Caspase-3反义寡核苷酸对γ-射线诱导的HL-60细胞中caspase-3表达与细胞凋亡的抑制作用

γ-射线可诱导人髓性白血病细胞株HL-60细胞凋亡,但其机制尚未完全明了。为了观察caspase-3在这种细胞凋亡模型中的作用,本研究设计合成针对caspase-3mRNA5′-非编码区和编码起始区的反义寡核苷酸(ASODNs),即ASODN-1和ASODN-2,以脂质体介导法将不同浓度ASODN-1和ASODN-2转染进入HL-60细胞,γ-射线照射。应用TUNEL法观察凋亡细胞形态学变化及检测凋亡细胞百分率,免疫细胞化学、Westernblotting和RT-PCR技术分别检测caspase-3及其mRNA在引入ASODNs前后的表达水平,并以错配寡核苷酸(MODN)转染及未转染细胞作为对照组。TUNEL法检测发现,当ASODN-1和ASODN-2转染终浓度≥3μmol/L时,γ-射线诱导的HL-60细胞凋亡率降低,与对照组相比均有显著性差异(P<0.01)。免疫细胞化学结果显示,与两对照组相比,转染ASODNs后各组caspase-3阳性细胞率显著下降,阳性细胞染色减弱,其平均灰度值显著增高(P<0.01)。Westernblotting检测显示,转染ASODNs组细胞caspase-3蛋白酶原表达降低,其中ASODN-1组显著低于ASODN-2组。RT-PCR结果显示两对照组细胞caspase-3mRNA均有明显表达,转染ASODNs后caspase-3mRNA表达丰度降低。另外,ASODN-1抑制细胞凋亡和caspase-3表达的作用显著强于ASODN-2(分别为P<0.05和P<0.01)。实验结果表明,caspase-3mRNAASODNs能够抑制γ-射线照射诱导的HL-60细胞凋亡,下调caspase-3蛋白和caspase-3mRNA的表达水平,其抑制作用在一定范围内呈剂量依赖性。     

High-Level Expression of the Antimicrobial Peptide Plectasin in Escherichia coli

Plectasin is a defensin-like antimicrobial peptide isolated from a fungus, the saprophytic ascomycete Pseudoplectania nigrella. Plectasin showed marked antibacterial activity in vitro against Gram-positive bacteria, especially Streptococcus pneumoniae, including strains resistant to conventional antibiotics. Plectasin could kill the sensitive strain as efficaciously as vancomycin and penicillin and without cytotoxic effects on mammalian cell viability. In order to establish a bacterium-based plectasin production system, in the present study, the coding sequence of plectasin was optimized, and then cloned into pET32a (+) vector and expressed as a thioredoxin (Trx) fusion protein in Escherichia coli. The soluble fusion protein collected from the supernatant of the cell lysate was separated by Ni²⁺-chelating affinity chromatography. The purified protein was then cleaved by Factor Xa protease to release mature plectasin. Final purification was achieved by Ni²⁺-chelating chromatography again. The recombinant plectasin exhibited the same antimicrobial activity as reported previously. This is the first study to describe the expression of plectasin in E. coli expression system, and these works might provide a significant foundation for the following production or study of plectasin, and contribute to the development and evolution of novel antimicrobial drugs in clinical applications.     

Novel 14S,21‐dihydroxy‐docosahexaenoic acid rescues wound healing and associated angiogenesis impaired by acute ethanol intoxication/exposure

Acute ethanol intoxication and exposure (AE) has been known to impair wound healing and associated angiogenesis. Here, we found that AE diminished the formation of novel reparative lipid mediator 14S,21‐dihydroxy‐docosa‐4Z,7Z,10Z,12E,16Z,19Z‐hexaenoic acid (14S,21‐diHDHA) and its biosynthetic intermediate 14S‐hydroxy‐DHA (14S‐HDHA) from docosahexaenoic acid (DHA) in murine wounds. However, AE did not reduce the formation of DHA and the intermediate 21‐HDHA. These results indicate that in the biosynthetic pathways of 14S,21‐diHDHA in wounds, AE suppresses the 14S‐hydroxy‐generating activity of 12‐lipoxygenase‐like (LOX‐like), but does not suppress the 21‐hydroxy‐generating activity of cytochrome P450 and DHA‐generating activities. The AE‐suppression of 12‐LOX‐like activity was further confirmed by the diminished formation of 12‐hydroxy‐eicosatetraenoic acid in wounds under AE. Supplementing 14S,21‐diHDHA to wounds rescued the AE‐impaired healing and vascularization. 14S,21‐diHDHA restored AE‐impaired processes of angiogenesis in vitro: endothelial cell migration, tubulogenesis, and phosphorylation of p38 mitogen‐activated protein kinase (MAPK). Taken together, the suppression of 14S,21‐diHDHA formation is responsible, at least partially, for the AE‐impairment of cutaneous wound healing and angiogenesis. Supplementing 14S,21‐diHDHA to compensate its deficit in AE‐impaired wounds rescues the healing and angiogenesis. These results provide a novel mechanistic insight for AE‐impaired wound healing that involves the necessary roles of 14S,21‐diHDHA. They also offer leads for developing 14S,21‐diHDHA‐related therapeutics to ameliorate AE‐impairment of wound healing. J. Cell. Biochem. 111: 266–273, 2010. © 2010 Wiley‐Liss, Inc.     

Synthesis and evaluation of nitric oxide-releasing derivatives of farnesylthiosalicylic acid as anti-tumor agents

Novel furoxan-based nitric oxide (NO)-releasing derivatives (11a–p) of farnesylthiosalicylic acid (FTA) were synthesized. Compounds 11d, 11f, 11k, and 11m–o displayed anti-tumor activities superior to FTA and sorafenib in most cancer cells tested. Analysis of six compounds revealed that 11d, 11f, 11n, 11o, and 11p, but not 11a that had low anti-tumor activity, produced high levels of NO, associated with their strong anti-tumor activity. Furthermore, the anti-tumor activity of 11f was partially mimicked by the furoxan moiety, but reduced by pre-treatment with hemoglobin. Importantly, treatment with 11f inhibited Ras-related signaling in cancer cells. Apparently, the high anti-tumor activity of 11f was attributed to the synergic effect of high levels of NO production and inhibition of Ras-related signaling in cancer cells. Our findings suggest that the furoxan/FTA hybrids may hold greater promise as therapeutic agents for the intervention of human cancers.     

Evaluation of the cytoprotective effects of bovine lactoferrin against intestinal toxins using cellular model systems

This study investigated the effects of bovine lactoferrin (BLf) on the growth of different groups of bacteria in vitro. BLf showed a significant inhibitory effect on the growth of selected pathogens but not probiotics. BLf, in combination with probiotics, has the potential to influence the composition of the gut microflora via inhibition of intestinal pathogens with no significant effect on probiotic bacteria.     

SAR and molecular mechanism study of novel acylhydrazone compounds targeting HIV-1 CA

We synthesized a series of acylhydrazone compounds bearing naturally occurring amino acids’ side chains as HIV assembly inhibitors. Biological evaluation indicated that the compounds had anti-SIV and capsid assembly inhibitory activities. The structure–activity relationship (SAR) study showed that compounds bearing proper aromatic side chains had potential antiviral activities. The molecular modeling experiments revealed the molecular mechanism that they could bind to CA in the same manner as CAP-1 and occupy two more grooves.     

Microscopic and Protein Expression Analysis of Grain Weight Variation within the Spike of Triticum aestivum

The kernels possess significant grain weight variation in one wheat (Triticum aestivum L) plant because of their different positions within the spike. In order to understand the molecular basis of weight, a proteomic approach, employing twodimensional electrophoresis and matrix-assisted laser desorptionfionization time of flight mass spectrometry (MALDI-TOF MS), was used to identify proteins between two kinds of kernels, the high weight kernel (large kernel) and the low weight kernel (small kernels) with different positions within spikes of one wheat cultivar, Shannong. Microscopic observation showed that the endosperm cells in large kernels enlarged their volume and accumulated storage materials at grain filling stage (17 days after anthesis, DAP), whilst those in small kernels were mainly in cell division with larger vacuoles during this period. Proteins were extracted from the kernels at this time, and resolved using 24-cm immobilized pH gradient strips with a pH 3–10 linear gradient in the first dimension and a 12.0% sodium dodecyl sulphate-polyacrylamide gel electrophoresis in the second dimension. About 750 protein spots in each gel were resolved after electrophoresis and 45 proteins were expressed significantly differently between the two kernels. MALDI-TOF MS characterization of the resolved spots in the two samples enabled us to identify 28 proteins whose levels were altered; 19 and 9 proteins were up-regulated in high and low weight kernels, respectively. In particular, proteins beneficial to materials synthesis and transmission increased distinctly in high weight kernels, while in low weight kernels, proteins involved in cell division were increased. The kernels with different position in spike might be at different physiological status, and this might be one of the causes resulting in grain weight differences within one spike.     

Molecular biology approaches for understanding microbial polycyclic aromatic hydrocarbons (PAHs) degradation

The known or suspected hazards of polycyclic aromatic hydrocarbons (PAHs) have provoked enormous concentration and endeavours to relieve or eliminate these precarious compounds from miscellaneous environments including soil, water and air. Among various interventions, biodegradation is an appealing approach for its comparative high efficiency and preferable safety. Microorganisms played crucial role in biodegradation of PAHs. Traditional access mainly including culture-dependent procedures has discovered and isolated PAHs-degrading microorganisms which could be subsequently applied to specific contaminated locus. Although certain progress has been achieved owing to traditional methods, much details in PAHs bioremedation leave pending because of the complexity nature of this process. As the rapid development of biology, molecular techniques such as PCR, fingerprinting technique (mainly DGGE), DNA hybridization technique and gene reporters technique have been intensively applied to gain further insight into the mechanism of PAHs degradation. These techniques not only proved the existence and role of uncultivable microorganisms in the whole population of PAHs degrading related microbials, but also made it possible to revealed the otherwise undetectable complex relationships between multi-microorganism concerned in PAHs biodegradation. Application of such techniques in the field of PAHs biodegradation were reviewed in this article.     

葡萄病毒B的RT-PCR检测技术研究

旨在建立适合葡萄的葡萄病毒B的RT-PCR检测技术。从感染葡萄病毒B的葡萄皮层中提取总RNA,以其为模板合成cDNA第一链,并利用两对引物分别进行PCR扩增;回收PCR特异扩增产物,与pUCm-T载体连接,并进行转化、重组克隆的筛选、重组质粒的酶切鉴定和序列测定。结果显示,两对引物均能获得与预期片段大小一致长约460 bp和470 bp的扩增产物扩增片段序列与已报道GVB序列(序列号:X75448)的核苷酸同源性均为81%,经反复多次试验证实,利用总RNA为模板合成cDNA并进行PCR扩增检测GVB是准确、可靠的。     

抗氧化酶过表达对趋磁螺菌MSR-1耐氧性的影响

在格瑞斯瓦尔德磁螺菌(Magnetospirillum gryphiswa ldense)MSR-1中分别过量表达3种抗氧化酶Fe-超氧化物歧化酶、谷胱甘肽过氧化物酶和过氧化氢酶HPII,并分析过量表达这3种酶对趋磁螺菌MSR-1耐氧性的影响。通过PCR分别扩增大肠杆菌DH5α的Fe-超氧化物歧化酶(sodB)、谷胱甘肽过氧化物酶(btuE)、过氧化氢酶HPⅡ(katE)基因序列,将前两个片段分别连接到广宿主质粒pBBR1MCS-2上,后一个片段连接到广宿主质粒pBBR1MCS-5上,构建成表达质粒pBBR1MCS-sodB,pBBR1MCS-btuE和pBBR1MCS-katE,将3个质粒通过双亲接合转移的方法分别转入趋磁螺菌MSR-1。3种抗氧化酶过表达对趋磁螺菌MSR-1耐氧性影响的试验结果为过量表达Fe-超氧化物歧化酶对菌体生长影响不明显;过量表达谷胱甘肽过氧化物酶、过氧化氢酶HPII使趋磁螺菌MSR-1致死。上述试验结果表明抗氧化酶系在菌体耐氧过程中的全局协调调控的重要性。