小叶女贞叶片转录组研究

目的 探讨小叶女贞中化学成分生物合成的遗传基础。方法 采用Illumina/Solexa Hiseq 2000测序平台在转录水平上对小叶女贞叶片进行高通量测序,结合生物信息学方法开展基因功能注释和SSR位点搜索。结果 共获得4 907 020 800个clean reads,含4.90 Gb的有效数据。利用生物信息学对clean reads进行拼接和组装共获得157 450条Unigene,总长度、平均长度和N₅₀分别为115.49 Mb,734 bp和1 510 bp。进一步进行基因功能预测,在GO数据库中共有17 756条Unigene可被分别注释到细胞组分、分子功能和生物学过程3大类别中,将Unigene与COG数据库进行比对,可获得25个功能类别,其中大部分Unigene与叶片中物质的合成与转运相关。在KEGG数据库中进行搜索比对,Unigene可定位到21类代谢途径中,涉及重要次生代谢产物苯丙素类、黄酮类和萜类生成的Unigene数目分别为860,216和806条。利用SSR分析软件,在所有的Unigene中共搜索到17 593个SSR位点,重复类型以二核苷酸为主。结论 本研究首次对小叶女贞转录组进行测序分析,获得的参与次生代谢的关键基因可为研究小叶女贞药用成分的生物合成和调控机制研究奠定基础。     

基于高通量测序技术探究白鲜碱对红色毛癣菌体外抑菌作用

目的 研究白鲜碱对皮肤浅部真菌红色毛癣菌的体外抑菌作用。方法 采用液基稀释法测定白鲜碱对红色毛癣菌的最低抑菌浓度（MIC）;采用高通量测序技术对白鲜碱（25.0、12.5 μg/mL）作用的红色毛癣菌进行转录组测序,对测序序列进行处理和生物学信息分析,进行差异表达基因、基因本体论（GO）功能显著性和京都基因与基因组百科全书（KEGG）富集分析等。结果 液基稀释法结果表明,白鲜碱对红色毛癣菌的MIC为50 μg/mL。高通量测序结果显示,对照组与白鲜碱 25.0 μg/mL组比较,差异基因总数为890个;其中上调表达384个,下调表达542个。白鲜碱作用后红色毛癣菌的差异基因主要表现于细胞膜结构、膜固有成分、氧化还原酶活性、对药物的反应和细胞色素复合体等。白鲜碱作用后红色毛癣菌在代谢途径、MAPK信号通路、ABC转运蛋白合成、色氨酸代谢等富集通路相关基因均有显著性改变（P<0.05）。结论 白鲜碱对红色毛癣菌的抑菌机制可能与抑制细胞膜结构的完整性、影响红色毛癣菌的能量代谢、降低耐药性等途径有关。     

羟基红花黄色素A生物合成途径短链还原酶基因的特征及功能研究

目的 探究参与红花黄酮类生物合成途径特别是羟基红花黄色素A（HSYA）关键短链脱氢还原酶基因（SDRs）的功能。方法 基于红花转录组数据库以及代谢组数据库,筛选参与HSYA生物合成途径的SDRs,用qRT-PCR法分析表达模式。采用无缝克隆技术构建过表达载体,以农杆菌GV3101介导遗传转化云南巍山红花品系,对转基因T₂代植株进行阳性验证,并对花冠SDRs的基因表达量进行分析,UPLC-Q-TOF/MS法测定次生代谢物的含量。结果 筛选出3个参与HSYA生物合成途径的关键短链脱氢还原酶基因CtSDR1、CtSDR2、CtSDR3,表达量从高到低依次为花冠>叶>茎>根。花冠中表达量随花冠发育逐渐升高。对转基因T₂代植株进行阳性验证后的花冠进行qRT-PCR分析发现：与空白对照组相比,转CtSDR3过表达T₂代阳性植株花冠中CtSDR3基因的转录水平增加了2~3倍,次生代谢物HSYA的含量提高了7.1%～16.6%（P＜0.05）。结论 CtSDR3可能参与了红花中黄酮类化合物特别是HSYA的生物合成,为阐释CtSDR3在HSYA生物合成途径中的功能提供了数据支撑。     

基于高通量测序的青叶胆转录组研究

目的 探讨青叶胆中獐牙菜苦苷生物合成的遗传基础。方法 采用Illumina Hiseq 2500高通量测序技术,对青叶胆全草进行转录组测序。结果 共获得68 643个unigene,平均长度901 bp,共有42 954条unigene注释到NR,SwissProt,COG,KOG,GO,Pfam数据库中,獐牙菜苦苷生物合成的3个阶段中,有43条unigene参与中间体的生成,3条unigene参与萜类骨架合成,25条unigene可能参与萜类最后的修饰。结论 首次对青叶胆转录组进行测序分析,获得了可能参与獐牙菜苦苷生物合成的候选基因,为后续功能基因的挖掘奠定了基础。     

槐耳颗粒联合DC-CIK细胞对乳腺癌MDA-MB-231干细胞体内外杀伤作用研究

目的 探究白屈菜红碱（CHE）对腺样囊性癌细胞（ACC2）生长的抑制作用及机制。方法 利用CCK8法、EdU法、Hoechst33342/PI双染色法、试剂盒法检测CHE对ACC2细胞活力、细胞增殖、细胞凋亡和活性氧（ROS）水平的影响;通过Western blotting技术检测CHE对Cleaved-Caspase 3、PARP、NF-κB、p-JNK、p-p38蛋白表达的影响;利用斑马鱼移植瘤模型检测CHE对斑马鱼体内ACC2细胞生长的抑制作用。结果 CCK-8结果显示：与对照组比较,2、3、4、5、6、7、8、9、10 μmol/L的CHE显著降低ACC2细胞的存活率（P<0.05、0.01）,且呈浓度相关性; ROS检测结果显示：与对照组比较,5、8 μmol/L的CHE导致ACC2细胞内的ROS水平显著上升（P<0.05、0.01）; EdU增殖检测结果表明：与对照组比较,5、8 μmol/L的CHE致使ACC2细胞的增殖能力显著下降（P<0.01）;Hoechst/PI染色结果显示：与对照组比较,CHE 5、8 μmol/L组ACC2细胞凋亡率显著上升（P<0.01）。抗氧化剂N-乙酰半胱氨酸（NAC）显著抑制CHE诱导的ROS水平升高、细胞凋亡增加（P<0.01）;Western blotting结果显示：2、5、8 μmol/L的CHE能够显著上调Cleaved-Caspase 3、PARP、NF-κB蛋白的表达（P<0.01）,且呈现浓度相关性,5、8 μmol/L的CHE能够显著上调p-JNK的蛋白表达（P<0.01）,8 μmol/L的CHE能够显著上调p-p38的蛋白表达（P<0.01）;NAC显著降低由CHE导致的Cleaved-Caspase 3、PARP、NF-κB、p-JNK、p-p38蛋白表达增加（P<0.01）,5、8 μmol/L CHE能够有效抑制斑马鱼体内肿瘤的生长（P<0.01）。结论 体外及斑马鱼移植瘤模型证明,CHE可以有效抑制ACC2细胞生长,其机制与提高细胞ROS水平,上调NF-κB、p-JNK、p-p38表达,从而抑制细胞增殖、诱导细胞凋亡相关。     

基于生物信息学手段筛选对肾透明细胞癌具有潜在干预作用的天然活性成分及中草药

目的 应用生物信息学及系统药理学的方法探索可干预肾透明细胞癌的潜在化学成分以及含有目的成分的中草药。方法 首先借助ＧＥＯ基因表达谱数据分析筛选得到肾透明细胞癌与癌旁正常组织的差异基因,同时分析得到差异基因的生物学功能,接着构建差异基因的蛋白互作网络,进一步分析差异基因的在TCGA及GTEx背景数据库中的表达及预后价值,得到目标基因后借助CTD数据库挖掘与目标基因相关化合物,剔除非天然化合物后经TCMSP工具寻找含有天然化合物的中草药。结果 得到肾透明细胞癌与癌旁正常组织的差异基因共有357个上调基因,353个下调基因,富集生物过程包括氧化还原过程、排泄、对药物的反应等,分子功能包括蛋白质均二聚活性、运输活动、受体结合等,细胞定位在细胞外泌体、质膜的组成部分、顶质膜等。在KEGG信号中富集到PPAR信号通路、碳代谢、甘氨酸,丝氨酸和苏氨酸的代谢等信号通路,利用cytohuba基于degree筛选排名前20的基因为核心基因,有明确差异以及有预后意义的BUB1、C3、KIF20A、RRM2、TOP2A基因确定为目标探究与其相关的化合物72个,包含每种化合物的中草药至少一种。结论 基于生物信息学挖掘到多种中草药可以影响目标基因从而干预肾透明细胞癌。     

催化菘蓝活性木脂素生物合成的漆酶基因家族生物信息学分析

目的 从转录组数据库中发掘并分析了参与菘蓝落叶松脂素生物合成的关键合成酶之一——漆酶（laccase）的基因序列,并进行生物信息学分析,为以后的功能研究提供可靠依据。方法 利用生物信息学分析软件,从已有菘蓝转录组数据库中寻找漆酶并分析其各项特征,主要包括理化性质、同源性分析、蛋白结构分析和茉莉酸甲酯（MeJA）诱导后表达特征等分析。结果 菘蓝漆酶基因（Iilacs）中的Iilac3和Iilac5在MeJA诱导下的转录水平与药效成分的积累相一致,推测其可能为参与落叶松脂素合成的潜在的功能基因。结论 通过对菘蓝漆酶进行详细的生物信息学分析,可为今后功能蛋白的筛选和进一步研究其生理生化机制和结构特征打下坚实的基础。     

白芍单萜苷成分与生物合成相关基因的时空表达特性及其相关性研究

目的 揭示白芍不同发育时期不同组织部位中单萜苷含量及其生物合成基因的表达差异和两者之间的相关性。方法 通过HPLC测定白芍叶、花、果、根及根中不同部位(韧皮部、木质部、须根和根皮)在不同发育时期氧化芍药苷、白芍内酯苷、芍药苷和苯甲酰芍药苷含量。采用RT-PCR对筛选出来的8个白芍单萜苷生物合成相关基因进行相对含量测定。应用R语言分析4种单萜苷含量与各生物合成相关基因表达量的相关性。结果 芍药苷和苯甲酰芍药苷主要分布于根中,而氧化芍药苷和白芍内酯苷主要分布于花中。氧化芍药苷、芍药苷和苯甲酰芍药苷主要分布于木质部中,白芍内酯苷主要在韧皮部和须根。4种单萜苷成分在根中花期和果期含量均较低,在黄枯期中最高,展叶期次之。单萜苷含量与其关键酶基因表达量相关性分析结果表明,HMGR2和IDI基因与芍药苷、苯甲酰芍药苷和氧化芍药苷含量呈高度正相关。结论 白芍单萜苷及其生物合成相关基因存在时空特异表达模式,甲羟戉酸途径是白芍中生物合成萜类骨架的主要途径。     

绝经后骨质疏松症免疫反应相关基因的鉴定及其中药活性成分的筛选

目的 采用生物信息学方法识别绝经后骨质疏松症中免疫反应相关的基因,并探讨其作用机制和筛选靶向中药活性成分。方法 在基因表达数据库(GEO)中检索"postmenopausal osteoporosis",下载基因数据集GSE230665,通过R软件中的"limma"数据包获取差异表达基因,使用David工具对差异基因进行基因本体(GO)功能富集分析并筛选出免疫反应相关的关键基因,然后对关键基因进行GO功能富集分析和京都基因与基因组百科全书(KEGG)通路富集分析。采用STRING平台和Cystoscope软件建立蛋白相互作用(PPI)网络,用MCODE和CytoNCA插件识别重要基因簇模块和核心基因。在CTD数据库搜索"postmenopausal osteoporosis"对核心基因进一步筛选并搜索靶向核心基因的中药活性成分,并在PubChem数据库中搜索中药活性成分的3D结构,Autodock 4软件进行分子对接,以及探索核心基因与绝经后骨质疏松症的相互作用。结果 在绝经后骨质疏松症中筛选出43个关键基因,GO富集分析显示主要参与涉及正向调节炎症反应、白细胞介素(IL)-12的产生、肿瘤坏死因子的产生、细胞迁移和趋化作用等;KEGG通路主要涉及趋化因子信号通路、磷脂酰肌醇3-激酶/蛋白激酶B(PI3K/Akt)信号通路、肿瘤坏死因子(TNF)信号通路、核因子-κB(NF-κB)信号通路等。鉴定出9个核心基因依次为IL-10、B2M、IL-17A、CXCR4、TLR4、CCR5、NOTCH1、TLR2、SMAD3。通过验证,在绝经后骨质疏松症患者中核心基因的表达均上调。核心基因对应的中药活性成分为雌二醇、白藜芦醇、槲皮素、姜黄素、双酚A和木黄酮等,这些中药活性成分可能通过抑制免疫反应来修复绝经后骨质疏松症。结论 利用生物信息学筛选出与免疫反应相关的核心基因(IL-10、B2M、IL-17A、CXCR4、TLR4、CCR5、NOTCH1、TLR2、SMAD3)以及其受靶向调控的主要中药活性成分(雌二醇、白藜芦醇、槲皮素、姜黄素、双酚A和木黄酮),为进一步探索绝经后骨质疏松症的分子发病机制以及新的治疗靶点提供新的理论依据。     

远志流浸膏中3种指标性成分的含量测定

目的 建立测定远志流浸膏中3种指标性成分的含量的方法。方法 采用高效液相色谱（HPLC）法同时测定8批远志流浸膏中远志𠮿酮Ⅲ,3,6’-二芥子酰基蔗糖这两种成分含量,和高效液相色谱法测定碱水解远志流浸膏中细叶远志皂苷成分含量。结果 远志𠮿酮Ⅲ、3,6’-二芥子酰基蔗糖、细叶远志皂苷在各自测定范围内线性关系良好（r＞0.999 9）,平均加样回收率为98.76~103.05%,RSD 0.82~3.79%。结论 本试验方法结果准确、专属性好、重复性好,稳定性好,可为远志流浸膏的质量控制提供依据。     

Investigation of the prevalence of tetQ, tetX and tetX1 genes in Bacteroides strains with elevated tigecycline minimum inhibitory concentrations

In this study, the antibiotic susceptibilities to tigecycline and tetracycline of 35 selected Bacteroides fragilis group strains were determined by Etest, and the presence of tetQ, tetX, tetX1 and ermF genes was investigated by polymerase chain reaction (PCR). tetQ was detected in all 12 B. fragilis group isolates (100%) exhibiting elevated tigecycline minimum inhibitory concentrations (MICs) (≥8 μg/mL) as well as the 8 strains (100%) with a tigecycline MIC of 4 μg/mL, whilst tetX and tetX1 were present in 15% and 75% of these strains, respectively. All of these strains were fully resistant to tetracycline (MIC ≥ 16 μg/mL). On the other hand, amongst the group of strains with tigecycline MICs < 4 μg/mL (15 isolates), tetQ, tetX and tetX1 were found less frequently (73.3%, 13.3% and 46.7%, respectively). All but two strains harbouring the tetQ gene in this group were non-susceptible to tetracycline, with a MIC > 4 μg/mL. These data suggest that in most cases tigecycline overcomes the tetracycline resistance mechanisms frequently observed in Bacteroides strains. However, the presence of tetX and tetX1 genes in some of the strains exhibiting elevated MICs for tigecycline draws attention to the possible development and spread of resistance to this antibiotic agent amongst Bacteroides strains. The common occurrence of ermF, tetX, tetX1 and tetQ genes together predicted the presence of the CTnDOT-like Bacteroides conjugative transposon in this collection of Bacteroides strains.     

Synthesis and evaluation of 2,6-piperidinedione derivatives as potentially novel compounds with analgesic and other CNS activities

New 2,6-piperidinediones 2_a–g and 4_a–d were prepared by initial condensation of aromatic aldehydes or cycloalkanones with cyanoacetamide to give α-cyanocinnamides l_a–g or cycloalkylidenes 3_a,b which underwent Michae1 addition with ethyl cyanoacetate or diethylmalonate. Compounds 4_a–d were alkylated by various alkyl halides to produce the N-alkylated 2,6-piperidinedione derivatives 5_a–m. Some new selected compounds 2_a–c,f, 4_a–d & 5_e,h,j were pharmacologically evaluated for potential anticonvulsant, sedative and analgesic activities. These compounds exhibited significant anticonvulsant and analgesic effects after a single I.P. administration 100 mg/kg b.wt. . On the other hand all the investigated compounds induced hypnotic activity and prolonged the phenobarbital sodium- induced sleep as compared with the control group and the most potent compound was found to be 2_f.     

Homocysteinemia,hypertension, and family history of diabetes in a smoking male population in Saudi Arabia

Arabs have a lower incidence of atherosclerosis than other ethnicities, but few studies have examined homocysteine (HCYS) as a risk factor for cardiovascular disease in this population. Here, we investigated the association between serum HYCS levels and risk factors for cardiovascular disease (smoking, hypertension, and family history of diabetes) in Saudi males. A total of 50 smokers and 72 nonsmokers completed a general health questionnaire. In addition, their lipid profiles were measured using routine methods and HCYS levels by high-performance liquid chromatograph with electrochemical detection. Regression analysis showed negative associations between HCYS and glucose (r = −0.22; P < 0.05) as well as family history of diabetes (r = −0.21; P < 0.05). HCYS levels were similar between hypertensive and nonhypertensive smokers, but they were significantly elevated in hypertensive nonsmokers (P = 0.027) and lower in smokers with family history of diabetes (P = 0.01). Levels of HCYS among nonsmokers inversely correlated with history of diabetes and elevated glucose. Nonsmokers’ HCYS levels were significantly elevated in the presence of hypertension and correlated with diastolic blood pressure. Thus, HCYS may be a predictor of hypertension among nonsmokers. Until further trials are conducted, we recommend vitamin B6/folic acid supplementation for the Saudi hypertensive population as an adjuvant therapy.     

Encephalopathy in toxicity tests on laboratory animals

Clinical signs of encephalopathy followed administration of lethal doses of a wide range of drugs, foods, pesticides and other agents in 92 reviewed studies on laboratory animals. Common signs of depression of the brain were anorexia, hypokinesia, hypothermia, prostration, ataxia, pallor, and hyporeflexia and of stimulation were convulsions, hyperreflexia, tremors, muscular spasms, and fever. Less common signs included catalepsy, exophthalmos, phonation, piloerection, bradypnea, tachypnea, automatism, flaccidity, a Straub reaction, erection of the penis, nystagmus, spacial disorientation, and hunch back. Aggressiveness took the form of fighting when animals were aggregated and occasionally automutilation when segregated. Certain signs such as vomiting and panting occurred only in cats and dogs. In chronic studies at sublethal daily doses, there were relatively few signs of encephalopathy. At autopsy, the brain was found relatively resistant to changes in weight, to hydration and to dehydration. Histologically there was no evidence of tissue encephalopathy in a third to a half of the studies. In the remaining studies, the brain was found to have participated in the general inflammatory reaction (capillary or capillary-venous congestion) to toxic doses of the agent under study. Less common histopathological reactions included thrombosis, edema, granulocytic infiltrative meningitis and cerebral abscesses.     

EFFECTS OF 2-GUANIDINE-4-METHYLQUINAZOLINE ON GASTRIC ACID SECRETION IN RATS

In this study 2-guanidine-4-methylquinazoline (2-GMQ) appeared to decrease basal and stimulated gastric acid secretion, while structurally related compounds as dimethyl- biguanide, cyanoguanidine and 2-cyanoamino-4-methylpyrymidine did not. Thus, there is an antisecretory effect when the biguanide group is associated with a lipophilic structure. The antisecretive effects exerted by 2-GMQ are associated with anti H₂-histamine activity.The anti H₂-histamine nature of the effects of 2-GMQ was confirmed by the capacity of this compound of depressing the chronotropic activity of the isolated guinea pig auricle increased by histamine, as well as relaxant activity in rat uterus contracted by histamine, since both preparations are rich in H₂-histamine receptors.     

Chemopreventive effect of celecoxib in gastric cancer

COX (cyclooxygenase) is one of the key enzymes involved in the synthesis of a variety of prostaglandins (PGs), some of which have been strongly linked to inflammation. One of its two well-known isoforms, COX-2, is an inducible enzyme whose induction and expression is dynamically regulated by growth factors, mitogens, and tumor promoters. Several animal and clinical studies have reported the chemopreventive effect of celecoxib, a selective COX-2 inhibitor; and in particular, a few studies have shown that celecoxib prevents the development of gastric cancer. Administration of celecoxib also showed increases in cardiovascular risk and disruption of renal physiology. Therefore, studies hoping to clarify how selective COX-2 inhibitors modulate gastric cancer must keep in mind that coxibs have also been linked to serious cardiovascular events and disruption of renal physiology. Received 12 July 2006; accepted 21 August 2006     

EFFECT OF POLICOSANOL SUCCESSIVE DOSE INCREASES ON PLATELET AGGREGATION IN HEALTHY VOLUNTEERS

Policosanol is a cholesterol-lowering drug with hypocholesterolemic effects demonstrated in experimental models, healthy volunteers and type II hypercholesterolemic patients. In addition, antiplatelet effects of policosanol have been shown in experimental models and healthy volunteers. The effect of successively increasing doses of policosanol on platelet aggregation was investigated in a randomized, placebo-controlled, double-blind study conducted in 37 healthy volunteers. The volunteers were on a placebo-baseline period (two tablets per day) for 7 days and thereafter they received randomly, under double-blind conditions, placebo or policosanol (10mgday⁻¹) for 7 days. After this period dosage was doubled to 20mgday⁻¹for the next 7 days and then again doubled to 40mgday⁻¹, while the control group received placebo tablets all the time. Platelet aggregation as well as coagulation time was measured at baseline and after each dosing step. Results showed that antiplatelet effects of policosanol were successfully enhanced throughout the study, thus suggesting a dose-dependent relationship. No significant effect was reached during the first dosing period, but significant reductions of epinephrine and ADP-induced platelet aggregation were observed after the second one. Finally, a significant inhibition of platelet aggregation induced by all the agonists was observed at the last dosing step. Coagulation time remained unchanged during the trial.     

NEURAMIDE STIMULATES ADRENOCORTICOTROPIN BUT NOT PROLACTIN RELEASE FROM RAT PITUITARY

Neuramide (NMD), a substance found in crude preparations of porcine stomach extract, is a viral inhibitor that also has putative immunostimulatory effects. The effects of NMD on stress-hormone (ACTH and prolactin—PRL) release were assessed inin vivoandin vitrostudies. In the former, blood levels of corticosterone and PRL were measured in NMD-treated male rats.In vitroexperiments were performed to evaluate the effects of NMD and three of its fractions (obtained with high performance liquid chromatography) on ACTH and PRL release from perfused rat pituitary slices. NMD increased plasma corticosterone levelsin vivoand produced dose-dependent increases inin vitropituitary release of ACTH. No effects on PRL secretion were observedin vivoorin vitro. The stimulatory effects on ACTH release were caused by the NMD fraction with a molecular weight of >5000<10000Da.     

Steroidal sapogenin contents in some domestic plants

In order to find out the values of the steroid resources for the future use. the compositions and contents of steroidal sapogenins from 13 domestic plants have been investigated. As a result,Dioscorea nipponica, D. quinqueloba andSmilax china were found to have large amount of diosgenin. And pennogenin inTrillium kamtschaticum andParis verticillata, yuccagenin inAllium fistulosum, hecogenin inAgave americana and neochlorogenin inSolanum nigum were appeared to be major steroidal sapogenins.