抗肿瘤血管三结构域单链抗体ＶＨ／Ｌ的构建与表达

以本室研制的一株抗肿瘤血管单克隆体ＡＡ９８为基础,采用ＰＣＲ扩增抗体ＡＡ９８基因的重链可变区（ＶＨ）和轻链（Ｌ）,以重链恒定区１（ＣＨ１）５′端１２个氨基酸的序列作为连接肽,并将连接肽中的Ｌys变为Ｓer,构建ＶＨ－连接肽－Ｌ三结构域单链抗体。重组ＶＨ／Ｌ单链抗体在大肠杆菌中得到了高效表达,其表达量占菌体总蛋白质的２０％。,表达的蛋白质在菌内形成包含体,经凝胶过滤法复性,获得了有抗原结合活性的ＶＨ／Ｌ。该三结构域单链抗体的成功构建和复性,为重组抗体片段的研制提供了借鉴。     

抗吗啡活性肽的分离纯化及一级结构测定

报道了一种新的具有抗吗啡镇痛活性的肽的分离纯化,并进行部分一级结构测定．狗脑先经醋酸提取,冷冻成干粉,然后上ＳｅｐｈａｄｅｘＧ－５０和Ｓ－ＳｅｐｈａｒｏｓｅＦ．Ｆ柱,最后经ＲＰ－ＨＰＬＣ纯化,鉴定纯度后,测定其抗吗啡镇痛活性,通过ＳＤＳ－ＰＡＧＥ法测得其分子量为８．９ｋＤ．氨基酸序列分析测得该肽的Ｎ端序列为：Ｖ－Ｉ－Ｓ－Ｖ－Ａ－Ｄ－Ｗ－Ｔ－Ｑ－Ｉ－Ｆ－Ｔ－Ｍ－Ｒ－Ｙ－Ｆ－Ｉ－Ｔ－Ｇ－Ｙ－Ｈ－Ｑ－Ｄ－Ｙ－Ｘ－Ｇ－Ｌ－Ｈ－Ｉ－Ｇ．经部分一级结构同源序列检索,未见与此有同源的蛋白质的报道,暂命名该肽为ＣＣ４肽     

2．5分辨率单斜Al－（L－丙氨酸）胰岛素晶体结构研究

空间群为Ｐ２１的Ａ１－（Ｌ－丙氨酸）胰岛素晶胞内,一个不对称单位含有一个六聚体,应用差值Ｆｏｕｒｉｅｒ技术,立体化学制最小二来技术和Ｘ—ＰＬＯＲ程序并辅以电子密度图的人工拟合,解析了分辨率ＡＩ—（Ｌ－丙氨酸）胰岛素（Ａｌ－Ｌ－ＡｌａⅠ）的晶体结构。最终Ｒ因子为２０．６％,与标准键长与键角的均方根偏差分别为和４．１９°,从电子密度图与模型的拟合来看,六聚体中每条Ａ链的Ａｌ位置替换的Ｌ—Ａｌａ清晰可见,每条Ｂ链Ｎ端Ｂ１—Ｂ８伏段都为α螺旋构象,形成了Ｂ１—Ｂ１９的连续α螺旋段。     

2．5分辨率单斜Al－（L－丙氨酸）胰岛素晶体结构研究

空间群为Ｐ２１的Ａ１－（Ｌ－丙氨酸）胰岛素晶胞内,一个不对称单位含有一个六聚体,应用差值Ｆｏｕｒｉｅｒ技术,立体化学制最小二来技术和Ｘ—ＰＬＯＲ程序并辅以电子密度图的人工拟合,解析了分辨率ＡＩ—（Ｌ－丙氨酸）胰岛素（Ａｌ－Ｌ－ＡｌａⅠ）的晶体结构。最终Ｒ因子为２０．６％,与标准键长与键角的均方根偏差分别为和４．１９°,从电子密度图与模型的拟合来看,六聚体中每条Ａ链的Ａｌ位置替换的Ｌ—Ａｌａ清晰可见,每条Ｂ链Ｎ端Ｂ１—Ｂ８伏段都为α螺旋构象,形成了Ｂ１—Ｂ１９的连续α螺旋段。     

海洋细菌Pseudomonas sp．抗菌代谢产物的研究

从海洋细菌Pseudomonas sp.发酵液中分离鉴定9个环二肽和2个苯环类化合物,经波谱鉴定为环(酪氨酸-脯氨酸)(1),环(酪氨酸-异亮氨酸)(2),环(苯丙氨酸-脯氨酸)(3),环(缬氨酸-脯氨酸)(4),环(异亮氨酸-脯氨酸)(5),环(亮氨酸-脯氨酸)(6),环(丙氨酸-脯氨酸)(7),环(缬氨酸-丙氨酸)(8),环(丙氨酸-亮氨酸)(9),对羟基苯甲醛(10),二-(2-乙基己基)邻苯二甲酸酯(11).其中化合物1～4对多种海洋细菌显示一定的抗菌活性.     

乌约活性组分LEF的化学成分及抗风湿作用

从乌药（Ｌｉｎｄｅｒａ ａｇｇｒｗｅｇａｔａ（Ｓｉｍｓ）Ｋｏｓｔｅｒｍ）根的镇痛、抗火活性组分（ＬＥＦ）中分离得到６个缩合鞣质烃化合物,鉴定出其中４个,分别为表儿茶素（１）－ｅｐｉｃａｔｅｃｈｉｎ、表榈儿茶素（－）－ｅｐｉｇａｌｌｏｃａｔｅｃｈｉｎ、ｐｒｏｃｙａｎｉｄｉｎＢ－２及ｔｒｉｍｅｒｉｃ ｐｒｏａｎｔｈｏｃｙａｎｉｄｉｎ（ｃｉｎｎａｍｔａｎｎｉｎｓ Ｂ１）,另得到一个黄酮苷,鉴定为橙皮甙ｈ     

北京鸭Apo A-I溴化氰裂解片段的分离、纯化及其功能域定位

溴化氰可裂解北京鸭ａｐｏＡ－Ｉ为１１个肽段,通过测定纯化后各片段分子量和Ｎ末端氨基酸序列确定片段３～１０在ａｐｏＡ－Ｉ分子中的位置,分别为６４～２４０,７４～２４０,６４～２０６,１～１３６,１～６３,１７１～２０６,２０７～２４０和１３７～１７０．并对上述片段功能进行研究,结果为：（１）这些片段均可与脂质结合形成大小不同的脂质体,其大小与片段长短成正比。（２）ＡｐｏＡ－Ｉ溴化氰片段３～１０激活ＬＣＡＴ活性分别为完整ａｐｏＡ－Ｉ的６５％、５２％、６０％、３９％、８％、７％、０％和２％,说明激活ＬＣＡＴ的活性主要存在于６４～１３６之间。（３）只有片段３、４和９形成的脂质体可与肝ＨＤＬ受体结合,其他均无明显结合力,显示氨基酸２０７～２４０是ａｐｏＡ－Ｉ与ＨＤＬ受体结合片段。     

亲和层析纯化HepG_2细胞分泌的PAI－1

本文报道用抗ＰＡＩ－１单克隆抗体（ＭｃＡｂ）亲和层析建立了纯化ＰＡＩ－１的简便方法。经免疫亲和层析,ＳｅｐｈａｃｒｙｌＳ２００凝胶过滤,从ＨｅｐＧ２细胞培液中分离到糖基化和非糖基化两种形式的ＰＡＩ－１,回收率为８４％,ＰＡＩ－１比活性６．１×１０４ＩＵ／ｍｇ。糖基化ＰＡＩ－１分子量为５０ｋＤ,比活性５．８×１０４ＩＵ／ｍｇ。非糖基化ＰＡＩ－１分子量４３ｋＤ,占总ＰＡＩ－１３０％,仍具有ＰＡＩ－１活性。用ＣｏｎＡ－Ｓｅｐｈａｒｏｓｅ亲和层析进一步纯化得到ＳＤＳ－ＰＡＧＥ纯的糖基化ＰＡＩ－１。     

滇重楼地上部分的配糖体

从滇重楼ＰａｒｉｓｐｏｌｙｐｈｙｌｌａＳｍ．ｖａｒ．ｙｕｎｎａｎｅｎｓｉｓ（Ｆｒ．）Ｈ－Ｍ,地上部分,分离出４个微量的配糖体,经光谱分析和化学降解证明其化学结构分别为２５Ｓ－异钮替皂甙元－３－Ｏ－α－Ｌ－鼠李吡喃糖基（１→２）［α－Ｌ－鼠李吡喃糖基（１→４）］－β－Ｄ－葡萄吡喃甙（Ａ）,２６－β－Ｄ－葡萄吡喃糖基－纽替皂甙元－３－Ｏ－α－Ｌ－鼠李吡喃糖基（１→２）［α－Ｌ－鼠李吡喃糖基（１→４）－β－Ｄ－葡萄吡喃糖甙（Ｂ）,山奈酚－３－Ｏ－β－Ｄ－葡萄吡喃糖基（１→６）－β－Ｄ－葡萄吡喃甙（Ｃ）,７－Ｏ－α－Ｌ－鼠李吡喃糖基－山奈酚－３－Ｏ－β－Ｄ－葡萄吡喃糖基（１→６）－β－Ｄ－葡萄糖甙（Ｄ）。     

麦冬的配糖体成分

从川麦冬（Ｏｐｈｉｏｐｏｇｏｎ ｊａｐｏｎｉｃｕｓ）根部乙醇提取物中分离得到四个已知化合物,经波谱分析确定其结构分别为：龙脑７－Ｏ－β－Ｄ－吡喃葡萄糖甙（１）,龙脑７－Ｏ－〔β－Ｄ－呋喃芹糖（１→６）〕－β－Ｄ吡喃葡萄糖甙（２）,４－烯丙基－１,２－苯本酚１－Ｏ－〔α－Ｌ－吡喃鼠李糖（１→６）〕－β－Ｄ－吡喃葡萄糖甙（３）,５－烯－１β,３β,１６β,２２Ｓ－胆甾四醇１－Ｏ－α－Ｌ－吡喃鼠李糖１６     

CRISPR/Cas9编辑大肠杆菌工程菌生产氨基葡萄糖

【背景】氨基葡萄糖(glucosamine, GlcN)及其衍生物N-乙酰氨基葡萄糖(N-acetylglucosamine,GlcNAc)是合成糖胺聚糖的重要前体物质,在医药、化妆品和保健品领域具有广泛的应用价值。传统的生产方式存在诸多弊端,如环境污染、原料限制、不适于海鲜易过敏人群等问题,因此利用微生物发酵法生产GlcN和GlcNAc越来越受到青睐。【目的】利用微生物发酵生产并提高N-乙酰氨基葡萄糖的产量,探索分子改造及发酵条件优化策略。【方法】以大肠杆菌MG1655为出发菌株,首先利用表达载体共表达大肠杆菌来源的glmS和酿酒酵母来源的gna1,构建GlcNAc的生物合成路径,然后利用CRISPR/Cas9技术敲除GlcNAc的分解代谢与转运途径,以提高GlcNAc的产量,最后结合发酵条件优化使GlcNAc的产量得到进一步提升。【结果】通过分子改造得到一株产GlcNAc菌株RY-5,发酵20 h后GlcNAc的产量达到了2.36 g/L,相较于初始构建的菌株RY-1提高了29倍,进一步对装液量和诱导剂IPTG的添加时间等条件进行发酵优化,GlcNAc产量达到了7.74g/L,与优...     

DNA binding properties of 9-substituted harmine derivatives

The beta-carboline alkaloids have been characterized as a group of potential antitumor agents. The underlying mechanisms of harmine and its derivatives were investigated by DNA binding assay and Topoisomerase (Topo) inhibition assay. Meanwhile, the DNA photocleavage potential of these compounds and their cytotoxicity were also examined by DNA photocleavage assay and cytotoxicity assay in vitro. Harmine and its derivatives exhibited remarkable DNA intercalation capacity and significant Topo I inhibition activity but no effect with Topo II. Introducing an appropriate substituent into position-9 of beta-carboline nucleus enhanced the affinity of the drug to DNA resulting in remarkable Topo I inhibition effects. These results suggested that the ability of these compounds to act as intercalating agents and Topo I inhibitors was related to the antitumor activity. Moreover, these data showing a correlation between cytotoxicity and Topo I inhibition or DNA binding capacity are very important as they strongly suggested that the Topo I-mediated DNA cleavage assay and DNA binding assay could be used as a guide to design and develop superior analogues for antitumor activities.     

Plant regeneration from protoplast-derived callus of rice (Oryza sativa L.)

Protoplasts isolated from cultured rice cells of an A-58 cytoplasmic male sterile line (A-58 MS)(Oryza sativa L.) were used to investigate the regeneration of rice plants. A cultured cell line (T₃) of A-58 MS with a high growth rate and dense cytoplasm was selected. About 10% of the protoplasts prepared from this established cell line plated in RY-2 (a new medium) formed colonies. The calli formed shoots and roots in the regeneration medium and developed into whole plants.Protoplasts also were prepared from suspension cultures of 25 other varieties of rice using the same methods. The protoplasts isolated from two of the 25 varieties, Fujiminori and Toyotama, had high rates of cell division in RY-2 medium. Only protoplastderived calli from Fujiminori, produced whole plants in the regeneration medium.Abbreviations LS Linsmaier and Skoog (1965) - 2,4-D 2,4-dichlorophenoxyacetic acid - BA 6-benzyladenine - MES 2-(N-Morpholino)ethanesulfonic acid, monohydrate     

CD8+ cytotoxic T-APC stimulate central memory CD8+ T cell responses via acquired peptide-MHC class I complexes and CD80 costimulation, and IL-2 secretion

We previously showed that naive CD4+ Th cells acquire peptide-MHC class I (pMHC I) and costimulatory molecules from OVA-pulsed dendritic cells (DC(OVA)), and act as Th-APCs in stimulation of CD8+ CTL responses. In this study, we further demonstrated that naive CD8+ cytotoxic T (Tc) cells also acquire pMHC I and costimulatory CD54 and CD80 molecules by DC(OVA) stimulation, and act as Tc-APC. These Tc-APC can play both negative and positive modulations in antitumor immune responses by eliminating DC(OVA) and neighboring Tc-APC, and stimulating OVA-specific CD8+ central memory T responses and antitumor immunity. Interestingly, the stimulatory effect of Tc-APC is mediated via its IL-2 secretion and acquired CD80 costimulation, and is specifically targeted to OVA-specific CD8+ T cells in vivo via its acquired pMHC I complexes. These principles could be applied to not only antitumor immunity, but also other immune disorders (e.g., autoimmunity).     

Synthesis and biological evaluation of N-(7-indolyl)-3-pyridinesulfonamide derivatives as potent antitumor agents

We herein report the synthesis and antitumor activity of E7070 analogues containing a 3-pyridinesulfonamide moiety. E7070 was selected from our sulfonamide-based compound collections, currently undergoing Phase II clinical trials because of its tolerable toxicity profile and some antitumor responses in the Phase I setting. Of the analogues examined, ER-35745, a 6-amino-3-pyridinesulfonamide derivative, demonstrated significant oral efficacy against the HCT116 human colon carcinoma xenograft in nude mice.     

Discovery of substituted biphenyl imidazoles as potent,bioavailable bombesin receptor subtype-3 agonists

We report SAR studies on a novel non-peptidic bombesin receptor subtype-3 (BRS-3) agonist lead series derived from high-throughput screening hit RY-337. This effort led to the discovery of compound 22e with significantly improved potency at both rodent and human BRS-3.     

红酵母RY-4中虾青素的分离纯化研究

本文主要研究了红酵母RY-4菌株中天然虾青素的提取和纯化工艺。通过各种破壁提取方法的比较分析,得出了酸热法破壁丙酮提取为较经济理想的粗色素提取方法,提取的粗色素油中虾青素含量达51.7%。通过对各种柱层析条件的摸索研究,得出了以硅胶为吸附剂,Ⅴ(石油醚)∶Ⅴ(乙酸乙酯)混和液为洗脱剂梯度洗脱从粗色素油中分离纯化虾青素的方法,所得虾青素纯度达到97%。     

Antitumor agents 294. Novel E-ring-modified camptothecin-4β-anilino-4'-O-demethyl-epipodophyllotoxin conjugates as DNA topoisomerase I inhibitors and cytotoxic agents

Two conjugates (1 and 2) of camptothecin (CPT) and 4β-anilino-4'-O-demethylepipodophyllotoxin were previously shown to exert antitumor activity through inhibition of topoisomerase I (topo I). In this current study, two novel conjugates (1E and 2E) with an open E-ring in the CPT moiety were first synthesized and evaluated for biological activity in comparison with their intact E-ring congeners. This novel class of CPT-derivatives exhibits its antitumor effect against CPT-sensitive and -resistant cells, in part, by inhibiting topo I-linked DNA (TLD) religation. An intact E-ring was not essential for the inhibition of TLD religation, although conjugates with an open E-ring were less potent than the closed ring analogs. This lower religation potency resulted in decreased formation of protein-linked DNA breaks (PLDBs), and hence, less cell growth inhibition. In addition to their impact on topo I, conjugates 1E, 2, and 2E exhibited a minor inhibitory effect on topo II-induced DNA cleavage. The novel structures of 1E and 2E may present scaffolds for further development of dual function topo I and II inhibitors with improved pharmacological profiles and physicochemical properties.     

Synthesis of new camptothecin analogs with improved antitumor activities

Novel hexacyclic camptothecin analogs containing cyclic amidine, urea, or thiourea moiety were designed and synthesized based on the proposed 3D-structure of the topoisomerase I (Topo I)/DNA/camptothecin ternary complex. The analogs were prepared from 9-nitrocamptothecin via 7,9-diaminocamptothecin derivatives as a key intermediate. Among them, 7c exhibited in vivo antitumor activities superior to CPT-11 in human cancer xenograft models in mice at their maximum tolerated doses though its in vitro antiproliferative activity was comparable to SN-38 against corresponding cell lines.     

The isolation and in vivo Potent Antitumor activity of clerodane diterpenoid from the oleoresin of the brazilian medicinal plant, copaifera langsdorfi desfon.

An extremely potent antitumor neo-clerodane diterpene was isolated from the oleoresin of the Brazilian medicinal plant, Copaifera langsdorfii Desfon. This compound was identified as (-)-kolavenol 1. The antitumor effect of 1 against IMC carcinomas as determined from the increase in lifespan (I.L.S.) in mice was twice that of 5-FU. The structure elucidation and the antitumor activity of the other related compounds 2 6 in this oleoresin were also described.