鲫鱼视网膜亮度型水平细胞上不同视锥信号的相互作用:实验及模型

本文应用胞内记录和动态模型分析方法,研究了离体鲫鱼视网膜视锥驱动的高度型水平细胞（ＬＨＣ）上不同视锥信号的相互作用。实验表明,绿背景光的作用可以提高ＬＨＣ的红光反应,这种增强作用与绿敏锥的活动程度密切相关,模型分析表明,背景光 的作用谷氨酸介导的前馈性通路和ＧＡＢＡ介导的反馈性通路活动同时得以增强,水平细胞对光反应的增强效应不能为外泊性ＧＡＢＡ所消除。则其程度为前馈性通路和反馈性通路活动增加的相对     

大鼠大脑微血管片段的分离纯化及鉴定

目的:分离大鼠大脑微血管并纯化去除完整的神经细胞,用于克隆血脑屏障上的特异表达基因.方法:采用液相合成法制备粒径200～500 nm的铁氧体磁珠,经两侧颈内动脉插管注入大鼠大脑半球.采用机械分离和酶消化相结合的方法解离脑组织,用筛网滤去组织块和大血管,再在磁场下分选标记磁珠的微血管片段,并从形态学、分子生物学和生物活性角度鉴定获得的脑微血管片段.结果:扫描电镜下没有发现微血管周围存在完整的神经细胞,但在部分区域有胶质的终足包裹.全脑组织微管相关蛋白2a、谷氨酰胺合成酶和CD31的RT-PCR产物均在相应位置出现阳性条带,分离纯化的脑微血管仅CD31阳性.微血管片段内皮细胞摄取的Rh123荧光强度显著低于传代培养的微血管内皮细胞荧光强度.结论:采用本方法可以获得高纯度的、不附带完整神经细胞的脑微血管片段.     

Improved specimen preparation for cryo-electron microscopy using a symmetric carbon sandwich technique

Image shift due to beam-induced specimen charging has become the most severe problem in electron microscopy for imaging two-dimensional (2D) crystals of biological macromolecules, especially in the case of highly tilted specimens. Image shift causes diffraction spots perpendicular to the tilt axis to disappear even at medium or low resolution. The yield of good images from tilted specimens prepared on a single layer of continuous carbon support film is therefore very low. In this paper, we have used 2D crystals of aquaporin-4 to investigate the effect of a carbon sandwich preparation method on specimen charging. We find that a larger number of images show sharp diffraction spots perpendicular to the tilt axis if crystals are placed in between two sheets of carbon film as compared to images taken from specimens prepared by the conventional single carbon support film technique. Our results demonstrate that the reproducible carbon sandwich preparation technique overcomes the severe specimen charging problem and thus has the potential to significantly speed up structure analysis by electron crystallography.     

Optimizing chemotherapy by measuring reversal of P-glycoprotein activity in plasma membrane vesicles

The appearance of multidrug resistance (MDR) of cancer cells is a major obstacle to successful chemotherapy. Several proteins have been identified that pump chemotherapeutic drugs out of cells, thus bringing about MDR. One representative pump is the P-glycoprotein, whose function can be inhibited by blockers (also known as reversers, modulators or chemosensitizers). In clinical application, many of these blockers are often not effective because they become bound to the plasma of the patients. The extent of plasma binding of the blocker varies in different persons and we have developed a 96-well kit to assay such inter-person differences. The assay uses membrane vesicles isolated from a human lymphoblastoid cell line (CEM Col1000). Uptake of rhodamine into the vesicles was measured with different concentrations of the blockers verapamil and XR9576 in presence of human plasma. The reverser XR9576 is nearly 30 times more effective than the classical blocker verapamil, the relevant K(m) values ranging from 2.66 to 45 nM for XR 9576 and 0.7 to 5.5 microM for verapamil. An even greater difference between these two drugs, nearly 1,000-fold, could be shown also in intact cells by calcein AM uptake experiments.     

Development of highly nutritive culture media

Summary A highly nutritive culture medium (MGM-464) was developed for insect cell primary culture. The new medium consists of 6 inorganic salts, 4 organic acids, 21 amino acids, 3 sugars, 10 vitamins, and 8 other chemicals, including natural substances. The complete medium was generated by adding 20 ml fetal bovine serum to 100 ml MGM-464. The detail of the composition of the medium is given in a table, and the protocol to prepare the medium is described in the text. Among the 15 kinds of cultures made with MGM-464, embryonic cells from a walking stick and ovarian cells from the common white were subcultured more than 70 times, and embryonic cells of a chrysomelid beetle were subcultured more than 15 times. Other cultures could not be subcultured. However, embryonic cells from the commercial silkworm and a cockroach, ovarial cells from the commercial silkworm and a sphingid moth, nervous cells from the commercial silkworm and two sphingid moths, and cells from the dorsal vessel plus surrounding tissue of the commercial silkworm survived for several mo. The cells from the honeybee embryos, aphid embryos, and planthopper embryos were rather short-lived, and deteriorated after about 1 mo.     

Cytopathological evaluations combined RNA and protein analyses on defined cell regions using single frozen tissue block

The co-existence of multiple cell components in tissue samples is the main obstacle for precise molecular evaluation on defined cell types. Based on morphological examination, we developed an efficient approach for paralleled RNA and protein isolations from an identical histological region in frozen tissue section. The RNA and protein samples prepared were sufficient for RT-PCR and Western blot analyses, and the results obtained were well coincident each other as well as with the corresponding parameters revealed from immunohistochemical examinations. By this way, the sampling problem caused by cell-cross contamination can be largely avoided, committing the experimental data more specific to a denned cell type. These novel methods thus allow us to use single tissue block for a comprehensive study by integration of conventional cytological evaluations with nucleic acid and protein analyses.     

A simple non-enzymatic method for the isolation of high yields of functional rat hepatocytes

A method for isolation of rat hepatocytes using liver perfusion by ethylenediamine tetra-acetic acid (EDTA)-containing sucrose solution and mechanical tissue disaggregation by controlled vibration (MVD) is described. The yields of hepatocytes produced by this method were similar to those obtained using collagenase perfusion. The cells had well preserved membrane integrity as judged by the trypan blue staining test (91 +/- 4%), ATP contents, rates of endogenous respiration and enzyme leakage that indicated they were functional cells. There was little evidence of expression of latent damage when the cells were stored either at 37 degrees C (by pre-incubation) or at 4 degrees C. This method can be used to isolate high yields of functional cells from rat liver if the collagenase perfusion technique is not available.     

Influence of inoculum preparation on the growth of Penicillium chrysogenum

AIMS: The influence of the spore preparation on subsequent fungal growth of Penicillium chrysogenum was assessed. METHODS AND RESULTS: The influence of four factors [the nature of the diluting solution (physiological water and physiological water added with Tween-80), the age of the sporulating culture (4, 8 and 12 days), the strain (737, 738 and 740) and the inoculum size (102, 103, 104 and 105 spores ml(-1)] on two responses (i.e. the radial growth rate, mu, and the lag time, lambda) was studied using an experimental screening methodology. CONCLUSIONS: The main conclusion was the strong effect of the inoculum size on lambda. In contrast, the diluting solution had no effect on both the experimental responses. In order to obtain the highest growth rates, it is recommended to use 4-day-old sporulating cultures with an inoculum size of 102 spores ml(-1). SIGNIFICANCE AND IMPACT OF THE STUDY: There is a need for standardizing spore preparation in predictive mycology. The screening methodology is a powerful tool to determine the influence of qualitative and quantitative factors on various biological responses and can be applied widely in microbiology.     

Localization of sucrose synthase and callose in freeze-substituted secondary-wall-stage cotton fibers

Summary.  Methods for cryogenic fixation, freeze substitution, and embedding were developed to preserve the cellular structure and protein localization of secondary-wall-stage cotton (Gossypium hirsutum L.) fibers accurately for the first time. Perturbation by specimen handling was minimized by freezing fibers still attached to a seed fragment within 2 min after removal of seeds from a boll still attached to the plant. These methods revealed native ultrastructure, including numerous active Golgi bodies, multivesicular bodies, and proplastids. Immunolocalization in the context of accurate structure was accomplished after freeze substitution in acetone only. Quantitation of immunolabeling identified sucrose synthase both near the cortical microtubules and plasma membrane and in a proximal exoplasmic zone about 0.2 μm thick. Immunolabeling also showed that callose (β-1,3-glucan) was codistributed with sucrose synthase within this exoplasmic zone. Similar results were obtained from cultured cotton fibers. The distribution of sucrose synthase is consistent with its having a dual role in cellulose and callose synthesis in secondary-wall-stage cotton fibers. Received August 19, 2002; accepted November 12, 2002; published online June 13, 2003 RID="*" ID="*" Correspondence and reprints: Department of Biological Sciences, Texas Tech University, Lubbock, TX 79409-3131, U.S.A. E-mail: candace.haigler@ttu.edu     

Intrinsic Regulation of Brain Inflammatory Responses

It is now well accepted that inflammatory responses in brain contribute to the genesis and evolution of damage in neurological diseases, trauma, and infection. Inflammatory mediators including cytokines, cell adhesion molecules, and reactive oxygen species including NO are detected in human brain and its animal models, and interventions that reduce levels or expression of these agents provide therapeutic benefit in many cases. Although in some cases, the causes of central inflammatory responses are clear—for example those due to viral infection in AIDS dementia, or those due to the secretion of proinflammatory substances by activated lymphocytes in multiple sclerosis—in other conditions the factors that allow the initiation of brain inflammation are not well understood; nor is it well known why brain inflammatory activation is not as well restricted as it is in the periphery. The concept is emerging that perturbation of endogenous regulatory mechanisms could be an important factor for initiation, maintenance, and lack of resolution of brain inflammation. Conversely, activation of intrinsic regulatory neuronal pathways could provide protection in neuroinflammatory conditions. This concept is the extension of the principle of central neurogenic neuroprotection formulated by Donald Reis and colleagues, which contends the existence of neuronal circuits that protect the brain against the damage initiated by excitotoxic injury. In this paper we will review work initiated in the Reis laboratory establishing that activation of endogenous neural circuits can exert anti-inflammatory actions in brain, present data suggesting that these effects could be mediated by noradrenaline, and summarize recent studies suggesting that loss of noradrenergic locus ceruleus neurons contributes to inflammatory activation in Alzheimer's disease.