Shikonin inhibits the proliferation and induces the apoptosis of human HepG2 cells

This study investigated the potential of shikonin as an anticancer agent against liver cancer and an in vitro human hepatoma cancer model system. The HepG2 cell line was the hepatoma cancer model in the present study. The inhibitory effect of shikonin on the growth of HepG2 cells was measured by MTT assay. To explore the underlying mechanism of cell growth inhibition of shikonin, the cell cycle distribution, DNA fragmentation, mitochondrial membrane potential (ΔΨm) disruption, and expression of Bax and Bcl-2 were measured in HepG2 cells. The activity of shikonin in inducing apoptosis was investigated through the detection of Annexin V signal and CD95 expression by flow cytometry and electron microscopy, respectively. Shikonin inhibited the growth of HepG2 cells in a dose-dependent manner. The IC50 value (inhibiting cell growth by 50%) was 4.30 mg/mL. Shikonin inhibited cell growth in a dose-dependent manner and blocked HepG2 cell cycle progression at the S phase. The changes in mitochondrial morphology, dose-dependently decreased in ΔΨm, were observed in different concentrations of the drug treatment group. Western blot analysis showed that cajanol inhibited Bcl-2 expression and induced Bax expression. Furthermore, we show that shikonin increases Annexin V signal and CD95 (Fas/APO) expression, resulting in apoptotic cell death of HepG2 cells. In addition, lump formation of intranuclear chromatin, pyknosis of cell nucleus, deletion of microvillus, vacuolar degeneration of mitochondria, reduction of rough endoplasmic reticulum, and resolution of free ribosome, etc., associated with apoptosis were discovered by electron microscopy in HepG2 cells after 48 h treatment. Shikonin inhibited HepG2 cells, possibly through the pathway of inducing early apoptosis, and was beneficial for restoring the apoptotic sensitivity of HepG2 cells by CD95, and should therefore be considered as a candidate agent for the prevention or treatment of human hepatoma.     

New triterpenoid saponins from the roots of Gypsophila pacifica Kom.

Six new triterpenoid saponins (1-6) have been isolated from the roots of Gypsophila pacifica Kom. Their structures were established on the basis of extensive NMR (¹H, ¹³C, TOCSY, HSQC, and HMBC) and ESIMS studies.     

Interactive effects of molybdenum and phosphorus fertilizers on photosynthetic characteristics of seedlings and grain yield of Brassica napus

A pot experiment with acid yellow–brown soil was conducted to investigate the interactive effects of molybdenum (Mo) and phosphorus (P) fertilizers on the photosynthetic characteristics of seedlings and grain yield of Brassica napus which is sensitive to soil P and Mo deficiency. Both Mo and P fertilizers were applied at three levels (0 mg Mo kg^?1, 0.15 mg Mo kg^?1, 0.30 mg Mo kg^?1 soil; 0 mg P kg^?1, 80 mg P kg^?1, 160 mg P kg^?1 soil). The results showed that P fertilizer application increased grain yield, soluble sugar concentrations of seedling leaves, DM and P accumulation of seedling shoots of Brassica napus in the absence or presence of Mo fertilizer. In contrast, Mo fertilizer increased these parameters only in the presence of P fertilizer. Mo accumulation in shoots, chlorophyll concentrations and net photosynthesis rate (P _n) of seedling leaves were increased by both Mo and P fertilizers, particularly with the combination of the two fertilizers. The results also showed that the Mo and P fertilizers increased photosynthetic rate through two different mechanisms, with Mo increasing photosynthetic activity of mesophyll cells, and P increasing stomatal conductance. The results demonstrate that there was a synergetic effect on photosynthesis and grain yield between Mo and P fertilizers and it is conducive for Brassica napus growth to co-apply the two fertilizers.     

Sleeping Sites of <Emphasis Type="Italic">Rhinopithecus brelichi</Emphasis> at Yangaoping,Guizhou

Arboreal primates spend about half of their lives at sleeping sites; hence, selection of sleeping sites is crucial for individual survival, and data concerning them is important for conservation efforts. We collected data on sleeping sites for a group of the endangered snub-nosed monkey (Rhinopithecus brelichi) at Yangaoping (27°58′N, 108°45′E) from January 2006 to December 2007. All sleeping sites were located in the mid-slope and in the shadow of ridges facing the northeast and southeast. The monkeys remained quiet while entering and occupying sleeping sites, and slept in evergreen species during the cold season (December–March). Trees in sleeping sites were similar in height and girth at breast height to those elsewhere, but some trees in lower areas were larger. The monkeys usually slept in close proximity to the last feeding spot, and their daily activities usually occurred around the sleeping site. Areas adjacent to sleeping sites were used more intensively than those not adjacent. Monkeys left the sleeping sites later in the morning in the cold season. These behavioral responses suggested that predation risk, thermoregulation, and climate stresses are the main determining factors in the selection of sleeping sites for this temperate monkey.     

Characterization of Hlcyst-3 as a member of cystatins from the tick <Emphasis Type="Italic">Haemaphysalis longicornis</Emphasis>

The gene encoding cystatin from the tick Haemaphysalis longicornis has been reported previously. In the study reported here, we characterized a member of cystatins and designated it as Hlcyst-3 (H. longicornis cystatin-3). Its full-length cDNA is 602 bp, and it encodes a putative 129 amino acid protein with an obvious signal peptide. Sequence analysis revealed that it has significant homology with the known secreted cystatin. The recombinant protein was expressed in a GST-fused soluble form in Escherichia coli and was purified by affinity chromatography. The inhibitory activity of the recombinant protein against papain and cathepsin L was identified by fluorogenic substrate analysis. Real-time PCR revealed that Hlcyst-3 was mostly expressed in the tick midgut.     

Mouse granulosa cells contribute more to the mRNA synthesis of mZP2 than oocyte does

In order to elucidate the biogenesis of mouse zona pellucida 2 (mZP2) protein, RT-PCR, and in situ hybridization were carried out to localize the expression of mouse ZP2 mRNA. Cumulus cells of the OCC (Oocyte-Cumulus cell Complex) were isolated from the oocytes after superovulation for the RNA extraction. The frozen sections of ovaries from adolescent and aged mice were prepared to hybridize with RNA probe of mouse ZP2. mRNA of ZP2 was detected in isolated cumulus cells by RT-PCR. Results of in situ hybridization showed that the mRNA of ZP2 was synthesized in both oocyte and granulosa cells at different folliculogenesis stages; and the expression of ZP2 mRNA in granulosa cells was stronger than that in oocyte; much weaker expression of mZP2 was detected in the follicles of aged mouse. These suggest that the entire amount of ZP2 mRNA generated in the granulosa cells layer should be much more than that in oocyte. Therefore, we think that the granulosa cells contribute more to the mZP2 mRNA synthesis than oocyte does.     

施用控释氮肥对稻田土壤微生物生物量碳、氮的影响

借助农业部望城红壤水稻土生态环境野外观测试验站的控释氮肥试验,研究了施用控释氮肥对水稻不同生育期间稻田土壤微生物生物量碳、氮动态变化的影响。试验共设5个处理:①CK,(不施氮肥);②Urea(施用尿素);③CRNF(施用与处理②等氮量的控释氮肥);④70%CRNF(施用控释氮肥,用氮量为处理②的70%);⑤50%CRNF+M(施用控释氮肥和猪粪,总氮量为处理②的70%,其中控释氮肥用量为处理②的50%,猪粪含氮量为处理②的20%)。结果表明,施肥后10 d,施氮处理土壤微生物生物量碳和氮均达最高,随生育进程推进逐渐下降,成熟期有一定的回升;施肥初期,施用等氮量的控释氮肥处理(CRNF)土壤微生物量碳、氮含量较尿素处理(Urea)分别增加5.4%和22.5%,而水稻生育中后期,控释氮肥处理(CRNF)土壤微生物量碳、氮含量较尿素处理(Urea)下降幅度大,该处理向地上部提供氮素营养较尿素处理高;施氮量较高的CRNF处理,土壤微生物生物量碳低于控释氮肥节氮处理(70%CRNF),但在大多数取样时期,土壤微生物量氮高于控释氮肥节氮处理(70%CRNF);控释氮肥配施有机肥的节氮处理较其他单施化肥处理显著增加土壤微生物生物量碳、氮含量。控释氮肥与有机肥配施,不仅能节约氮肥用量,而且能明显地提高土壤微生物生物量碳、氮的含量。     

HIV－1载体中存在着提高转录和翻译基因的元件

目的研究HIV-1载体中的一些元件如Rev和Tat蛋白对其骨架的转录及外源基因表达水平的影响。方法将HIV-1表达GFP载体（FUGW）单独或分别与Rev蛋白表达质粒（pLP2）、Tat蛋白表达质粒（pcDNA3.1-Tat）,及表达Rev和Tat蛋白的质粒（△8．9）等摩尔共转染人293T细胞后,经实时定量RT-PCR、FACS、荧光显微镜镜检等方法检测,比较其表达量。结果Rev与RRE结合后,载体骨架及外源基因的转录是单独转染FUGW时的3倍,Tat与TAR结合后,则提高其骨架及外源基因的转录近4倍,而Rev和Tat蛋白的协同作用,其转录本则可提高至6倍。FACS和荧光显微镜镜检也显示GFP蛋白表达量明显提高。F-TPO载体（HIV-1载体乳腺特异表达促血小板生成素）与△8．9在小鼠乳腺上皮细胞HC-11共转染和表达,则TPO蛋白的表达量接近pcDNA3．1-TPO载体的8倍。结论HIV-1载体中存在着提高转录和翻译基因的元件,可提高其骨架的转录和外源基因的表达,且该现象并不依赖于细胞类型和外源基因的种类。     

蛋白芯片载体表面修饰对探针固定影响的研究

采用3-氨基丙基-三甲氧基硅烷((3-aminopropyl)trimethoxysilane,APTES)、戊二醛(glutaraldehyde,GA)、多聚-L-赖氨酸(poly-L-lysine,PLL)修饰芯片载体表面,对3种不同修饰方法制备的蛋白质芯片进行对比研究。将Cy3标记羊抗鼠IgG固定在修饰后片基上,选择蛋白探针的固定率作为检测指标;将小鼠IgG作为探针固定在芯片上,靶蛋白为Cy3标记羊抗鼠IgG,通过生物芯片扫描仪检测反应后荧光强度,选择蛋白探针的反应性作为检测指标,探讨制备蛋白质芯片较佳的表面修饰方法。结果显示,戊二醛修饰玻片对蛋白固定较好,有较高的反应活性,检测限较宽,但背景噪声较高。     

弓形虫膜表面抗原SAG2蛋白的高效表达及免疫活性分析

在大肠杆菌中以可溶性形式高效表达弓形虫膜表面抗原SAG2蛋白,并对其免疫活性进行分析。应用PCR技术从刚地弓形虫RH株的基因组DNA中扩增编码SAG2的基因片段,亚克隆至原核表达载体pET32a(+),在大肠埃希菌(E.coli)BL21内表达,并对其表达条件进行优化,Western blotting和ELISA分析纯化蛋白的免疫原性;纯化的重组蛋白免疫小鼠制备多抗,用间接免疫荧光试验(IFA)分析表达蛋白的免疫反应性。成功构建重组质粒pET32a(+)-tSAG2,所表达的融合蛋白大小约为38kD。在IPTG终浓度为0.1mmol/L、诱导时间4-6h和培养温度32℃条件下,重组SAG2蛋白主要以可溶性形式在大肠杆菌中高效表达,每升培养菌液约获得可溶性重组SAG2蛋白16mg。Western blotting及ELISA结果显示纯化蛋白具有良好的免疫原性。IFA显示重组蛋白的抗血清能够识别刚地弓形虫表面的SAG2天然蛋白,所表达蛋白具有良好的免疫反应性。截断的SAG2基因在大肠杆菌中得到了高效表达,重组蛋白保持了天然蛋白的免疫活性,为进一步利用该重组蛋白进行弓形虫病免疫诊断及基因工程亚单位疫苗的研制奠定基础。