Comparison of retinal pigment epithelium cell preparations from the bovine eye

Retinal pigment epithelium (RPE) cells were collected from bovine eyes using a new method. The cells were harvested by vortexing the RPE and underlying choroid in 0.05 M citrate phosphate buffer, pH 5. RPE cells recovered by this method were compared to a standard method by microscopic examination of cell integrity, estimation of total protein, and assay of 11-cis and all-trans retinyl ester hydrolase (REH) activities. Results suggest that this method collects RPE cells of good integrity and with a significantly higher protein yield than the conventional method. Additionally, a much higher retinyl ester hydrolase activity was noted. Therefore we propose that this procedure offers a new and convenient method in the collection of RPE proteins for certain purposes such as enzyme purification.     

Comparison of retinal pigment epithelium cell preparations from the bovine eye

Summary Retinal pigment epithelium (RPE) cells were collected from, bovine eyes using a new method. The cells were harvested by vortexing the RPE and underlying choroid in 0.05 M citrate phosphate buffer, pH 5. RPE cells recovered by this method were compared to a standard method by microscopic examination of cell integrity, estimation of total protein, and assay of 11-cis and all-trans retinyl ester hydrolase (REH) activities. Results suggest that this method collects RPE cells of good integrity and with a significantly higher protein yield than the conventional method. Additionally, a much higher retinyl ester hydrolase activity was noted. Therefore we propose that this procedure offers a new and convenient method in the collection of RPE proteins for certain purposes such as enzyme purification.     

小型猪骨髓间充质干细胞经心导管冠状动脉移植及其迁移和分化的研究

目的建立小型猪心导管介入冠状动脉治疗模型,为骨髓间充质于细胞（MSCs）移植治疗心血管疾病提供新的方法,观察骨髓间充质干细胞经心导管介入冠状动脉移植后在心肌内的迁移及分化。方法选用冠状动脉解剖生理特点与人类相似的小型猪,应用心导管介入技术将体外培养扩增的小型猪自体MSCs移植进入左冠状动脉前降支,用免疫荧光检测即刻移植和移植后6W移植细胞的肌钙蛋白T（cTnT）、缝隙连接蛋白43（Cx43）、anti－Ⅷfactor的表达。结果成功的完成了小型猪心导管介入冠状动脉进行自体骨髓间充质干细胞移植。移植细胞存活,向心肌组织迁移,并向心肌细胞和毛细血管方向分化。结论该方法稳定,技术先进。可进行准确的定位和动态观测,为临床应用提供了一个可靠的技术平台。MSCs移植后在心肌内发生迁移及分化。     

Resting potential of excitable neuroblastoma cells in weak magnetic fields

The mechanism by which static and low-frequency magnetic fields are transduced into biological signals responsible for reported effects on brain electrical activity is not yet ascertained. To test the hypothesis that fields can cause a subthreshold change in the resting membrane potential of excitable cells, we measured changes in transmembrane current under voltage clamp produced in SH-SY5Y neuroblastoma cells, using the patch-clamp method in the whole-cell configuration. In separate experiments, cells were exposed to static fields of 1, 5, and 75 G, to time-varying fields of 1 and 5 G, and to combined static and time-varying fields tuned for resonance of Na+, K+, Ca2+, or H+. To increase sensitivity, measurements were made on cells connected by gap junctions. For each cell, the effect of the field was evaluated on the basis of 100 trials consisting of a 5-s exposure immediately followed by a 5-s control period. In each experiment, the field had no discernible effect on the transmembrane current in the vicinity of zero current (- 50 mV voltage clamp). The sensitivity of the measuring system was such that we would have detected a current corresponding to a change in membrane potential as small as 38 microV. Consequently, if sensitivity of mammalian cells to magnetic fields is mediated by subthreshold changes in membrane potential, as in sensory transduction of sound, light, and other stimuli, then the ion channels responsible for the putative changes are probably present only in specialized sensory neurons or neuroepithelial cells. A change in transmembrane potential in response to magnetic fields is not a general property of excitable cells in culture.     

Production of transgenic birds

The avian embryo presents a tremendous challenge for those interested in accessing and manipulating the avian germ line. By far the most successful method of gene transfer is by retrovirus vector. The efficacy of retrovirus vectors has been demonstrated by germ line insertion of replication-competent retroviruses as well as the insertion of replication-defective retrovirus vectors carrying bacterial marker genes. Retroviral vectors have also been shown to be useful for the transfer and expression of genes in somatic cells. Further, germ line transgenesis has been reported in both the chicken and the Japanese quail. In addition, several alternative gene transfer methods are under development. These include transfection of avian sperm, development of germ line chimeras using primordial germ cells and blastodermal cells, and the development of embryonic stem cell lines. Potentially, basic research and the poultry industry will derive substantial benefit from this revolutionary technology.     

Production of transgenic birds

The avian embryo presents a tremendous challenge for those interested in accessing and manipulating the avian germ line. By far the most successful method of gene transfer is by retrovirus vector. The efficacy of retrovirus vectors has been demonstrated by germ line insertion of replication-competent retroviruses as well as the insertion of replication-defective retrovirus vectors carrying bacterial marker genes. Retroviral vectors have also been shown to be useful for the transfer and expression of genes in somatic cells. Further, germ line transgenesis has been reported in both the chicken and the Japanese quail. In addition, several alternative gene transfer methods are under development. These include transfection of avian sperm, development of germ line chimeras using primordial germ cells and blastodermal cells, and the development of embryonic stem cell lines. Potentially, basic research and the poultry industry will derive substantial benefit from this revolutionary technology.     

Adenyl cyclase activity of the cell membrane throughout the cell cycle: ultrastructural study of the cell line BHK 21/C 13]

Adenylate cyclase activity in the surface membrane of baby Hamster kidney (BHK 21/C13) cells is ultrastructurally studied as a function of the cell cycle. Cells are synchronized by a physical method suited to structural and functional investigations. The activity is highest through G1, decreases in S, markedly drops as cells traverse G2 and is very low or often seems not present at all in M.     

Simple method for a medium-cell separation.

The present method makes possible a quick separation of cells from the medium with the aid of strips of filter paper and physiological solution. Cell suspension is put on the strip and all water soluble components are washed away by soaking in saline solution, while cells remain on the spot. The experiment on 14-C-uridine uptake proved the suitability of the method for membrane-transport studies.     

A new rapid method for collection and preparation of cell suspensions for electrom microscopy

Summary We describe a new rapid and simple method for collection and preparation of cell suspensions for electron microscopy; the cells are prefixed with glutaraldehyde in their culture medium, and are then compacted on a filter disc. Post-fixation in osmium, staining and dehydration are performed by transferring the filter disc and the cell pellet from one solution to the next. The pellet is easily separated from the filter disc just before treatment in propylene oxide. This method preserves the fine structure as well as the classical technique. Advantages are that numerous cells have the same orientation in the sections and that many samples can be taken in a very short time.Acknowledgments. The authors are indebted to Prof. H. Leclerc, Director of I.N.S.E.R.M. laboratory U 146 in which part of this work has been performed, and to Dr J.F. Dubremetz, Dr G. Prensier and Dr P.A. Trinel for their advices.     

Immigration of lymphoid precursor cells into the thymic rudiment inXenopus

Summary The origin of thymic lymphocytes was investigated, using a new reliable method to mark cells inXenopus. It was easily observed that extraneous cells immigrated into the thymic rudiment 4 days after fertilization and differentiated into a cell population identified as thymic lymphocytes in a fully developed thymus. Clearly, lymphoid precursor cells are of extrinsic origin.This work was supported in part by a grant from the Ministry of Education, Science and culture of Japan (No. 59770026).     

Anti-myosin stains chromaffin cells.

The presence in fixed chromaffin cells of antigenic sites for a myosin antibody was demonstrated using immunofluorescence techniques. Tests on viable cells showed that at least some of the antigenic sites seem to be localized on or close to the cell surface and explained the cell agglutination that occurred with the addition of the myosin antibody to cells isolated by a method described in this paper.     

Anti-myosin stains chromaffin cells

Summary The presence in fixed chromaffin cells of antigenic sites for a myosin antibody was demonstrated using immunofluorescence techniques. Tests on viable cells showed that at least some of the antigenic sites seem to be localized on or close to the cell surface and explained the cell agglutination that occurred with the addition of the myosin antibody to cells isolated by a method described in this paper.Acknowledgments. This work was supported by grants from the Medical Research Council of Canada. We thank Dr B. G. Livett for advice in the preparation of isolated cells, Drs J. Lowenthal and B. Collier for comments and Mrs J. Ritchie for technical assistance.     

Colony hybridization method for screening in situ of eukaryotic amplified genes

A new method for autoradiographic screening of amplified genes in cellular clones is described. The main feature of the device is to keep viable cells from each clone, which can subsequently be regrown. The availability of this biochemical screening method allows screening for recombinants harboring unselectable markers as well.     

Colony hybridization method for screening in situ of eukaryotic amplified genes

Summary A new method for autoradiographic screening of amplified genes in cellular clones is described. The main feature of the device is to keep viable cells from each clone, which can subsequently be regrown. The availability of this biochemical screening method allows screening for recombinants harboring unselectable markers as well.     

Isolation of type II epithelial cells from rabbit fetal lungs by adherence on an IgG-coated surface

The lung is comprised of about 40 different cell types, of which only 15% are type II cells. These are the major, if not the sole, source of synthesis and secretion of lung surfactant. To date a large number of methods have been described for the isolation of pure populations of type II cells using a wide variety of techniques, but most of these have employed differential centrifugation methods and have used adult rodents. The present study reports the isolation of type II cells from fetal rabbit lungs by the immunoglobin G plating method. Pure populations of fetal type II cells in high yield and with good viability were obtained by the procedure for the first time from rabbit fetal tissue.     

用于细胞培养的聚乙烯醇水凝胶力学性能模拟及定量控制

采用化学交联法制备聚乙烯醇（PVA）水凝胶,以此作为细胞体外培养基底。通过改进的拉伸法测量材料的杨氏模量（E）,并讨论交联剂与羟基单体的摩尔比（d）、PVA的初始浓度（C0）对E的影响。利用回归分析建立E与C0、d关系的数学模型,实现对E的定量控制,模拟细胞在体内生长的生物物理环境。结果表明：该数学模型模拟的相对误差不超过0．08,可实现对PVA力学性能的定量控制。此外,研究了基底硬度对L02细胞铺展及形态学特征的影响,细胞更倾向于在硬基底上铺展,在软的基底上,细胞的形态更趋近于圆形。     

Differenzierungsmethode metachromatischer Zellen nach ihrem Säuregrad

Summary A method is described for histochemical definition of the metachromatic substance of mast cells. A slide of tissue containing mast cells is treated with substances dissolving metachromasia, such as hyaluronidase and water, and later on stained in aqueous solutions of gradually heightened pH. A marked spot of the slide is counted at each pH. Concentrations of hydrogen and numbers of mast cells result in a characteristic curve.     

姜黄素逆转人结肠癌细胞HCT-8／VCR的双向凝胶电泳技术的建立及优化

目的建立并优化姜黄素逆转人结肠癌细胞HCT-8／VCR蛋白质纽的双向电泳技术。方法姜黄素作用于人结肠癌细胞HCT-8／VCR,提取蛋白质。对双向凝胶电泳中蛋白质样品的处理,蛋白上样量,IPG胶条的选择,以及聚焦条件等进行调整和每化。结果采用瓶内刮取的方法裂解细胞,pH3-10NL的胶条,上样量200ug,延长除盐时间,聚焦60000伏小时,可以得到分辨率较高、重复性较好的双向电泳图谱。结论初步建立了分辨率较高且重复性较好的姜黄素逆转人结肠癌细胞HCT-8／VCR蛋白质双向凝胶电泳图谱。     

Methods for determining ploidy in amphibians: Nucleolar number and erythrocyte size

Summary Diploid and triploidXenopus can be easily and reliably distinguished by the size of their erythrocytes. This method has several advantages over other methods, such as counting metaphase chromosomes and counting nucleoli. One problem with the latter method is the reduction in cells with a full complement of nucleoli when regenerating tissue is used.Research supported by NIH grant EY 01662.