不同年份生产的乙型脑炎减毒活疫苗SA14-14-2基因稳定性研究

为了研究不同年份生产的乙型脑炎减毒活疫苗病毒E蛋白基因稳定性,从分子水平控制乙型脑炎减毒活疫苗质量,确保疫苗安全性,本研究分析了不同年份生产的乙脑活疫苗病毒E蛋白基因核苷酸序列及编码的氨基酸序列,并与该疫苗原始种子、主种子、工作种子、乙脑病毒强、弱毒株进行比较。结果显示不同年份生产的乙脑活疫苗病毒E蛋白基因核苷酸序列与其原始种子、主种子、工作种子和基因库中登录的乙脑病毒弱毒株SA14-14-2的相应序列完全一致,与乙脑病毒强毒株SA14的E蛋白氨基酸序列比较有9个位点氨基酸发生了改变。不同年份生产的乙脑活疫苗病毒E蛋白基因稳定性表明该疫苗质量稳定、安全。     

A differential interaction of daunomycin,adriamycin and their derivatives with human erythrocytes and phospholipid bilayers

Drug-membrane association of daunomycin, adriamycin and three of its derivatives, adriamycin-14-octanoate (AD-14-OCTA), adriamycin-14-acetate (AD-14-ACE) and $\text{N-}\text{trifluoroacetyladriamycin-14-valerate}$ (AD32), was studied using phospholipid bilayers and human erythrocytes. The various drugs exhibited a differential affinity to membrane-lipid domains.Lipid-incorporated drugs exhibit a marked change in the shape of the emission spectrum which was utilized for the evaluation of the apparent dielectric constant, ?, of the environment surrounding the anthracycline moiety, as well as for the determination of the partitioning constant. By measuring the fluorescence polarization and the fluorescence lifetime of the incorporated drugs, rotational relaxation times of 4–8 ns were derived. These parameters provide a supportive evidence for the association of the fluorophore of the drugs with membrane-lipid domains.The anthracycline derivatives interact to a different degree with dipalmitoyl phosphatidylcholine and phosphatidylserine as reflected by changes in their thermotropic properties assessed by differential scanning calorimetry. Daunomycin was the most effective in decreasing the temperature of the phase transition and brought about a comparable reduction in the enthalpy of melting as AD32 and AD-14-OCTA. Adriamycin was the least potent of the series.AD-14-ACE and AD32 protected erythrocytes against hypotonic lysis, adriamycin and daunomycin had no significant effect on the susceptibility to hypotonic lysis, whereas AD-14-OCTA proved to be hemolytic even at low concentration (approx. 10^?7 M).The interaction of erythrocytes with daunomycin, AD-14-ACE and Ad-14-OCTA resulted in a shape change from biconcave discs to cups. Adriamycin and AD32 did not affect erythrocyte shape.The differential drug-membrane interactions may be an important determinant in the antitumor differential efficiency of the drugs, especially in view of the fact that derivatives that do not intercalate into the DNA (AD32) are at least as potent as those that do.     

急性肺损伤大鼠肺组织TOLL样受体4及CD14mRNA的表达

目的 探讨内毒素致急性肺损伤大鼠肺组织TOLL样受体4及CD14 mRNA表达的变化.方法 24只雄性Wistar大鼠随机分为2组:对照组、LPS模型组,每组再分为4 h和8 h两个亚组.尾静脉注射脂多糖(LPS)(10 mg/kg)建立大鼠急性肺损伤模型.检测大鼠动脉血气、肺体指数,实时荧光定量PCR测定肺组织TOLL样受体4及CD14 mRNA的表达,并观察肺组织病理变化.结果 与对照组相比,模型组4 h和8 h时大鼠肺组织中的TLR4及CD14 mRNA表达均显著增高(P＜0.05或P＜0.01).病理学观察显示,模型组大鼠肺组织出现出血及坏死.结论 内毒素致急性肺损伤的发病机制可能与TLR4及CD14 mRNA的表达升高有关.     

华北高产农田生态系统中冬小麦分蘖期生长与碳截留

模拟华北高产施肥条件盆栽冬小麦,采用^14C同位素对分蘖期冬小麦进行脉冲标记,并分别在分蘖期、拔节期、花期和成熟期取样测定冬小麦地上部、根和土壤中的^14C含量,研究冬小麦光合固定碳对土壤碳库的输入机制。结果表明：冬小麦冬前植株地上部分的生长可以用三次方程较好地拟合;冬小麦出苗后36d停止生长,植株地上部分平均干质量为0．219g·株^-1,折合为0．09gC·株^-1。冬小麦分蘖期光合固定的碳在植株地上部分、地下根部和土壤中的分配比例分别占光合同化HC总量的35.2％、10．4％和51．3％。到冬小麦生长期末,植株地上部分、地下根部和土壤中的^14C含量分别降至最初光合同化^14C总量的3．9％、4．6％和26．4％。通过呼吸作用所释放的^14C总量随作物生长时间的推移不断增大,到生长期末,通过呼吸作用输出的^14C的量占分蘖期冬小麦固定总量的65．2％。     

CTX-M-14型超广谱β-内酰胺酶的序列分析与原核表达

目的对大肠埃希菌所产CTX-M-14型超广谱β-内酰胺酶（ESBLs）进行基因克隆和重组表达,探讨其特性。方法以产CTX-M-14型超广谱β-内酰胺酶大肠埃希菌12号菌总基因组DNA为模板,PCR扩增CTX-M-14,将其克隆入pUCm-T Vector载体后测定该核苷酸序列;再将基因编码区克隆到原核表达载体pET-28α,构建含CTX-M-14基因的重组表达质粒,转化到大肠埃希菌BL21中进行IPTG诱导表达。SDS-PAGE电泳鉴定表达的酶蛋白后再过Ni-NTA柱纯化。结果PCR扩增出大小为876bp的基因片段,与GenBank上同类酶的基因序列同源性为100％。大肠埃希菌BL21转化pET-28a／CTX-M-14重组质粒后,ESBLs试验为阳性。此基因能在大肠埃希菌中大量表达,SDS-PAGE电泳显示蛋白分子质量大约为30KD。结论成功表达重组的CTX-M-14型酶,为进一步做酶动力学及酶的其他分子生物学特性研究奠定基础。     

水牛CD14 cDNA的克隆、表达及鉴定

目的:克隆水牛白细胞分化抗原14(buffalo cluster of differentiation antigen14,bCD14)基因,表达bCD14蛋白,并进行Western Blot鉴定.方法:采用RT-PCR方法从水牛外周血白细胞中扩增bCD14基因,构建重组质粒pET28a-bCD14,转化入E coli BL21,IPTG诱导表达,对表达蛋白进行可溶性分析及Western blot鉴定.结果:bCD14基因含有一个1 122bp的开放阅读框,编码373个氨基酸;与印度水牛、挪威大鼠和人CD14的cDNA序列同源性分别为97.95%、68.78%、78.60%,氨基酸同源性分别为96.78%、61.27%、72.34%;主要以包涵体形式表达,表达蛋白经Western Blot鉴定,得到了一条约46 kD的特异性条带.结论:该文成功克隆了bCD14基因,表达了bCD14蛋白,为进一步揭示水牛抵抗革兰氏阴性菌感染的免疫机制奠定了基础.     

Comparative studies of somatostatin-14 and some of its fragments on passive avoidance behavior, open field activity and on barrel rotation phenomenon in rats

Behavioral effects of somatostatin-14, and some of its fragments [somatostatin(3–8), somatostatin(9–14), somatostatin(7–10)] after intracerebroventricular (ICV) administration have been investigated in male rats. In a passive avoidance learning test, somatostatin-14 (0.6 nM) given immediately after the learning session increased the avoidance latency at 24 hr after the injection, when compared to a somatostatin(3–8) (0.6 nM)-treated group. However, compared to a saline-treated group, the peptides did not significantly influence the avoidance latency. Somatostatin-14 administered in higher dose (6.0 nM) decreased the avoidance latency compared to the saline-treated group, while its fragments did not influence it. In an open field behavioral test, immediately after the 24-hr passive avoidance test, 6 nM of somatostatin-14 decreased the rearing activity, while the fragments did not influence this behavior. Somatostatin-14 produced barrel rotation in a dose-related manner, but after the injection of a high dose of the peptide (12 nM) all of the animals died in cardiorespiratory failure (apnea, pulmonary oedema). The fragments did not produce barrel rotation.     

14C-Metabolism in cultured cotyledon explants of radiata pine

Excised cotyledons of radiata pine ( Pinus radiata D. Don), cultured under shootforming (plus cytokinin) and elongating (minus cytokinin) conditions, were incubated in ¹⁴C-glucose, ¹⁴C-acetate or ¹⁴C-bicarbonate at different stages of growth and differentiation. ¹⁴CO₂ was produced when the cotyledons were fed ¹⁴C-glucose and ¹⁴C-acetate (no measurement was made for ¹⁴C-bicarbonate feeding). Label from these precursors was incorporated into ethanol-soluble and -insoluble fractions. The largest percentage of radioactivity was associated with the ethanol-soluble portion, which was further fractionated into lipids, amino acids, organic acids and sugars. The amount of label and the pattern of labelling associated with each of the above classes of metabolites varied with time in culture and morphogenetic behaviour of the cotyledons. In general, there was a tendency towards a high rate of incorporation of label in elongating cotyledons during the period of rapid elongation. On the other hand, a high rate of incorporation of label in shoot-forming cotyledons coincided with the period of meristematic tissue formation. The data obtained support the hypothesis that organized development in vitro involves a shift in metabolism, which precedes and is coincident with the initiation of the process.     

Development of a purification procedure for the placental protein 14 involving metal-chelate affinity chromatography and hydrophobic interaction chromatography

Placental protein 14 was isolated from the biological material of patients undergoing legal abortions. The major part of ballast protein was removed by ion-exchange chromatography on DEAE-Sepharose and CM-Sepharose. Albumin was separated by chromatography on Blue-Sepharose. Complete purification was obtained by metal-chelate affinity chromatography on Nickel-Chelate Sepharose and hydrophobic interaction chromatography on Phenyl-Sepharose and Octyl-Sepharose. The protein was not exposed to denaturing agents or extreme pH.     

The directional control of sucrose and asparagine transport in lupin by abscisic acid

Injections of exogenous abscisic acid into the primary flowerhead of Lupinus luteus cv. Weiko III reduced the movement of ¹⁴C-sucrose into the flowerhead from the uppermost leaves. Sucrose transported from below the lateral branches subtending the flowerhead, was diverted into the lateral branches by injection of the exogenous abscisic acid into the flowerhead. ¹⁴C-sucrose was also diverted from a lateral branch injected with exogenous abscisic acid to all other parts of the plant, particularly the main stem and leaves, and the roots. Transport of ¹⁴C-asparagine administered at the cotyledonary node was directed from the flowerhead into the subtending lateral branches by injection of abscisic acid into the flowerhead. Transport of both ¹⁴C-sucrose and ¹⁴C-asparagine into the flowerhead was reduced at least three fold at physiological levels of abscisic acid. No significant correlation was found between the amount of ¹⁴C-asparagine entering a sink and the dry weight of the tissues of that sink. It is concluded that distribution of ¹⁴C-sucrose and ¹⁴C-asparagine between the flowerheads and lateral branches of L. luteus is actively and dynamically controlled and that abscisic acid levels play a significant part in that control. It is suggested that the relative levels of endogenous abscisic acid in plant organs could serve as an important factor in the directional control of assimilate transport in plants.     

Alkaloids from Delphinium pentagynum

Aerial parts of a collection of Delphinium pentagynum Lam. from Niebla, Southern Spain, furnished one diterpene alkaloid, 2-dehydrodeacetylheterophylloidine, two norditerpene alkaloids, 14-demethyl-14-isobutyrylanhweidelphinine and 14-demethyl-14-acetylanhweidelphinine, the known alkaloids 14-deacetylnudicauline, methyllycaconitine, 14-deacetyl-14-isobutyrylnudicauline, 14-acetylbrowniine, browniine, delcosine, lycoctonine, 18-methoxygadesine, neoline, karakoline and the aporphine alkaloid magnoflorine. Structures of the alkaloids were established by MS, 1D and 2-D NMR techniques.     

RT-PCR检测HUTP14A mRNA时排除假基因HUTP14C干扰的方法

人类U3蛋白14C基因(HUTP14C)是人类U3蛋白14A基因(HUTP14A)的假基因。两者转录本序列同源性高达95%。常规RT-qPCR技术在检测HUTP14A mRNA丰度时,HUTP14C的存在会影响检测结果。本研究旨在建立检测HUTP14A mRNA时排除HUTP14C干扰的RT PCR方法。本研究设计出能分别从多种肿瘤细胞DNA和RNA中特异性扩增HUTP14A和HUTP14C的引物,避免假基因HUTP14C对其同源基因HUTP14A检测的干扰。在检测细胞系HUTP14A mRNA时,通过DNaseⅠ消除RNA中污染的HUTP14C DNA,用靶向HUTP14C 3′-UTR的siRNA沉默HUTP14C mRNA后,再用RT PCR检测HUTP14A mRNA丰度,使结果更加准确。在18对肝癌及癌旁组织中,利用特异性引物进行RT PCR检测,HUTP14A和HUTP14C mRNA的表达略高于癌旁组织。本研究提示,针对有假基因存在的功能基因,对其mRNA丰度进行检测时,在提取细胞或组织总RNA后,用DNaseⅠ处理,再用RNA直接进行PCR扩增,排除DNA污染后,再进行RT-PCR或RT-qPCR扩增。大多假基因具有较长的3′-UTR区,在该区域设计siRNA特异性沉默假基因的mRNA后,用RT-qPCR检测功能基因的mRNA丰度,可以排除假基因mRNA的影响。在病理组织中检测功能基因的mRNA丰度时,可以根据假基因和其功能基因的序列差异设计出特异扩增功能基因的引物,从假基因的3′-UTR区设计特异扩增假基因的引物,通过RT-qPCR技术分别检测二者的mRNA。     

动态监测Toll样受体4 CD14在大鼠急性肝衰竭中的变化

目的观察toll样受体4(toll like receptor 4,TLR4),CD14在D-氨基半乳糖介导的急性肝衰竭模型中的动态变化律规。方法用D-氨基半乳糖剂量造成大鼠急性肝衰竭模型,用RT-PCR方法检测toll样受体,CD14 mRNA在D-氨基半乳糖介导的大鼠急性肝衰竭6、12、24、48、72、120和148 h的表达。结果Toll样受体4mRNA在急性肝衰竭的12 h有显著升高,与正常组差异有显著性(P<0.05),CD14 mRNA在6 h与正常组差异有非常显著性(P<0.01),两者均在48 h达到高峰,72、120和148 h逐渐下降。LPS、ALT和AST在6 h有轻度升高,至24 h与正常组差异有显著性(P<0.05),48 h(P<0.01)达高峰,72 h(P<0.01)、120 h和148 h逐渐下降。结论Toll样受体4,CD14与LPS、ALT和AST有显著正相关。Toll样受体4,CD14与D-氨基半乳糖介导的急性肝衰竭有着密切的联系,可能成为临床上治疗急性肝衰竭的新靶点。     

SIMS microscopy: a tool to measure the intracellular concentration of carbon 14-labelled molecules.

Monolayer cultures of human fibroblasts were incubated for 24 h with 14C-arginine and observed by means of SIMS microscopy (ion microscopy). Carbon 14 imaging showed the intracellular distribution of labelled arginine which featured high nuclear incorporation. The local concentration of this amino acid in different cells and intracellular structures was assessed through local isotopic 14C/12C ratio measurement. This relates the signal intensity of the labelling isotope carbon 14 to that of the corresponding natural isotope (carbon 12) of known tissular concentration. Using this method we were able to measure minor variations in the molecular concentration of arginine (expressed as mumol/g of tissue) between different fibroblasts. Results of this study indicate that SIMS microscopy is well adapted to carbon 14 detection and can provide quantitative maps of the cellular and subcellular distribution of 14C-labelled molecules.     

青少年牙周炎相关基因SEC14L5的生物信息学分析

用基因芯片技术来检测青少年牙周炎患者白细胞中表达异常的基因,检测中发现了一个未知基因表现为显著下调,其名称是SEC14L5,并推测该基因与白细胞的功能异常有着重要的作用。因此,利用生物信息学的方法对SEC14L5基因及其蛋白产物进行各种分析,在分析中我们发现SEC14L5编码的蛋白与SPF蛋白具有很大的相似性。     

乙型脑炎SA14-14-2减毒株灭活与不灭活以及P3株死疫苗在小鼠体内的免疫原性比较

将乙型脑炎SA14 14 2减毒株 (简称 14 2株 )病毒液和灭活 14 2株病毒液皮下和腹腔免疫小白鼠 ,P3株死苗作对照 ,测免疫效价 ,结果 14 2株病毒液的效力为 1 8× 10 -5～ 6 .1× 10 -6ID50 /ml,灭活 14 2株病毒液为 9 9× 10 -1～ 7 2× 10 -3 ID50 /ml,P3株死苗为 4 0× 10 -3 ～ 2 2× 10 -4 ID50 /ml。同时免后 14天采血用PRNT法测抗体 ,结果为 14 2株病毒液 1∶2 (皮下和腹腔 ) ;灭活 14 2株病毒液 <1∶2 ,P3株死苗为皮下 1∶2、腹腔 1∶10。结果表明 :14 2株在小白鼠体内的免疫原性远远高于灭活 14 2株病毒液和P3株死疫苗 ,14 2株经灭活后对小白鼠基本无保护作用。 14 2株抗体水平不高 ,而免疫力远高于P3株死苗 ,说明 14 2株与P3株死疫苗免疫性质不同 ,有细胞免疫参与。     

Oxo fatty acid esters from Cryptocoryne spiralis rhizomes

Two new compounds, isolated from the rhizomes of Cryptocoryne spiralis, have been characterized as ethyl 14-oxotetracosanoate and 15-oxoeicosanyl 14-oxoheptadecanoate by spectral data and chemical studies. Hentriacontane and sitosterol have also been isolated and identified.     

Alkaloids of Papaver bracteatum: 14-β-hydroxycodeinone, 14-β-hydroxycodeine and N-methylcorydaldine

Three new alkaloids have been identified from Papaver bracteatum, 14-β-hydroxycodeinone, 14-β-hydroxycodeine and N-methylcorydaldine. The presence of alpinigenine was also confirmed.     

流行性乙型脑炎强毒株和弱毒疫苗株SA14-14-2对猴子和小鼠的致病性和病理学研究

为了解乙型脑炎 (乙脑 )减毒活疫苗弱毒株SA14 14 2神经毒力的减弱程度 ,本文对弱毒株及其原株SA14强毒株进行了猴体和小白鼠的致病性和病理学变化的比较试验。SA14强毒株病毒 (原始滴度 6 15× 10 8/ml) ,以10 -2 和 10 -4 ～ 10 -7不同稀释度于丘脑两侧合并脊髓注射恒河猴 ,每组除 10 -4 1只外其余均为 2只。另以 10 -4 和10 -6～ 10 -8不同稀释度脑内注射小鼠 ,每组 8只。结果猴子除 10 -4 1只外其余全部发病死亡 ,小鼠则全部死亡。SA14 14 2以 1∶5稀释病毒 (原始滴度为 8× 10 6/ml)按同样方法注射 4只猴和 30只小鼠 ,结果全部存活。另以SA1410 -2 病毒皮下注射 3只猴未死亡 ,而以 10 -1皮下注射 30只小鼠时则全部死亡。SA14 14 2以 1∶5稀释病毒皮下注射小鼠时则全部存活。病理组织学结果显示二种动物接种SA14株强毒后主要表现为弥散性脑脊髓炎 ,以神经细胞坏死为其主要特征和最突出的病变。猴子的病变以脊髓前角、丘脑和中脑黑质为重 ,小鼠的病变则以大脑皮质、海马部最重 ,脊髓的病变却比脑轻。接种弱毒株的动物则仅有轻微炎症反应、神经细胞坏死极少出现。以上结果表明以脑内接种时恒河猴和小鼠对乙脑病毒均高度敏感 ,以皮下接种时小鼠的敏感性高于猴子。乙脑SA14 14 2弱毒株的神经毒力包括致     

NMR spectral mapping of Lipid A molecular patterns affected by interaction with the innate immune receptor CD14

Soluble CD14 (sCD14) is a serum glycoprotein that binds to the Lipid A moiety of lipopolysaccharides (LPS) with high affinity as part of the innate immune response to bacterial endotoxins. In order to investigate structural interactions of Lipid A with sCD14, we have prepared an isotopically labeled form of a fully active and chemically defined endotoxin, Kdo₂-Lipid A, which allowed us to carry out detailed NMR spectral mapping of this agonist ligand bound to sCD14 and identify for the first time structural regions that are strongly affected during complex formation with sCD14. These map to two adjacent areas comprising the lower portions of the sugar headgroup and upper half of the acyl chains I, III, and V, which are spatially proximal to the 1- and 4′-phosphate ends. Additionally, we have detected for the first time, presence of differential dynamic behavior for the affected resonances, suggesting a likely role for dynamics in the mechanism of Lipid A pattern recognition by sCD14.