Electron microscopic study of mouse embryonic stem cell-derived cardiomyocytes

Differentiation of embryonic stem cell (ESC)-derived embryoid bodies (EBs) is a heterogeneous process. ESCs can differentiate in vitro into different cell types including beating cardiomyocytes. The main aim of the present study was to develop an improved preparation method for scanning electron microscopic study of ESC-derived cardiac bundles and to investigate the fine structural characteristics of mouse ESCs-derived cardiomyocytes using electron microscopy. The mouse ESCs differentiation was induced by EBs’ development through hanging drop, suspension and plating stages. Cardiomyocytes appeared in the EBs’ outgrowth as beating clusters that grew in size and formed thick branching bundles gradually. Cardiac bundles showed cross striation even when they were observed under an inverted microscope. They showed a positive immunostaining for cardiac troponin I and α-actinin. Transmission and scanning electron microscopy (TEM & SEM) were used to study the structural characteristics of ESC-derived cardiomyocytes. Three weeks after plating, differentiated EBs showed a superficial layer of compact fibrous ECM that made detailed observation of cardiac bundles impossible. We tried several preparation methods to remove unwanted cells and fibers, and finally we revealed the branching bundles of cardiomyocytes. In TEM study, most cardiomyocytes showed parallel arrays of myofibrils with a mature sarcomeric organization marked by H-bands, M-lines and numerous T-tubules. Cardiomyocytes were connected to each other by intercalated discs composed of numerous gap junctions and fascia adherences.     

Electron microscopic study of isolated proteoglycans]

Chemically isolated separate preparations of the non-aggregating protein-chondroitin-keratin sulphate (PCKS) fraction from the hyaline cartilage and hyaluronic acid (HUA) of the vitreous body and of the umbilicus were investigated by electron microscopy. PCKS and HUA in films without cytochrome c were present in the form of granules and differed by structural organization. The proteoglycans of the cytochrome c films were seen as finely-filamentous-cellular network, and were distinctly differentiated by their macromolecular organization. A mixture of both proteoglycans formed complexes as a result of a noncovalent interaction. Uranyl acetate ensured a good contrasting of proteoglycans, especially of PCKS, without cytochrome c.     

Electron microscopic study of the interaction between neoplastic fibroblasts and the substrate in tissue culture]

Electron microscope study of neoplastic L fibroblasts was carried out in 15, 30, 60, and 90 min after their attachment to solid substratum. Comparative analysis of neoplastic and normal fibroblasts at the same stages was carried out. Spreading rate of L fibroblasts proved slower than that of normal fibroblasts. Primary reaction of neoplastic cells was disturbed on the contact with substratum: it was morphologically manifested in changing of structure of the cell lower surface. Bundles of microfilaments were absent from the neoplastic fibroblasts' cytoplasm. The above changes, apparently, may entail the lesser degree of spreading and the weaker attachement of neoplastic fibroblasts to the substratum.     

Electron microscopic demonstration of cholinesterases in nervous tissue

Summary Acetylcholinesterase was demonstrated at ultrastructural level in the motor nerve cells of rat's spinal cord using the Karnovsky-Roots modification of Koelle's thiocholine method. Selective inhibitors were employed to check the validity of the reaction.Prolonged formaldehyde fixation improved the poor penetration of the reactive agents and diminished the relatively large crystal size of the end product, which were the two main difficulties of the method. The preservation of ultrastructure was highly improved, when thin sections were made without freezing using a tissue chopper.Acetylcholinesterase was localized in the nuclear envelope, in the rough-surfaced endoplasmic reticulum, in medium-sized vesicles of the Golgi apparatus, and around synaptic terminals. Synaptic vesicles were found negative.     

Electron microscopic characterization of chick embryonic skeletal muscle proteoglycans

In this article, proteoglycans from embryonic chick leg muscle are quantitatively and qualitatively compared with day 8 high density cell culture cartilage proteoglycans by electron microscopy of proteoglycan-cytochrome c monolayers. The visualized proteoglycan profiles were separated into four categories according to shape, size, and complexity. The two major categories were further characterized by lengths of core proteins, lengths of side projections, and distance between side projections. Two large proteoglycans are identifiable in spread leg muscle preparations. One group has a core protein (mean length of 205 nm) from which extend long thin side projections that we interpret to be groups of chondroitin sulfate glycosaminoglycans with a mean length of 79 nm. This large chondroitin sulfate proteoglycan is the only type found in muscle cultures as determined both biochemically in the past and now by electron microscopy and is referred to as muscle proteoglycan. The second large proteoglycan has a mean core protein length of 250 nm and side projections that are visibly shorter (mean length of 38 nm) and thicker than those of the muscle proteoglycan. This group is referred to as the mesenchymal proteoglycan since its biosynthetic origin is still uncertain. We compare these two profiles with the chick cartilage chondroitin sulfate proteoglycan that has a mean core protein length of 202 nm and side projections with a mean length of 50 nm. The data presented here substantiate the earlier biochemical characterization of these noncartilage proteoglycans and establish the unique structural features of the muscle proteoglycan as compared with the similar profiles of the cartilage and mesenchymal proteoglycans.     

Electron microscopic study of troponin

Troponin and its components or fragments were observed in an electron microscope by the use of the rotary shadowing technique. In freshly prepared troponin with low viscosity, globular particles were mainly observed. The size of the long axis of the particles was 13.2 +/- 1.3 nm and the size perpendicular to the long axis was 9.5 +/- 1.2 nm. The mean axial ratio was 1.4 +/- 0.3. Most of the particles observed in a stored troponin preparation, having a higher viscosity than that of fresh troponin, had a globular head with a thin tail, with the total length of 25.4 +/- 1.4 nm (head-tail type particles). The axial size of the globular portion was 8.3 +/- 1.2 nm and the tail length was 17.1 +/- 1.6 nm. Observation of various particles during the transitional stages indicated that, in the globular particles, the tail region of head-tail type particle was associated along the globular head region. Troponin T was a filamentous particle with 16.9 +/- 1.5 nm length. The 26K fragment of troponin T, which was devoid of the N-terminal 45 residues from troponin T, was a filamentous particle with the length of 14.4 +/- 1.3 nm. Troponin T1, one of two chymotryptic subfragments of troponin T, was a filamentous particle of 11.6 +/- 1.4 nm length. Troponin C.T in the presence of Ca2+ was a particle with a globular head (7 nm in size) and a tail of about 17 nm length. The Fab fragment of anti-troponin T1 formed regular transverse striations along the thin filament of rabbit skeletal muscle with a 38 nm period.(ABSTRACT TRUNCATED AT 250 WORDS)     

Statistical study of rapid mechanodependent cell movements in deformed explants of African clawed frog Xenopus laevis embryonic tissues

Computer analysis of artificially deformed (stretched or compressed) double explants (sandwiches) of the blastocoel roof (BRs) and suprablastoporal region (SBRs) of African clawed frog Xenopus laevis early gastrula has been performed using frames of time-lapse microfilming. During the first 14 min after cutting off, the velocities and displacement angles of several hundreds of cells relative to one another, as well as to fixed points and the extension axis, were measured in the control and deformed samples. It has been found that the deformation of samples leads to a rapid reorientation of large cell masses and increase in the velocities of movements along the extension axes or perpendicularly to the compression axes. In addition, an increase in the velocities of mutual cell displacements in the stretched BRs and cell convergence to the extension axes have been observed. Comparison of different angular sectors demonstrates a statistically significant positive correlation between the mean velocities of cell movements and the number of cells moving within an individual sector. This suggests cooperativity of mechanodependent cell movements. In general, these results demonstrate an important role of mechanical factors in regulation of collective cell movements.     

Electron microscopic study of chromatin in maturing lymphocyte nuclei using ammoniacal silver]

By means of the ammoniacal silver reaction, cytochemical properties of the chromatin in white rat lymph node lymphocytes were investigated at different stages of their maturation. Electron dense granules of the reaction product are shown to be localized over the compact chromatin region. The number of granules increases as the amount of compact chromatin enlarges. A possible role of arginine histones in the process of chromatin condensation is suggested, this suggestion being based on the assumption that ammoniacal silver binds with active arginine groups of histones.     

Electron microscopic study of DNA compactization under the effect of putrescine]

DNA-putrescine complexes were studied by electron-microscopy with the use of protein-free method. The latter gives the opportunity to investigate the interaction of DNA molecules spread on the surface layer of hypophase and the polyamine molecules in the thick layer of hypophase. Polyamine concentration varied from 5 x 10(-4) mM to 5 x 10(-1) mM. Under the low concentration of putrescine the complexes are represented by agglomerations of kinked knobbed fibres 10 to 20 nm thick, consisting of several fibres of duplex DNA. Upon increasing of putrescine concentration from 5 x 10(-4) to 1.5 x 10(-1) mM, the fibres become more thick (up to 25 nm), highly twisted and have the appearance of cylinders. Very often in the composition of complexes, it is possible to encounter the circular structures, which were formed at the expense of intermolecular interaction of different parts of the complex. The circular structures can serve as "embryos" of toroids of different sizes, that is of different degree of saturation with DNA and putrescine. At the concentration of putrescine 5 x 10(-1) mM the complexes have the appearance of toroids and structures on the basis of toroids, cylinders. The scheme of possible transitions of fibres of various thickness is proposed. The regularities of the compactization process, stimulated by polyamines, don't depend on the degree of compactization (the thickness of compacting fibre), that is they are similar for duplex DNA and for the fibres 25 nm thick, consisting of dozens of DNA molecules.     

Electron microscopic study of forest soil

Scanning electron microscopy was used to evidence the aggregated structure of a forest soil as well as the presence of fungal hyphae external to soil aggregates. The supernatant of soil suspension in water mainly contained isolated bacteria, while ultrathin sections of aggregates frequently revealed groups of bacteria surrounded by a sheath of mucilage with adhering clay minerals on the outside. These results confirm the existence of two particular biotopes in the soil studied: one is located inside aggregates, and the other, in the inter-aggregate spaces.     

Electron microscopic study of alpha-actinin.

Electron microscopic studies of the structure of purified α-actinin alone and in complex with F-actin have determined the molecular shape and size of this protein. α-Actinin molecules represent rods of about 300 Å in length and about 20 Å in diameter.     

Electron microscopic study of the innervation of the heart conduction system in rats]

A systemic quantitative electron microscopic analysis on innervation of the sinus node, the atrioventricular node, the bundle of His and its pedicles within the interventricular septum has been performed in intact hearts of mature rats. The data have been obtained on the size of nonmyelinated and myelinated nerve fibres, efferent and afferent terminals within different parts of the cardiac conductive system, their interconnection with specialized cardiomyocytes have been described. Application of certain methods for electron microscopic investigation on the innervation of mammalian cardiac conductive system has been discussed.     

Electron microscopic study of the division processes in cysts of the sarcosporidian, Sarcocystis, ovifelis

The metrocyte stage in the cysts of S. ovifelis has been found to divide not by endodyogeny, but by a different mode, not yet completely understood. The nucleus of the dividing metrocyte displays no nuclear envelope, the cytoplasm is full of large vacuoles. During the metrocyte division, some kind of nucleoplasm spreading occurs over the cytoplasm and between the large vacuoles, thus making any morphological limits between the cytoplasm and the nucleus invisible. The cytokinesis is accomplished due to the deep invaginations of the metrocyte pellicle. The metrocyte division first gives rise to a large multicellular (4--6) body lying in the peripheral zone of the cyst. The very impossibility of endodyogenetic division in the metrocyte stage may be due to its vaery peculiar morphofunctional organization, much different from what is characteristic of any asexual penetrative stage (= zoite) of Apicomplexa. The progeny of a metrocyte are interstitial cells which eventually, through the number of generations, evolve towards the metrozoite stage. Step by step, the interstitial cells aquire polarity due to the establishment of a conoid, rhoptries and micronnemes on the anterior pole, the pellicle becoming more rigid thus making, together with subpellicular microtubules, the cytoskeleton of the parasite. The study performed enables us to conclude that it is interstitial cells of advanced generations that divide by endodogeny. Merozoites being homologous to gamonts of other coccidia undergo no asexual division at all.