DEVELOPMENT OF CHLOROPLAST FINE STRUCTURE IN ASPEN TISSUE CULTURE

Chloroplast ontogeny has been examined in 42-day etiolated triploid aspen callus (Populus tremuloides Michx.) subjected to two different light conditions. White and low-intensity red illumination showed little differences in their stimulatory effects on plastid development, the red light-irradiated plastids developing only slightly more slowly. Asynchronous plastid development was noted in both lighting systems. Etioplasts contained an interconnected tubular net, phytoferritin aggregates, electron-transparent vesicles which seem to invaginate from the inner plastid membrane, membrane-bound homogeneous spheroids and starch grains. Irradiation caused various morphological changes within the proplastids; the tubular complex became transformed into the more ordered prolamellar body-like structure from which radiated membrane-bound sacs filled with electron-dense material. These sacs, characterized as thylakoid precursors, were transformed into a thylakoidal system typical of mature chloroplasts. This ontogenetic scheme represents an additional pathway for the development of photosynthetic lamellae. Other light-induced changes in the developing plastid include disappearance of phytoferritin particles and homogeneous spheroids, decrease in starch content, and appearance of osmiophilic droplets.     

FINE STRUCTURE OF DIFFERENTIATING MOUSE PANCREATIC EXOCRINE CELLS IN TRANSFILTER CULTURE

Fine structural observations have been made in the 11-day embryonic mouse of exocrine cells in pancreatic epithelium developing in tissue culture transfilter from salivary gland mesenchyme of the 13-day embryonic mouse. After 2 days in culture, the exocrine cells show increased cytoplasmic density, abundant ribosomes in aggregate or "rosette" form, and expanded profiles of rough-surfaced endoplasmic reticulum. After 3 and 4 days in culture, the cells exhibit continued expansion of the profiles of endoplasmic reticulum, increased amounts of Golgi membranes, and large areas of light density (prozymogen granules). After 5 days in culture, dense zymogen granules are present in the most highly differentiated cells. In addition, at the filter-epithelial surface, at 2 days, small fibers can be discerned which, after 4 days in culture, show obvious periodicity and are thought to be collagen. The significance of these changes, in relation to the mesenchymal effect, to the onset of specific synthesis and to the stabilization of differentiation is discussed.     

THE FINE STRUCTURE OF EMBRYONIC CHICK SKELETAL MUSCLE CELLS DIFFERENTIATED IN VITRO

Dissociated myoblasts from 12-day chick embryos were cultured in monolayer, and the differentiation of skeletal muscle cells was studied by electron microscopy. The results have revealed a striking ultrastructural similarity between the in vivo and the in vitro developing muscle, particularly with respect to the myofibrils and sarcoplasmic reticulum. This study demonstrates that all the characteristic organelles of mature skeletal muscle can develop in vitro in the absence of nerves.     

DISEASED MUSCLE CELLS IN CULTURE

(1) Cultures of differentiated muscle cells have been grown from diseased human, mouse and chick skeletal muscle, and from cardiac muscle of the myopathic hamster. (2) Methods of culture established for normal embryonic and adult skeletal muscle cells have proved suitable for cultures of diseased muscle cells. (3) Myoblasts obtained from dy^2J mouse muscle crushed in vivo before explanting fuse in culture and form morphologically normal myotubes. Studies of the effects of innervation by dy^2J spinal cord neurones on the differentiation of normal, dy^2J and dy myotubes have been inconclusive but it is probable that innervation does not play a part in the pathogenesis of this disorder. (4) Myoblasts prepared by trypsinization of embryonic dy muscle behave normally in culture and fuse to form myotubes that appear normal. It is not clear if myoblasts that migrate from explants of adult muscle in vitro fuse. Aggregates of non-fusing cells have been described, but under other culture conditions normal and abnormal forms of myotube have been observed. dy muscle fibres fail to regenerate even when cultured with normal spinal cord explants and dy nerves are without effect on regenerating normal muscle fibres. These tissue-culture studies suggest that the dy mouse mutation is a myopathic disorder. (5) Embryonic mdg myoblasts have a normal cell cycle in vitro and fuse to form well-differentiated myotubes with cross-striations. mdg myotubes have normal electro-physiological properties but do not contract spontaneously or on depolarization. The defect in the muscle of the mdg mutant appears to be a failure of excitation-contraction coupling. (6) Cells migrate earlier from explants of adult dystrophic chick muscle than from normal muscle but dystrophic chick myotubes appear morphologically normal. Myotubes prepared from embryonic dystrophic chick muscle become vacuolated and degenerate, changes that can be prevented by anti-proteases such as antipain. Lactic dehydrogenase isozyme subunit M4 is absent from dystrophic muscle in vivo but reappears in cultured myotubes. Dystrophic myotubes innervated in culture by either normal or dystrophic neurones exhibit bi-directional lcoupling and multiple innervation. These results suggest that there are changes in dystrophic myotubes and that chick muscular dystrophy is a myopathy. (7) Cardiac muscle cells from the cardiomyopathic hamster synthesize less actin and myosin than normal cells, and Z lines in dystrophic cells are irregularly arranged. The beat frequency of myopathic cardiac cells is lower than that of normal cells and declines more rapidly. Tissue-culture studies have not been made of hamster skeletal muscle. (8) Human dystrophic myotubes do not show degenerative changes in culture and have normal histochemical reactions. RNA synthesis appears normal in dystrophic myotubes but there may be changes in adenyl-cyclase activity and protein synthesis in dystrophic cells. Morphological and biochemical changes have been found in muscle cells cultured from a case of acid-maltase deficiency but phosphorylase activity re-appeared in myotubes cultured from biopsies of phosphorylase-deficient muscle. Innervation by normal mouse nerves does not induce degenerative changes in dystrophic myotubes. (9) Studies on the origins of myoblasts in explants of muscle fibres in culture suggest that in these conditions myoblasts are derived only from satellite cells and that this process may be the same in normal and diseased muscle.     

CORRELATION OF FINE STRUCTURE AND PHYSIOLOGY OF THE INNERVATION OF SMOOTH MUSCLE IN THE GUINEA PIG VAS DEFERENS

An electron microscope study of the innervation of smooth muscle of the guinea pig vas deferens was undertaken in order to find a structural basis for recent electrophysiological observations. The external longitudinal muscle coat was examined in transverse section. Large areas of the surfaces of adjacent muscle cells were 500 to 800 A apart. Closer contacts were rare. A special type of close contact suggested cytoplasmic transfer between neighbouring cells. Groups of non-myelinated axons from ganglia at the distal end of the hypogastric nerve ramified throughout the muscle. Some small axon bundles and single axons lay in narrow fissures within closely packed muscle masses. Many axons contained "synaptic vesicles." About 25 per cent of the muscle fibres in the plane of section were within 0.25 µ of a partly naked axon; of these 15 per cent were within 500 A of the axon, and about 1 per cent made close contact (200 A) with a naked axon. It is unlikely that every muscle fibre is in close contact with an axon, and it is not possible for every fibre to have many such contacts. Muscle fibres are probably activated by both diffusion of transmitter from naked portions of axons a fraction of a micron distant, and electrotonic spread of activity from neighbouring cells.     

人脐静脉血管平滑肌促内皮细胞组织因子表达的体外实验研究

目的研究血管平滑肌细胞对血管内皮细胞组织因子表达的影响并探讨其临床意义.方法用贴块法培养人脐静脉平滑肌细胞;酶消化法培养人脐静脉内皮细胞;用培养平滑肌细胞条件培养液(SMC-CM)刺激培养的内皮细胞,一步凝固法检测内皮细胞组织因子的活性;Northern blot检测内皮细胞组织因子的mRNA表达;并用酶联免疫吸附试验检测SMC-CM中IL-1α、IL-1β、TNF-α和VEGF的含量.结果 SMC-CM使内皮细胞组织因子活性呈剂量依赖性增强,作用6h增至最高,最高增强约38倍;SMC-CM使内皮细胞组织因子mRNA表达显著增强;SMC-CM中的组织因子诱导剂不耐热,且并非IL-1α、IL-1β、TNF-α和VEGF等已知的组织因子诱导剂.结论血管平滑肌细胞能促进血管内皮细胞组织因子的表达,提示体内增生的平滑肌细胞,如动脉再狭窄新内膜中的平滑肌细胞可能诱导局部血管内皮细胞活化及表达组织因子,在局部血栓形成中起一定作用.     

MYOSIN-LIKE AGGREGATES IN TRYPSIN-TREATED SMOOTH MUSCLE CELLS

Segments of the lower small intestine of the toad Bufo marinus were excised and soaked for approximately 2 hr in Ringer's solution (pH 7.4 or 7.8) containing crystalline trypsin and then fixed for electron microscopy at approximately the same pH. Thin sections of the tunica muscularis of these specimens show smooth muscle cells ranging in appearance from severely damaged at one extreme to apparently unaffected at the other. Among these are cells at intermediate stages, including some which exhibit large and conspicuous populations of thick filaments closely resembling artificially prepared aggregates of smooth muscle myosin. The thick filaments have the form of tactoids ~ 250–300 A in diameter in their middle regions and are ~ 0.5–1.0 µ in length. In some preparations they also display an axial periodicity approximating 143 A. They are usually randomly oriented and segregated from the thin filaments, which tend to form closely packed, virtually crystalline bundles at the periphery of these cells. "Dense bodies" are absent from cells showing these changes. The simplest interpretation of these data is that smooth muscle myosin normally exists among the actin filaments in a relatively disaggregated state and that trypsin induces aggregation by altering the conformation of the myosin molecule. Alternatively, trypsin may act indirectly through an effect on some other smooth muscle protein which normally forms a stable complex with relatively disaggregated myosin.     

大鼠主动脉内皮细胞和平滑肌细胞的原代培养及生物学特性比较

目的培养大鼠主动脉平滑肌细胞和内皮细胞,细胞纯化与鉴定,比较生物学特性的差异。方法采用血管环贴壁法培养动脉内皮细胞,组织块贴壁法培养动脉平滑肌细胞,并采用有限稀释法挑选内皮细胞单克隆,免疫细胞荧光鉴定二者的特异性标志,相差显微镜观察二者单个细胞及细胞群体在形态上的差异性,CCK-8试剂盒检测细胞的增殖,比较二者对胰酶消化,粘附,冻存后复苏的情况。结果血管环贴壁法成功培养血管内皮细胞,组织块培养法成功培养出血管平滑肌细胞,内皮细胞能够形成单克隆集落,培养的细胞均表达相应的特异性标志,内皮细胞增殖速度和平滑肌细胞有差异,内皮细胞对胰酶的耐受性较差,内皮细胞粘附所需时间短,对冻存后的耐受性较好。结论组织块贴壁法适合内皮细胞和平滑肌细胞的培养,有限稀释法能够纯化原代培养的内皮细胞,大鼠主动脉平滑肌细胞和内皮细胞在细胞形态、增殖、粘附、对胰酶的反应、冻存后复苏均存在差异。     

FINE STRUCTURE IN FROZEN-ETCHED YEAST CELLS

The freeze-etching technique, which is a special kind of freeze-drying, allows electron microscopic investigation of cells and tissues in the frozen state. In regard to yeast cells (Saccharomyces cerevisiae) a freeze-fixation technique has been developed which does not kill the object. The electron micrographs therefore are considered to impart an image of high fidelity. The cutting of the frozen object, which actually consists of a fine splintering, produces not only cross-sectional views (cross-fractures) of the structures but also surface views of the membranes and organelles. Many surface structures are described which have not been shown by the usual sectioning techniques. The cytoplasmic membrane contains hexagonal arrangements of particles which are apparently involved in the production of the glucan fibrils of the cell wall. Alterations of the distribution of nuclear pores are shown in cells of different ages. Freeze-etching enables a clear distinction of endoplasmic reticulum and vacuoles in yeast cells. The membranes of the vesicular systems are covered by ribosomes arranged in circular patterns. The mitochondrial envelope shows small perforations which could allow the exchange of macromolecules. The storage granules consist of concentric layers of lipid, presumably phosphatide. A Golgi apparatus has been detected which may be involved in the storage of lipid. The structure of the unit membrane and the membrane structures of all organelles as revealed by chemical fixation are confirmed in principle. Glycogen agglomerations are identified in the ground plasm of older cells. Insight into artifacts introduced by common chemical fixation and embedding techniques is obtained and discussed.     

LOCUS AND STATE OF AGGREGATION OF MYOSIN IN TISSUE SECTIONS OF VERTEBRATE SMOOTH MUSCLE

Structures with the characteristics of molecular myosin were identified by electron microscopy in tissue sections of vertebrate smooth muscle. No thick filaments of myosin were found regardless of preparative procedures, which included fixation at rest and in contraction, glycerine extraction, and storage at low pH prior to fixation. Absence of thick myosin filaments and presence of what appear to be myosin molecules is in accord with conclusions based on X-ray diffraction (3, 12) and birefringence data (4) from living smooth muscles at rest and in contraction. Explanations are provided for appearances thought by others (6, 20, 21) to represent thick myosin filaments. Our present observations are in accord with the model for smooth muscle contraction which we have previously proposed (1).     

ARCHITECTURE AND NERVE SUPPLY OF MAMMALIAN SMOOTH MUSCLE TISSUE

Smooth muscle tissue from mouse urinary bladder, uterus, and gall bladder has been studied by means of the electron microscope. The smooth muscle cells are distinctly and completely separated from each other by a cytolemma comparable to the sarcolemma of striated muscle. The tissue is thus cellular and not syncytial. With this evidence, supported by electron microscopy of other tissues, we question the existence of true syncytia in animal tissues. Individual cell membranes necessary for the electrophysiologic events exist in smooth muscle, and its nerve and conduction in a tissue such as uterus or bladder can occur at the cellular level as well as at the tissue area level. The smooth muscle cell contains myofilaments, nucleus, endoplasmic reticulum, mitochondria, Golgi complex, centrosome, and pinocytotic vesicles. These structures are described in some detail, and their probable interrelations and functions are discussed. The autonomic nerves innervating smooth muscle cells are composed of axons and lemnoblasts. The axon is suspended by the mesaxon formed by the infolded plasma membrane of the lemnoblast. The respective plasma membranes separate axon and lemnoblast from each other and from surrounding muscle cells. The axons of autonomic nerves never penetrate the plasma membrane of the muscle cell, but pass or intrude into muscle cell pockets, forming a contact between axonal plasma membrane and smooth muscle plasma membrane. The lemnoblast shows well developed endoplasmic reticulum with Palade granules, mitochondria, and a long, elliptical nucleus. The axon contains neurofilaments, mitochondria, and synaptic vesicles; the quantity of the latter two being significantly greater in the periphery of lemnoblasts and near axon-muscle contact regions. We regard the contact regions as the synapses between the autonomic nerves and the smooth muscle cells.     

FORMATION OF BONE TISSUE IN CULTURE FROM ISOLATED BONE CELLS

A system is described for the formation of bone tissue in culture from isolated rat bone cells. The isolated bone cells were obtained from embryonic rat calvarium and periosteum or from traumatized, lifted periosteum of young rats. The cells were cultured for a period of up to 8 wk, during which time the morphological, biochemical, and functional properties of the cultures were studied. Formation of bone tissue by these isolated bone cells was shown, in that the cells demonstrated osteoblastic morphology in light and electron microscopy, the collagen formed was similar to bone collagen, there was mineralization specific for bone, and the cells reacted to the hormone calcitonin by increased calcium ion uptake. Calcification of the fine structure of the cells and the matrix is described. Three stages in the calcification process were observed by electron microscopy. It is concluded that these bone cells growing in vitro are able to function in a way similar to such cells in vivo. This tissue culture system starting from isolated bone cells is therefore suitable for studies on the structure and function of bone.     

ULTRASTRUCTURAL INTERRELATIONSHIPS OF NERVE PROCESSES AND SMOOTH MUSCLE CELLS IN THREE DIMENSIONS

The muscularis externa of the intestinal wall of frogs was fixed in osmium tetroxide, embedded in Vestopal-W, serially sectioned for electron microscopy, and stained with uranyl acetate. A method to obtain individually mounted and properly positioned serial sections is described. The three-dimensional techniques used during the course of this investigation demonstrate that it is possible to examine carefully relatively large areas of tissue on individual serial sections with the electron microscope and subsequently to construct montages of electron micrographs of pertinent areas from each section. Several carefully rendered interrelationships of nerve processes and smooth muscle cells in three dimensions are exhibited and described. Recent studies of other neuro-effector relationships are discussed in relation to the present status of the nature and organization of the autonomic nervous system in visceral organs.