Plastid structure and carotenogenic gene expression in red- and white-fleshed loquat (Eriobotrya japonica) fruits

Loquat (Eriobotrya japonica Lindl.) can be sorted into red- and white-fleshed cultivars. The flesh of Luoyangqing (LYQ, red-fleshed) appears red-orange because of a high content of carotenoids while the flesh of Baisha (BS, white-fleshed) appears ivory white due to a lack of carotenoid accumulation. The carotenoid content in the peel and flesh of LYQ was approximately 68 μg g(-1) and 13 μg g(-1) fresh weight (FW), respectively, and for BS 19 μg g(-1) and 0.27 μg g(-1) FW. The mRNA levels of 15 carotenogenesis-related genes were analysed during fruit development and ripening. After the breaker stage (S4), the mRNA levels of phytoene synthase 1 (PSY1) and chromoplast-specific lycopene β-cyclase (CYCB) were higher in the peel, and CYCB and β-carotene hydroxylase (BCH) mRNAs were higher in the flesh of LYQ, compared with BS. Plastid morphogenesis during fruit ripening was also studied. The ultrastructure of plastids in the peel of BS changed less than in LYQ during fruit development. Two different chromoplast shapes were observed in the cells of LYQ peel and flesh at the fully ripe stage. Carotenoids were incorporated in the globules in chromoplasts of LYQ and BS peel but were in a crystalline form in the chromoplasts of LYQ flesh. However, no chromoplast structure was found in the cells of fully ripe BS fruit flesh. The mRNA level of plastid lipid-associated protein (PAP) in the peel and flesh of LYQ was over five times higher than in BS peel and flesh. In conclusion, the lower carotenoid content in BS fruit was associated with the lower mRNA levels of PSY1, CYCB, and BCH; however, the failure to develop normal chromoplasts in BS flesh is the most convincing explanation for the lack of carotenoid accumulation. The expression of PAP was well correlated with chromoplast numbers and carotenoid accumulation, suggesting its possible role in chromoplast biogenesis or interconversion of loquat fruit.     

Inhibition of CLIC4 enhances autophagy and triggers mitochondrial and ER stress-induced apoptosis in human glioma U251 cells under starvation

CLIC4/mtCLIC, a chloride intracellular channel protein, localizes to mitochondria, endoplasmic reticulum (ER), nucleus and cytoplasm, and participates in the apoptotic response to stress. Apoptosis and autophagy, the main types of the programmed cell death, seem interconnected under certain stress conditions. However, the role of CLIC4 in autophagy regulation has yet to be determined. In this study, we demonstrate upregulation and nuclear translocation of the CLIC4 protein following starvation in U251 cells. CLIC4 siRNA transfection enhanced autophagy with increased LC3-II protein and puncta accumulation in U251 cells under starvation conditions. In that condition, the interaction of the 14-3-3 epsilon isoform with CLIC4 was abolished and resulted in Beclin 1 overactivation, which further activated autophagy. Moreover, inhibiting the expression of CLIC4 triggered both mitochondrial apoptosis involved in Bax/Bcl-2 and cytochrome c release under starvation and endoplasmic reticulum stress-induced apoptosis with CHOP and caspase-4 upregulation. These results demonstrate that CLIC4 nuclear translocation is an integral part of the cellular response to starvation. Inhibiting the expression of CLIC4 enhances autophagy and contributes to mitochondrial and ER stress-induced apoptosis under starvation.     

猪链球菌2型与宿主细胞体外相互作用研究进展

猪链球菌2型(Streptococcus suis serotype2,SS2)是一种世界范围内的引起猪链球菌病的重要的人兽共患病原菌,人感染SS2后多呈现败血症型和脑膜炎型。2005年猪链球菌在我国四川省的感染暴发和近年来不断出现的零散病例给我国的公共卫生和食品安全带来了威胁,但迄今为止,对SS2致病机制研究尚不够深入。SS2的致病过程和宿主细胞相互作用密切相关,SS2-体外宿主细胞相互作用模型的建立对于揭示SS2致病分子机理具有重要的意义。目前的研究报道主要是从猪链球菌黏附与侵袭上皮细胞或内皮细胞、猪链球菌抗吞噬细胞的吞噬和猪链球菌激活免疫细胞炎症反应方面研究猪链球菌与宿主细胞的相互作用,但与其他常致病性链球菌如化脓性链球菌和肺炎链球菌相比,有些SS2-体外宿主细胞相互作用模型还缺乏统一的标准,由于感染模型的实验参数不尽相同得到结果也有所不同。本文对近年来猪链球菌2型与宿主细胞体外相互作用及其致病机理研究进展作一综述。     

rmlB基因在单核细胞增生李斯特菌耐药性、生物被膜形成和毒力方面的作用

【目的】探究单核细胞增生李斯特菌(Listeria monocytogenes,Lm)rmlB基因在细菌耐药、生物被膜形成和毒力方面的作用。【方法】通过同源重组的方法敲除Lm染色体上的rmlB基因,比较野生株与rmlB缺失株在耐药性方面的差异;利用微孔板法观测rmlB缺失菌株生物被膜形成能力的变化;利用RT-PCR检测缺失菌株中主要毒力基因转录表达,并观察rmlB缺失对细菌溶血活性的影响。【结果】同野生菌株相比,rmlB缺失菌株对头孢菌素和杆菌肽等作用位点在细菌细胞壁和细胞膜的敏感性显著增加(P≤0.01),生物被膜形成能力显著降低(P≤0.01),细菌主要毒力基因hly的转录表达及溶血活性也发生显著降低(P≤0.01)。【结论】rmlB基因在Lm生物被膜形成和耐受作用位点位于细胞壁和细胞膜的抗生素及细菌毒力方面具有重要作用。     

Asymmetric reduction of ketopantolactone using a strictly (<Emphasis Type="Italic">R</Emphasis>)-stereoselective carbonyl reductase through efficient NADPH regeneration and the substrate constant-feeding strategy

Objectives

To characterize a recombinant carbonyl reductase from Saccharomyces cerevisiae (SceCPR1) and explore its use in asymmetric synthesis of (R)-pantolactone [(R)-PL].

Results

The NADPH-dependent SceCPR1 exhibited strict (R)-enantioselectivity and high activity in the asymmetric reduction of ketopantolactone (KPL) to (R)-PL. Escherichia coli, coexpressing SceCPR1 and glucose dehydrogenase from Exiguobacterium sibiricum (EsGDH), was constructed to fulfill efficient NADPH regeneration. During the whole-cell catalyzed asymmetric reduction of KPL, the spontaneous hydrolysis of KPL significantly affected the yield of (R)-PL, which was effectively alleviated by the employment of the substrate constant-feeding strategy. The established whole-cell bioreduction for 6 h afforded 458 mM (R)-PL with the enantiomeric excess value of >99.9% and the yield of 91.6%.

Conclusions

Escherichia coli coexpressing SceCPR1 and EsGDH efficiently catalyzed the asymmetric synthesis of (R)-PL through the substrate constant-feeding strategy.

     

啮齿动物及凋落物存留对天山野果林野生樱桃李种子命运的影响

2010年9月至2013年4月通过设置人工土壤种子库,研究了野生樱桃李土壤种子库的动态及啮齿动物和凋落物覆盖对种子库中种子命运的影响.结果表明: 在有取食动物扰动下,48.3%的种子萌发输出为幼苗,50%的种子被动物搬运或当场取食,4%的种子腐烂.在排除了取食动物干扰的条件下,樱桃李种子形成了短期持久的土壤种子库,3年后依然有部分种子萌发并输出为幼苗.凋落物覆盖和对照处理中,被搬运和当场取食种子的比例均显著低于清除凋落物的裸地.地表凋落物存留能减少动物搬运、取食,但不足以导致新生幼苗的大量出现,而啮齿类动物的搬运或取食是影响野生樱桃李种子命运和种子库动态的主要因素.
     

Regulation of cyp26a1 on Th17 cells in mouse peri‐implantation

Cytochrome P450 26A1 (cyp26a1) is expressed in the mouse uterus during peri‐implantation. The repression of this protein is closely associated with a reduction in implantation sites, suggesting a specific role for cyp26a1 in pregnancy and prompting questions concerning how a metabolic enzyme can generate this distinct outcome. To explore the effective downstream targets of cyp26a1 and confirm if its role in peri‐implantation depends on its metabolic substrate RA (retinoic acid), we characterized the changes in the peripheral blood, spleen and uterine implantation sites using the cyp26a1 gene vaccine constructed before. Flow cytometry results showed a significant increase in CD4⁺RORγt⁺ Th17 cells in both the peripheral blood and spleen in the experimental group. The expression of RORγt and IL‐17 presented the Th17 cells reduction in uterus followed by the suppression of cyp26a1 expression. For greater certainty, cyp26a1 antibody blocking model and RNA interference model were constructed to determine the precise target immune cell group. High performance liquid chromatography results showed a significant increase in uterine at‐RA followed by the immunization of cyp26a1 gene vaccine. Both the ascertain by measuring RARα protein levels in peri‐implantation uterus after gene vaccine immunization and researches using the specific agonist and antagonist against RARα suggested that RARα may be the main RA receptor for signal transduction. These results provided more evidence for the signal messenger role of RA in cyp26a1 regulation from the other side. Here, we showed that the cyp26a1‐regulated Th17 cells are dependent on at‐RA signalling, which is delivered through RARα in mouse peri‐implantation.     

Molecular mechanism of ERK dephosphorylation by striatal‐enriched protein tyrosine phosphatase

Striatal‐enriched tyrosine phosphatase (STEP) is an important regulator of neuronal synaptic plasticity, and its abnormal level or activity contributes to cognitive disorders. One crucial downstream effector and direct substrate of STEP is extracellular signal‐regulated protein kinase (ERK), which has important functions in spine stabilisation and action potential transmission. The inhibition of STEP activity toward phospho‐ERK has the potential to treat neuronal diseases, but the detailed mechanism underlying the dephosphorylation of phospho‐ERK by STEP is not known. Therefore, we examined STEP activity toward para‐nitrophenyl phosphate, phospho‐tyrosine‐containing peptides, and the full‐length phospho‐ERK protein using STEP mutants with different structural features. STEP was found to be a highly efficient ERK tyrosine phosphatase that required both its N‐terminal regulatory region and key residues in its active site. Specifically, both kinase interaction motif (KIM) and kinase‐specific sequence of STEP were required for ERK interaction. In addition to the N‐terminal kinase‐specific sequence region, S245, hydrophobic residues L249/L251, and basic residues R242/R243 located in the KIM region were important in controlling STEP activity toward phospho‐ERK. Further kinetic experiments revealed subtle structural differences between STEP and HePTP that affected the interactions of their KIMs with ERK. Moreover, STEP recognised specific positions of a phospho‐ERK peptide sequence through its active site, and the contact of STEP F311 with phospho‐ERK V205 and T207 were crucial interactions. Taken together, our results not only provide the information for interactions between ERK and STEP, but will also help in the development of specific strategies to target STEP‐ERK recognition, which could serve as a potential therapy for neurological disorders.