DAB致大鼠肝癌过程中免疫组化及免疫电镜研究

本文采用二甲氨基偶氮苯(DAB)诱发的大鼠肝癌模型,运用组织病理学、血清学、免疫组化及免疫电镜技术相结合的方法,对诱癌过程中各个不同时期肝脏的组织病理学变化及肝癌阳性标志物AFP的表达进行了动态观察。结果显示:早在血清AFP浓度上升和由卵圆细胞转变而来的小肝细胞表达AFP之前,肝小叶中就出现了散在的AFP阳性肝细胞,我们认为这种AFP阳性肝细胞可作为肝细胞癌前病变的早期征象之一。在AFP阳性的肝细胞内,AFP主要定位于核周间隙、粗面内质网和高尔基复合体。     

Chemical probes for investigating protein liquid-liquid phase separation and aggregation

Protein liquid-liquid phase separation drives the dynamic assembly of membraneless organelles for fulfilling different physiological functions. Under diseased condition, protein may undergo liquid-to-solid condensation to form pathological amyloid aggregates closely associated with neurodegenerative diseases. Chemical probe serves as an important chemical tool not only for exploring the basic principle of the dynamic assembly of different protein condensates in vitro and in cell but also for clinical diagnosis and therapeutics of the related diseases. In this review, we first introduce chemical probes to image and regulate protein condensates. Then, we summarized three different categories of chemical probes including general amyloid dye, selective positron emission tomography tracer, and disaggregating binder, which feature distinct interaction pattern and activity upon binding to different pathological amyloid fibrillar aggregates. Next, we discuss the development of chemical probes for tracking protein amorphous aggregates in cells. Finally, we point out future direction in expanding the probes’ chemical space and applications.     

Mg~(2+)对线粒体H~+-ATP酶的F_O在脂质体重建时的影响

线粒体ATP合成酶是由具有H~+转运活性的F_0亚基,可溶性的催化中心F_1和连接二者的致寡霉素敏感蛋白(OSCP)所组成. 将纯化的猪心线粒体H—ATP酶复合体的F_0亚基,用胆酸盐透析法在有Mg~(2+)和无Mg~(2+)条件下在大豆磷脂脂质体上重建得脂酶体(L·F_0).用探剂9-AA荧光淬灭法和电权法测定了两种脂酶体的质子转运活力.由两种方法所得的实验结果均表明,在透返介质中加入1mmolmg~(2+)条件下形成的脂酶体(L·F_0)+Mg~(2+)较无Mg~(2+)者的质子转运活性明显增加.前者的荧光强度变化较后者增加约30%;由电极法测得的质子转运的初速度,前者为5nmolH~+′sF_0,后者为3nmolH~+′s·nmolF_O,质子转运活性高约一倍.这进一步支持Mg~(2+)通过调节脂的物理状态而诱导F_O具有较适合的构象,并进而将这一影响传递至F_1,使整个H~+—AhP酶具有较高活性的假设.     

Influence of phosphate ion on the fluorescence of 3-fluorotyrosine

Summary 3-Fluorotyrosine fluorescence is quenched effectively by phosphate ions not only by a dynamic but also by a static mechanism owing to H-bond complex formation in ground state. 3-Fluorotyrosine pK_a values both in the ground and first excited state (8.3 and 4, respectively) are appreciably lower than those of tyrosine, thus promoting 3-fluorotyrosinate ion formation in the excited state. Additional emission owing to 3-fluorotyrosinate ion (near 350 nm) may be taken erroneously for tryptophan fluorescence.     

嗜热蓝藻层理鞭枝藻（Mastigocladus laminosus）藻胆体在解离过程中荧光发射光谱和光能传递的研究

研究了层理鞭枝藻藻胆体在不同浓度磷酸缓冲溶液中解离过程中荧光发射光谱的变化和光能传递。完整藻胆体的７７Ｋ荧光光谱中只有一个峰,位于６８５ｎｍ它是末端发射体（核心－膜连接多肽和别藻蓝蛋白－Ｂ）的荧光峰。部分解离藻胆体的荧光光谱的主峰位移至６５２ｎｍ：次峰位于６８５ｎｍ;６６０ｎｍ为一弱荧光发射肩。它们依次为Ｃ－藻蓝蛋白,末端发射体和别藻蓝蛋白的荧光。严重解离藻胆体的荧光主峰移６４４ｎｍ;次峰由６８５ｎｍ移至６８２ｎｍ;６６０ｎｍ荧光发射肩消失。这表明Ｃ－藻蓝蛋白所捕获的光能已不能传递给别藻蓝蛋白,但可传递给末端发射体洞时又表明Ｃ－藻蓝蛋白不仅与别藻蓝蛋白相连接而且还与末端发射体相连接。提出该藻胆体光能传递链如下：核心－膜连接多肽藻红蓝蛋白→Ｃ－藻蓝蛋白→别藻蓝蛋白别藻蓝蛋白－Ｂ     

油麻藤凝集素的荧光光谱研究

用化学修饰、内源荧光和荧光淬灭等方法研究了油麻藤凝集素（ＭＳＬ）的溶液构象变化和微环境的构象特征。研究发现ＭＳＬ分子中总共有９个色氨酸（Ｔｒｐ）残基,它们的荧光能被丙烯酰胺淬灭,但不易为ＫＩ接近而淬灭,ＭＳＬ经Ｎ－溴代琥珀酰亚胺（ＮＢＳ）修饰后,其内源性荧光发射谱发生相应变化,结果表明ＭＳＬ分子中部分Ｔｒｐ残基埋藏于分子内部,而位于分子表面的Ｔｒｐ残基可能处于分子的疏水袋中。     

Cyclic AMP induces rapid increases in gap junction permeability and changes in the cellular distribution of connexin43

The rapid effects of cAMP on gap junction-mediated intercellular communication were examined in several cell types which express different levels of the gap junction protein, connexin43 (Cx43), including immortalized rat hepatocyte and granulosa cells, bovine coronary venular endothelial cells, primary rat myometrial and equine uterine epithelial cells. Functional analysis of changes in junctional communication induced by 8-bromo-cAMP was monitored by a fluorescence recovery after photobleaching assay in subconfluent cultures in the presence or absence of 1.0 mm 1-octanol (an agent which uncouples cells by closing gap junction channels). Communicating cells treated with 1.0 mm 8-bromo-cAMP alone exhibited significant increases in the percent of fluorescence recovery which were detected within 1–3 min depending on cell type, and junctional communication remained significantly elevated for up to 24 hr. Addition of 1.0 mm 8-bromo-cAMP to cultured cells, which were uncoupled with 1.0 mm octanol for 1 min, exhibited partial restoration of gap junctional permeability beginning within 3–5 min. Identical treatments were performed on cultures that were subsequently processed for indirect immunofluorescence to monitor Cx43 distribution. The changes in junctional permeability of cells correlated with changes in the distribution of immunoreactive Cx43. Cells treated for 2 hr with 10 m monensin exhibited a reduced communication rate which was accompanied by increased vesicular cytoplasmic Cx43 staining and reduced punctate surface staining of junctional plaques. Addition of 1.0 mm 8-bromo-cAMP to these cultures had no effect on the rate of communication or the distribution of Cx43 compared to cultures treated with monensin alone. These data suggest that an effect of cyclic AMP on Cx43 gap junctions is to promote increases in gap junctional permeability by increasing trafficking and/or assembly of Cx43 to plasma membrane gap junctional plaques.We acknowledge the technical assistance of Richard Lewis and Meghan Abella. We thank Dr. Hugh Dookwah for contributions to the myometrial cell isolation protocol and Drs. Stephen H. Safe, Timothy D. Phillips, and Evelyn Tiffany-Castiglioni for helpful discussions. This work was funded by NIH (HD-26182, P42-ES04917, ES05871-01A1), the March of Dimes Birth Defects Foundation Basic Research grant #1-0796, and USDA 92-37203-7952.     

The sodium concentration of the lateral intercellular spaces of MDCK cells: A microspectrofluorimetric study

MDCK cell monolayers grown on glass coverslips were used to examine the Na⁺ concentration in individual lateral intercellular spaces (LIS) by video fluorescence microscopy. The LIS was filled with the Na⁺-sensitive fluorescent dye SBFO by incubation of the monolayers for 75–90 min with 250 m of the membrane impermeant form of the dye. After dye loading, the monolayers were perfused at 37°C with solutions buffered with HEPES or bicarbonate/CO₂ containing 142 mm Na⁺. Ratios of the fluorescence images after sequential excitation with 340 nm and 380 nm light were performed and in situ calibration of LIS Na⁺ was accomplished after blocking the Na⁺ pump with 5 × 10^–4 m ouabain. Measurements of Na⁺ along the basolateral-to-apical axis of the LIS at 1.0 or 1.5 m intervals did not reveal a Na⁺ gradient when the perfusate was either HEPES or bicarbonate/CO₂ solutions. In bicarbonate solutions, the mean Na⁺ concentration (mm) was 157.2 ± 2.3, 15 mm higher than the bath Na⁺ concentration. In HEPES solutions, however, the Na⁺ concentration was not different from the bath concentration (142.7 ± 3.1 mm). The time course of Na⁺ changes in LIS was investigated by rapidly switching the perfusate from 142 to 80 mm Na⁺ and measuring the Na⁺ changes at one focal plane.We would like to thank P.H. Tran and C. Gibson for their technical and computational assistance as well as Dr. B.-E. Persson (University of Uppsala, Sweden) for his contribution in the early phases of the study.     

Transport and dynamics of molecules dissolved in maize root cortex membranes

Translational diffusion of a fluorescent sterol probe was measured in the plasma membranes of protoplasts isolated from cortical cells of the primary root of maize seedlings. The apparent lateral diffusion coefficient was typically observed to be nearly insensitive to temperature, while the mobile fraction increased with increasing temperature. These fluorescence photobleaching recovery (FPR) measurements were compared with the electron paramagnetic resonance (EPR) spectra of the methyl ester of 13-doxyl palmitic acid in membranes of corn root tissue in situ. The complex spectra observed with this probe were analyzed as weighted sums of simpler spectra of various order parameters and rotational correlation times. The reconstituted spectra calculated from the model show that EPR also detects a mobile (less ordered, fluid) fraction, distinguished by the order parameter S=0.1 to 0.2, which becomes more abundant as temperature increases and is qualitatively comparable to the mobile fraction determined by the FPR method. The observed results on the mobile fractions and the diffusion rates for translational (FPR) as well as rotational (EPR) motions are interpreted in terms of membrane organization, thus providing information on the population and structural patterns of the coexisting domains with a special emphasis on the response of the membrane to temperature changes.This work was supported in part by grants from the Ministry of Science and Technology of the Republic of Slovenia and the International Research Program of the U.S. Department of Agriculture (USDA-JF 814-51) to M.S., and by grants from the Competitive Grants Program of the U.S. Department of Agriculture (88-37264-3807 and 90-37264-5471) to E.A.N.