基因重组人肿瘤坏死因子-α突变体的构建

在详细分析ＴＮＦ α结构及有关ＴＮＦ α结构与功能关系研究的基础上,设计并人工合成了一对ＴＮＦ α结构基因点突变引物。应用ＰＣＲ和分子克隆技术构建了一种新型人肿瘤坏死因子（ＴＮＦ α）分子的编码基因,将该编码基因插入表达质粒,转化大肠杆菌,通过温度诱导获得了表达蛋白,对突变体克隆进行了ＤＮＡ电泳、酶切位点检测以及基因序列测定,并对突变体蛋白进行了ＳＤＳ ＰＡＧＥ电泳检测。     

肿瘤坏死因子与其受体相互作用的计算机模拟研究

利用同源模建方法,以ＴＮＦＲ５５受体胞外区的晶体结构为参考模板,预测了ＴＮＦＲ７５受体胞外区Ｃｙｓ１８～Ｐｈｅ１４７片段的三维结构．根据Ｒ５５受体胞外区与ＬＴ相结合的复合物的晶体结构,预测了ＴＮＦ与Ｒ５５及Ｒ７５胞外区的复合物的三维结构,模拟了ＴＮＦ与受体之间的相互作用．由于ＴＮＦ与受体的作用形式是三聚体对三聚体,因此在模拟ＴＮＦ与受体相互作用时选择了包括一个非对称的ＴＮＦ三聚体和一个受体（Ｒ５５或Ｒ７５）单体的模拟系统．结合已有的突变体实验结果,利用计算机模拟分析手段,发现了一些ＴＮＦ突变体之所以具有受体选择性的三维结构基础和发挥了关键作用的氨基酸残基以及这些残基之间的主要作用形式．研究深化了对已有的突变体实验结果的认识,建立了不同的实验结果之间的内在关联,为以后有目的的新型突变体设计和实验研究打下了基础．     

3种人肿瘤坏死因子衍生物的研制

本文在详细分析肿瘤坏死因子（ＴＮＦ）结构的基础上应用ＰＣＲ技术和基因工程手段改造人ＴＮＦ分子,构建了３种人ＴＮＦ的衍生物。３种人ＴＮＦ衍生物是;Ｎ端缺失７个氨基酸且８、９、１０位的ＰｒｏＳｅｒＡｓｐ改为ＡｒｇＬｙｓＡｒｇ的ＴＮＦ（简写为ｈＴＮＦＤ１）：Ｃ端１５７位Ｌｅｕ改为Ｐｈｅ的ＴＮＦ（简写为ｈＴＮＦＤ２）;Ｎ端和Ｃ端同时作上述改变的ＴＮＦ（简写为ｈＴＮＦＤ３）。这３种衍生物在大肠杆菌中均获得较高表述,它们对Ｌ９２９细胞的细胞毒活性较重组天然人ＴＮＦ有很大升高,尤其是ｈＴＮＦＤ１和ｈＴＮＦＤ３,升高达３个数量级。文中对３种衍生物活性升高的原因作了分析。     

玉米叶绿体ATP合成酶ε亚基的定点突变

利用突变引物和ＰＣＲ方法对玉米叶绿体ＡＴＰ合成酶的ε在进行定点突变,使ε亚基４２位上的Ｔｈｒ分别替换成Ｃｙｓ、Ａｒｇ、Ｉｌｅ和Ｐｒｏ。Ｔｈｒ变为Ｐｒｏ的ε亚基突变体不能表达,其他突变体与野生型一样,都能正常表达。Ｔｈｒ突变为Ｃｙｓ和Ａｒｇ后,突变体对ＡＴＰａｓｅ活力的抑制比野生型略高一些,但突变为Ｉｌｅ后,其抑制程度则极大地增强了。     

3‘端非翻译区影响TNFa cDNA在大肠杆菌中的表达

采用ＰＣＲ方法改变了表达构建物ｐＲＬ－ｒｈＴＮＦ中的结构,即去掉ｒｈ ＴＮＦａ ｃＤＮＡ ３’端非翻译区序列１１０ｂｐ（命名为ｐＲＬ－ｒｈ ＴＮＦａ２）,转入大肠杆菌后,观察数个阳性转化子的表达情况,并与原型ｐＲＬ－ｒｈ ＴＮＦａ的表达进行比较。结果表明,去掉３’端非翻译区的ｐＲＬ－ｒｈＴＮＦａ２能稳定表达ｒｈＴＮＦａ,其发酵及纯化产品的生物学特征均等同于原型的,且其表达量比原型的有提高,提示ｅ‘     

凝乳酶β-转角突变体的构建、表达和性质分析

为了阐明牛凝乳酶的第二个发夹结构中的Ⅵ型β转角的功能,利用基因突变技术,将该转角的氨基酸残基（Ｌｅｕ２０Ｇｌｙ２１Ｔｈｅ２２Ｐｒｏ２３Ｐｒｏ２４Ｇｌｎ２５）改为（Ｌｅｕ２０ＧｌｙＧｌｙＧｌｎ２５）、（Ｌｅｕ２０ＳｅｒＧｌｙＧｌｎ２５）或（Ｌｅｕ２０ＧｌｙＳｅｒＧｌｎ２５）,得到３个突变牛凝乳酶原表达质粒ｐＭＣＧＧ,ｐＭＣＳＧ和ｐＭＣＧＳ。除ｐＭＣＧＳ不能在大肠杆菌中表达外,ｐＭＣＧＧ和ｐＭＣＳＧ均能以包含体的形式进行表达,且表达水平与野生型相当。像野生型凝乳酶原一样,ｐＭＣＧＧ和ｐＭＣＳＧ的表达产物经过变性复性,均能自活化为假凝乳酶,而且复性后的酶原突变体在ＤＥＡＥＳｅｐｈａｒｏｓｅＣＬ６Ｂ上的柱行为与野生型相类似,但突变体假凝乳酶的凝乳及水解酪蛋白活性均明显低于野生型。说明突变对酶原的折叠影响较小,但对酶的催化效率影响较大。     

hTNF受体在BHK细胞中的表达及其与hTNF—α的相互作用

将两种人肿瘤坏死因子（ｈＴＮＦ）受体ｈＴＮＦＲ５５和ｈＴＮＦＲ７５基因分别克隆到表达载体ｐｃＤＮＡ３、ｐＤＲ２以及ｐＸＪ４１中,以脂质体介导的方法转染ＢＨＫ细胞,用［１２５Ｉ］ＴＮＦα筛选出阳性克隆,并进一步鉴定了表达两种ｈＴＮＦＲｓ的细胞株;反转录（ＲＴ）ＰＣＲ和ＥＬＩＳＡ结果表明两种ｈＴＮＦＲｓ在ＲＮＡ转录和蛋白质合成水平均获得了表达。但ｈＴＮＦα不能引发这些细胞株的细胞毒活性;结合野生型ｈＴＮＦα及其突变体Ｒ２Ｋ的细胞毒活性和体内毒性的测定结果,利用这些细胞株进一步测定了突变体ｈＴＮＦαＲ２Ｋ和野生型ｈＴＮＦα分别与两种受体的竞争结合活性,从而为两种受体以及ｈＴＮＦα的结构和功能研究的进一步深入奠定了一定的基础。     

Fas与FasL的分子及细胞生物学研究进展

Ｆａｓ是Ｉ型膜蛋白,属ＴＮＦ受体家庭成员,许多细胞表达Ｆａｓ。而Ｆａｓ配体（ＦａｓＬ）只在激活Ｔ淋巴细胞表达,是Ⅱ型膜蛋白,属ＴＮＦ家庭成员,当ＦａｓＬ与Ｆａｓ结合时,Ｆａｓ可向细胞传递“死亡信号”,细胞在数小时内凋亡,Ｆａｓ－ＦａｓＬ介导的细胞凋亡在免疫调失及免疫病理中,可能有重要作用。     

地塞米松、噻庚啶和山莨菪碱对LPS诱导大鼠肝脏TNFα表达的影响

地塞米松（Ｄｅｘ）、噻庚啶（Ｃｙｐ） 和山莨菪碱（Ａｎｉ） 对脂多糖（ＬＰＳ） 诱导的大鼠肝脏ＴＮＦα表达的影响。Ｗｉｓｔａｒ大鼠４０ 只, 静脉注射ＬＰＳ（ＥｃｏｌｉＯ１１１Ｂ４ ５ｍ ｇ／ｋｇ） 后, 立即静脉给予Ｄｅｘ ５ｍ ｇ／ｋｇ、Ｃｙｐ５ｍ ｇ／ｋｇ 或Ａｍ ｉ１０ｍ ｇ／ｋｇ,于ＬＰＳ攻击后２ｈ 取动物的肝脏,ＡＰＡＡＰ法进行ＴＮＦα免疫组织化学研究,Ｎｏｒｔｈ－ｅｒｎ 杂交分析ＴＮＦαｍ ＲＮＡ 表达水平。结果发现ＬＰＳ攻击后２ｈ, 肝脏ＴＮＦαｍ ＲＮＡ 表达水平显著增高, 肝脏枯否氏细胞胞浆内有大量的ＴＮＦα红染颗粒。Ｄｅｘ、Ｃｙｐ 或Ａｎｉ均能显著降低大鼠肝脏ＴＮＦαｍ ＲＮＡ 水平和ＴＮＦα含量。结果表明Ｄｅｘ、Ｃｙｐ 和Ａｎｉ均显著抑制ＬＰＳ诱导的ＴＮＦα基因表达, 可能有抗感染性休克作用。     

人组织型纤溶酶原激活剂（t－PA）突变体FrGGI的构建、表达及特性分析

为了获得半衰期延长,特异活性提高及具有ＰＡＬ－１抗性的新型ｔ－ＰＡ溶栓剂,利用基因重组及定位突变技术构建了ｔ－ＰＡ的Ｋ１、Ｋ２区糖基化位点消除,ＰＡＩ－１结合位点缺失,Ｆ与Ｅ区连接序列Ｈｉｓ４４～Ｓｅｒ５０置换为纤粘蛋白Ⅰ型Ｆ区间连接序列ＧｌｕＳｅｒＬｙｓＰｒｏＧｌｕＡｌａＧｌｕＧｌｕ的ｔ－ＰＡ组合突变体ＦｒＧＧＩ,并在中国仓鼠卵巢细胞中获得了高效表达。对表达产物的生物学特性分析表明,ＦｒＧＧＩ在大鼠血浆中的半衰期延长了１５倍,并获得了ＰＡＩ－１抗性,是一株很有希望的新型溶栓剂候选株。     

Ectogenesis and the case against the right to the death of the foetus

Ectogenesis, or the use of an artificial womb to allow a foetus to develop, will likely become a reality within a few decades, and could significantly affect the abortion debate. We first examine the implications for Judith Jarvis Thomson’s violinist analogy, which argues for a woman’s right to withdraw life support from the foetus and so terminate her pregnancy, even if the foetus is granted full moral status. We show that on Thomson’s reasoning, there is no right to the death of the foetus, and abortion is not permissible if ectogenesis is available, provided it is safe and inexpensive. This raises the question of whether there are persuasive reasons for the right to the death of the foetus that could be exercised in the context of ectogenesis. Eric Mathison and Jeremy Davis have examined several arguments for this right, doubting that it exists, while Joona Räsänen has recently criticized their reasoning. We respond to Räsänen’s analysis, concluding that his arguments are unsuccessful, and that there is no right to the death of the foetus in these circumstances.     

On the diversity of the Cladocera in the tropics

The mythical concept of an impoverished tropical cladoceran fauna is refuted. On a planetary scale, around half of the cladoceran species presently known occur exclusively in the tropics-subtropics, often with considerable restriction to particular geographical subzones. On a regional (political) scale, the situation is often unclear because of the continued fragmentary nature of studies, and because political units are not a good basis for biogeographical comparisons. At the finest level of resolution (lake-perlake comparisons), there appears to be an upper limit of c. 50 cladoceran species per individual lake. No significant difference between lakes in the temperate zone and in the tropics could be established here. Daphnia is largely absent from the tropics, but is replaced by more Sidids, Moinids, and Bosminids, such that the average cladoceran community in the limnetic zone of a tropical lake is not characterized by less species but rather by lower population densities. This, in turn, is considered a consequence of higher prevalent predation levels in the tropics.     

Tubular networks in the terminal endings of the visual receptor cells in the human,the monkey,the cat and the dog

Summary A tubular network was found in the terminal endings of the visual receptor cells in the human, the monkey (Macaca mulatta), the cat and the dog. These tubules are arranged in close groups in the vicinity of the synaptic lamellae and the invaginated dendrites. According to the form, diameter, density of the tubules and to the consistence of the network formed by them one can distinguish at these places an initial type (type I), a transitory (type II) and a vesicular one (type III). In the the type III branching, bizarre forms are frequent. The diameter of all the tubules reaches 500–600 Å, their density and walls being the same as in the synaptic vesicles.Similar networks also occur in the axons of the visual receptor cells of the monkey.

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Zusammenfassung In den Endigungen der Photorezeptorzellen von Mensch, Affe (Macaca mulatta), Katze und Hund kommen aus Tubuli bestehende Komplexe vor. Organellenartig in geschlossenen Gruppen angeordnet, liegen sie in Nähe der synaptischen Lamellen und der invaginierten Dendriten. An diesen Stellen kann man nach Form, Durchmesser, Dichte und Konsistenz der von den Tubuli gebildeten Komplexe drei Typen unterscheiden: 1. einen initialen (Typus I), 2. einen Übergangstypus (Typus II) und 3. einen vesiculären Typus (Typus III). In letzterem kommen häufig verzweigte, bizarre Formen vor. Der Durchmesser sämtlicher Tubuli erreicht 500–600 Å. Ihre Dichte und ihre Wand gleicht denen der synaptischen Vesikel.Ähnliche Komplexe fanden wir auch in den Axonen der Photorezeptorzellen vom Affen.

     

Assessing the Physiological State of the Fucoids from the Barents Sea Littoral by the End of the Polar Night

Methods of amperometry and potentiometric titration were used to follow dark respiration (DR) and apparent photosynthesis (AP) in the fucoids Ascophyllum nodosum (L.) Le Jol, Fucus vesiculosus L., and F. serratus L. from the Barents Sea littoral by the end of the 40-day-long polar night. The macroalgae were shown to manifest species-specific low rates of photosynthesis and respiration. However, in spite of their low photosynthetic status due to the effects of subzero temperature and prolonged low or zero illumination, the macroalgae have been able to restore DR and AP to the initial level already by the day 9; the ability to restore AP depended on the level of illumination. The study of the changes in the carbonate–bicarbonate system in the light and darkness demonstrated that the macroalgae grown in darkness, in contrast to those grown in twilight, could absorb bicarbonate in darkness; however, they lost this capacity after two-day-long illumination at an irradiance of 7 mol/(m² s). Bicarbonate uptake in darkness and the capacity to restore the systems of photosynthesis and respiration in fucoid cells are discussed in the context of algal energy metabolism under the polar night conditions.     

Arranging the elements of the potassium channel: the T1 domain occludes the cytoplasmic face of the channel

The voltage-gated potassium channel is currently one of the few membrane proteins where functional roles have been mapped onto specific segments of sequence. Although high-resolution structures of the transmembrane portions of three bacterial potassium channels, the tetramerization domain and the cytoplasmic ball are available, their relative spatial arrangement in mammalian channels remains a matter of ongoing debate. Cryo-electron microscopic images of the six transmembrane voltage-gated Kv channel have been reconstructed at up to 18 Å resolution, revealing that the T1 domain tetramerizes and is suspended below the transmembrane segments. However, the resolution of these images is insufficient to reveal the location of the third piece of the puzzle, the inactivating ball domain. We have used the aberrant interactions observed in a series of chimæric channels to establish that an assembled T1 domain restricts access to the cytoplasmic face of the channel, suggesting that the N-terminal ball and chain may be confined in the space between the T1 domain and the transmembrane portion of the channel.     

Feeding the vertebrate retina from the Cambrian to the Tertiary

The retina of the vertebrate eye is metabolically active and requires nutritive support. During the last 540 million years it has evolved into forms as complicated and nutritionally demanding as those found in avian or primate eyes. Diffusion from the choroid is generally able to supply the metabolic needs of thin retinae. However, when the thickness exceeds the limits of diffusion, structures are needed to supplement the vascular supply from the choroid. These supplemental nutritive devices include the choroidal gland, the falciform process and preretinal vascular plexus of fish, the conus papillaris of lizards, the pecten oculi of birds, the intraretinal vessels of mammals and a few novel systems that remain difficult to classify. These vascular systems are among the most variable features of the vertebrate eye. Here, we review classical and recent findings regarding such retinal nutrition systems, propose a three category classification for them based on histologic origins and speculate on the evolutionary forces which drove their development.     

Observations on the permeability of the choriocapillaris of the eye

Summary The choriocapillaris is a fenestrated capillary bed located posterior to the retinal pigment epithelium. It serves as the main source of supply to the photoreceptors, retinal pigment epithelium, and other cells of the outer retina. The permeability of these capillaries to intravenously injected ferritin (MW — approx. 480,000; mol. diam. 11 nm) was examined in the mouse, rabbit, and guinea pig, each of which is characterized by a different type of retinal vascularization. In all three species, the bulk of the ferritin remained in the capillary lumina, where it appeared to be blocked at the level of the diaphragmed fenestrae. Some ferritin was present in endothelial cell vacuoles. The results confirm previous work on the rat choriocapillaris and indicate that the barrier function of the choriocapillary endothelium is present even among species in which the retinal circulation differs significantly.Supported by NIH grant EY03418     

Separation of the pineal vesicle from the wall of the third ventricle during the post-hatching development of the chicken

Summary According to light- and electron-microscopic observations the pineal organ of the 3-day-old chicken consists of a prominent end vesicle and a tapering parenchymal stalk. During this stage the pineal lumen is in open communication with the third ventricle. However, in the 40-day-old chicken, which still possesses a well-developed end vesicle, the proximal portion of the pineal stalk displays regressive changes leading to local fragmentation. At this stage the pineal stalk is reduced, and the pineal lumen is missing. In 1-year-old chickens the parenchyma of the proximal portion of the stalk is further diminished, and in 3-year-old domestic fowl is completely displaced by bundles of collagenous fibers, only some nerve fibers being present. This post-hatching pineal development may reflect the sequence of changes leading from pineal sense organs to pineal glands.This work was supported by a grant-in-aid for Scientific Research from the Ministry of Education, Science and Culture of Japan