洋葱伯克霍尔德菌发酵产抗菌物质CF66I温度控制策略

在3.7 L发酵罐中考察了不同温度23℃～33℃对洋葱伯克霍尔德菌CF-66发酵产抗菌物质CF66I的影响,并对不同温度下的发酵过程进行了动力学特性分析。在此基础之上,提出抗菌物质CF66I合成的分阶段控制策略：发酵过程0～20 h控制温度在30℃,20 h后温度控制在25℃至发酵结束。采用此温度控制策略进行了CF66I的发酵,CF66I的活性达到了3.783 u/mL,比采用单一温度下的最大值提高了26.1%。     

Nogo-66 and myelin-associated glycoprotein (MAG) inhibit the adhesion and migration of Nogo-66 receptor expressing human glioma cells

Malignant gliomas are common and aggressive brain tumours associated with significant morbidity and mortality. We showed in this report that substratum adherence and migration by human U87MG glioma cells in culture were significantly attenuated by the extracellular domains of Nogo-A (Nogo-66) and the myelin-associated glycoprotein (MAG). U87MG cells contained significant amounts of endogenous Nogo-66 receptor (NgR), and treatment of the cells with phosphatidylinositol-specific phospholipase C (PI-PLC) or NgR antibodies resulted in an increase in their ability to adhere to, or migrate through, Nogo-66- and MAG-coated substrates. Nogo-66 and MAG may therefore modulate glioma growth and migration by acting through the NgR, a phenomenon that has potential therapeutic implications.     

洋葱伯克霍尔德菌（Burkholderia cepacia）CF-66发酵生产新型抗菌物质CF66I培养基的优化

利用基于统计学的实验设计RSM（Response surface methodology）优化了Burkholderia cepacia CF-66产新型抗菌活性物质CF66I的发酵培养基组成。首先,用部分重复因子实验对培养基组分NH4Cl,MgSO4·7H2O,柠檬酸钠及酵母粉浓度对菌株产CF66I的影响进行评价,找出主要影响因子为柠檬酸钠和酵母粉。两者均为正影响,其他组分对CF66I活性的影响不显著。其次用最陡爬坡路径逼近最大响应区域。最后用中心组合设计及响应面分析确定主要影响因子的最佳浓度。菌株在优化培养基中培养较初始培养基CF66I活性提高了约两倍。     

Age-related changes in levels of p66Shc and serine 36-phosphorylated p66Shc in organs and mouse tissues

Mammalian life span can be controlled by p66Shc protein through regulation of cellular response to oxidative stress. We investigated age-related changes in the amount of p66Shc and its Ser36-phosphorylated form in various mouse organs and tissues and correlated it with the level of antioxidant enzymes. Comparing to the newborn, in adult 6-month-old mice, the level of p66Shc was increased particularly in liver, lungs, skin and diaphragm. In older animals the level of p66Shc decreased while signaling pathway responsible for Ser36 phosphorylation of p66Shc protein seemed to be continually enhanced. The amount of p66Shc phosphorylated at Ser36, significantly increased with age, resulted in higher free radical production and, in consequence accumulation of damages caused by free radicals. The increased amount of Ser36-phosphorylated p66Shc in livers of 12- and 23-month-old mice was correlated with the decreased level of antioxidant enzymes. Moreover, we found that p66Shc is a resident of mitochondria- and plasma membrane-associated membranes and that its level there depends on the age of animal.     

Chromatographic characterization of synthetic peptides: SPf66 malaria vaccine

The development and validation of a quantitative size-exclusion chromatography (SEC) method for SPf66 malaria vaccine was achieved. The results show the reliability of the analytical method for the intended use. SPf66 malaria vaccine characterization was perforrmed using both relative techniques such as the conventional SEC and absolute techniques: mass spectrometry and multi-angle laser-light scattering detection. The relative and absolute molecular masses were in the 4600-18,000 Da range. The results clearly indicate the presence of the monomer and dimer species, whereas the third species could be the trimer or tetramer.     

The crystal structure of novel chondroitin lyase ODV-E66, a baculovirus envelope protein

Chondroitin lyases have been known as pathogenic bacterial enzymes that degrade chondroitin. Recently, baculovirus envelope protein ODV-E66 was identified as the first reported viral chondroitin lyase. ODV-E66 has low sequence identity with bacterial lyases at <12%, and unique characteristics reflecting the life cycle of baculovirus. To understand ODV-E66’s structural basis, the crystal structure was determined and it was found that the structural fold resembled that of polysaccharide lyase 8 proteins and that the catalytic residues were also conserved. This structure enabled discussion of the unique substrate specificity and the stability of ODV-E66 as well as the host specificity of baculovirus.     

Nogo-A and Nogo-66 receptor in amyotrophic lateral sclerosis

Nogo/reticulon (RTN)-4 has been strongly implicated as a disease marker for the motor neuron disease amyotrophic lateral sclerosis (ALS). Nogo isoforms, including Nogo-A, are ectopically expressed in the skeletal muscle of ALS mouse models and patients and their levels correlate with the disease severity. The notion of a direct involvement of Nogo-A in ALS aetiology is supported by the findings that Nogo-A deletion in mice reduces muscle denervation and prolongs survival, whereas overexpression of Nogo-A destabilizes motor nerve terminals and promotes denervation. Another intriguing, and somewhat paradoxical, recent finding revealed that binding of the Nogo-66 receptor (NgR) by either agonistic or antagonistic Nogo-66-derived peptides protects against p75 neurotrophin receptor (p75(NTR))-dependent motor neuron death. Ligand binding by NgR could result in subsequent engagement of p75(NTR), and this association could preclude pro-apoptotic signalling by the latter. Understanding the intricate interplay among Nogo isoforms, NgR and p75(NTR) in ALS disease progression may provide important, therapeutically exploitable information.     

P66Shc mediates increased platelet activation and aggregation in hypercholesterolemia

Background and hypothesis

Hypercholesterolemia leads to a prothrombotic phenotype. Platelet hyperactivity associated with hypercholesterolemia has been attributed, in part, to oxidative stress. P66Shc is a well-known determinant of cellular and organismal oxidative stress. However, its role in platelet biology is not known. We hypothesized that p66Shc mediates platelet hyperactivation and hyperaggregation in hypercholesterolemia.

Methods and results

P66Shc was expressed in both human and mouse platelets, as determined by qRT-PCR and immunoblotting. Mouse platelet p66Shc expression was upregulated by hypercholesterolemia induced by high-fat diet feeding. Compared to wild-type mice, high-fat diet-induced p66Shc expression in platelets was suppressed in transgenic mice expressing a short hairpin RNA targeting p66Shc (p66ShcRNAi). High-fat diet feeding of wild-type mice amplified surface P-selectin expression on platelets stimulated by the thrombin receptor agonist protease-activated receptor-4 (PAR4), and increased aggregation of platelets induced by thrombin. These exaggerated platelet responses induced by high-fat diet feeding were significantly blunted in p66ShcRNAi mice. Finally, thrombin-stimulated platelet reactive oxygen species were suppressed in p66ShcRNAi mice.

Conclusions

Hypercholesterolemia stimulates p66Shc expression in platelets, promoting platelet oxidative stress, hyperreactivity and hyperaggregation via p66Shc.     

suhB基因对绿针假单胞菌HT66生防能力的影响

【背景】绿针假单胞菌(Pseudomonas chlororaphis) HT66是一株兼具生防安全性和吩嗪-1-甲酰胺(Phenazine-1-Carboxamide,PCN)高产的植物根际促生菌,在生物防治、生态农业及可持续发展农业领域具有广阔的应用前景。非编码RNA (ncRNA) SuhB参与了细胞中多个过程的代谢调控。【目的】探究suhB基因对绿针假单胞菌HT66生防能力的影响。【方法】以同源重组的方法无痕敲除suhB基因构建突变菌株HT66ΔsuhB,利用质粒回补suhB基因构建突变菌株HT66ΔsuhB-pBBR-suhB,研究suhB基因对菌株生长状态、生物膜形成、群集运动及PCN合成的影响。【结果】缺失suhB基因后,菌株HT66生长缓慢,平台期滞后12 h,而且生物量减少为野生型的61.6%;在KMB培养基中单位细胞PCN产量最高达109.5mg/g,为野生株的2.1倍;生物膜形成量明显增加,为野生型的1.8倍;在运动性检测平板上,野生株的运动半径为21 mm,而suhB突变株的运动半径缩减至9.7 mm,群集运动能力明显下降。suhB基因回补突变株上述生物学功能同野生株相似。在突变株HT66ΔsuhB中,pME6015-phzI-lacZ融合质粒表达的LacZ酶活与野生型差异不显著;pME6015-phzR-lacZ融合质粒表达的LacZ酶活显著上升,为野生型的3.1倍;pME6522-phzAp-lacZ融合质粒表达的LacZ酶活为野生型的1.8倍。【结论】绿针假单胞菌HT66中suhB基因参与了菌株生长、生物膜形成、群集运动及PCN合成等多个过程的调控。本研究为该菌株的代谢改造与生防应用提供了理论基础。     

丝氨酸蛋白酶HtrA2/Omi可参与调节线粒体中的适配蛋白p66Shc

目的:p66Shc在线粒体内积累和HtrA2/Omi的功能缺陷都能导致线粒体损伤,诱导细胞凋亡.探讨在线粒体中HtrA2对p66Shc的调控作用.方法:构建p66Shc和成熟型HtrA2的真核表达质粒,共转染HEK293T细胞,免疫印迹法(Western blot)检测p66Shc蛋白;构建原核表达质粒,大肠杆菌纯化蛋白,体外切割实验,SDS-PAGE分离后考马斯亮蓝染色检测;提取HtrA2功能缺陷小鼠( mnd2)大脑组织的线粒体,检测线粒体内p66Shc的蛋白水平.结果:细胞实验和体外实验证明HtrA2可以切割p66Shc,且在mnd2小鼠大脑中,线粒体内p66Shc的蛋白水平明显升高(P＜0.05).结论:p66Shc是HtrA2的直接底物,且HtrA2参与调节线粒体中p66Shc的蛋白水平,揭示了HtrA2发挥神经保护功能新的可能机制.     

Nogo receptor 3, a paralog of Nogo-66 receptor 1 （NgR1）, may function as a NgR1 co-receptor for Nogo-66

Nogo-A is a major myelin associated inhibitor that blocks regeneration of injured axons in the central nervous system (CNS).Nogo-66 (a 66-residue domain of Nogo-A) expressed on the surface of oligodendrocytes has been shown to directly interact with Nogo-66 receptor 1 (NgR1).A number of additional components of NgR1 receptor complex essential for its signaling have been uncovered.However,detailed composition of the complex and its signaling mechanisms remain to be fully elucidated.In this study,we show that Nogo receptor 3 (NgR3),a paralog of NgR1,is a binding protein for NgR1.The interaction is highly specific because other members of the reticulin family,to which Nogo-A belongs,do not bind to NgR3.Neither does NgR3 show any binding activity with Nogo receptor 2 (NgR2),another NgR1 paralog.Majority of NgR3 domains are required for its binding to NgR1.Moreover,a truncated NgR3 with the membrane anchoring domain deleted can function as a decoy receptor to reverse neurite outgrowth inhibition caused by Nogo-66 in culture.These in vitro results,together with previously reported overlapping expression profile between NgR1 and NgR3,suggest that NgR3 may be associated with NgR1 in vivo and that their binding interface may be targeted for treating neuronal injuries.     

MiR-124 attenuates doxorubicin-induced cardiac injury via inhibiting p66Shc-mediated oxidative stress

Previous studies showed that miR-124 had a protective role by reducing oxidant stress and preventing cell apoptosis and autophagy. However, its role in doxorubicin-induced cardiomyopathy was less known. In our study, we confirmed increased ROS and decreased expression of miR-124 in doxorubicin-treated heart tissues and primary cardiomyocytes. The oxidative stress and cell apoptosis were alleviated by overexpressing miR-124, characterized by decreased activity of MDA and increased activity of SOD. While inhibiting miR-124 generated opposed effects. Mechanistically, our bioinformatic prediction and luciferase assay confirmed that miR-124 inhibited the expression of p66Shc, a proapoptotic signaling pathway. Our results suggested that miR-124 was hopeful to become a therapeutic target in doxorubicin-related cardiomyopathy.     

Negative regulation of chemokine receptor signaling and B-cell chemotaxis by p66Shc

Shc (Src homology 2 domain containing) adaptors are ubiquitous components of the signaling pathways triggered by tyrosine kinase-coupled receptors. In lymphocytes, similar to other cell types, the p52 and p66 isoforms of ShcA/Shc participate in a self-limiting loop where p52Shc acts as a positive regulator of antigen receptor signaling by promoting Ras activation, whereas p66Shc limits this activity by competitively inhibiting p52Shc. Based on the fact that many signaling mediators are shared by antigen and chemokine receptors, including p52Shc, we have assessed the potential implication of p66Shc in the regulation of B-cell responses to chemokines, focusing on the homing receptors CXCR4 (C-X-C chemokine receptor type 4) and CXCR5 (C-X-C chemokine receptor type 5). The results identify p66Shc as a negative regulator of the chemotactic responses triggered by these receptors, including adhesion, polarization and migration. We also provide evidence that this function is dependent on the ability of p66Shc to interact with the chemokine receptors and promote the assembly of an inhibitory complex, which includes the phosphatases SHP-1 (Src homology phosphatase-1) and SHIP-1 (SH2 domain-containing inositol 5''-phosphatase-1), that results in impaired Vav-dependent reorganization of the actin cytoskeleton. This function maps to the phosphorylatable tyrosine residues in the collagen homology 1 (CH1) domain. The results identify p66Shc as a negative regulator of B-cell chemotaxis and suggest a role for this adaptor in the control of B-cell homing.     

肝癌细胞(AH_(66))甲胎蛋白基因结构的一些变化

对AFP基因重新表达的分子机制的研究,有助于了解癌变的本质。我们以AFPmRNA反转录合成的~3H-cDNA为探针,进行液相杂交;用RAF 65和RAF_(87)与体外染色质转录系统转录的~(32)P-RNA进行点杂交,测出移植性大鼠肝癌AH_(66)细胞核和离体染色质的AFP基因转录水平远远高于正常大鼠肝。以BamHI,EcoRI,HindⅢ和PstI酶解基因组DNA,然后与缺口翻译标记的~(32)P-RAF_(65)和~(32)P-RAF_(87)探针杂交,测知BamHI和EcoRI的酶谱相同,而HindⅢ和Pst I的带型明显不同,表明结构上存在某些变化。用HpaⅡ和MspI测定了AH_(66)和正常大鼠肝AFP基因的甲基化程度,结果表明,AH_(66)的AFP基因甲基化不足。AFP基因的染色质构型,由它对DNaseI的敏感性来测定,AH_(66)对DNaseI比正常大鼠肝更敏感,表明基因处于活性状态。所有这些结果表明,AH_(66)的AFP基因存在某些结构上的变化,这种变化对于AFP基因从掩盖到活性状态可能是重要的。     

D,L-sulforaphane-induced apoptosis in human breast cancer cells is regulated by the adapter protein p66Shc

Cancer chemopreventive response to D,L-sulforaphane (SFN), a synthetic racemic analogue of broccoli constituent L-sulforaphane, is partly attributable to apoptosis induction, but the mechanism of cell death is not fully understood. The present study demonstrates a critical role for adapter protein p66(Shc) in SFN-induced apoptosis. Immortalized mouse embryonic fibroblasts (MEF) derived from p66(shc) knockout mice were significantly more resistant to SFN-induced apoptosis, collapse of mitochondrial membrane potential, and reactive oxygen species (ROS) production compared with MEF obtained from the wild-type mice. Notably, a spontaneously immortalized and non-tumorigenic human mammary epithelial cell line (MCF-10A) was resistant to SFN-induced ROS production and apoptosis. Stable overexpression of manganese superoxide dismutase in MCF-7 and MDA-MB-231 human breast cancer cells conferred near complete protection against SFN-induced apoptosis and mitochondrial membrane potential collapse. SFN treatment resulted in increased S36 phosphorylation and mitochondrial translocation of p66(shc) in MDA-MB-231 and MCF-7 cells, and SFN-induced apoptosis was significantly attenuated by RNA interference of p66(shc) in both cells. SFN-treated MDA-MB-231 and MCF-7 cells also exhibited a marked decrease in protein level of peptidyl prolyl isomerase (Pin1), which is implicated in mitochondrial translocation of p66(shc) . However, stable overexpression of Pin1 failed to alter proapoptotic response to SFN at least in MCF-7 cells. Finally, SFN-induced S36 phosphorylation of p66(Shc) was mediated by protein kinase Cβ (PKCβ), and pharmacological inhibition of PKCβ significantly inhibited apoptotic cell death resulting from SFN exposure. In conclusion, the present study provides new insight into the mechanism of SFN-induced apoptosis involving PKCβ -mediated S36 phosphorylation of p66(shc).     

T66I突变提高HIV-1整合酶可溶性研究

在研究HIV-1整合酶（IN）抗药性突变T66I时,发现这一突变同时可以提高整合酶的溶解性。原核表达了IN1–288/T66I和野生型（WT）,取菌体破碎后的上清, SDS-PAGE和his标签蛋白质染色进行分析,结果表明IN1–288/T66I可溶性约是WT的2.4倍。600 ml培养基中诱导表达IN1–288/T66I/BL21,亲和层析纯化共收获蛋白质4.72 mg。用改进的ELISA方法测定IN1–288/T66I和IN1 288/F185K /C280S链转移催化活性,结果显示两种蛋白质活性基本相当。提供了有别于F185K /C280S突变的另外一种整合酶可溶性表达的途径,IN1–288/T66I重组蛋白还可以应用到整合酶抑制剂筛选中,以获取避开T66I抗药性突变的抑制剂。     

Ectodomain shedding of human Nogo-66 receptor homologue-1 by zinc metalloproteinases

The Nogo-66 receptor (NgR) plays a pivotal role in the inhibition of neuroregeneration as the receptor for multiple neurite outgrowth inhibitors such as Nogo-A. We have previously shown that NgR undergoes zinc metalloproteinase-mediated ectodomain shedding in neuroblastoma cells. Here, we demonstrate that the NgR-related protein NgR homologue-1 is released from neuroblastoma cells as a full-length ectodomain (NgRH1-ecto) and an N-terminal fragment (NTF-NgRH1) containing the leucine-rich repeat region of the protein. Inhibitors of the major protease classes failed to block the release of NgRH1-ecto, suggesting that this occurs via a protease-independent mechanism, presumably by a phospholipase-like enzyme. The release of NTF-NgRH1 was blocked by a hydroxamate-based zinc metalloproteinase inhibitor and tissue inhibitor of metalloproteinases-2 and -3, but not -1, implicating the involvement of membrane-type matrix metalloproteinases in this process. Our findings thus highlight the parallels between the ectodomain shedding of NgRH1 and that previously described for NgR.     

Nogo-66 inhibits adhesion and migration of microglia via GTPase Rho pathway in vitro

Nogo-66 is a 66-amino-acid-residue extracellular domain of Nogo-A, which plays a key role in inhibition neurite outgrowth of central nervous system through binding to the Nogo-66 receptor (NgR) expressed on the neuron. Recent studies have confirmed that NgR is also expressed on the surface of macrophages/microglia in multiple sclerosis, but its biological effects remain unknown. In the present study, our results demonstrated that Nogo-66 triggered microglia anti-adhesion and inhibited their migration in vitro, which was mediated by NgR. We also assessed the roles of small GTP (glycosyl phosphatidylinositol)-binding proteins of the Rho family as the downstream signal transducers on the microglia adhesion and mobility induced by Nogo-66. The results showed that Nogo-66 activated RhoA and reduced the activity of Cdc42 in the meanwhile, which further triggered the anti-adhesion and migration inhibition effects to microglia. Nogo-66 inhibited microglia polarization and membrane protrusion formation, thus might eventually contribute to the decreasing capability of cell mobility. Taken together, the Nogo-66/NgR pathway may modulate neuroinflammation via mediating microglia adhesion and migration in addition to its role in neurons. Better understanding the relationship between Nogo-66/NgR and neuroinflammation may help targeting NgR for treating central nervous system diseases related with inflammation.     

HIV P66蛋白的表达、纯化及活性检测

此研究的目的是体外表达HIV-1逆转录酶P66亚单位蛋白,并得到纯度较高且具有活性的重组蛋白。首先通过PCR方法从含HIV-1 HXB2标准株全基因的质粒中扩增出全长p66基因,插入表达载体pTWIN1,构建pTWIN-p66质粒。再将该质粒转化到表达菌BL21,在IPTG诱导下表达P66蛋白。表达产物经几丁质柱一步纯化后得到P66蛋白纯品。纯化的P66蛋白分别催化B95-8细胞总RNA、RT试剂盒中对照RNA进行逆转录;同时以试剂盒中的M-MLV RT为对照进行相同的逆转录,PCR后电泳比较活性。结果显示我们构建的pTWIN-p66质粒在IPTG诱导下在原核细胞中P66亚单位蛋白表达量较高,经一步纯化后蛋白凝胶扫描纯度达88%,并具有较好的逆转录活性。与M-MLV RT体外活性比较,两者活性相当。该研究为进一步开发国产HIV确证诊断试剂及抗AIDS药物的研究打下了初步基础。