FERTILIZATION PROCESS IN THE HEART-URCHIN,CLYPEASTER JAPONICUS OBSERVED WITH A DIFFERENTIAL INTERFERENCE MICROSCOPE*

The observations of the fertilization process in the heart-urchin, Clypeaster japonicus with a differential interference microscope indicate that the sperm pronucleus is carried to the center of the egg by the growth of the sperm aster as stated by Chambers (5), and that the egg pronucleus is carried to the center of the aster by a filamentous structure formed between them. The curved path of egg pronucleus in the fertilized egg is interpreted as the combination of the movement of the center of the aster and the movement of the egg pronucleus toward the center of the aster. The movement and the rotation of the sperm head result from pushing by the tail being engulfed in the egg.     

花鲈消化道内分泌细胞的鉴别和定位

应用免疫组织化学Elivision二步法 ,用 5 羟色胺 (5 HT)、生长抑素 (SST)、胃泌素 (Gas)、胰高血糖素 (Glu)、胰多肽 (PP)等 5种抗哺乳动物血清对花鲈 (Lateolabraxjaponicus)消化道内分泌细胞 ,进行免疫组织化学定位的研究。结果表明 :5 HT和SST细胞分布于食管、胃贲门部、胃体部和胃幽门部 ,而肠道各段均未检出。Gas细胞分布于胃幽门部和前肠、中肠 ;食管、胃贲门部、胃体部和后肠未检出。Glu细胞分布于前肠和中肠 ,其余各段均未检出。PP细胞在消化道各段均未检出。本文将对花鲈消化道内分泌细胞的分布密度、分布型及形态进行描述和讨论。     

DEVELOPMENT OF ENDOTHELIA AND ERYTHROID CELLS IN THE MOUSE METANEPHROGENIC MESENCHYME IN CULTURE WITH THE FETAL LIVER

Histogenetic response in vitro of cells of the mouse metanephrogenic mesenchyme to different kinds of tissues was studied by means of transfilter induction technique. When the metanephrogenic mesenchyme obtained from 11-day mouse embryos was cultivated for 7 days in combination with the fetal liver or the primary differentiated hepatoma tissue, cell islets in which cells were arranged in a pavement-like or radial fashion, sinusoid endothelia and erythroid cells were induced in the culture, while in combination with the adult liver, no particular structures were. The number of the cell islets, which were absolutely absent in the initial culture, increased with time of the fetal liver-combined cultivation.
When the mesenchyme was cultivated for 7 days in combination with the spinal cord and simultaneously with the fetal liver, new structures which were somewhat different from but faintly reminiscent of tubules and glomeruli were formed. Such structures seemed to be intermediate in appearance between the tubules and the sinusoids, and were formed largely at the expense of normal development of cell islets, sinusoid endothelia, erythroid cells, tubules and glomeruli.     

THE RELATIONSHIP BETWEEN DIURNAL VARIATION OF THE NUMBER OF CELLS IN MITOSIS AND OF THE NUMBER OF CELLS SYNTHESIZING DNA IN THE EPITHELIUM OF THE HAMSTER CHEEK POUCH

1. The numbers of cells in mitosis and in DNA synthesis in the epithelium of the hamster cheek pouch have been studied at different times of the day and night. 2. By accumulation of mitotic cells using colcemid, both the rate of entry of cells into mitosis and the duration of mitosis have been estimated at two different times of day. 3. A diurnal variation has been demonstrated in both the mitotic index and in the tritiated thymidine labelling index. Although these variations are of different amplitude and timing, the experimental data fit closely to the hypothesis that the diurnal mitotic variation is the result of a partially synchronous population moving through the DNA synthetic period. No direct action on the mitotic process need be postulated. 4. From the results of mitotic accumulation, it is clear that the rate of entry of cells into mitosis depends on the time of day at which this is studied. There is also the possibility that the duration of mitosis is slightly longer when the mitotic index is high. 5. It is concluded that, at least in the epithelium of the hamster cheek pouch, the diurnal rhythm in the number of mitoses present is a reflection of the diurnal variation in the number of cells synthesizing DNA at some time earlier. Small fluctuations in the mitotic pattern imposed by this partially synchronous population moving from S into mitosis, could be caused by slight variations in the duration of mitosis.     

INCREASE IN SIZE AND CELL NUMBER OF LATERAL ROOT PRIMORDIA IN THE PRIMARY OF INTACT PLANTS AND IN EXCISED ROOTS OF PISUM SATIVUM AND VICIA FABA

Increase in cell number, and in anlage volume and length have been investigated during the development of lateral root primordia in roots of intact plants of Pisum sativum and Vicia faba and in excised roots of both species cultured in White's medium supplemented with 2% sucrose. With the exception of primordia in excised roots of Vicia, the general equation which best described increase in each aspect of primoridium growth measured against time was that for exponential growth. When the times necessary for cell number and primordium volume and length to double were determined at intervals over the period of development studied, however, they were found to vary. Similarly, estimates of the size of the proliferative fraction of cells at different times during anlage development indicated that this index of meristematic activity also fluctuated over the developmental period investigated, i.e., increase in cell number and in primordium volume and length do not occur in a truly exponential fashion as the primordia increase in size and cell number. One difference between anlage development in the roots of intact plants and in those grown in culture was that whereas the former primordia completed their development and emerged as lateral roots over the period of the investigation, the latter did not. Moreover, cell doubling time and anlage volume and length doubling times were longer, and the proliferation fraction of cells lower, over the whole period of, and at intervals during, primordium development in the excised roots compared with the results obtained for the roots of the corresponding intact plants.     

麦冬花药绒毡层和乌氏体的细微结构

麦冬(Ophiopogon japonicus)的绒毡层发育为分泌型。在小孢子母细胞时期,绒毡层细胞达到了发育的高峰。此时,绒毡层细胞中细胞器非常丰富,具大量线粒体、高尔基体和质体,尤以肉质网含量最多;原乌氏体出现较早,在小孢子母细胞时期绒毡层细胞中就已出现;四分体时期,大量原乌氏体被排入内切向面的质膜和纤维素壁之间;到了小孢子早期,绒毡层细胞失去细胞壁,原乌氏体分布在质膜的凹陷处,孢粉素物质在其上沉积,发育为乌氏体,乌氏体有单个和复合两种类型;当花粉成熟时,绒毡层细胞完全解体。     

THE NUMBER AND POSSIBLE FUNCTIONS OF DNA-SYNTHESIZING CELLS IN HUMAN BLOOD

The number of DNA-synthesizing cells in the blood of patients with various disorders was studied autoradiographically after incubation of blood in vitro with [³H]thymidine. The DNA-synthesizing cells were cytologically assigned to the following categories: erythroid, myeloid, lymphoplasmacytoid and unidentifiable (monocytoid or blast-like) cells. The following patient categories were studied: mitral valvular disease (samples obtained from peripheral vein, pulmonary artery and left auricle), ‘autoimmune diseases’(systemic lupus erythematosus, schleroderma, Hashimoto's thyroiditis, immunohaemolytic anaemia), patients with depressed haemopoiesis (aplastic anaemia, nitrogen-mustard induced bone-marrow hypoplasia) and with increased haemopoiesis (haemolytic anaemia, pernicious anaemia before and during initial vitamin-B₁₂ therapy, red-cell mass regeneration after haemorrhage or iron deficiency) and patients with bacterial infection. In all conditions studied, the number of labelled monocytoid and blast-like cells varied between 0 and 4/μl. Similarly, the number of labelled lympho-plasmo-cytoid cells was consistently low (0–8/μl) in all cases studied except two, where values of 37 and 63/μl were found. Both these patients had severe bacterial infections. The function(s) and potential(s) of these cells are discussed. The fate of the blast-like and monocytoid cells remains obscure. The lympho-plasmocytoid cells probably serve an immunological function, perhaps by disseminating immune responses. Whether or not some DNA-synthesizing cells in the blood are haemopoietic stem cells cannot be decided from the available evidence.     

RELATIONSHIP BETWEEN THE NUMBER OF DIVIDING AND NONDIVIDING CELLS OF CYANOBACTERIA IN NORTH ATLANTIC PICOPLANKTON1

In the North Atlantic over a wide geographic region that includes various oceanic regimes and a temperature range from 10 to 22° C, an increase in the number of nondividing Synechococcus cells (X) was generally accompanied by a greater-than-proportional increase in the number of dividing cells (Y). As a result, the fraction of dividing cells (FDC = Y · (Y + X)^?1) was positively related to population size (Y + X). Recognizing that FDC is generally greater in a rapidly growing population than in a slowly growing one, our empirical finding implies a positive correlation between specific growth rate and standing stock for Synechococcus. One notable exception occurred during winter (T < 5°C) in a eutrophic coastal embayment when a decrease in cell abundance was not matched by a decrease in FDC.     

MICROTUBULES IN THE FORMATION AND DEVELOPMENT OF THE PRIMARY MESENCHYME IN ARBACIA PUNCTULATA : I. The Distribution of Microtubules

Prior to gastrulation, the microtubules in the presumptive primary mesenchyme cells appear to diverge from points (satellites) in close association with the basal body of the cilium; from here most of the microtubules extend basally down the lateral margins of the cell. As these cells begin their migration into the blastocoel, they lose their cilia and adopt a spherical form. At the center of these newly formed mesenchyme cells is a centriole on which the microtubules directly converge and from which they radiate in all directions. Later these same cells develop slender pseudopodia containing large numbers of microtubules; the pseudopodia come into contact and fuse to form a "cable" of cytoplasm. Microtubules are now distributed parallel to the long axis of the cable and parallel to the stalks which connect the cell bodies of the mesenchyme cells to the cable. Microtubules are no longer connected to the centrioles in the cell bodies. On the basis of these observations we suggest that microtubules are a morphological expression of a framework which opeartes to shape cells. Since at each stage in the developmental sequence microtubules appear to originate (or insert) on different sites in the cytoplasm, the possibility is discussed that these sites may ultimately control the distribution of the microtubules and thus the developmental sequence of form changes.     

POLARITIES, CELL DIFFERENTIATION AND PRIMARY INDUCTION IN THE AMPHIBIAN EMBRYO

1. Amphibian eggs are spherical, while the embryos are bilaterally symmetrical. The latter is manifested morphologically when gastrulation begins with the formation of the blastopore at a bilaterally symmetrical (vegetal-dorsal) location on the surface of the embryo. To account for this change in symmetry two polarities (vectors or axes) are required. These need not go through the centre, but if they do, one will go through two poles, called ‘animal’ and ‘vegetal’ in the amphibian embryo, and the other will pass through two points on opposite sides of the egg, one at the ‘dorsal’ and one at the ‘ventral’ side. Together these two polarities define a plane of bilateral symmetry. 2. It may be assumed that one polarity determines that gastrulation begins in the vegetal hemisphere, and the other that it begins at the dorsal side. 3. Judging from the distribution of pigment in the cortex of the egg and that of the yolk-hyaloplasm in the interior, an animal-vegetal polarity is already present in the unfertilized egg. That cytoplasmic components are actually part of the material substrate of this polarity is evident from the fact that the pattern of gastrulation may be upset if the distribution of yolk-hyaloplasm is deranged. 4. At fertilization the pigment border is raised at the side opposite the fertilizing sperm, giving rise to the ‘grey crescent’. The latter confers the first visible bilateral symmetry on the egg, and in fact it determines the presumptive median plane, for blastopore formation begins in the midline of the grey crescent. The dorso-ventral polarity imposed by the sperm is not irreversibly determined. By various experimental means, e.g. restriction of the oxygen supply, it may be inverted. 5. In order to understand the mechanism of the polarities it is necessary to study the processes on which the effects of the polarities are exerted, viz. the process of invagination associated with the formation of the blastopore. It has been known for a long time that at the bottom of the blastoporal groove are located some large flask-shaped cells, called ‘Ruffini's cells’. Various arguments can be mobilized to support the notion that these cells actually are engaged in pulling in the embryonic surface. 6. These cells are the first representatives of a cell type different from the spherical cells which are typical of the early embryo. It may therefore be presumed that Ruffini's cells are the products of the first cell differentiation occurring during amphibian embryogenesis. And it may further be assumed that the polarities somehow control this process. 7. A number of observations suggest that the animal-vegetal polarity is in direct control of the differentiation, ensuring that Ruffini's cells are formed only in the vegetal hemisphere. This point has been corroborated by isolating in cultures small aggregates from various regions of the blastula. When this is done it is found that the only path of differentiation available to animal cells is the formation of small spherical aggregates composed of a mixture of ciliated and non-ciliated cells. In contrast, in cultures of vegetal cells an outgrowth of cells occurs, and these cells share a number of properties with Ruffini's cells, and it is suggested that they are representatives of this cell type. 8. The formation of these cells is suppressed by inhibitors of RNA synthesis and by anaerobiosis induced by KCN. Since oxidative metabolism is apparently required for the differentiation of Ruffini's cells - gastrulation in the intact embryo is suppressed by anaerobiosis - a number of carbohydrate metabolites were scrutinized for their effect on the formation on Ruffini's cells. It was found that at 10 mm lactate completely suppresses their appearance, and indeed all the other cell differentiations that can otherwise be observed in our cell cultures. Since there is a very steep animal-vegetal cytoplasmic gradient in carbohydrate, the content being lowest at the vegetal pole, lactate might potentially be the agent of the animal-vegetal polarity, but there are a number of facts which do not readily support this idea. 9. If animal cells are explanted together with a few vegetal cells, some of the aggregates do not become ciliated, but rather exhibit an outgrowth similar to the one observed with vegetal cells. These animal cells have the same general shape as the vegetal Ruffini's cells, but they are smaller and more pigmented, typical ‘animal’ features. When the cultures are preserved, the cells undergo further differentiation, becoming either ‘mesenchyme’ cells, nerve cells, pigment cells and sometimes even muscle cells may be observed. In the normal embryo these differentiation patterns occur in that part of the animal hemisphere which becomes induced through contact with the vegetal material entering the blastocoel during gastrulation. Thus there is reason to assume that the induction occurring in our cultures is a miniature of the normal induction process. 10. Just as in the sea-urchin embryo, the animal cells in amphibia may become ‘vegetalized’ by addition of Li⁺ to the culture medium. 11. For various reasons it is likely that Ruffini's cells contain heparan sulphate, and in the belief that this substance might be the inductor proper, its effect was tested on animal cells. It turned out that in a concentration of 0·1 ppm it can alter the differentiation pattern of these cells, and we suggest that heparan sulphate, for the time being, is the most likely candidate for the role of primary inductor in the amphibian embryo. 12. The edges of the blastoporal groove, and hence the formation of Ruffini's cells, proceeds gradually around the circumference of the embryo. The effect of the dorso-ventral polarity therefore appears to be concerned with the time at which the cells undergo differentiation, imposing a spatial and a temporal gradient on this phenomenon. The second overt manifestation of the dorso-ventral polarity, next to the formation of the grey crescent, concerns the size of the embryonic cells, the dorsal ones being always smaller than the ventral. This fact suggests the possibility that the polarity may exert its effect by interfering with the process of cell division. 13. The cell divisions in the early embryo are distinguished by being synchronous; all cells are either undergoing mitosis or they are in interphase. The duration of the latter is typically very short. After a certain number of cell divisions, around 10, when the embryos are in the mid-blastula stage, the synchrony is gradually lost, while the interphase becomes considerably prolonged. This peculiar behaviour suggests that the cytoplasm of the early embryonic cells contain some factor which ensures the synchrony. The well-known presence in the early embryo of deoxyriboside-containing material, in an amount corresponding roughly to the total amount of DNA residing in the cell nuclei after 10 cell divisions hinted that deoxyribosides might indeed be the ‘synchrony factor’. 14. This idea was tested first on intact embryos. An excess of deoxyribonucleotides was injected into very early embryos. The result was developmental arrest at a pregastrula stage (no Ruffini's cells formed) in a large percentage of embryos. However, the number of cells was greater than in the controls, and the rate of cell division higher, indicating a delay in the transition to synchrony, thus supporting the proposed mechanism. Furthermore, the deoxynucleotides inhibited cell differentiation and an explanation of this was found in the fact that they also strongly inhibited RNA synthesis. 15. The studies were extended to cell cultures. It was found that deoxyribosides inhibit the differentiation of animal as well as vegetal cells; instead, the cells go on dividing at least for another two rounds. The utilization of added deoxyribosides does not demonstrate that the endogenous substances are similarly utilized. That they are, was indicated by the following experiment: In the presence of cytosine arabinoside, an inhibitor of DNA synthesis de novo, the explanted cells go on dividing an unknown number of times, and then they, animal as well as vegetal cells, undergo differentiation. But in either case these cells are larger (about four times) than the controls. This result suggests that in the experimental cultures the cells go on dividing as long as the cytoplasmic deoxyribosides last and then stop, while the controls synthesize their own DNA for two rounds of division before they undergo differentiation. 16. It is now possible to suggest a mechanism for the dorso-ventral polarity. First it affects the cell size such that the dorsal cells are the smallest. If the cytoplasmic deoxyribosides are evenly distributed at the outset, then small cells must be nearer exhaustion than large ones. A dorso-ventral gradient in cell sue will therefore automatically imply a dorso-ventral gradient in the time at which the cells reach the state in which they can undergo differentiation.     

MESENCHYME CELLS IN STARFISH DEVELOPMENT: EFFECT OF TUNICAMYCIN ON THEIR DIFFERENTIATION, MIGRATION AND FUNCTION

The effect of tunicamycin, an inhibitor of protein glycosylation, on starfish development was investigated. Specific developmental events such as 1) bulging of the archenteron tip, 2) migration of mesenchyme cells, 3) formation of coelomic pouches and 4) mouth formation, are inhibited in the presence of this drug. These events are discussed in connection with differentiation, migration and function of mesenchyme cells. The possibility is discussed that tunicamycin exerts its effect by interfering with de novo synthesis of a cell surface factor(s) supporting dynamic cell surface activities.     

THE SYNTHESES OF TOTAL MACRONUCLEAR PROTEIN, HISTONE, AND DNA DURING THE CELL CYCLE IN EUPLOTES EURYSTOMUS

Increased alkaline phosphatase activity is induced in certain epithelial cell cultures by hormones with adrenal glucocorticoid activity or their analogues such as prednisolone (Δ^I-hydrocortisone). Enzyme induction occurs in two distinct phases. During the first 12 hr after the addition of prednisolone, there is a small increase in alkaline phosphatase levels. After 15 to 24 hr, the enzyme activity shows a sudden, marked linear rise, reaching a maximum at 60 to 80 hr. Puromycin blocks enzyme induction immediately, even when added during the period of rapid increase of enzyme. Actinomycin D blocks induction when added no later than 8 hr after the addition of prednisolone. On the other hand, Actinomycin D added during the phase of rapid enzyme induction has no effect for at least 12 hr. These findings suggest that de novo protein synthesis is involved in prednisolone induction of alkaline phosphatase and that the RNA messenger for this enzyme is relatively stable.     

上皮性卵巢肿瘤组织MKP-1及p-ERK1/2蛋白表达的研究

研究丝裂原活化蛋白激酶（mitogen activated protein kinase, MAPK） 相关蛋白丝裂原活化蛋白激酶磷酸酶（mitogen activated protein kinase phosphatase-1, MKP-1）和磷酸化细胞外信号调节激酶（phosphorylation extracellular signal-regulated kinases, p-ERK1/2）曲在上皮性卵巢肿瘤组织及正常卵巢组织中的表达差异,并探讨其在卵巢癌发生、发展中的作用,为卵巢癌的治疗提供新的思路及实验依据。选取64例上皮性卵巢癌、35例卵巢上皮性交界瘤及32例卵巢上皮性良性肿瘤患者的组织,另选取26例正常卵巢组织作对照,进行MKP-1及p-ERK1/2的免疫组化分析,并同时对其中部分病例进行上述蛋白的Western—blot研究。结果显示正常卵巢、良性肿瘤、交界瘤及卵巢癌组织MKP—1的表达依次递减,各组之间进行两两比较均有显著性差异（P〈0．01）,FIGOⅢ期与Ⅳ期卵巢癌组织MKP-1的表达显著低于Ⅰ期与Ⅱ期卵巢癌组织（P〈0．01）;而p-ERK1/2在正常卵巢、良性肿瘤、交界瘤及卵巢癌组织的表达依次递增．各组之间进行两两比较也均有显著性差异（P〈0．01）。FIGOⅢ期与Ⅳ期卵巢癌组织P～ERK1/2的表达显著高于Ⅰ期与Ⅱ期卵巢癌组织（P〈0．01）。且免疫组化及western—blot均显示MKP-1与p—ERK1/2在卵巢癌组织中的表达存在显著的负相关性,（r=-0．90,P〈0．01及r=-0．78,P〈0．01）。本研究结果表明MKP-1与p—ERK1/2的异常表达可能跟上皮性卵巢肿瘤的发生、发展有关,它们之间的表达失衡可能是卵巢癌发生、发展的原因之一．     

A MITOCHONDRIAL PUMP IN THE CELLS OF THE ANAL PAPILLAE OF MOSQUITO LARVAE

Mosquito larvae were raised to fourth instar in distilled water in order to maximally stimulate the salt-absorbing function of the anal papillae. Two exceptional features are observed, at the fine structure level, in the epithelial lining of the papillae. At the basal (cuticular) surface of the cells, the cell membrane is thrown into deep, narrow, parallel folds. The folds not only follow a rigid pattern at the cellular level but are also arranged at right angles to the long axis of the entire organ. A complicated pattern of canaliculi connects to the distal (plasma) surface of the cells. At this surface, paired mitochondria (sometimes triplets) are clamped about the membranes of the canaliculi to form structures referred to as "mitochondrial pumps." Mitochondria are also oriented in rather precise relation to the basal folds. Glycogen granules are found throughout the cytoplasm. The endoplasmic reticulum is sparse. The Golgi zones are flew and not well developed. Unidentifiable, irregular vesicles with lipid-like membranes are found associated with the basal folds. The distal (plasma) surfaces of the cells are covered by a homogeneous granular layer the composition of which is unknown.     

汉坦病毒在原代人胚肾与人胚肺细胞中增殖及其特性的研究

近年来研究发现,汉坦病毒能在多种细胞株及人体细胞中增殖、适应.国内学者用人胚肺二倍体细胞(2BS)体外适应该病毒成功,但有关原代人胚肺、肾细胞的报道尚少.作者选用两株病毒及两种原代人胚细胞用于体外感染、观察,应用免疫荧光间接法及胶体金包埋前染色电镜技术对宿主细胞中增殖的病毒进行了动态观察及特异性定位研究,现报告如下.1 材料和方法1.1 细胞的分离和培养1.1.1 人胚细胞:取正常孕妇5—7个月水囊引产胚肾及肺组织,按常规方法分散细胞,5—7天后长满单层.1.1.2 非洲绿猴肾上皮细胞(VeroE6):由安徽省医学科学研究所倪大石惠赠.1.2