled water. Serum and liver samples were collected at 6 hours, 3 days and 7 days after treatment. Liver damage was measured using a liver function test and histological analyses. Liver fibrogenesis and hepatic stellate cell (HSC) activation were evaluated using Sirius Red and anti-alpha-smooth muscle actin (α-SMA) immunohistochemical stains.

Results

TACE caused liver injury with greater increases in serum alanine aminotransferase and aspartate aminotransferase levels on day 3 (P<0.05). Histological analyses revealed increased hepatic necrosis in adjacent non-tumorous liver tissue from day 3 compared to the TAE group (Suzuki score of 2.33±1.29 versus 1.13±1.18, P = 0.001). HSC activation and proliferation were significantly increased in the TACE group compared to the control group at 3 and 7 days after treatment (0.074±0.014 vs. 0.010±0.006, and 0.088±0.023 vs. 0.017±0.009, P<0.05). Sirius Red staining demonstrated a statistically significant increase in collagen deposition in the livers in the TACE group 7 days after embolization compared to the control group (0.118±0.012 vs. 0.060±0.017, P = 0.05).

Conclusion

The results of this animal study revealed that TACE induced prominent hepatocellular damage and hepatic fibrogenesis, which compromised liver function and may be responsible for chronic liver decompensation.     

CSTP1, a Novel Protein Phosphatase,Blocks Cell Cycle,Promotes Cell Apoptosis,and Suppresses Tumor Growth of Bladder Cancer by Directly Dephosphorylating Akt at Ser473 Site

Akt/protein kinase B is a pivotal component downstream of phosphatidylinositol 3-kinase (PI3K) pathway, whose activity regulates the balance between cell survival and apoptosis. Phosphorylation of Akt occurs at two key sites either at Thr308 site in the activation loop or at Ser473 site in the hydrophobic motif. The phosphorylated form of Akt (pAkt) is activated to promote cell survival. The mechanisms of pAkt dephosphorylation and how the signal transduction of Akt pathway is terminated are still largely unknown. In this study, we identified a novel protein phosphatase CSTP1(complete s transactivated protein 1), which interacts and dephosphorylates Akt specifically at Ser473 site in vivo and in vitro, blocks cell cycle progression and promotes cell apoptosis. The effects of CSTP1 on cell survival and cell cycle were abrogated by depletion of phosphatase domain of CSTP1 or by expression of a constitutively active form of Akt (S473D), suggesting Ser473 site of Akt as a primary cellular target of CSTP1. Expression profile analysis showed that CSTP1 expression is selectively down-regulated in non-invasive bladder cancer tissues and over-expression of CSTP1 suppressed the size of tumors in nude mice. Kaplan-Meier curves revealed that decreased expression of CSTP1 implicated significantly reduced recurrence-free survival in patients suffered from non-invasive bladder cancers.     

玉米ZmSOD基因克隆及其在干旱胁迫下的表达

为探讨超氧化物歧化酶(superoxide dismutase,SOD)基因在玉米抗逆反应中的作用,研究选用2个抗旱性有明显差别的玉米品种为试验材料,采用RT-PCR、实时荧光定量PCR(qRT-PCR)及二维凝胶电泳(2-DE)耦联的基质辅助激光解吸电离/飞行时间质谱(MALDI-TOF)法,对ZmSOD基因的序列结构及其在干旱胁迫下的表达特性进行分析。结果表明：(1)从干旱敏感品种‘登海605’(DH605)和抗旱品种‘蠡玉35’(LY35)玉米叶片中分别成功克隆出ZmSOD1和ZmSOD2基因。DH605中ZmSOD1开放阅读框(ORF)全长456 bp,编码151个氨基酸,编码的蛋白等电点为5.76,分子量为15.05 kD。LY35中ZmSOD2ORF全长459 bp,编码152个氨基酸,编码的蛋白等电点为5.65,分子量为15.11 kD;ZmSOD1和ZmSOD2是亲水性稳定蛋白,都含有Cu/Zn-SOD结构域,蛋白序列N端无信号肽及无跨膜结构域。同源性和系统进化分析表明,玉米ZmSOD和谷子Cu/Zn-SOD2的亲缘关系最近,相似性高达96.05%。(2)在干旱条件下,ZmSOD1在DH605中转录水平明显降低,LY35中ZmSOD2转录水平显著升高;2-DE耦联的质谱分析显示：ZmSOD1在DH605中表达量明显降低,LY35中ZmSOD2表达量无显著性变化,却检测到Mn-SOD蛋白表达量显著增加。(3)相关分析显示,干旱条件下,DH605叶片中ZmSOD1转录水平和其蛋白丰度及SOD酶活性呈极显著性正相关,LY35叶片中ZmSOD2转录水平与SOD酶活性呈显著性正相关。研究结果为进一步探索SOD基因在调节玉米抗性以及逆境胁迫应答过程中的作用机制提供了基础。     

Exosome-mediated miR-7-5p delivery enhances the anticancer effect of Everolimus via blocking MNK/eIF4E axis in non-small cell lung cancer

Everolimus is a kind of mammalian target of rapamycin (mTOR) inhibitors. Activated mitogen-activated protein kinase interacting kinases/eukaryotic translation initiation factor 4E (MNK/eIF4E) axis plays a crucial role in resistance to Everolimus in non-small cell lung cancer (NSCLC). The eIF4E phosphorylation increased by mTOR inhibitors is mainly mediated by MNKs. However, the mechanisms are poorly understood. Recently, extensive reprogramming of miRNA profiles has also been found after long-term mTOR inhibitor exposure. Our previous studies have confirmed that tumor suppressor miR-7-5p is decreased in A549 cells after treatment with Everolimus. Exactly, MNK1 is the target of miR-7-5p. In this study, we investigated the biological functions and potential molecular mechanisms of miR-7-5p in the NSCLC undergoing treatment with Everolimus. We confirmed that Everolimus targeted mTORC1 inducing NSCLC cells to secrete miR-7-5p-loaded exosomes in Rab27A and Rab27B-dependent manners. Loss of intracellular miR-7-5p induced phosphorylation of MNK/eIF4E axis, but a supplement of extra exosomal miR-7-5p could reverse it. Of note, both low expression of miR-7-5p and elevated MNK1 protein were associated with a poor prognosis of NSCLC. Both endogenous miR-7-5p and exo-miR-7-5p enhanced the therapeutic efficacy of Everolimus by inhibiting the proliferation, migration, and metastasis of NSCLC in vitro and in vivo. The combination of miR-7-5p with Everolimus induced apoptosis to exhibit a synergistic anticancer therapeutic efficacy through dual abrogation of MNK/eIF4E and mTOR in NSCLC. In conclusion, Everolimus decreases the intracellular miR-7-5p by releasing of miR-7-5p loaded exosomes from NSCLC cells in Rab27A and Rab27B dependent manners. Either endogenous miR-7-5p or exo-miR-7-5p combined with Everolimus can enhance the anticancer efficacy by targeting MNK/eIF4E axis and mTOR. Besides, both low levels of miR-7-5p and positive expression of MNK1 act as independent poor prognostic biomarkers for NSCLC. Therefore, restoring miR-7-5p carried by exosome may be a promising novel combined therapeutic strategy with Everolimus for NSCLC.Subject terms: Drug development, Growth factor signalling, Oncogenesis     

Deregulation of Cdc2 kinase induces caspase-3 activation and apoptosis

Progression of the cell cycle and control of apoptosis are tightly linked processes. It has been reported that manifestation of apoptosis requires cdc2 kinase activity yet the mechanism(s) of which is largely unclear. In an attempt to study the role of human MDM2 (HDM2) in interphase and mitosis, we employed the Xenopus cell-free system to study HDM2 protein stability. Interestingly, HDM2 is specifically cleaved in Xenopus mitotic extracts but not in the interphase extracts. We demonstrate that HDM2 cleavage is dependent on caspase-3 and that activation of cdc2 kinase results in caspase-3 activation in the Xenopus cell-free system. Furthermore, expression of cdc2 kinase in mammalian cells leads to activation of caspase-3 and apoptosis. Taken together, these data indicate that deregulation of cdc2 kinase activity can trigger apoptotic machinery that leads to caspase-3 activation and apoptosis.