一份条斑和颖花异常水稻双突变体的形态特征和细胞学观察

在水稻遗传转化过程中发现一个不含外源基因的条斑和颖花异常的双突变体。该突变体的茎、叶、穗出现条斑。在分蘖盛期,一些叶片开始分岔或卷曲;花器官数目增多,表现为多内外稃,叶片状浆片,或浆片增大,雌雄蕊增多,颖花开裂。透射电镜对叶片白色组织细胞超微结构观察,发现细胞壁内陷,质体结构异常,不能发育出正常叶绿体所具有的片层和类囊体。叶绿素总含量和净光合速率明显低于野生型。突变体绿色组织部分中的细胞生长正常,但细胞较大。利用扫描电镜对花器官形态发生过程进行观察,雄蕊原基发育严重不同步,原基大小也不一样;心皮原基较小。     

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.     

基于粪便DNA的马鹿种群数量和性比

为分析黑龙江省完达山林区马鹿种群生存状态,制定科学有效地保护措施,从分子水平研究了种群数量和性比。2006、2007两年冬季跟踪马鹿足迹链,于五泡林场共搜集210份粪便,以成功提取DNA的167份作为分析样本。通过多态性较高的7个微卫星位点进行了基因分型分析,显示167份粪便DNA分属66只个体。基于非损伤性标志重捕法,统计出林场内马鹿数量2a平均47(39—60)只,密度0.302(0.251—0.386)只/km2,与2002年大样方法调查结果相比有减无增。SRY基因性别鉴定显示,种群雌雄性比1.00∶2.00(22♀,44♂),存在较多雄性个体,分析认为偷猎是导致性比失衡的最主要原因。数量的持续下降和性比失衡提示完达山林区马鹿种群数量的恢复需要更好地保护工作。