Cibacron Blue亲和层析及应用

以Ｆ３ＧＡ（Ｃｉｂａｃｒｏｎ　Ｂｌｕｅ　Ｆ３ＧＡ）为配基建立了一种可用于免疫毒素（ＩＴ）分离纯化的亲和层析方法。实验中用三种不同来源的核糖体灭活蛋白（ＲＩＰ）,即蓖麻毒素Ａ链（ＲＴＡ）,苦瓜毒素（ｍｏｍｏｒｄｉｎ,ＭＴ）和Ｓａｐｏｒｉｎ,以探讨ＲＩＰ与Ｆ３ＧＡ的相互作用。分析显示三种ＲＩＰ均能引起Ｆ３ＧＡ吸收光诸明显红移,提示ＲＩＰ均可与Ｆ３ＧＡ发生特异结合。将Ｆ３ＧＡ与Ｓｅｐｈａｄｅｘ交联可获得Ｂｌｕｅｄｅｘ。Ｂｌｕｅｄｅｘ亲和层析是一种经济有效,简单易行,便于在各类实验室中使用的蛋白质亲和层析技术。结果表明：在低盐溶液中ＲＴＡ和ＭＴ均可迅速地与Ｂｌｕｅｄｅｘ结合,而在高盐溶液中（０．６５ｍｏｌ／ＬＮａＣｌ）又极易被洗脱回收。这一技术用于免疫毒素的研究可有效地去除游离抗体,而不影响其杀伤活性。     

黄瓜细胞中水杨酸的信号传递研究

应用放射性标记、薄层层析和阴离子交换柱层析技术,证明黄瓜（ＣｕｃｕｍｉｓｓａｔｉｖａＬ．）细胞中存在肌醇脂质信使系统,并且水杨酸（ＳＡ）能够促进肌醇磷脂代谢,激活磷脂酶Ｃ（ＰＬＣ）,促进磷脂酰肌醇降解,导致第二信使肌醇１,４,５三磷酸（ＩＰ３）和双酰甘油（ＤＡＧ）含量增加,表明ＳＡ信号传递有可能通过肌醇脂质信使系统的介导来完成。ＳＡ的生理功能尤其在诱导植物抗病性中的作用很可能是通过肌醇脂质信使系统介导的。     

Cibacron Blue亲和层析及应用

以Ｆ３ＧＡ（Ｃｉｂａｃｒｏｎ ＢｌｕｅＦ３ＧＡ）为配基建立了一种可用于免疫毒素（ＩＴ）分离纯化的亲和层析方法,实验中用三种不同来源的核糖体灭活蛋白（ＲＩＰ）,即蓖麻毒素Ａ链（ＲＴＡ）,苦撤毒素（Ｍｏｍｏｒｄｉｎ,ＭＴ）和Ｓａｐｏｒｉｎ,以探讨ＲＩＰ与Ｆ３ＧＡ的相互作用。分析三种ＲＩＰ均能引起Ｆ３ＧＡ吸收光谱明显红移,提示ＲＩＰ均可与Ｆ３ＧＡ发生特异结合,将Ｆ３ＧＡ与Ｓｅｐｈａｄｅｘ交联可获得Ｂｌｕ     

二甲亚砜与维甲酸对人卵巢癌细胞COC1的生长抑制及转化生长因子β_1表达的影响

本实验以人卵巢癌细胞株（ＣＯＣ１）为模型,观察诱导分化剂二甲亚砜（ＤＭＳＯ）与维甲酸（ＲＡ）对该细胞生长增殖、ＤＮＡ合成和转化生长因子β１（ＴＧＦβ１）在细胞内表达的影响。结果显示：ＤＭＳＯ与ＲＡ对人卵巢细胞ＣＯＣ１生长有明显的抑制作用,生长曲线表明作用５天后其生长抑制率分别为６２．７％和４２．１％;３Ｈ－胸腺嘧啶核苷（３Ｈ－ＴｄＲ）掺入实验说明ＤＭＳＯ组与ＲＡ组的单位时间计数率（ＣＰＭ）明显低于对照组（Ｐ＜０．０１）。用药３天后百分掺入抑制率分别为６０．４％与３７．９％,表明ＤＭＳＯ与ＲＡ抑制ＣＯＣ１细胞的ＤＮＡ合成;免疫细胞化学反应表明,ＤＭＳＯ或ＲＡ处理５天后,对照组细胞ＴＧＦβ１表达为阳性,定位于胞浆,而处理组细胞ＴＧＦβ１呈阴性或弱阳性反应。以上结果提示ＤＭＳＯ和ＲＡ对人卵巢癌细胞有一定的诱导分化作用。     

中枢应用β-内啡肽对大鼠血浆唾液酸水平的影响以及与免疫功能的关系

实验选择体重２００－２５０ｇ健康Ｗｉｓｔａｒ大鼠６４只,采用麻醉大鼠中枢微量注射,分光光测定法及免疫组织化学法,研究侧脑室,弓状核（ＡＲＣ）区注射β－内啡肽（β－ＥＰ）对大鼠血浆唾液酸（ＳＡ）水平的影响及与免疫功能的关系,结果表明：（１）侧脑室注射β－ＥＰ可明显降低血浆ＳＡ水平;（２）血浆ＳＡ水平在ＡＲＣ区注射β－ＥＰ后明显降低,此效应可被Ｍ胆碱受体阻断剂阿托品或切断双侧颈迷走神经所阻断;（３）Ａ     

组胺H_1受体激动剂PEA抑制胃酸分泌效应的中枢机制

本文分析了中枢注射ＰＥＡ抑制胃酸分泌效应的脑内过程。雄性Ｗｉｓｔａｒ大鼠,摘除双侧肾上腺,用３７℃的生理盐水通过恒流泵进行连续胃液流。第三脑室给药,观察其对五肽促胃液素（１６０μｇ／ｋｇ,ｓ．ｃ．）诱导的胃酸分泌的影响。结果如下：（１）预先脑室注射抗ＣＲＦ血清（２．５μｌ,１：２００００）可阻断１ＯμｇＰＥＡ的中枢抑酸效应;（２）分别脑室给予ＣＲＦ（１．０和２．０μｇ）和β－内啡肽（０．９４和１．２５μｇ）均明显抑制胃酸分泌;（３）预先注射纳洛酮（５．０μｇ,ｉ,ｃ．ｖ,）可取消ＣＲＦ（１．０μｇ）的中枢抑酸效应,而预先注射抗ＣＲＦ血清（２．５μｌ）对β－内啡肽（０．９４μｇ）的中枢抑酸效应无明显影响。结果提示：ＰＥＡ可能首先引起ＣＲＦ释放,后者再刺激内源性阿片肽释放,从而导致迷走介导的胃酸分泌抑制效应。     

磷脂酶C-γ2与信号转导

细胞间信息传递及细胞外信号对靶细胞的调控,多年来一直是生物学和医学界研究的焦点。随着Ｃａ２＋和二酰基甘油（ｄｉａｃｙｌｇｌｙ－ｃｅｒｏｌ,ＤＡＧ）第二信使地位的确定,对磷脂酶Ｃ（ＰＬＣ）的研究引起了人们的关注。ＰＬＣ可水解磷脂酰肌醇４,５－二磷酸（ｐｈｏｓ－ｐｈａｔｉｄｙｌｉｎｏｓｉｔｏｌ４,５－ｂｉｓｐｈｏｓｐｈａｔｅ,ＰＩＰ２）产生肌醇１,４,５－三磷酸（ｉｎｏｓｉｔｏｌ１,４,５－ｔｒｉｐｈｏｓｐｈａｔｅ,ＩＰ３）和ＤＡＧ。ＩＰ３可导致细胞内储存Ｃａ２＋的释放。因此,ＰＬＣ处于Ｃａ２＋和Ｄ…     

钙调素对花粉萌发和花粉管生长的效应

牛脑和玉米胚ＣａＭ能显著促进花粉萌发和花粉管生长（图１）,而ＣａＭ抑制剂ＴＦＰ、ＣＰＺ及另外两个专一性更强的抑制剂Ｃｏｍｐｏｕｎｄ４８／８０和Ｗ７均严重抑制甚至阻止花粉的萌发（图２,３）。用对ＣａＭ亲和性较低的Ｗ７同系物Ｗ５,在与Ｗ７同样浓度下,对花粉萌发和花粉管生长无明显影响。此外,Ｗ７对花粉萌发和花粉管生长的抑制效应可被外源ＣａＭ所消除（图４）。在花粉萌发过程中,其内源ＣａＭ含量显著上升,在花粉萌发率接近最大值时,花粉ＣａＭ含量达最高水平（图５）。上述结果表明ＣａＭ对花粉萌发和花粉管生长的调控起重要作用。     

药物诱导后的人大肠癌细胞的[Ca^2＋]i的变化

采用荧光分光光度计法检测维甲酸（ＲＡ）、１,２５（ＯＨ）２ＶＤ３及佛波酯（ＰＭＡ）诱导ＣＣＬ２２９细胞分化后［Ｃａ２＋］ｉ变化,并观察内质网（ＥＲ）特异的Ｃａ２＋－ＡＴＰａｓｅ抑制剂Ｔｈａｐｓｉｇａｒｇｉｎ（ＴＧ）、ＩＰ３受体抑制剂Ｈｅｐａｒｉｎ对ＲＡ诱导［Ｃａ２＋］ｉ变化的影响,从而探讨ＲＡ诱导［Ｃａ２＋］ｉ变化与ＥＲ的关系。结果显示：ＲＡ和１,２５（ＯＨ）２ＶＤ３在数秒内引起［Ｃａ２＋］ｉ显著升高。在ＥＧＴＡ和Ｖｅｒａｐａｍｉｌ预处理细胞条件下,ＴＧ不能抑制ＲＡ引起Ｃａ２＋从细胞内钙池中外流,ＲＡ作用后ＴＧ仍能升高［Ｃａ２＋］ｉ。另外,Ｈｅｐａｒｉｎ也不能完全抑制ＲＡ升高［Ｃａ２＋］ｉ。提示ＲＡ诱导大肠癌细胞升高［Ｃａ２＋］ｉ可能通过ＥＲ上ＩＰ３敏感性和非敏感性钙池,亦可能细胞内存在除ＥＲ外对ＲＡ敏感的钙池。     

胞壁钙在红光抑制绿豆下胚轴伸长中的作用

研究了胞壁钙在红光抑制黄化绿豆（ＰｈａｓｅｏｌｕｓｒａｄｉａｔｕｓＬ．）下胚轴切段伸长生长中的作用。培养在有Ｃａ２＋介质中的切段胞壁钙含量比无Ｃａ２＋介质中的高３倍多,但不论介质中有无外源Ｃａ２＋,红光对下胚轴伸长的抑制程度都为２０％～２５％。乙醇双乙胺醚Ｎ,Ｎ,Ｎ′,Ｎ′四乙酸（ＥＧＴＡ）减少胞壁钙含量,相应地抵消红光对伸长的抑制;ｖｅｒａｐａｍｉｌ、Ｌａ３＋处理的切段胞壁钙含量与黑暗对照接近,但削弱红光的抑制作用;Ａ２３１８７减少胞壁钙,相应地抵消红光作用,甚至促进伸长生长。此外,氯丙嗪不影响胞壁钙含量,却阻止红光抑制伸长。表明红光无需外源Ｃａ２＋也能抑制切段伸长生长,但并非完全不需要Ｃａ２＋,可能胞壁自身的Ｃａ２＋基本能满足伸长生长所需。胞壁Ｃａ２＋的作用很复杂,它既可作为钙库起内流Ｃａ２＋信号的作用,也可在壁区起生长调节作用。     

Mammalian blood-mediated mutagenicity tests using a multipurpose strain of Escherichia coli K-12

Cytogenetic studies have been performed to determine the effect of 6-mercaptopurine (6-MP) on mammalian chromosomes in vivo. An elevated level of chromosome breakage is present in mouse bone marrow cells within 12 h after oral or parenteral dosing with 200 mg/kg. Damage is most severe at 24 h after oral dosing and at 72 h following intraperitoneal injection. A dose-response curve was constructed using oral doses of 10 to 200 mg/kg. The no-effect dose level lies within the 10–25 mg/kg range when administered as a single dose. In subacute studies, positive results have been obtained with as little as 2.5 mg/kg/day administered orally for 5 days.     

Mapping the interaction of bradykinin 1-5 with the exodomain of human protease activated receptor 4

The angiotensin converting enzyme breakdown product of bradykinin, bradykinin 1-5 (RPPGF), inhibits thrombin-induced human or mouse platelet aggregation. RPPGF binds to the exodomain of human protease-activated receptor 1 (PAR1). Studies determined if RPPGF also binds to the exodomain of human PAR4. RPPGF binds to a peptide of the thrombin cleavage site on PAR4. Recombinant wild-type and mutated exodomain of human PAR4 was prepared. The N-terminal arginine on RPPGF binds to the P2 position or proline46 on PAR4 to block thrombin cleavage. These data indicate that RPPGF influences thrombin activity by binding to the thrombin cleavage site on both PAR4 and PAR1.     

Time-dependent inhibition of inositol-1,4,5-trisphosphate-5-phosphatase by calmidazolium chloride in rat GH3 cells.

The calmodulin inhibitor calmidazolium chloride inhibited the activity of soluble and particulate Ins(1,4,5)P₃-5-phosphatase from GH₃ cells, with an ₅₀ value of 100 μM following a 10-min preincubation with enzyme. The inhibition was time-dependent and could not be reversed by washing of the particulate fraction. It is concluded that although the inhibitory effect of calmidazolium chloride cannot be related per se to inhibition of calmodulin function, effects of this compound unrelated to actions upon calmodulin function may be found when concentrations that are only moderately supramaximal are used.     

Analysis of the reduction of nitroxides by flavin mononucleotide

This article describes a simle method to prepare hydroxylamines from nitroxides by photo-activated flavin mononucleotide. The half-time of reduction varied from 2 to 38.4 s for a series of nitroxides. For most nitroxides short exposures to light (min) were sufficient to produce significant amounts of hydroxylamine; longer periods of exposure increased the yields of other products. Proxyl (2,2,5-trimethyl-5-alkylpyrrolidine-N-oxy) nitroxides were unsually reactive with a much higher yield of products which could not be reoxidized by ferricyanide to the nitroxides. Optimum conditions for reversible reduction depend on the nitroxide and the amounts of other reducible substances such as oxygen and ferricyanide that may be present.     

Quinone-enhanced ascorbate reduction of nitric oxide: role of quinone redox potential

The quinones 1,4-naphthoquinone (NQ), methyl-1,4-naphthoquinone (MNQ), trimethyl-1,4-benzoquinone (TMQ) and 2,3-dimethoxy-5-methyl-1,4-benzoquinone (UQ-0) enhance the rate of nitric oxide (NO) reduction by ascorbate in nitrogen-saturated phosphate buffer (pH 7.4). The observed rate constants for this reaction were determined to be 16±2,215±6,290±14 and 462±18 M^-1 s^-1, for MNQ, TMQ, NQ and UQ-0, respectively. These rate constants increase with an increase in quinone one-electron redox potential at neutral pH, E₇¹. Since NO production is enhanced under hypoxia and under certain pathological conditions, the observations obtained in this work are very relevant to such conditions.     

The Involvement of PLC-IP3 Signaling Pathway in Pollen Tube Growth

The effect of phospholipase C (PLC) signaling pathway on lily (Lilium davidii Duch.) pollen tube elongation was examined by means of microinjection. Pollen tube elongation was inhibited by microinjecting antibodies against animal PLCβ1-3 or inositol-1,4,5-triphosphate receptor （IP3R2, 3）, but was not affected by antibodies against animal PLCβ4 or IP3R1. Pollen tube elongation was also stimulated significantly by microinjecting IP3. The results suggest that PLC-IP3 signaling pathway might present in pollen system and be involved in pollen tube growth.     

A role for EHD4 in the regulation of early endosomal transport

All four of the C-terminal Eps15 homology domain (EHD) proteins have been implicated in the regulation of endocytic trafficking. However, the high level of amino acid sequence identity among these proteins has made it challenging to elucidate the precise function of individual EHD proteins. We demonstrate here with specific peptide antibodies that endogenous EHD4 localizes to Rab5-, early embryonic antigen 1 (EEA1)- and Arf6-containing endosomes and colocalizes with internalized transferrin in the cell periphery. Knock-down of EHD4 expression by both small interfering RNA and short hairpin RNA leads to the generation of enlarged early endosomal structures that contain Rab5 and EEA1 as well as internalized transferrin or major histocompatibility complex class I molecules. In addition, cargo destined for degradation, such as internalized low-density lipoprotein, also accumulates in the enlarged early endosomes in EHD4-depleted cells. Moreover, we have demonstrated that these enlarged early endosomes are enriched in levels of the activated GTP-bound Rab5. Finally, we show that endogenous EHD4 and EHD1 interact in cells, suggesting coordinated involvement in the regulation of receptor transport along the early endosome to endocytic recycling compartment axis. The results presented herein provide evidence that EHD4 is involved in the control of trafficking at the early endosome and regulates exit of cargo toward both the recycling compartment and the late endocytic pathway.     

Induction of porphyrin synthesis in chick embryo liver cell culture by synthetic polychlorobiphenyl isomers

The effects of a series of synthetic di-tetra- and hexachlorobiphenyl isomers and commercial polychlorinated biphenyls on the porphyrin biosynthesis in chick embryo liver cells in culture were examined.It was found that 3,4,3′,4′-tetra- and 3,4,5,3′,4′,5′-hexachlorobiphenyl isomers were the most active inducers, which were approximately 20 times as active as 1,4-dihydro-3,5-dicarbethoxy-2,4,6-trimethylpyridine (DDC) in porphyrin production. 3,5,3′,5′-Tetra- and 2,3,4,2′,3′,4′-hexachlorobiphenyl isomers were moderate inducers, which were approximately 2.0 to 2.5 times as active as DDC. 2,4,6,2′,4′,6′-Hexachlorobiphenyl showed the same activity as DCC. Compounds such as 4,4′-di-, 2,3,2′,3′-, 2,4,2′,4′- and 2,6,2′,6′-tetrachlorobiphenyl were weak inducers and 2,5,2′,5′-tetrachloro- and decachlorobiphenyl isomers were found to be inactive. Kanechlor-400 was the strongest inducer among the commercial polychlorinated biphenyls investigated.The structural requirements for potent porphyrin-inducing activity of chlorobiphenyl isomers were found to be the para and meta substituted structure causing a more highly conjugated and nearly coplanar conformation. It was found that induction caused by some chlorobiphenyls was subject to feed-back repression by end-product heme. In addition, the metabolism of chlorobiphenyls in mice was influenced by the unsubstituted pairs of carbon atoms in the molecule. These results lead us to postulate the following hypothesis, namely, that strong inducers may displace heme directly and incorporate into a hydrophobic pocket of the apo-represor protein, thus causing an induction of δ-aminolevulinic acid synthetase.     

Nuclear pool of phosphatidylinositol 4 phosphate 5 kinase 1α is modified by polySUMO-2 during apoptosis

Phosphatidylinositol 4 phosphate 5 kinase 1α (PIP5K) is mainly localized in the cytosol and plasma membrane. Studies have also indicated its prominent association with nuclear speckles. The exact nature of this nuclear pool of PIP5K is not clear. Using biochemical and microscopic techniques, we have demonstrated that the nuclear pool of PIP5K is modified by SUMO-1 in HEK-293 cells stably expressing PIP5K. Moreover, this SUMOylated pool of PIP5K increased during apoptosis. PolySUMO-2 chain conjugated PIP5K was detected by pull-down experiment using affinity-tagged RNF4, a polySUMO-2 binding protein, during late apoptosis.