酶消化结合组织块培养法用于颞下颌关节盘组织工程细胞扩增的初步研究

目的采用酶消化结合组织块培养法对山羊颞下颌关节（temporomandibular joint，TMJ）盘细胞进行体外培养和扩增，探索TMJ关节盘细胞体外培养及扩增的新方法。方法在无菌条件下，切取一月龄山羊TMJ关节盘，剪至1．0mm^3的碎块，用0．25％胰酶、0．01％I型胶原酶消化关节盘组织块，将消化好的组织块置入6孔板中培养。在倒置显微镜下连续观察细胞的形态变化及贴壁率，甲苯胺蓝染色、I型胶原免疫组化染色进行细胞鉴定，测定其生长曲线。结果原代培养的关节盘纤维软骨细胞4天可观察到贴壁细胞，7天贴壁细胞逐渐增多，第10天时细胞彼此相连，铺满平底，细胞以梭形为主，部分多角形。传代后12小时贴壁率达90％，大部分为多角形，4～5天即可长满瓶底。甲苯胺蓝染色可见异染颗粒，胶原免疫纽化染色胞浆内可见棕黄色颗粒。结论酶消化结合组织块培养法培养的山羊TMJ关节盘细胞具有较强的增殖能力，可作为TMJ关节盘组织工程中获取大量原代细胞的实用方法。     

An improved electrophoretic method for a screening program for haemoglobinopathies

Summary A method for a screening program for haemoglobinopathies in a starch agar gel mixed with saponin is presented. Normal and abnormal blood containing haemoglobins S, C, I, M Boston, D Punjab, beta thalassaemia major and beta thalassaemia minor, were applied, in a tray with the capacity for 100 samples. The electrophoresis was performed in 45 min using 300 V. This method offers special advantages for the examination of a large number of samples, using a small amount of whole blood and without the previous preparation of haemoglobin solution.Acknowledgments. We would like to express our gratitude to Dr C. Daghlian and Dr Lewis J. Greene for their valuable suggestions. We also thank the Conselho Nacional do Desenvolvimento Científico e Tecnológico, CNPq, for financial support.     

A study of irrigation fluids for neurosurgery on brain primary cell cultures

Summary Primary cell cultures from newborn rat brain hemispheres were exposed to different irrigation fluids used in neurosurgery. The cells died after incubation for 5 min with hydrogen peroxide, and the number of cells was drastically decreased after 10 sec of incubation. They shrank after incubation in Elliott's artificial cerebrospinal fluid for 3 h, but the viability as determined by trypan blue exclusion test was not affected. Physiological sodium chloride, Ringer's solution and the culture medium 199 with Hank's salt had no noticeable effect on the viability or morphology of the cells.Acknowledgments. This study was supported by grants from the Medical Faculty of Göteborg and from Statens Naturvetenskapliga Forskningsråd. We want to thank Dr Åke Sellström and Dr Hans-Arne Hansson for valuable discussions.     

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.     

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.     

Establishment of normal diploid and malignant heteroploid cell lines from non-treated and benzo(a)pyrene treated hamster embryo cell cultures

Summary Two normal diploid control cell lines and a heteroploid malignant transformed cell line from B(a) P treated hamster embryo cell cultures were established. The 14-month-old B(a)P transformed cell line grew 8-times faster than the 20-month-old control cell line. The control cell line showed normal diploid chromosome complement in 93% cells and heteroploidy in 7% cells while B(a)P treated line showed 83% heteroploid cells and only 17% diploid cells. This is the first report on the establishment of diploid hamster cell cultures grown for extended period. Acknowledgments. This work was supported by the French Ministry of Quality of Life, by the Institut National de la Santé et Recherche Médicale, by the S.E.I.T.A. and by Contract No. 12-14-7001-297 of the Agricultural Research Service, U.S. Dept. of Agriculture, Administered by the Athens, Georgia Area, Richard B. Russell Agricultural Research Center, Athens, Georgia 30604, USA. We thank Mr.B. R. Sharma, Mr.E. J. Radin, Dr.R. Hakim and Dr.Ved Brat for their help during this work.     

Different marrow cell number requirements for the haemopoietic colony formation and the cure of the W/Wv anemia

Summary The lowest cell number in the normal marrow transplant, which allows the cure of W/W^v anemia was found to be between 10⁴ and 10⁵. This exceeds by several times the lowest cell number necessary for the haemopoietic colony formation. Therefore, either the colony forming cell is not the haemopoietic stem cell but rather its progeny, or this cell requires an aid from some other cells to exert its activity.Acknowledgments. We are indebted to Doc. Dr Maksymilian Siekierzynski for his help and advice and to Miss Elzbieta Szewczyk for expert technical assistance.     

Cortisol resistant RPMI-1788 lymphocytes become sensitive to cortisol subsequent to a 24-h incubation period in medium containing purified human transcortin

Summary RPMI-1788 lymphocytes (a human cell line) are resistant to cortisol in vitro. Prior incubation for a minimum of 24 h a medium which contains purified human transcortin at a concentration of 50 g/ml renders these cells sensitive to the inhibitory action of cortisol as regards the synthesis of DNA. Only the transcortin-exposed cells contain a cortisol binding species whose sedimentation behavior in a sucrose gradient is identical to that of transcortin.This work was supported in part by grant ET-47 from the American Cancer Society. I thank Mr D. Pina and Dr R. Khaund for their excellent technical assistance.     

Involvement of prolyl endopeptidase in ascidian fertilization

Summary Inhibitory effects on fertilization of the ascidian of three benzyloxycarbonyl(Z)-aminoacyl prolinals and Z-Gly-Pro-chloromethyl ketone added before and after insemination were examined. The results suggest that the prolyl endopeptidase is involved in the process of fertilization, especially in a process taking place between chorion elevation and cell cleavage.Acknowledgment. We are indebted to Dr T. Numakunai of the Marine Biological Station of Tohoku University for collecting the ascidians and to Dr M. Hoshi of the Tokyo Institute of Technology for his helpful discussion. We are also grateful to Dr T. Yoshimoto and Prof. D. Tsuru of Nagasaki University for their generous gift of Z-Gly-Prochloromethyl ketone. This work was supported by a Grant-in-Aid from the Ministry of Education, Science and Culture of Japan.     

Radioresistance in cells with high content of metallothionein

Summary An endogenous cytoplasmic protein, metallothionein (MT) apparently gave rise to radioresistance in 2 different cell lines. A dose reduction factor of 1.9 was achieved in MT-containing cells. MT accounted for a 3–4-fold increase of total sulfhydryl groups in the resistant cell strains, compared to the non-resistant lines from which they were derived. The protein is rich in cysteine (30%), an amino acid known to give radioprotection when administered exogeneously. Glutathione levels and cell-cycle phase distribution showed no marked difference between resistant and corresponding non-resistant cells.We thank Dr F. Devik, Dr L. Eldjarn and Dr E. Jellum for stimulating discussions, and Miss A. K. Syversen and Mrs T. Nysted for skillful technical assistance. This work was partly supported by the Norgwegian Council for Science and the Humanitites. LE is a fellow of the Norwegian Society for Fighting Cancer.     

Collagen synthesis of cultured fibroblast from Werner's syndromes of premature aging

Summary The difference of collagen producibility between 2 groups of skin fibroblasts from patients with Werner's syndrome with skin change and with normal skin, and the difference of collagen accumulation to cell layer between skin fibroblast from Werner's syndrome and controls were studied.Acknowledgments. We are grateful to Dr M. Ohkido and Dr I. Matsuo of the Department of Dermatology of Tokai University for their supply of materials and generous advice and Mr K. Takeichi of the Department of Pathology of Tokai University for his technical assistance.     

On fragmentation and elimination of ovarian oocytes

Summary Examination of numerous fragmented oocytes in the ovary revealed an asynchronous relationship between nuclear and cell divisions. The asynchrony was observed in non-fragmented oocytes as well and was considered to be one of the common processes leading towards oocyte elimination in the ovary. The present study additionally demonstrates the morphology of follicle cell degeneration observed on the surface of fragmented oocytes.The authors are very grateful to Mr T. Horii, Dr K. Takeda, Dr S. Morisawa, Mr K. Takahashi, Mr Y. Matsumoto and Mrs M. Shinohara for their laboratory help.     

Effects of loading density on cafish blood

Summary Results from this study indicate that a fish density stress syndrome exists for red blood cell morphology. Smaller (by 3.5%) and rounder (by 0.6%) red blood cells were consistently found in intensive fish cultures.Acknowledgments. This work was conducted as part of the Federal Interagency Energy/Environmental Research and Development Program with funds administered through the Environmental Protection Agency (EPA Contract No. EPA-IAG-D8-E721-DR, TVA Contract No. TV-41967A). Dr Ralph H. Brooks and Billy G. Isom are acknowledged for making this study possible. The data were kindly donated by Dr R.W. Williams and W.E. Barksdale, both formerly with the Department of Biology, University of North Alabama, Florence, Alabama.     

Slice cultures of cerebellar,hippocampal and hypothalamic tissue

Summary Cerebellar, hippocampal and hypothalamic slices prepared from newborn and 7-day-old rats were cultured by means of the roller-tube technique. Identification of cells was made easier by the fact that at least part of the characteristic cytoarchitecture of the tissue was preserved in vitro. Cerebellar Purkinje cells and neurones of the deep cerebellar nuclei were recognized on the basis of their size, their location within the culture and their dendritic arborization. Pyramidal cells of all hippocampal subfields displayed their characteristic basal and apical dendritic trees with numerous spinous processes. Hippocampal granule cells gave rise to a monopolar dendritic arbor; their axons terminated in the dentate hilus and CA3 region. Golgi-like immuniperoxidase staining allowed localization of groups of neurophysin-positive neurones in slices prepared from the anterior hypothalamus. These neurones, bilaterally bordering the third ventricle, usually displayed a simple dendritic arborization and fine beaded axons.—Cultivation of brain slices prepared from young rats offers particular advantages in that the cultured cells are organized in an organotypic monolayer and individual living neurones may be directly visualized.Acknowledgments. I would like to thank Dr J. Zimmer and Dr M. Sofroniew for their valuable help with the Timms and immunoperoxidase staining techniques and Mr R. Schlichter and L. Wohlfart for illustrating the culture procedure. The excellent technical assistance of Ms L. Rietschin and E. Hoffmann is gratefully acknowledged.     

Zur Wasserpermeabilität des Protoplasmas

Summary The permeability of cells to dissolved molecules is given by the permeation constant ofCollander (cm·h^–1), whilst the permeability to water is measured by a filter constant (cm·Atm.^–1·h^–1). Therefore these constants are not comparable, and it is impossible to calculate quantitative data on the semipermeability of a cell.In this paper the permeation process is considered as a counterdiffusion of dissolved molecules into the cell and water out of the cell. From the two resulting diffusion equations formulae and a graphic method are derived, which permit to calculate the permeation constant of water, when the permeation constant of a given substance entering the cell is known. These constants are comparable and their ratio is a conclusive measure of the semipermeability.     

The use of a Quantimet image analysis system to analyse trypsin banding patterns of V79/4 (AH1) Chinese hamster chromosomes

Summary A Quantimet image-analysis system was programmed to identify and print out banding patterns from G-banded chromosome spreads of a clone derived from the established cell line V79/4 (Chinese hamster fibroblasts), designated V79/4(AH1).Acknowledgments. We thank Dr. J.R.K. Savage and Dr J. Thacker, MRC Harwell for their advice, also Dr. P.D. Holt for useful discussions. We also thank D. Coates for his assistance with the Quantimet. This work was partly supported by Euratom grant No. 134-74-1 BIOUK.     

A Laser-Doppler-Velocimeter using an optical fiber and its application to local velocity measurement in the coronary artery

Summary A Laser-Doppler-Velocimeter with an optical fiber has been developed to measure arterial blood velocity accurately in a small sample volume. After fundamental experiments to evaluate the accuracy of the present method, blood flow velocity was measured in canine coronary arteries.Acknowledgment. We thank Dr J. Koyama, Vice President of the Institute of Electronics and Communication Engineering of Japan, for his keen interest and continuous encouragement throughout the work. Thanks are also due to Dr H. Kita for English revision, and Dr K. Mito and Mr O. Hiramatsu for their excellent technical assistance. This investigation was supported by the Grants from the Ministry of Education of Japan (No. 521125 and No. 337049 and No. 587008).     

Electrophoretic profiles of proteins and glycoproteins of chick embryo fibroblasts during development]

The variations of proteins and glycoproteins of Chick embryo fibroblasts are studied during development. This investigation is carried out using polyacrylamide disc gel electrophoresis in SDS. Two glycoproteins of high apparent molecular weight (250,000 and 200,000) undergo quantitative modification: they increase from the 8th to 12th day of development and then remain unchanged to the 16th day. They are cell surface components as suggested by fluorescamine labelling and trypsin sensitivity. The results are discussed in terms of relationship between tumor- and embryo cells.     

Narciclasine,an inhibitor of protein synthesis. Action onAllium cepa L. root meristems

Summary Shortening of nucleologenesis time in a synchronous cell population, labelled as binucleate, by a caffeine pulse and fall in frequency of prophases are related to narciclasine inhibition of protein synthesis inAllium cepa L. meristems.Acknowledgments. We gratefully acknowledge Dr D. Vázquez and Dr A. Jiménez, from the Instituto de Bioquímica de Macromoléculas, for calling our attention to this protein synthesis inhibitor and providing us samples both of narciclasine andNarcissus bulbs. We also greatly appreciate Dr B. Rodriguez and Dr F. M. Panizo of the Instituto de Química Orgánica (Departamento de Productos Naturales) for their valuable help in isolation and characterization of narciclasine, and for laboratory facilities. The work has been partially supported by the Comisión Asesora para la Investigación Científica (Spain). A.S. has a fellowship awarded by the Instituto de Cultura Hispánica, Spain.     

动物外周血单个核细胞分离方法探讨

目的 探索用人淋巴细胞分离液分离多种属动物外用血单个核细胞（PBMCs）的可行性及评价其分离效果,建立一种适于野外大规模多种属动物PBMCs的分离方法.方法 采用人淋巴细胞分离液分离来源于新疆的伊犁马、新疆驴、不同品种犬、新疆双峰驼、天山马鹿和来源于重庆的荣昌猪、中国荷斯坦奶牛、简阳大耳黑山羊外周血PBMCs.随机抽样检测分离的动物PBMCs细胞总数、细胞纯度和细胞活率.结果 用人淋巴细胞分离液成功分离上述8种动物PBMCs,每毫升动物外周血分离的PBMCs细胞总数为0.52×106～2.03×106个,纯度为67%～93%,细胞活率为92.5%-98.0%.结论 用人淋巴细胞分离液分离多种动物PB-MCs的方法宜在动物的病毒分子流行学调查中进一步推广.