DEVELOPMENT OF JUNCTIONAL COMPLEX IN CULTURED CELLS

The development of specialized intercellular junctions in cultured cells was studied ultrastructurally. MDCK cells, derived from dog kidney, were fixed in situ at proper times after replating, and the sections were cut perpendicular to the plane of the monolayer. In three or four days, the apposition of cell membranes and condensation of extracellular flocculent material were observed between the neighboring cells, and such were regarded as the early signs of desmosome formation. In many cases, a desmosome was formed first, and the formation of a tight junction followed on the apical (medium facing) side. Finally, all intercellular spaces were closed by a junctional complex at the apical edge. In the complex, a tight junction, (intermediate junction) and desmosome(s) succeeded each other in a medium-substratum direction in all cases. In glutaraldehyde-O^sO⁴ fixed specimens, the intermediate dense line in the desmosome was ascertained from the infant stage of development, while in O^sO⁴ fixed material, the structure was obscure throughout the observation but side-arm-like projections were more prominent.     

高尔基体在人胃腺癌细胞诱导分化过程中的变化

MGc 80-3细胞高尔基体呈发育差、结构不典型状态,但经dBcAMP诱导后,细胞内高尔基体组数增多、分布集中、体积增大,高尔基囊数目增多、排列规则,囊的膜内颗粒增多、分布较为均匀,恢复为与其相应正常细胞相似、发育良好的典型高尔基体结构。这种变化不仅抑制了胃癌细胞的恶性分泌活动,同时对细胞表面成份的变化也起着一定的调节作用。认为高尔基体结构与功能向典型方向的转??变是癌细胞恶性表型逆转的一种重要表现,对于癌细胞由恶性向正常方向的分化具有重要影响。     

SPHEROIDAL BODIES IN THE JUNCTIONAL SARCOPLASMIC RETICULUM OF LIZARD MYOCARDIAL CELLS

The sarcoplasmic reticulum (SR) of lizard (Anolis carolinensis) myocardial cells has been examined, with particular attention being paid to the structural details of the peripheral couplings (junctional SR). Spheroidal bodies are present within the opaque core of junctional SR; these can be seen both in sections made en face and in sections cut to show the apposition of the junctional SR with the sarcolemma. Opaque junctional processes extend between the sarcolemma and the peripheral junctional SR. The myocardial cells in addition contain some SR cisternae deep within the cells which also possess opaque cores composed of spheroids. Although the significance of the junctional SR spheroidal bodies is unknown, it is thought that they could act as a matrix on which enzymes such as calcium-specific ATPase may be located.     

CHANGES IN CELL SURFACE MORPHOLOGY DURING DIFFERENTIATION OF MICROMERE- AND MESOMERE-DERIVED CELLS OF THE SEA URCHIN EMBRYO

Micromeres and mesomeres isolated from 16-cell embryos of the sea urchin, Strongylocentrotus intermedius , were cultured in vitro , and changes in the cells surface architecture during the differentiation of the micromere- and mesomere-derived cells were observed using scanning electron microscopy. Two types of the distribution of the surface microvilli were observed in both blastomere-derived cell masses. One type showed a uniform distribution of the microvilli and the other type showed an uneven one. Though many microvilli were observed in most of both mesomere and micromere-derived cells at the 64-cell stage and the early blastula stage (16 hr after the 16-cell stage at 6°C) respectively, the microvilli decreased in number at the later stages in both blastomere-derived cell masses as compared with the 64-cell stage and the early blastula stage respectively. Rapid disappearance of the surface microvilli was observed in the micromere-derived cells in contrast with the mesomere-derived cells which still had many microvilli even at the midmesenchyme stage.     

DIFFERENTIATION OF INTRACAPSULAR CELLS IN THE SPOROPHYTE OF SPHAEROCARPOS DONNELLII

Ultrastructural and histochemical changes during intracapsular cell differentiation in the premeiotic sporophyte of the liverwort Sphaerocarpos donnellii Austin were studied. From an initially undifferentiated meristematic tissue, spore mother cells and nutritive cells become differentiated. The first indications of ultrastructural differentiation into two cell types are the accumulation of lipid within spherosomes and the occurrence of plastid tubules in the presumptive spore mother cells. Once differentiated the two cell types are clearly distinguishable on the basis of cytoplasmic vacuolation, stored food reserve, and cell and nuclear size. The mature spore mother cell contains many spherosomes, small vacuoles, starch-containing plastids, and a large central nucleus. The mature nutritive cell, on the other hand, is extremely vacuolate and contains large, starch-filled plastids, a few spherosomes, and a small nucleus. A previously undescribed type of cell was observed in developing sporophyte capsules. This cell is located peripherally in the capsule and degenerates during differentiation of spore mother cells and nutritive cells.     

THE KINETICS OF GRANULOSA CELLS IN DEVELOPING FOLLICLES IN THE MOUSE OVARY

This investigation describes the kinetics of the granulosa cells in medium-sized follicles type 3b, 4 and 5a in ovaries of 28-day-old Bagg mice. the method of labelling with ³H-thymidine followed by high resolution autoradiography is used in the experimental work, which consist of determining percentage labelled mitosis (PLM-) and continuous labelling (CL-) curves. In order to analyse the data by computer two alternative hypotheses A and B are set up. Both include the assumptions of no cell loss, exponential growth and a resting compartment Q. In hypothesis A cells from Q re-enter the mitotic cycle via the normal DNA-synthesis compartment S_p. Hypothesis B includes beside compartment S_p a special DNA-synthesis compartment S_q where only cells from Q are synthesizing DNA, and these cells re-enter the mitotic cycle via the G₂ compartment. the mean transit time in S_q is considered to be longer than the mean transit time in S_q. On the basis of the hypothesis mathematical expressions for the PLM- and CL-curves are obtained, and by means of a computer the theoretical curves are fitted to the experimental values: thereby all relevant cell kinetical parameters are estimated. Hypothesis B seems to give the best fit between the theoretical and experimental curves. the estimated parameters are: mean cycle times, μ_c= (56.1 hr, 56.1 hr and 22.3 hr for type 3b, 4 and 5a respectively), doubling times, T _D= (96.4 hr, 118.6 hr and 59.1 hr) and the proportion of cells in Q, p _Q = (0.60, 0.71 and 0.69).     

MITOSIS AND THE PROCESSES OF DIFFERENTIATION OF MYOGENIC CELLS IN VITRO

The relation between the mitotic cycle and myoblast fusion has been studied in chick skeletal muscle in vitro. The duration of the cell cycle phases was the same in both early and late cultures. By tracing a cohort of pulse-labeled cells, it was found that myoblast fusion does not occur in S, G₂, or M. Cell surface alterations required for fusion are dependent upon the position of the cell in the division cycle. In early cultures, fusion takes place only after a minimum delay of 5 hr from the time the cell has entered G₁. The mitosis preceding fusion may condition the cell for the abrupt shift in synthetic activity that occurs in the subsequent G₁. In older cultures fusion of labeled cells is diminished. Two factors account for the cessation of fusion in older cultures. First, the number of myogenic stem cells declines, but these cells do not disappear as the cultures mature. Their persistence was demonstrated by labeling dividing mononucleated cells in older cultures and challenging them with nascent myotubes. Some of these labeled cells were incorporated into the forming myotubes. Second, a block to fusion develops during myotube maturation. Well developed myotubes challenged with labeled competent myogenic cells failed to incorporate the labeled nuclei.     

DIFFERENTIATION OF NEUROBLASTOMA CELLS IN CULTURE

1. An elevation of the intracellular level of cyclic AMP in neuroblastoma cells by prostaglandin E₁ by an inhibitor of cyclic AMP phosphodiesterase, or by analogues of cyclic AMP irreversibly induces many differentiated functions which are characteristic of mature neurones. These include formation of long neurites, increase in size of soma and nucleus associated with a rise in total RNA and protein contents, increase in activities of specific neural enzymes, loss of malignancy, increase in sensitivity of adenylate cyclase to catecholamines and blockade of cells in G₁-stage of the cell cycle. 2. Other agents, including serum-free medium, X-irradiation, 6-thioguanine, cytosine arabinoside, methotrexate, 5-bromodeoxyuridine, nerve growth factor, glial extract and hypertonic medium can induce some of the differentiated functions which are induced by high intracellular cyclic AMP. 3. Morphological differentiation and differentiated biochemical functions can each be expressed in the absence of the other. 4. Many of the responses of normal embryonic nerve cells to cyclic AMP are similar to those of neuroblastoma cells. 5. A working hypothesis for the malignancy of nerve cells has been proposed. This states that an abnormal regulation of cyclic AMP phosphodiesterase activity which allows the expression of high amounts of this enzyme in neuroblastoma cells, may be one of the early lesions during a malignant transformation of nerve cells. 6. A new experimental therapeutic model for the treatment of neuroblastoma is proposed. This involves the administration of sodium butyrate followed by the injection of l -dihydroxyphenylalanine (l-dopa) and prostaglandin E₁ in the presence of cyclic AMP phosphodiesterase inhibitor. 7. Recent studies have elucidated the control mechanisms of some differentiated functions in neuroblastoma cells. Cyclic AMP may become an important biological tool to probe the regulation and expression of many other differentiated functions in these cells. In addition to neuroblastoma cells, other neuronal culture systems are now available for investigating the problems of differentiation and maturation in nerve cells.     

A UNIQUE JUNCTIONAL STRUCTURE FOUND IN BLASTULA CELLS OF THE NEWT, TRITURUS PYRRHOGASTER

The outermost cell layer of the animal half of the newt blastula ( Triturus pyrrhogaster ) was examined to investigate intercellular junctions by transmission and scanning electron microscopy. A unique structure is observed at the terminal region of the intercellular junction. The structures are cytoplasmic ridges elevated from the cell surfaces, and their inner part is filled with spaces of various sizes. It is supposed that these ridges result from the encounter of cytoplasmic folds protruding from two neighboring cells.
Below the ridges, there is a short close junctional area which is followed by a long region of intercellular space intermittently bridged by cytoplasmic projections. Microvillus-like cytoplasmic processes on the apical cell surfaces, and microfilaments and microtubules in subsurface regions are observed in this material as in many other embryonic cells of amphibians.     

鸡Ⅹ期胚盘细胞体外培养

为证实经遗传修饰的鸡X期胚盘细胞具有参与受体胚胎发育和形成嵌合体的能力 ,本研究将由鸡X期胚盘制成的细胞悬液与经脂质体包埋的抗鸡传染性支气管炎病毒基因重组质粒PGS1共孵育后 ,直接显微注入同期受体胚盘 (14 0枚 ) ;或对转染后供体细胞进行G418抗性筛选后显微注入同期受体鸡胚盘 (14 0枚 ) ;或将供体细胞体外培养 4 8h ,再与脂质体 PGS1复合物共孵育后显微注入同期受体鸡胚盘 (190枚 ) ,制备转基因嵌合体鸡 ,并应用PCR和RAPD方法 ,对鸡胚和雏鸡不同组织或血液中的DNA进行检测。结果表明 :直接注射组孵化率(5 7% )显著 (P <0 0 1)高于G418筛选处理组 (1 4 % )和培养 4 8h处理组 (2 1% ) ;G418筛选处理组不同胚龄鸡胚组织、器官中外源DNA的PCR检测阳性率均高于其它二个组。实验结果证明 ,体外培养 4 8h并经遗传修饰的胚盘细胞仍然具有形成嵌合体的能力 ,利用早期胚盘细胞途径制备转基因鸡是可行的。     

THE DIFFERENTIATION OF PIGMENT CELLS IN CULTURES OF CHICK EMBRYONIC NEURAL RETINAE

Neural retinal cells of 8–9 day-old chick embryos were differentiated into pigment cells in the conditions of cell culture for about 25 days. The increase of pigment cells in vitro was semi-quantitatively shown, by counting the number of black foci of pigmented cells per plate throughout the culture period. The increase paralleled the increase in the activity of tyrosinase. The addition of a small number of pigment cells freshly dissociated from tapeta to the cultures of neural retinae did not increase the number of black foci in vitro . Electron microscopic observations revealed the morphological differences of melanin granules between those in pigment cells of the neural retinal cultures and those in cultured tapetum cells. It was discussed that pigment cells appearing in the neural retinal cultures were derived from neural retinal cells, but not from contaminated cells of the tapetum.     

大鼠原生殖细胞培养和分化的研究

研究大鼠胚胎原生殖细胞（primordial germ cells,PGCs)的培养及分化,取受精后11-12.5天大鼠PGCs进行原代培养,光、电镜观察PGCs及其分化细胞的微细结构,碱性磷酸酶染色检测细胞的分化程度,结果显然显示大鼠PGCs大而圆,散在分布,或多个聚集成团,胞质中含有椭圆形的线粒体和丰富的核糖体,在鼠胚成纤维细胞饲养层存在的情况下,PGCs保持未分化状态,碱性磷酸酶反应呈强阳性,在缺乏饲养层的条件下PGCs很快分化,形态不规则,有伪足,碱性磷酸酶反应减弱,进一步分化可形成具有细长突起的神经元样细胞,胞质中含有细丝束的表皮细胞,可见节律性跳动的心肌细胞,具有分泌颗粒的分泌细胞及似血管,心脏形状的管腔结构等,由PGCs分化来的细胞碱性磷酸酶反应均呈阴性,结果表明大鼠PGCs能够分化形成三个胚层的衍生物,生殖嵴来源的PGCsp是一种具有发育全能性的胚胎多能干细胞,本研究同时证明鼠胚饲养层能抑制大鼠PGCs的分化。     

DETECTION OF COMPLEX CARBOHYDRATES IN THE GOLGI APPARATUS OF RAT CELLS

Two methods used for the electron microscopic detection of glycoproteins were applied to a variety of cell types in the rat; one involved successive treatment of sections with periodic acid, chromic acid, and silver methenamine; and the other, a brief treatment with a chromic acid-phosphotungstic acid mixture. The results obtained with the two methods were identical and, whenever the comparison was possible, similar to those obtained with the periodic acid-Schiff technique of light microscopy. In secretory as well as in nonsecretory cells, parts of the Golgi apparatus are stained. The last saccule on one side of each Golgi stack is strongly reactive (mature face), and the last saccule on the other side shows little or no reactivity (immature face); a gradient of reactivity occurs in between these saccules. The more likely explanation of the increase in staining intensity is that carbohydrate is synthesized and accumulates in saccules as they migrate toward the mature face. In many secretory cells, the mature face is associated with strongly stained secretory granules. Other structures stained are: (1) small vesicles, dense and multivesicular bodies, at least some of which are presumed to be lysosomal in nature; (2) cell coat; and (3) basement membrane. The evidence suggests that the Golgi saccules provide glycoproteins not only for secretion, but also for the needs of the lysosomal system as well as for incorporation into the cell coat and perhaps basement membrane.     

LOW RESISTANCE CONNECTIONS BETWEEN CELLS IN THE DEVELOPING ANTHER OF THE LILY

Low resistance junctions were demonstrated between cells in anthers from young buds of Lilium longiflorum Croft by standard electrophysiological techniques. Electrodes containing a dye were used to stain impaled cells for later histological identification. Electrical coupling is widespread; germinal cells are coupled to one another; coupling is also observed between somatic elements, and germinal and somatic cells are similarly interconnected. Cytoplasmic bridges are implicated in the first case; plasmodesmata are probably responsible for the interactions in the other two. Although the physiological role of the low resistance junctions shown here and present in embryonic animal tissues is unknown, the possible function of this form of intercellular communication in the development of the anther is discussed.