CONTACT-INHIBITED REVERTANT CELL LINES ISOLATED FROM SV40-TRANSFORMED CELLS : I. Biologic, Virologic, and Chemical Properties

Two contact-inhibited "revertant" cell lines were isolated from an SV40-transformed mouse 3T3 cell line (SV-3T3) after exposure to 5-fluoro-2'-deoxyuridine. Revertant cells resembled 3T3 cells morphologically and grew to saturation densities which were similar to those of 3T3 cells; however, revertant cells readily formed both single and multinucleated giant cells in confluent cultures. SV40 virus was rescued from revertant cells by fusion with permissive monkey cells. The rescued virus transformed 3T3 cells with the same efficiency as wild type virus, and produced transformed colonies which were phenotypically similar to those produced by wild type virus. The revertant cells also resembled normal 3T3 cells in that they contained higher quantities of sialic acid than SV-3T3 cells. An inverse correlation was found between the saturation density of cells and their sialic acid content. Collagen content, however, of revertant cells was similar to that of SV-3T3 cells. The data presented suggest that the property of contact inhibition in revertant cells is related to the sialic acid content of the plasma membrane and that changes in sialic acid content of transformed cells are not directly specified by the viral genome.     

CONTACT-INHIBITED REVERTANT CELL LINES ISOLATED FROM SV40-TRANSFORMED CELLS : IV. Microfilament Distribution and Cell Shape in Untransformed, Transformed, and Revertant Balb/c 3T3 Cells

A comparison is made of the ultrastructure of the cell periphery in three cloned cell lines: untransformed Balb/c 3T3 cells, SV40-transformed Balb/c 3T3 cells, and revertant cells obtained from the transformed cell line by a selection technique utilizing concanavalin A. Both thin-section and surface replication techniques are used for in situ examination of the cell lines. Microfilaments, 70 Å in diameter (called alpha filaments), are abundant in untransformed and revertant cell lines, particularly in the anterior expansions of the cells, which tend to have many microvilli and small pseudopodia. Alpha filaments are diminished in the anterior expansions of transformed cells, which contain large blunt pseudopodia and relatively few microvilli. Surface replicas confirm the impression gained from thin sections that transformed cells have a greater proportion of their cell surface involved in bulging pseudopodia than either untransformed or revertant cells. Since alpha filaments are shown to bind heavy meromyosin and are similar to F-actin, these filaments are thought to be important in cell motility. These observations suggest that a close relationship exists between decreased alpha filaments, bulging pseudopodia, and loss of contact inhibition of movement in transformed cells.     

小剂量DNA毒剂诱导SV40转化的人细胞中的SV40DNA的增强复制

本文介绍了用~(32)P标记的pSV_2质粒为探针,通过DNA分子原位杂交研究小剂量DNA毒剂诱导被SV40转化的人细胞NB—E中SV40DNA的增强复制.最佳条件下,最大增强比可达4-5.而且,这种病毒DNA增强复制与病毒的增强复活与增强致突有相似的动力学过程和剂量响应关系.此结果为哺乳类细胞中可能存在SOS功能提供了进一步证据.被诱导细胞的Hirt沉淀与Hirt上清液中都存在SV40DNA片段,且Hirt上清液中SV40DNA片段大小均一,反映此过程与λ原噬菌体的诱导的起始过程有相似之处.小剂量紫外线可诱导被SV40转化的人XP细胞中的SV40 DNA的增强复制.说明这一诱导过程可能与切除修复功能有不同的遗传学过程.     

小鼠原生殖细胞建系过程及其分化特性的研究

以小鼠8.5dpc、10.5dpc、12.5dpc胚胎为材料,分离其中包含PGC的胚胎组织,使其生长于饲养层细胞上,在生长因子LIF、SCF和bFGF的共同作用下存活增殖,形成PGC克隆,经过几次分散转移至新的饲养层细胞,产生稳定增殖的EG干细胞克隆,共建成5株EG细胞系,AKP染色以及oct-4基因表达产物的免疫荧光检测均显示阳性。EG1、EG2、EG3、EG4、EG5,分别来自8.5、10.5dpc的胚胎,没有得到长期培养的12.5dpc的EG细胞系。EG细胞系在有饲养层细胞或添加LIF的环境中可稳定传代,保持不分化状态,至少15代内正常核型细胞所占比例80%以上。去除抑制分化因素的前提下,悬浮培养的EG细胞形成胚体,分化出类似胚胎内胚层和外胚层的细胞结构;贴壁生长的胚体能产生不同类型的分化细胞,包括上皮细胞、成纤维细胞、神经细胞等。EG细胞在裸鼠体内形成畸胎瘤。以上结果证实我们建立的EG细胞系具发育多能性,为研究早期胚胎和生殖细胞生长分化提供了模型。     

PARENCHYMAL CELLS FROM ADULT RAT LIVER IN NONPROLIFERATING MONOLAYER CULTURE : II. Ultrastructural Studies

Hepatic parenchymal cells from adult rats, established in vitro as a monolayer, have been evaluated by electron microscopy. Within 24 h after the initial seeding, the incubated cells were polygonal and in close apposition with three to six neighboring cells. The ultrastructure of the monolayer cells was examined at this time and after 3 and 10 days of incubation. With the exception of a few enlarged mitochondria, organelles in both the 1- and 3-day monolayer cells were indistinguishable quantitatively and morphologically from those found in the intact liver. After 10 days of incubation, however, the rough-surfaced endoplasmic reticulum (RER) had become dilated and vesiculated. In all cells studied, portions of RER were found in a close spatial relationship to mitochondria. From its frequency, this association appeared to be more than fortuitous, and the organelle complex may represent a functional unit necessary for new membrane formation, as suggested previously. The Golgi complexes of 1- and 3-day cells contained very low density lipoprotein-sized particles, which suggests that the monolayer cells synthesize lipoproteins. These electron microscope observations demonstrate that adult hepatic parenchymal cells in monolayer retain for several days the subcellular structural elements characteristic of normally functioning hepatocytes.     

Binding studies of SV40 T-antigen to SV40 binding site II.

SV40 T-Antigen binding site II was synthesized, cloned and analyzed for its ability to bind purified SV40 T-antigen. We report the binding constant of T-antigen for isolated site II. Using a filter binding assay the calculated binding constant was 6-8 fold less efficient than site I previously reported. Binding constants were calculated using two methods. The first was a direct calculation using a protein titration curve (KD). The second was by the ratio of measured association and dissociation rates. Both methods gave similar constants. Protection studies with SV40 T-antigen on the T-antigen binding sites in the wild-type array demonstrated that the binding constants of site I and site II are similar to those calculated for the individual sites. These results demonstrate that SV40 T-antigen does not bind cooperatively to sites one and two as earlier believed and are in agreement with recent observations emanating from several laboratories.     

NEW ARGININE-CONTAINING PEPTIDES ISOLATED FROM CHLORELLA CELLS

Seven kinds of arginine-containing peptides were isolated fromthe cells of Chlorella ellipsoidea, and their structures wereinvestigated. Their amino acid make-ups and quantities presentin randomly grown algal cells were found to be as follows (indecreasing order of contentin moles per dry weight of cells): Arg-Arg, Arg-Arg-Glu, Arg-Arg-Arg, Arg-Glu> Arg-(Arg₂, Glu)-Glu>Arg-(Arg₃,Glu), Arg-(Glu, Asp) Using synchronously mass-cultured algal cells, the quantitiesof these peptides as they changed during the algal life cyclewere followed. It was found that, except in the case of Arg-Arg-Arg,the contents (in moles per dry weight of cells) of the peptides(i) markedly increased during the stages from D_n to L₁, (ii)remained almost constant or more or less appreciably increasedduring the stages from L₁ to L₃, and (iii) decreased sharplyduring the transformation of L₃-cells (via L₄) into D_n-cellsin the dark. The content of Arg-Arg-Arg remained almost constantduring the period from D_n to L₃, and on transference of L₃-cellsin the dark it increased temporarily and then decreased duringthe transformation of L₄-cells into D_n-cells. Significance andpossible roles of these peculiar peptides in the life cycleof Chlorella were discussed. (Received May 10, 1965; )     

PROTEIN SYNTHESIS IN FRACTIONS FROM ISOLATED BRAIN CELL NUCLEI

Abstract— 1. The incorporation in vivo and in vitro of isotopically labelled leucine into fractions of nuclear proteins from young and adult rat brain was investigated.
2. During post-natal cerebral maturation, the ability of nuclei from brain cells to synthesize proteins decreased. The specific activities of all the fractions of nuclear protein were highest in 3-day-old rats and declined thereafter. Nuclei from adult brain cells exhibited only 10 per cent of the activity found in nuclei from brain cells of 3-day-old rats.
3. The 'residual protein' fraction was most rapidly labelled, peak activity being reached within 30 min after injection. In vitro , the 'residual protein' fraction attained maximum activity within 40 min.
4. The specific activity of the chromatin acidic proteins (HCl-insoluble) was considerably higher than that of the histones both in vivo and in vitro. Histones were the most inert of all the nuclear protein fractions studied.
The possible functional significance of the various protein fractions during the process of cerebral maturation and in the adult brain is discussed.     

人工分离花粉生殖细胞的细胞生物学研究

采用压片法从三科七种植物的成熟花粉粒中分离出生殖细胞。干涉差显微术和视频增差显微术观察表明,分离的生殖细胞有完整结构和清晰的图象,并呈现由纺锤形到球形的各种形态;细胞形态的变化与培养基渗透压、微管稳定剂等因素有关。首次获得了生殖细胞的扫描电镜的立体形象。细胞壁化学成分的检测否定细胞壁的存在,微管蛋白的免疫荧光鉴定显示了微管系统的空间分布,生活力的荧光测定证实分离的细胞是生活的。讨论了分离生殖细胞研究的前景。     

ACTION OF CYTOCHALASIN D ON CELLS OF ESTABLISHED LINES : II. Cortex and Microfilaments

Cells in culture exposed to cytochalasin D (CD) rapidly undergo a long-sustained tonic contraction. Coincident with this contracture the thin microfilaments of the cortex become compacted into feltlike masses. The ravelled filaments of these masses remain actinlike and bind heavy meromyosin; they are not disrupted or disaggregated, but rather, appear to represent a contracted state of the microfilament apparatus of the cell cortex. On continued exposure to CD, ‘myoid’ bundles, containing thick, dense filaments, and larger fusiform or ribbonlike, putatively myosinoid, aggregates may appear. These appearances are interpreted as consequences of a state of hypercontraction without relaxation induced by CD. They do not occur in CD-treated cells prevented from contracting by inhibitors of energy metabolism, and are readily reversible on withdrawal of CD. Extensive ordered arrays of thin microfilaments develop in cells which are reextending during early recovery.     

PROPERTIES OF CELL NUCLEI ISOLATED FROM VARIOUS REGIONS OF RAT BRAIN: DIVERGENT CHARACTERISTICS OF CEREBELLAR CELL NUCLEI

Abstract— Cell nuclei were isolated in yields ranging from 38 to 61 per cent from six anatomically defined brain regions of the albino rat. To provide basic information for further studies of altered genomic activity in brain cell nuclei, various properties of these isolated nuclei were measured, including counts of their number, estimates of the distribution of sizes, amounts of RNA, DNA and protein, and endogenous RNA polymerase activity. DNA content per nucleus approximated the accepted value of 6 pg per diploid set of chromosomes. Distributions of nuclear size showed a sensitivity to the concentration of divalent cation, with a shift toward larger nuclear diameters as the Mg concentration was reduced. Cell nuclei from hippocampus, hypothalamus-preoptic region, cerebral cortex, amygdala and midbrain plus brainstem were generally similar in yield, distribution of size, and RNA, DNA and protein content. Cell nuclei from cerebellum differed from those of other brain regions, in all of these parameters. The cerebellum contained a high content of DNA and had an enormous number (8 × 10⁸ per g wet wt.) of cell nuclei of predominantly very small size and characterized by lower ratios of RNA, histones and non-histone protein to DNA and lower endogenous activity of RNA polymerase than nuclei from other brain structures. These properties correlated well with properties of cerebellar tissue, namely, high content of small granule neurons and low ratio of RNA to DNA, and suggest that the small cerebellar nuclei may have relatively inactive genomes. The relationship of 'large' and 'small' cell nuclei to cell types in the brain is discussed.     

ESTABLISHMENT AND CHARACTERIZATION OF ETHIDIUM BROMIDE RESISTANCE IN SIMIAN VIRUS 40-TRANSFORMED HAMSTER CELLS : Effects on Mitochondria In Vivo

This study describes the isolation and subsequent characterization of four mammalian cell lines resistant to ethidium bromide (EB). Treatment of the simian virus 40- (SV40) transformed hamster cell line F5-1 first led to the establishment of the F2 cell line, which is resistant to 2 µg EB/ml. At this concentration cytochromes c and b are present in almost normal or only slightly diminished amounts, whereas cytochromes a + a₃ show an obvious decrease. The mitochondria of the F2 cell show a normal ultrastructure, not distinct from the parental cell line F5-1, and contain closed circular DNA. The sensitive parental F5-1 cells, however, when exposed to the same dye concentration exhibit the typical EB-induced ultrastructural changes in the mitochondria, and no more component I mitochondrial DNA can be demonstrated. 1 yr after establishment we derived from the F2 cell three more cell lines, resistant against 4, 8, and 16 µg of EB/ml. These cell lines, termed F4, F8, and F16, respectively, also revealed relatively intact-appearing mitochondria, although distinguishable from F5-1 and F2 mitochondria by a more condensed or unorthodox cristae conformation. F4, F8, and F16 cell lines contained closed circular mitochondrial DNA in the same position as that of the parental F5-1 cells, when analyzed in an isopycnic CsCl-EB gradient. A small shoulder at the lower density side of the DNA I peaks was observed. The newly acquired drug resistance of the F cells is hereditarily transmitted to the progeny cells and retained even after a period of growth in EB-free medium.     

Host-Pathogen Interactions : XVII. HYDROLYSIS OF BIOLOGICALLY ACTIVE FUNGAL GLUCANS BY ENZYMES ISOLATED FROM SOYBEAN CELLS

The ability of β-glucosylase I, a soybean cell wall β-glucosyl hydrolase, to degrade elicitors of phytoalexin accumulation was studied. Extensive β-glucosylase I treatment of the glucan elicitor isolated from the mycelial walls of Phytophthora megasperma var. sojae results in hydrolysis of 77% of the glucosidic bonds of the elicitor and destruction of 94% of its activity. Soybean cell walls contain some additional factor, probably one or more additional enzymes, which can assist β-glucosylase I in hydrolyzing the glucan elicitor. This was demonstrated by the more rapid hydrolysis of the glucan elicitor by a mixture of soybean cell wall enzymes (containing β-glucosylase I). In a single treatment, the mixture of cell wall enzymes hydrolyzed 91% of the glucosidic bonds and destroyed 85% of the activity of the elicitor. The enzymes from soybean cell walls will also hydrolyze elicitor-active oligoglucosides prepared from the mycelial walls of Phytophthora megasperma var. sojae. The active oligoglucosides are more susceptible than the glucan elicitor to hydrolysis by these enzymes. The mixture of cell wall enzymes or β-glucosylase I, by itself, hydrolyzes more than 96% of the glucosidic bonds and destroys more than 99% of the activity of the oligoglucoside elicitor. Two possible advantages for the existence of these enzymes in the walls of soybean cells are discussed.     

向日葵Ti T—DNA转化系的原生质体培养和细胞培养

根癌农杆菌离体感染向日葵子叶、下胚轴外植体形成的Ti T-DNA转化组织在激素条件下长期继代培养后,用来进行原生质体培养和细胞培养。适于B6S3转化系和T37转化系原生质体培养的培养基分别为附加不同激素和糖类的C81V和DPD培养基。用液体浅层法培养3～5天时,原生质体开始分裂。10天后形成细胞团。B6S3转化系还可直接从原生质体产生原胚状结构。转化系的细胞克隆均保持着激素自主型生长特性和冠瘿碱合成酶合成特性。     

IN VITRO PROLIFERATION OF MOUSE LYMPHOBLASTOID CELL LINES: GROWTH MODULATION BY VARIOUS POPULATIONS OF ADHERENT CELLS

The in vitro growth pattern of a number of mouse lymphoblastoid tumour cell lines was modified in the presence of adherent cell layers from various sources. The AVRij-1 and ST-4b cell lines exhibited a concentration—dependent growth pattern, i.e., they would only grow well when seeded at high starting cell concentrations. Better growth of these cells from low cell concentrations was observed in the presence of adherent cell layers from syngeneic or allogeneic bone marrow. Adherent cell layers derived from mouse spleen and pleural or peritoneal cavity could also promote the growth of the above tumour cells, but in a narrower range of cell concentrations and to a lower extent. Moreover, confluent adherent layers from the pleural and peritoneal cavities completely inhibited the growth of AVRij-1 and ST-4b cells, while adherent cell layers from the bone marrow did not inhibit growth at any cell concentration tested. The in vitro growth of concentration—independent cell lines was also affected by the presence of adherent cells from the bone marrow. Under syngeneic conditions, a slight increase in the growth of the ‘null’ or pre-B lymphoma cell line ABLS-8.1 was observed. On the other hand, the growth of tumour cells expressing more differentiated properties, such as the thymus T lymphoma tumour cell line ST-1.3 and the plasma cell tumour MPC-11.45.6.2.4, was inhibited in the presence of syngeneic bone marrow derived adherent cell layers. This inhibition was more pronounced under allogeneic conditions. Growth inhibition was also observed when concentration—independent cell lines were co-cultured with adherent cells from the pleural and peritoneal cavities. Thus, adherent cell layers from non-haemopoietic sources inhibited the growth of all cell lines tested. On the other hand, adherent cells from the bone marrow had a differential effect on growth of lymphoblastoid tumour cell lines. This depended on the in vitro growth properties of each tumour cell line and on some additional specific tumour cell properties. The latter could relate to the differentiation stage characterizing each tumour cell line. The culture method described here may serve as a model system for studies on interaction of leukaemic cell and the haemopoietic microenvironment.     

DIVISION AND GROWTH OF SINGLE MESOPHYLL CELLS ISOLATED ENZYMATICALLY FROM TOBACCO LEAVES

Single mesophyll cells isolated enzymatically from tobacco leaves divided and grew into aggregates of several cells in defined media. Morphological changes accompanying the division and the growth suggested that these cells are in the course of dedifferentiation. Some of the potentialities of these cells as an experimental material for developmental and genetical studies are discussed.     

A MUCILAGE-FORMING LACTOBACILLUS SPECIES ISOLATED FROM CIDER. PART II. POLYSACCHARIDE FORMATION

SUMMARY: Cultures grown at 25° under an atmosphere of CO₂ in a medium containing maltose, inorganic salts and ethanolic extracts of peptone and yeast autolysate produced a polysaccharide, which was isolated and purified. Examination by a variety of chemical and biological methods indicated that it was a dextran of low molecular weight.