两栖动物线粒体基因组结构特征分析

对2007年6月13日以前公布于GenBank上的78种两栖动物的线粒体基因组全序列进行了总结、比较和分析。78种基因组中基因的数量从35~41个不等;根据基因的数量、种类及其排列顺序的差别将其分为22种基因组类型,其进而聚为3组,其中类型4为两栖纲与其它脊椎动物的常见类型,类型8为两栖纲中现生3个目的公有类型。与类型4比较,其余21种线粒体基因组类型涉及基因变动的基因共有18个,其中变动比较多的是tRNA基因,移位、增多和缺失的发生频率都较大,而蛋白编码基因比较稳定,主要是移位。78种两栖动物中,蚓螈目的线粒体基因组均小于18000bps,多数在15000~16000bps;有尾目和无尾目均大于16000bps,其中有尾目多数在16000~17000bps,无尾目的多数在17000~18000bps。     

THE PERMEABILITY OF THE AMPHIBIAN OOCYTE NUCLEUS, IN SITU

Ultralow temperature radioautography, suitable for the quantitative localization of diffusible solutes, was used to study the permeability of the nuclear envelope in the intact amphibian oocyte Sucrose-³H solutions were injected into mature oocytes, in volumes of 0 016–0 14% of that of the cell, and the subsequent movement of the solute was recorded. The resultant radioautographs show diffusion gradients in the cytoplasm and nucleus, and concentration gradients across the nuclear envelope Analysis of these gradients discloses that the nuclear envelope is as permeable as a comparable structure composed of cytoplasm, and is about 10⁸ times more permeable than the oocyte plasma membrane The diffusion coefficient of sucrose in cytoplasm is 2 x 10^-6 cm²/sec, or about one-third its diffusivity in pure water. This reduction can probably be accounted for by an effective lengthening of the diffusional path because of obstruction by cytoplasmic inclusions. The nuclear: cytoplasmic sucrose concentration ratio at diffusional equilibrium is about 3 05, or 1.6 times as great as expected from the water content of the two compartments This asymmetry is attributed to an unavailability of 36% of the cytoplasmic water as solvent Finally, sucrose entry into oocytes from a bathing solution was monitored by whole cell analysis and radioautography. These and the microinjection results are consistent with a model in which sucrose entry into the cell is entirely limited by the permeability of the plasma membrane. The results are inconsistent with cell models that hypothesize a short-circuit transport route from the extracellular compartment to the nucleus, and with models in which cytoplasmic diffusion is viewed as limiting the rate of solute permeation.     

HETEROLACTIC GLYCOLYSIS IN THE SEGMENTING AMPHIBIAN EGG *

Bufo arenarum segmenting eggs have been found to exhibit, under anaerobic conditions, a glycolytic process of the heterolactic type, synthesizing 1.49 moles of lactic acid and 0.46 moles of alanine per mole of glucose broken down. These results would account for the lack of stoichiometry between glucose consumed and lactic acid synthesized reported by other authors. Experiments with radioactive glucose showed that the radioactivity recovered in alanine/radioactivity recovered in lactic acid ratio amounts to 6.2 when incubating after 3-hour anaerobiosis, and to 3.8 when eggs are incubated with radioactive glucose before submission to anaerobiosis. Results are discussed.     

佛波酯对蝾螈肌细胞分化的影响(英文)

佛波酯(TPA)是一种有效的皮肤癌促变剂。有不少实验事实指出,它的原始作用部位是质膜,而且对间隙连接的影响也已从各方面得到了证实。本文利用这种药物研究细胞通讯在肌细胞分化中的重要性。割取蝾螈神经胚中期躯干部体节中胚层,用组织培养的方法培养。实验组在培液(Steinberg+Leibovitz's L-15,9∶1)中加适量TPA,共试用了4种浓度,5、10、20和40ng/ml,以10ng/ml为适当,本文报道用这一浓度得到的结果。处理时间为4天,天天换新鲜配制的TPA溶液,连续三天,以后在正常培液中培养。对照组和实验组一样,在20℃下培养10—16天。比较对照组和实验组的结果,看到TPA的处理使预定体节细胞分化为肌细胞的过程受到了明显的阻抑。对照组在10天左右的培养中,可以观察到从预定体节细胞转变为成肌细胞,再融合为肌管。但是,实验组培养早期有一定量的脱落细胞,它们大多为不规则形,分散在外植块周围;还有一些不规则形的细胞,成片地自外植块铺展出来。不论是前者还是后者,在10天左右的培养过程中都未能表现进一步分化。除了看到一些梭形或长形的细胞夹杂在分散或成片的不规则细胞之间,没有找到过多核的肌管细胞。我们的实验结果结合过去形态的观察,说明细胞间通讯的时间性在肌细胞分化中是十分重要的。TPA处理的时间正相当于正常肌细胞分化中间隙连接大量存在并发挥作用的期间,很可能,TPA使间隙连接失去作用,处理之后再恢复正常培液,错过了时间,间隙连接的作用以后也不再能得到补偿。     

NATURE OF THE RETARDING INFLUENCE OF THE THYMUS UPON AMPHIBIAN METAMORPHOSIS

1. Though thymus-fed salamander larvæ often metamorphose normally, thymus feeding sometimes retards and in rare cases inhibits metamorphosis completely. 2. The addition of normal food to a thymus diet abolishes the inhibitory effect of the thymus. 3. Addition of a small quantity of iodothyrin leads rapidly to precocious metamorphosis of thymus-fed larvæ. 4. The inhibitory effect of the thymus is not due to a specific inhibiting substance in the thymus, but to the absence from the thymus of a substance required to develop the thyroid to the secretory state.     

THE GEOLOGICALLY YOUNGEST ALBANERPETONTID AMPHIBIAN, FROM THE LOWER PLIOCENE OF HUNGARY

Abstract:  The Albanerpetontidae are salamander-like, Middle Jurassic to Neogene lissamphibians from Laurasia and North Africa. Extensive series of albanerpetontid bones recently identified in collections from the Csarnóta 2 locality, south-central Hungary, extend the temporal range of the clade forward about seven million years from the middle Miocene to the early Pliocene. The Hungarian material is diagnostic for the Euramerican type genus Albanerpeton and pertains to a new species, A. pannonicus sp. nov., which differs from the seven previously reported congeners (Early Cretaceous–Miocene) in a distinctive combination of primitive and derived character states of the jaws and frontals, including a unique ventromedian keel on the azygous frontals. Some of the Hungarian specimens are articulated sets of skull bones, including ones containing the first three-dimensional examples of a nasal and jugals known for albanerpetontids, that help clarify some details of cranial osteology in these amphibians. Cladistic analysis nests A. pannonicus within the robust-snouted clade, as the sister taxon to an unnamed late Palaeocene species from Canada and A. inexpectatum from early–middle Miocene deposits in France, Austria and Germany. This phylogeny and recent reports of diagnostic Albanerpeton material from the Campanian of France and Maastrichtian of Romania suggest the evolutionary history of Albanerpeton was more complex than previously hypothesized, with Europe having played a larger role. The 25 fossiliferous layers at Csarnóta 2 record a shift from forest to grassland palaeoenvironments. Fossils of A. pannonicus are present in all layers, implying that this species was not adversely affected by the change in palaeoenvironments.     

纳络酮对东方蝾螈胚胎表皮传导的阻断作用

在胚胎发育的一定时期,表皮细胞呈现较强的β-内啡肽阳性免疫反应,而这时期正是表皮传导最活跃的时期。为了探索胚胎表皮传导和β-内啡肽-类阿片样多肽之间是否有关系,本实验采用纳络酮处理,发现表皮传导消失,待纳络酮作用消除后,表皮传导现象又再出现,说明纳络酮在胚胎表皮细胞传导中起了阻断的作用。     

一种在两栖类胚胎的免疫组织化学研究中有多种用途的方法

本文介绍了我们最近发展的一项用于两栖类胚胎的免疫组织化学研究的技术。两栖类胚胎经过适当的化学固定以后,用振动切片机可以得到50—100 μ的切片。我们用这样的切片进行免疫萤光和免疫酶标染色,均得到满意的结果,可以进行光镜(共聚焦显微镜,普通显微镜)及透射电镜的观察。由于在整个过程中避免了使用有机溶剂及包埋剂,所以最大限度地保存了抗原性。与传统的各种免疫组化技术比较,切片的各部分组织均能迅速与抗体反应,组织保存相当完好,可以满足电镜观察的要求。运用这种方法,还可以将同一胚胎的不同切片分别用于光镜和电镜观察,使结果更具说服力。     

THE EFFECTS OF ACTINOMYCIN D ON THE ULTRASTRUCTURE OF THE NUCLEUS OF THE AMPHIBIAN EMBRYONIC CELL

Observations have been made of ultrastructural modifications induced in the nuclei of differentiating amphibian embryonic cells cultured in the presence of Actinomycin D. Of particular interest are regions within the nucleus (regions otherwise rather empty) containing loose groupings of uniform threads having a diameter of around 200 A. These threads have been observed in continuous lengths up to 0.5 µ, and appear to be composed of subfilaments. It is suggested, after taking account of some recent work on lampbrush chromosomes, that these threads are lengths of uncoiled chromosome in a condition of heterosynthetic inhibition. It is further suggested that active and inactive portions of the genome may be distinguishable by the facility with which they can be induced to undergo this ultrastructural modification.     

两栖肢场的插值再生和图式调节

French-Bryant-Bryant的增生场图式调节机制被合理地作了修正,从而自洽地测决了同侧嫁接和异侧嫁接致生正常重肢的形成问题:(1)指出细胞插值生长和远向变换约束之间的平衡是一种自发对称性破损机制,会导致全区捷径插值被分区捷径插值局部地(在异侧嫁接)或整体地(在同侧嫁接)取代,分别在全区和分区极大周值差位置致生重肢.(2)关于重肢的手性和细胞来源的理论预测与实验结果吻合.(3)对于异常重肢的结构复杂性也提出了一种可行的解释.     

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.