Intranuclear and cytoplasmic annulate lamellae in the polyploid giant cells of the trophoblast

Intranuclear and cytoplasmic annulate lamellae in polyploid giant cells of the trophoblast have been studied in rat placenta on days 12--17 of development. The annulate lamellae are present in the cytoplasm within a limited time, being visible on day 12 only. These are arranged in bundles near the nucleus to be moving then to the cytoplasm. The end parts of annulate lamellae are broadened to make cisterns of rough endoplasmic reticulum. Unlike the cytoplasmic annulate lamellae, those found within the nucleus are seen in part of the nuclei investigated throughout the whole period examined to look as single structures (not gathered in bundles), they can be branching, separating closed spaces within the nucleus (making local swellings in the loci of branching; the latter having electron dense or transparent vesicles). Association with nuclear chromatin in some regions is a peculiar feature of the intranuclear annulate lamellae. This association is especially obvious at endoprophase in the cycle ofthe polytene nucleus during the somatic conjugation--chromonemes unite in a bundle and condense. Ultrastructural changes of the annulate lamellae is noted throughout the polytene nucleus cycle and during the cell differentiation. It is supposed that in the case of temporary labile chromosome polyteny in the nuclear cycle, which is characteristic of mammalian trophoblasts, annulate lamellae can well compare, in their function, with the synaptonemal complex--these prevent from too tight associations of homologues in the course of somatic conjugation of chromosomes.     

Intranuclear annulate lamellae in oocytes of the tunicate,Styela partita

Summary Intranuclear annulate lamellae have been observed with the electron microscope in oocytes of the tunicate, Styela partita. Morphological evidence suggests that the annulate lamellae may arise by a specialized fusion process of individual vesicles. Intranuclear vesicles appear to be formed, in time, before differentiated annulate lamellae. It is also suggested that the position and structure of an annulus is in large part determined by the fusion of the vesicles. An annulus may be present as soon as two vesicles have completed their fusion process. Finally, it is again suggested on the basis of morphological evidence that the intranuclear vesicles are derived by the blebbing activity of the inner layer of the nuclear envelope.This investigation was supported by grants (RG-9229, 9230) from the National Institutes of Health, Public Health Service. The electron microscope facilities used were also supported by a grant (GM-05479) from the National Institutes of Health to Professor H. W. Beams.     

Intranuclear and cytoplasmic annulate lamellae in grasshopper spermatocytes (Genus Melanoplus)

Intranuclear and cytoplasmic annulate lamellae were studied in grasshopper spermatocytes (Melanoplus) with the electron microscope. Although cytoplasmic annulate lamellae were observed in all three species examined, intranuclear annulate lamellae were found in only one species. The intranuclear annulate lamellae encompass certain nuclear material adjacent to the nuclear envelope forming a vesicle that is extruded into the spermatocyte cytoplasm. In this same species, cytoplasmic annulate lamellae are seen contiguous with granular masses of varying size. These structures were noted as being morphologically indistinguishable from the "yolk nuclei" of dragonfly oocytes (Kessel and Beams, 1969; Kessel, 1973).     

Long-distance interactions between Ehrlich ascites tumour cells.

In previous work, electron micrographs were made of adjacent surfaces of aldehyde-fixed, Ehrlich ascites tumour cells cultured on coverslips, after reacting some of their negatively charged surface sites with colloidal iron hydroxide (CIH) particles. It was observed that microvilli from one cell were aligned with intermicrovillus regions on another, where the density of the adsorbed CIH particles was significantly lower than in adjacent regions. Alignment, which was considered to represent interactions between the two peripheral cellular regions, took place when these regions were apparently separated by more than 200 nm, in an environment of physiologic ionic strength ( 0·145 m NaCl).In this communication we attempt to find feasible mechanisms for the alignment phenomenon in physical terms, in cases where the observed separation of 200 nm is correct, and in cases where the distances are overestimated due to preparative artifacts.It is concluded, that at distances of separation in excess of 200 nm, one feasible mechanism for alignment is that net negatively charged macromolecules diffusing out of cells in the region of their microvilli, electrostatically repel CIH-binding anionic sites in the lipid-rich “fluid” matrix of the periphery of the opposed cell, causing gaps in their distribution. The role of electrostatic and electrodynamic (van der Waals') forces in causing alignment is also discussed in terms of distance of separation.This communication is concerned with the interpretation in terms of various interactions, of electron micrographs showing evidence of alignment between microvilli from one cell with specific areas of another.     

Glycolysis and glutaminolysis in perifused Ehrlich ascites tumour cells

A perifusion system was designed in order to study glucose and glutamine metabolism by freshly harvested Ehrlich ascites tumour cells in steady state conditions. Cells were perifused in the presence of 5 mM glucose, 0.5 mM glutamine or 5 mM glucose and 0.5 mM glutamine. The results in steady state reveal that both substrates glucose and glutamine are continuously wasted by tumour cells, excreting two moles of lactate per mol of glucose and one mol of glutamate and ammonia per mol of glutamine consumed into the medium. Glutamine consumption in the presence of glucose was higher than with glutamine alone.     

Cytoplasmic 3H-arginine polypeptides of Ehrlich ascites tumour cells

Summary Low molecular weight ninhydrin positive peptide fractions of the Ehrlich tumour cell cytoplasm were isolated and characterized. After preliminary gel filtration of the cytoplasm on Sephadex G-25 column, the peptide mixture was fractionated on cationic exchanger SP-Sephadex C-25 column and eluted with increasing pH gradient. Five peaks were obtained. Only the first peak contained sugar component. All five peptides were studied with respect to molecular weight, isoelectric point and electrophoretic homogeneity. The cytoplasm of Ehrlich tumour cells contains one peptide of acidic (pI - 5.0), two slightly basic (pI - 7.7 and pI - 7.7) and two strongly basic nature (pI - 8.7 and pI - 8.9). Molecular weights varied from 8 500 to 18 500 daltons. The origin of these peptides is briefly discussed.     

Observations on the association of annulate lamellae with vinblastine-induced paracrystals in tumour cells in vitro.

Tumour cells in vitro responded to prolonged treatment with colchicine with the formation of annulate lamellae. Following the colchicine treatment, paracrystals were induced with vinblastine. A close association of annulate lamellae with vinblastine-induced crystals was observed. The hypothesis that annulate lamellae and the related unclear membrane with the pore complexes may play a role in the control of the cytoplasmic microtubule complex is discussed.     

Regulation of anaerobic glycolysis in Ehrlich ascites tumour cells.

1) In intact Ehrlich ascites tumour cells the anaerobic glycolytic flux rate and pattern of intermediates have been investigated at different pH values of the extracellular medium. 2) As predicted from the dependence of the lactic acid dehydrogenase equilibrium on pH a strong negative correlation between log ([lactate]/[pyruvate]) and pH has been found. 3) The steady state fluxes of glycolysis at pH 8.0 and 7.4 are rather equal, despite significant differences in the intracellular concentrations of glycolytic intermediates. At pH 8.0 the concentrations of ATP, glucose 6-phosphate, and fructose 6-phosphate are lower, and the concentrations of ADP, AMP, fructose 1,6-bisphosphate, triose phosphates, phosphoglycerates, and phosphoenolpyruvate are higher than at pH 7.4. 4) From the analysis of the pH dependent changes of metabolites it follows that different mechanisms are responsible for maintaining equal actual activities of hexokinase, phosphofructokinase and pyruvate kinase at pH 7.4 and 8.0. 5) From an application of the linear theory of enzymatic chains and a calculation of the control strength of the regulatory important enzymes results that hexokinase is evidently rate-limiting for glycolysis, and phosphofructokinase is also significantly influencing the glycolytic flux. Pyruvate kinase and glyceraldehyde phosphate dehydrogenase, on the other hand, do not significantly affect the rate of the overall glycolytic flux in ascites.     

Glucose regulates its own transport in Ehrlich ascites tumour cells

The capacity of Ehrlich ascites tumour cells to take up 2-deoxy-D-glucose and to bind cytochalasin B varies adaptatively with the level of glucose in the plasma or culture medium. The effect of glucose is exerted directly on the cells and does not necessarily require the participation of hormones such as insulin, glucagon or corticosterone, although glucagon and the glucocorticoids, but not insulin, can also increase the number of glucose carrier molecules administered in vitro. Cycloheximide suppresses the acute inductive effect of glucose, suggesting that protein synthesis might be required for the increased transport activity.     

NAD(P) redox compartmentation in Ehrlich ascites tumour cells

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1. Normoxia-anoxia transitions and use of glycolytic inhibitors and substrates have made possible a distinction between NAD(P)H redox changes in the mitochondrial and cytosolic compartments of ELD (Ehrlich-Lettré hyperdiploid) ascites tumour cells.     

Cytoplasmic annulate lamellae in human spermatogenesis

Summary Electron microscopic examination of normal human testicular tissue revealed annulate lamellae (AL) in the cytoplasm of primary spermatocytes and spermatids. AL of primary spermatocytes are encountered in the perinuclear region, parallel to the nuclear envelope and form single or multiple membranous profiles containing numerous annuli (500–600 Å in diameter) frequently associated with a fibrillar electron dense material. Spermatids contain numerous layers of AL either continuous with the nuclear envelope and caudal to the acrosome or peripherally positioned in the cytoplasm. Individual lamellae possess terminal dilations and display continuities with the endoplasmic reticulum. The interlamellar space in spermatid AL is entirely filled with a fine granular electron dense material. Additionally, the break-down of AL in spermatozoan residual bodies is indicated by a dilation of AL cisternae to form vacuoles following the dissolution of pore complexes.Supported in part by grant (AT-(40-1)-4002) from the U.S. Atomic Energy Commission     

Zinc-, copper- and cadmium-binding protein in Ehrlich ascites tumour cells.

The properties of Ehrlich ascites tumour cells exposed in vivo to cadmium were investigated as a function of the zinc status of the host animals. Tumour-cell growth was inhibited by cadmium in both zinc-sufficient and zinc-deficient animals. However, cells in zinc-sufficient tumours accumulate much less cadmium than those in deficient tumours. The subcellular distributions of cadmium and zinc do not depend on zinc status. Cadmium and zinc are bound to a low-molecular-weight protein with properties similar to metallothionein. Without exposure to cadmium, a zinc- and copper-binding protein is still present that behaves like a metallothionein. This protein can rapidly bind cadmium added to Ehrlich cells in vitro. It is shown that the zinc- and copper-binding protein contains free thiol groups. Ehrlich cells isolated from cadmium-treated animals are viable and show normal incorporation of uridine into RNA, but the cellular uptake of thymidine and its incorporation into DNA are inhibited.     

Quantification of ATP-producing and consuming processes of Ehrlich ascites tumour cells

ATP production of Ehrlich ascites tumour cells was estimated on the basis of their coupled respiration and lactate formation. ATP-consuming processes were assessed from the effects of selective inhibitors of RNA synthesis, protein synthesis and proteolysis, Na+/K+-ATPase on respiration. The extent of protein synthesis and proteolysis were also determined directly. From these values and those of the inhibition of respiration by selective inhibitors, a P/O ratio of 2.2 was calculated. About 75% of the total ATP consumption could be assigned to specific processes. The major ATP-consuming processes of tumour cells in an amino-acid-enriched medium, in which they are in an approximate steady state, are protein synthesis with about 30% of total ATP consumption, and Na+/K+-ATPase with about 20%, while RNA synthesis, ATP-dependent proteolysis and Ca2+-ATPase contribute about 10% each. In an amino-acid-free glucose medium, protein synthesis is reduced to a third, with a corresponding decrease of respiration, whereas the rate of the other ATP-consuming processes is unchanged.     

Interaction of calcium with mitochondria isolated from Ehrlich ascites tumour cells

Calcium ions are accumulated by intact mitochondria isolated from Ehrlich ascites tumour cells in a buffered system supplemented with ATP or succinate. In the ATP-supplemented system, the tumour mitochondria, in contrast to rat liver mitochondria, retain the accumulated Ca²⁺, do not exhibit a marked “irreversible” ATPase and do not swell. In the succinate-supplemented system, added Ca²⁺ stimulates respiration in either the absence or presence of added inorganic phosphate. Whereas respiration by rat liver mitochondria, measured in the presence of added phosphate, remains continuously activated after the addition of only a small amount of Ca²⁺, that by the tumour mitochondria can be stimulated by several successive additions of 100 μM Ca²⁺ and at all times exhibit appreciable activation ratios.