DNA synthesis decline involved in the developmental arrest of the limb buds in the embryos of the slow worm, Anguis fragilis (L.).

The present study was carried out to try and detect the biochemical mechanism involved in the developmental arrest of the limb bud in a serpentiform Reptile. Autoradiograpy, following tritiated thymidine incorporation, in embryos of the slow-worm (Anguis fragilis, L.) reveals a strong decrease in the rate of DNA synthesis in the mesodermal cells of the limb bud, after the degeneration of the apical ectodermal ridge (AER); the curve (a function of Gompertz) visualizing this decline shows that the drop in DNA synthesis becomes accentuated just after the degeneration of the AER. This decrease precedes the reduction of the mitotic index, the cell degeneration in the mesoderm and the other regressive changes occurring in the limb bud; it thus appears as the main causative factor of the developmental arrest of the limb bud. Furthermore, these results suggest that one of the functions of the AER would be to maintain a high level of DNA synthesis in the mesoderm underlying the AER in a normal limb bud.     

Kinematic analysis of drinking by the lacertid lizard, Lacerta viridis (Squamates, Scleroglossa)

The kinematics of drinking of Lacerta viridis were analysed. A drinking bout is composed of four phases: approach, immersion, emersion and withdrawal. The tongue and gravity are central to moving water through successive compartments of the buccal cavity and into the oesophagus. Upon the basis of formifunction analysis of water intake and transport, a kinematic model of drinking in lizards is proposed.     

Properties of cells from inverted embryos ofXenopus laevis investigated by scanning electron microscopy

Summary Xenopus embryos held inverted from the one cell stage show a partial reversal of the pattern of cleavage: the blastocoel forms towards the new upper pole, and the non-pigmented cells forming the blastocoel roof are smaller than normal endoderm cells. Two properties of the cells from inverted embryos have been studied: their capacity to form cilia when cultured for 48 h, normally a property of ectoderm cells; and their scanning electron microscopical appearance when isolated and cultured for shorter periods, which differs for normal ectoderm and endoderm cells. Groups of the upper, non-pigmented cells from inverted embryos do not form cilia in a longerterm culture, whereas groups of the lower, pigmented cells do. In contrast, the scanning electron microscopical appearance of the upper, non-pigmented cells of inverted embryos is more like that of normal ectoderm cells; the appearance of lower, pigmented cells is more like that of normal endoderm. Thus the determination to form cilia is not reversed by inversion, whereas the control of cell morphology is.     

Scanning electron microscopy of the aggregation of head mesoderm cells from chick embryos

Head mesoderm cells from chick embryos at different stages of development were dissociated and cultured on plastic coverslips. In all cultures several cellular aggregates were described by means of scanning electron microscopy. Isolated cells present filopodia and lamellipodia. However, when mesoderm cells make contact with one another the filopodial and lamellipodial activity in the contact cellular edge disappear. Thus, the cells into cellular clusters do not present projections. The clusters were circular and bidimensional in character. The scanning electron microscopic observations showed that it is the type 1 variant of "contact inhibition of locomotion" which occurs. By means of these mechanisms the bidimensional aggregates are formed and cellular overlapping is not present. Since the behaviour of the mesoderm cells "in vitro" in some way could be comparable to their behaviour "in situ", the results here observed are discussed in relation to the conduct of mesoderm cells "in vivo".     

Lectin teratogenesis. II: Demonstration of increased binding of concanavalin A to limb buds of rabbit embryos during the teratogenically sensitive period

The plant lectin concanavalin A (con A) causes malformations of rabbit embryos when 160 micrograms (in 40 microliter) are injected into the exocoelom on gestational days 12-15 but does not cause malformations on days 10-11. The purpose of this study was to investigate the mechanism for increased susceptibility of day 12-15 embryos to con A teratogenicity. Light microscopy of day 11 embryos 15-20 hr after treatment with con A revealed no observable difference from controls. Day 13 embryos at similar times exhibited limb buds with large areas that were denuded of ectoderm. Concurrent addition of alpha-methyl-D-mannoside (alpha MM), a specific inhibitor of con A, to the injection solution of day 13 embryos resulted in limb buds that appeared normal. The regions of con A binding to day 11 and day 13 embryos were visualized through epifluorescent microscopy of untreated embryos stained with fluorescein-labelled con A. Day 11 embryos exhibited moderate fluorescence on the surface of limb buds and the pericardial region. Day 13 embryos exhibited strong fluorescence of limb bud surfaces; the pericardial region remained moderately fluorescent. Addition of alpha MM to the incubation medium resulted in no fluorescence above background. Visualization of con A receptors was accomplished by ultrastructural analysis of forelimb buds stained with ferritin-labelled con A. Ferritin label was observed only on the surfaces of the ectoderm and was sparse over all regions of day 11 limb buds. In contrast, ferritin label was moderately heavy in all regions of the day 13 limb buds. No labelling occurred when the ferritin-labelled con A was preincubated with alpha MM. These observations indicate that the number of exposed con A receptors on limb buds of teratogenically sensitive embryos (day 13) is increased, compared with the number of exposed receptors on limb buds of younger, insensitive (day 11) embryos. The increased number of exposed con A receptors on limb buds during the teratogenically sensitive period provides not only increased binding of the lectin to sensitive embryos but also a potential mechanism for the anomalous attachment of distal regions of the limb buds to the body wall.     

An electron microscopic study of penetration by Trypanosoma rangeli into midgut cells of Rhodnius prolixus

The process of interaction of the Choachi strain of Trypanosoma rangeli with intestinal epithelial cells of Rhodnius prolixus was analyzed in experiments carried out in vitro and in vivo. For the in vitro experiments small fragments of the anterior region of the posterior midgut were incubated in the presence of the parasites, fixed, and processed for observation with the scanning electron microscope. Parasites attached to the surface of some epithelial cells, especially to the extracellular membrane layers (perimicrovillar membranes), were observed. For the in vivo experiments insects were infected with cultures of T. rangeli, sacrificed at different time intervals, and then processed for scanning and transmission electron microscopy. An intimate contact between the parasites and the membrane layers was observed. The parasites penetrated into cells that showed an electronlucent cytoplasm and a damaged surface, moved within the cytoplasm of the epithelial cell, reached the basal region, crossed the basal lamina, and entered the hemocoel.     

Prenatal diagnosis of genetic disorders of the skin by means of electron microscopy

Summary This paper summarizes the ultrastructural aspects of prenatal diagnosis of inherited skin disorders by means of electron microscopy on fetal skin biopsies obtained under fetoscopy. The investigation comprises skin samples of a series of 26 fetoscopies performed at the Departments of Gynecology at the Universities of Giessen and Lund. Among them, seven fetoscopies were performed for prenatal diagnosis in pregnancies at risk of epidermolysis bullosa or ichthyosis during the 19th and 22nd week of gestational age. Positive prenatal diagnosis was made in one fetus at risk of epidermolysis bullosa of unknown genetic type; this was based on dermolytic blister formation and collagenolysis diagnostic of EB dystrophica Hallopeau-Siemens. In a pregnancy with a fetal risk of bullous congenital ichthyosiform erythroderma, positive prenatal diagnosis was based on cytolysis and tonofilament clumping in the fetal epidermis and verified clinically and ultrastructurally after interruption; in another pregnancy bullous ECI was excluded ultrastructurally by the normal ultrastructural constitution of the fetal epithelium. In three fetuses at risk of the Herlitz syndrome and in one at risk of epidermolysis bullosa atrophicans inversa with pronounced sites of predilection, prenatal exclusion of the disorder was possible; this was based on the ultrastructural demonstration of the regular presence of normal hemidesmosomes with well-developed sub-basal dense plates at the dermo-epidermal junction in all four cases. One of these children has since been born and has normal skin; no scarring was found after birth on the sites of fetal skin biopsies.Possible application of this kind of prenatal diagnosis to other groups of genetic disorders is discussed in which biochemical defects are still unknown. Even more important is the possibility of excluding genetic disorders by demonstration of normal ultrastructural features in fetal skin biopsies and so avoiding abortion of healthy children.Awarded the Hans-Nachtsheim-Preis 1981In cooperation with R. Rauskolb and V. Jovanovic, Zentrum für Frauenheilkunde der Universität Giessen, 6300 Giessen, Federal Republic of Germany, and B. Gustavii, E. Cordesius, and L. Löfberg, Department of Gynecology, University of Lund, Sweden     

Analysis of replicating DNA molecules from embryos of the sea urchin, Hemicentrotus pulcherrimus, by electron microscopy

DNA was extracted from embryos of the sea urchin, Hemicentrotus pulcherrimus, at the S phase and examined by electron microscopy. We detected replication microbubbles with a mean size of 404 bases, in addition to replication macrobubbles of more than 1.0 kilobase (kb) in length. Seventy-five percent of the center-to-center distances of the microbubbles were 0.6-1.8 kb with a mean of 1.2 kb. Forty-five percent of the microbubbles were arranged as clusters of four or five microbubbles. These results suggest that at least 34% of the initiation sites for DNA replication are present on a DNA molecule in clusters in which the sites are arranged at 1.2-kb intervals.     

Acetylcholine's inhibitory effect on the calcium-dependent depolarization in the heart cells of a reptile (Lacerta sicula campestris). Preliminary notes.

The results of experiments performed with high - K+ solutions suggest that in the lizard atrial cells Ach normally displays its negative inotropic effects by a double mechanism = an "indirect" inhibition, mediated by the increase of the membrane permeability to K+ ions; and a "direct" inhibition of the Ca- carried slow inward current. In the ventricular fibres there isn't a direct-effect of this drug, under normal conditions.     

Role of mitochondrial swelling in the energetic effectiveness of intact myocardial cells (according to electron microscopy data)]

The myocardium of the left and the right cardiac ventricles was studied at various seasons on 20 rabbits. There was found a distinct seasonal dynamics of a number of quantitative indices of electronograms. In particular, there was found a relationship between the coefficients of energy efficacy of the mitochondria (integral value--calculated by multiplying the mitochondrial area by the number of mitochondrial cristae) and the mitochondrial area. With increase of the latter to 0,85 mc2 this relationship is positive and linear. With further swelling of the mitochondria this relationship disappears, however.     

Determination of bacterial cell number and cell volume by means of flow cytometry, transmission electron microscopy, and epifluorescence microscopy

Comparative measurements of bacterial total counts and volumes of flow cytometry (FCM), transmission electron (TEM), and epifluorescence microscopy (EFM), were undertaken during a four week mesocosm experiment. Total counts of bacteria measured by TEM, EFM, and FCM were in the range of 1 · 10⁶−6 cells ml⁻¹, 1 · 10⁶−3 · 10¹⁶ cells ml⁻¹, and 5 · 10⁵ cells ml⁻¹ respectively. The mean volume of the bacterial community, measured by means of EFM and TEM, increased from 0.12–0.15 μm³ at the start of the experiment to 0.39–0.53 μm³ at the end. Generally, there was good agreement between the two methods and regression analyses gave r = 0.87 (p < < 0.01) for cell volume and r = 0.97 (p < < 0.01) for cell number. DAPI stained bacteria with volumes less than 0.2 μm³ were not detected by flow cytometry and these were generally an order of magnitude lower than counts made by TEM and EFM. For samples where the mean bacterial cell volume was longer than 0.3 μm³, all three methods were in agreement both with respect to counts and volume estimates.     

Serial section reconstruction of the black yeast,Aureobasidium pullulans by means of high voltage electron microscopy

Summary Cultures ofAureobasidium pullulans were grown on a defined medium which enhanced hyphal growth over yeast-type growth. The hyphae at 24 hours postinoculation were prepared for high voltage electron microscopy (HVEM). Serial thick sections (0.5 m) were observed by means of HVEM operating at 1,000 kV. Tracings of cell walls and major cytoplasmic organelles were made on clear acetate sheets which were subsequently spaced at appropriate vertical heights in order to facilitate cell reconstruction. It was found that the majority of mitochondrial profiles were interconnected forming a highly reticulate mitochondrial network. No cells were found to contain a single mitochondrion, but several large mitochondrial networks were consistently located along the cell periphery and around nuclei and with only a few extensions into the central region of the cell.To a great extent vacuoles were connected by channels. A few individual small isolated vacuoles were also present. The vacuole system, unlike that of the mitochondria, was prominently located in the central region of the cell. Also located in the central region were from three to seven spherical nuclei which showed no indication of interconnections.