Myotube-like structures in cultures of rat liver cells

Branching and budding myotube-like structures developed in primary cultures of rat liver cells and in the JH1 cell line derived from them. Elongated uninuclear cells aligned in chains and fused into multinuclear tubes of varying length and thickness. The tubes contained thick and thin filaments running in all directions. The filaments were occasionally linked with M lines and formed incomplete hexagonal patterns resembling those of skeletal muscle myofilaments, but a regular arrangement of the filaments and organelles was lacking. Cross-striation and contractions were never observed. Both uninuclear cells and multinuclear tubes contained numerous lysosomes, myelin figures and lamellated bodies as well as electronlucent or content-filled vacuoles and cisternae of variable size, sometimes reminding the sarcoplasmic reticulum in early stages of its development. Endocytotic caveolae and vesicles were present in all elements. These features together with interdigitated cell processes and specialized cell contacts suggested a possible relationship of the cells to the reticuloendothelial system.     

Ultrastructure of ascosporogenesis in Nannizzia gypsea.

Ascosporogenesis in Nannizzia gypsea was studied by electron microscopy. Development of ascospores began with the formation of an ascus vesicle composed of two paired unit membranes. Myelin figures consisting of coiled or concentric membranes were regularly connected with the growing ascus vesicle. Both the ascus vesicle and the myelin figures possessed an electron-dense line between paired membranes, and both were stained by the periodic acid-silver methenamine technique. Invagination of the ascus vesicle about the haploid nuclei resulted in eight uninucleate prospores bounded by two concentric membranes. Spore wall material was deposited between the two membranes of the prospores, and deposition was greatest in areas of the wall overlying stacked elements of endoplasmic reticulum. A single myelin figure surrounded by a polysaccharide halo was observed in the spore.     

Monomolecular surface film and tubular myelin figures of the pulmonary surfactant in hamster lung

Summary Perfusion fixation via pulmonary trunk was applied to the alveolar lining layer in situ at different lung volumes using a fixative containing tannic acid-ferrocyanide osmium. The monomolecular surface film and hypophasic tubular myelin figures were enhanced. In the range of transpulmonary pressure (1–10 cmH₂O), the surface film appeared in the form of a single, electron-dense leaflet, 2.7±0.6 nm (M±SD) in thickness while trilaminar membrane structure was retained in all parts of the tubular myelin figures of the hypophase. The surface film was attached underneath at right angles with trilaminar membranes which formed the outermost parts of the tubular myelin. Such structural continuity was taken to support a view that the phospholipid unit membrane of the tubular myelin figure would be transformed at the hydrophobic phase into a pair of monomolecular leaflets, eventually forming the surface film.     

Demonstration of bacterial antigen in macrophages in experimental pyelonephritis

The immunomorphological characteristics of interstitial macrophages with PAS-positive granules were studied in experimental Escherichia coli pyelonephritis in rats, using an anti-E. coli antibody. Immunohistochemical, immunoelectron microscopical, as well as light- and electron microscopical findings were compared at twelve time points between 2 days and 13 weeks after infection. Macrophages with PAS-positive granules were present in the inflammatory infiltrates from the 7th day. The granules were phagolysosomes, filled predominantly with myelin figures. The myelin figures originated mainly from the constituents of the bacterial wall and reacted with the anti-E. coli antibody even 13 weeks after infection. The storage of bacterial residues with preserved antigenic structures for several weeks after infection indicates disturbed phagolysosomal elimination of the bacterial substances in the PAS-positive macrophages. In the formation of macrophages with PAS-positive granules, lysosomal overloading with large amounts of bacteria and cell debris is assumed, leading to consumption of the lysosomal enzymes, consequent incomplete breakdown and retention of the bacterial residues.     

Chemically mediated mechanical expansion of the pollen tube cell wall

Morphogenesis of plant cells is tantamount to the shaping of the stiff cell wall that surrounds them. To this end, these cells integrate two concomitant processes: 1), deposition of new material into the existing wall, and 2), mechanical deformation of this material by the turgor pressure. However, due to uncertainty regarding the mechanisms that coordinate these processes, existing models typically adopt a limiting case in which either one or the other dictates morphogenesis. In this report, we formulate a simple mechanism in pollen tubes by which deposition causes turnover of cell wall cross-links, thereby facilitating mechanical deformation. Accordingly, deposition and mechanics are coupled and are both integral aspects of the morphogenetic process. Among the key experimental qualifications of this model are: its ability to precisely reproduce the morphologies of pollen tubes; its prediction of the growth oscillations exhibited by rapidly growing pollen tubes; and its prediction of the observed phase relationships between variables such as wall thickness, cell morphology, and growth rate within oscillatory cells. In short, the model captures the rich phenomenology of pollen tube morphogenesis and has implications for other plant cell types.     

Exocytosis Precedes and Predicts the Increase in Growth in Oscillating Pollen Tubes

We examined exocytosis during oscillatory growth in lily (Lilium formosanum and Lilium longiflorum) and tobacco (Nicotiana tabacum) pollen tubes using three markers: (1) changes in cell wall thickness by Nomarski differential interference contrast (DIC), (2) changes in apical cell wall fluorescence in cells stained with propidium iodide (PI), and (3) changes in apical wall fluorescence in cells expressing tobacco pectin methyl esterase fused to green fluorescent protein (PME-GFP). Using PI fluorescence, we quantified oscillatory changes in the amount of wall material from both lily and tobacco pollen tubes. Measurement of wall thickness by DIC was only possible with lily due to limitations of microscope resolution. PME-GFP, a direct marker for exocytosis, only provides information in tobacco because its expression in lily causes growth inhibition and cell death. We show that exocytosis in pollen tubes oscillates and leads the increase in growth rate; the mean phase difference between exocytosis and growth is –98° ± 3° in lily and –124° ± 4° in tobacco. Statistical analyses reveal that the anticipatory increase in wall material predicts, to a high degree, the rate and extent of the subsequent growth surge. Exocytosis emerges as a prime candidate for the initiation and regulation of oscillatory pollen tube growth.     

Fabrication of elastomer arterial models with specified compliance.

A simple way of making elastic tubes using a mechanical lathe for precise control of the wall thickness is proposed in this study. These tubes are particularly useful for modeling properties of large arteries. Tubes with different geometric parameters and hence different elastic behavior have been made with a silicon elastomer (Rhodorsil RTV 1556). They have been created to be used for compliance measurements in hemodynamics research. Within a limited range of pressures, depending on the wall thickness, such tubes can be used to study models in which the compliance value is assumed to be constant.     

ISOLATION AND CHARACTERIZATION OF LAMELLAR BODIES AND TUBULAR MYELIN FROM RAT LUNG HOMOGENATES

Three surface-active fractions which differ in their morphology have been isolated from rat lung homogenates by ultracentrifugation in a discontinuous sucrose density gradient. In order of increasing density, the fractions consisted, as shown by electron microscopy, primarily of common myelin figures, lamellar bodies, and tubular myelin figures. The lipid of all three fractions contained approximately 94% polar lipids and 2% cholesterol. In the case of the common myelin figures and the lamellar bodies, the polar lipids consisted of 73% phosphatidylcholines, 9% phosphatidylserines and inositols, and 8% phosphatidylethanolamines. In the case of the tubular myelin figures, the respective percentages were 58, 19, and 5. Over 90% of the fatty acids of the lecithins of all three fractions were saturated. Electrophoresis of the proteins of the fractions in sodium dodecyl sulfate or Triton X-100 revealed that the lamellar bodies and the tubular myelin figures differed in the mobilities of their proteins. The common myelin figures, however, contained proteins from both of the other fractions. These data indicate that, whereas the lipids of the extracellular, alveolar surfactant(s) originate in the lamellar bodies, the proteins arise from another source. It is further postulated that the tubular myelin figures represent a liquid crystalline state of the alveolar surface-active lipoproteins.     

Effect of pH on visualization of fatty acids as myelin figures in mouse adipose tissue by freeze-fracture electron microscopy

We studied the effect of pH on visualization of fatty acids as myelin figures in young mouse epididymal adipose tissue. Fatty acid content of the tissue was increased to 12.4 nmol/mg wet weight by treating the tissue with 380 microM isoproterenol at pH 7.4 for 15 min in the absence of glucose and albumin. Myelin figures were found in freeze-fracture replicas of isoproterenol-treated tissue fixed with glutaraldehyde at pH 7.4 and then incubated and glycerinated at pH 8.1. Myelin figures were seen in replicas as concave or convex laminated sheets and long cylindrical multilamellar structures in fat cells and extracellular space. Myelin figures were sometimes seen in cells extending from the surface of intracellular lipid droplets, the site of lipolysis, to the cell surface and extracellular space. Myelin figures were not found in isoproterenol-treated tissue fixed at pH 7.4 and processed at pH 7.0. Smooth-surfaced droplets, instead, were found in these tissues in the extracellular space. Neither myelin figures nor smooth-surfaced droplets were found in tissues treated with insulin and glucose (to reduce fatty acid content to 1.4 nmol/mg), fixed at pH 7.4 and processed at either pH 8.1 or pH 7.0. Lowering pH of the media to 4.5 during processing of tissues treated with isoproterenol at pH 9.0 caused disappearance of myelin figures and appearance of smooth-surfaced droplets in the extracellular space. Myelin figures were found in replicas of tissue treated with isoproterenol for 15 min at pH 7.4, incubated 10 min at pH 8.4, quick-frozen and then freeze-fractured, indicating that formation of myelin figures was not dependent on glutaraldehyde fixation and glycerol infiltration of the tissue. Our findings show that excess fatty acids in adipose tissue can be visualized as myelin figures if the tissue is exposed to pH 8.1-9.0 and maintained at or above pH 7.4, or as smooth-surfaced droplets if the tissue is processed at pH 7.0 or 4.5. We conclude that myelin figures formed under these conditions are composed primarily of partially ionized fatty acids (acid-soaps), and that the smooth-surfaced droplets in the extracellular space are composed of un-ionized (protonated) fatty acids.     

General tube law for collapsible thin and thick-wall tubes

Modeling the complex deformations of cylindrical tubes under external pressure is of interest in engineering and physiological applications. The highly non-linear post-buckling behavior of cross-section of the tube during collapse attracted researchers for years. Major efforts were concentrated on studying the behavior of thin-wall tubes. Unfortunately, the knowledge on post-buckling of thick-wall tubes is still incomplete, although many experimental and several theoretical studies have been performed. In this study we systematically studied the effect of the wall thickness on post-buckling behavior of the tube. For this purpose, we utilized a computational model for evaluation of the real geometry of the deformed cross-sectional area due to negative transmural (internal minus external) pressure. We also developed an experimental method to validate the computational results. Based on the computed cross-sections of tubes with different wall thicknesses, we developed a general tube law that accounts for thin or thick wall tubes and fits the numerical data of computed cross-sectional areas versus transmural pressures.     

Demonstration of bacterial antigen in macrophages in experimental pyelonephritis

The immunomorphological characteristics of interstitial macrophages with PAS-positive granules were studied in experimentalEscherichia coli pyelonephritis in rats, using an anti-E. coli antibody. Immunohistochemical, immunoelectron microscopical, as well as light- and electron microscopical findings were compared at twelve time points between 2 days and 13 weeks after infection. Macrophages with PAS-positive granules were present in the inflammatory infiltrates from the 7th day. The granules were phagolysosomes, filled pre-dominantly with myelin figures. The myelin figures originated mainly from the constituents of the bacterial wall and reacted with the anti-E. coli antibody even 13 weeks after infection. The storage of bacterial residues with preserved antigenic structures for several weeks after infection indicates disturbed phagolysosomal elimination of the bacterial substances in the PAS-positive macrophages. In the formation of macrophages with PAS-positive granules, lysosomal overloading with large amounts of bacteria and cell debris is assumed, leading to consumption of the lysosomal enzymes, consequent incomplete breakdown and retention of the bacterial residues. Presented in part at the 73rd Congress of the German Pathological Society, Mainz, October 1989     

ELECTRON MICROSCOPE STUDIES ON ENZYME ACTIVITY AND THE ISOLATION OF THIOHYDANTOIN-INDUCED MYELIN FIGURES IN RAT LIVER

Characteristic cytoplasmic inclusions (myelin figures), consisting of concentric multilaminar paired membranes surrounding one or more lipid bodies, were produced in rat liver parenchymal cells by incorporating high doses of an anticonvulsant agent (Bax 422Z) into the animals' diet. Enzymatic reaction product (presumably lead phosphate) was found around the central fat of these myelin figures in liver which had been fixed in glutaraldehyde, incubated in Wachstein and Meisel's medium containing adenosine triphosphate or inosine tri- or diphosphate, postosmicated, embedded in epoxy resin, and examined in the electron microscope. In an attempt to isolate myelin figures, fresh liver from medicated rats was homogenized and differentially centrifuged. Thin sections of osmium tetroxide-fixed, Epon-embedded pellets from each fraction were examined with the electron microscope. The concentric membranous whorls, which are probably derived from cisternae of the endoplasmic reticulum, broke up as the cells were disrupted and became inextricably mixed with the microsomal fraction. However, when liver previously fixed in formalin for 24 hours was homogenized, the myelin figures remained intact.     

Progressive white matter pathology in the spinal cord of transgenic mice expressing mutant (P301L) human tau

Transgenic mice expressing mutant (P301L) tau develop paresis, neurofibrillary tangles and neuronal loss in spinal motor neurons beginning at 4 to 6 months of age. Astrocytes and oligodendrocytes acquire filamentous tau inclusions at later ages. Here we report pathology in the spinal white matter of these animals. Progressive white matter pathology, detected as early as 2 months of age, was most marked in lateral and anterior columns, with sparing of posterior columns until late in the disease. Early changes in Luxol fast blue/periodic acid Schiff (LFB/PAS) and toluidine blue stained sections were vacuolation of myelin followed by accumulation of myelin figures within previous axonal tubes and finally influx of PAS-positive macrophages. Myelin debris and vacuoles were found in macrophages. At the ultrastructural level, myelinated axons showed extensive vacuolation of myelin sheaths formed by splitting of myelin lamellae at the intra-period line, while axons were atrophic and contained densely packed neurofilaments. Other axons were lost completely, resulting in collapse and phagocytosis of myelin sheaths. Also present were spheroids derived from swollen axons with thin myelin sheaths containing neurofilaments, tau filaments and degenerating organelles. Many oligodendrocytes had membrane-bound cytoplasmic bodies composed of tightly stacked lamellae capped by dense material. The vacuolar myelopathy in this model to some extent resembles that reported in acquired immune deficiency syndrome and vitamin B12 deficiency. The progressive axonal pathology is most consistent with a dying-back process caused by abnormal accumulation of tau in upstream neurons, while vacuolar myelinopathy may be a secondary manifestation of neuroinflammation.     

Model for calcium dependent oscillatory growth in pollen tubes

Experiments have shown that pollen tubes grow in an oscillatory mode, the mechanism of which is poorly understood. We propose a theoretical growth model of pollen tubes exhibiting such oscillatory behaviour. The pollen tube and the surrounding medium are represented by two immiscible fluids separated by an interface. The physical variables are pressure, surface tension, density and viscosity, which depend on relevant biological quantities, namely calcium concentration and thickness of the cell wall. The essential features generally believed to control oscillating growth are included in the model, namely a turgor pressure, a viscous cell wall which yields under pressure, stretch-activated calcium channels which transport calcium ions into the cytoplasm and an exocytosis rate dependent on the cytosolic calcium concentration in the apex of the cell. We find that a calcium dependent vesicle recycling mechanism is necessary to obtain an oscillating growth rate in our model. We study the variation in the frequency of the growth rate by changing the extracellular calcium concentration and the density of ion channels in the membrane. We compare the predictions of our model with experimental data on the frequency of oscillation versus growth speed, calcium concentration and density of calcium channels.     

IAA stimulates pollen tube growth and mediates the modification of its wall composition and structure in Torenia fournieri

The effects of several hormones on pollen tube growth were compared in Torenia fournieri and it was found that IAA was the most effective, stimulating pollen tube growth and causing the shank part of pollen tubes to be slender and straighter. The role of IAA was investigated by studying the changes in ultrastructure and PM H(+)-ATPase distribution in the pollen tubes and the modification of the tube wall. Using the fluorescent marker FM4-64, together with transmission electron microscopy, it was shown that secretory vesicles and mitochondria increased in IAA-treated tubes. Immunolocalization and fluorescence labelling, together with Fourier-transform infrared analysis, detected that IAA enhanced the level of PM H(+)-ATPase and the synthesis of pectins, and reduced the cellulose density in pollen tubes. Importantly, to observe the orientation of cellulose microfibrils in pollen tubes in situ, atomic force microscopy was used to examine the 'intact' tube wall. Atomic force microscopy images showed that cellulose microfibrils were parallel to each other in the subapical region of IAA-treated tubes, but disorganized in control tubes. All results provided new insights into the functions of cellulose microfibrils in pollen tube growth and direction, and revealed that the IAA-induced changes of pollen tubes were attributed to the increase in secretory vesicles, mitochondria, and PM H(+)-ATPase, and the modification of pectin and cellulose microfibrils in the tube wall.     

Growth rate of myelin figures of egg-yolk phosphatidylcholine

The growth length of myelin figures was measured in detail using a video tape recording system of optical microscopy. In the beginning stage of growth, the growth process was not adapted to the diffusion-limited process of lipid molecules, which has been recently proposed. Another interpretation for the growth mechanism was proposed, where the growth results from swelling. The initial growth rate measured was in good agreement with the estimated value in consideration with the water flux in the first approximation of the lipid concentration gradient.     

Fine Structure of Quiescent and Germinating Aeciospores of Cronartium fusiforme

Aeciospores of the long-cycle heteroecious rust fungus, Cronartium fusiforme, were found to have an extremely thick cell wall with striking spicules protruding from it. The wall was readily degraded by commercial chitinase, but spicules were unaffected. Quiescent spores contained two nuclei with distinct nuclear membranes possessing many pores. Numerous membrane-bounded lipid bodies were found both in wild-type orange and in white mutant aeciospores. An abundance of irregularly ovoid mitochondria was present in quiescent spores. After glutaraldehydeosmium fixation, the surface of the mitochondria appeared to be covered with ribosomes or microtubules in a paracrystalline array, whereas after permanganate fixation only smooth outer mitochondrial membranes were noted. The latter fixative revealed abundant vesicular endoplasmic reticulum in the spore. Spores incubated at 20 C on agar produced one to five distinct germ tubes within 65 to 180 min. These thin-walled tubes exhibited varying degrees of branching, and reached a total hyphal length of 300 to 500 mu prior to rupturing. Emergence of germ tubes took place through a pore in the spore wall and appeared to be mainly a physical flowing of cytoplasm from the spore into the germ tube without division of nuclei or other cell organelles. On completion of germination, the protoplasm of the germ tube contained both nuclei and nearly all of the other spore contents. Mitochondria had smooth outer membranes, were greatly elongated, and possessed distinct longitudinal cristae. A limited amount of rough endoplasmic reticulum was arranged parallel to the germ tube wall. Other organelles seen in germ tubes were lipid bodies, concentric membrane figures, and numerous ribosomes. Lipid bodies appeared smaller and fewer in number than in quiescent spores.     

The fatty acid composition of sphingolipids from bovine CNS axons and myelin

Abstract— Cerebrosides, sulphatides and sphingomyelin were isolated from bovine CNS myelin and from myelin-free axons derived from myelinated axons. The fatty acid composition of each sphingolipid was determined by gas-liquid chromatography of the fatty acid methyl esters. In each case the fatty acids of the axonal sphingolipids were of shorter average chain length than those from the corresponding myelin lipids. These differences, however, were small and the fatty acids of the axonal cerebrosides and sulphatides were similar in average chain length to those reported previously for bovine myelin. The principal unsubstituted acid of both cerebroside and sulphatide from axons was 24: 1, with the total long chain acids (> C₁₈) amounting to 80 and 85 per cent, respectively. The corresponding figures for myelin galactolipids were 94 and 95 per cent long chain acids. The principal α-hydroxy acid of both axonal galactolipids was 24 h:0, with cerebroside having 80 per cent and sulphatide 92 per cent long chain acids, compared to the figures of 87 and 97 per cent for the corresponding myelin lipids. In axonal sphingomyelin the major acid was 18:0 (compared to 24:1 in myelin) and the long chain acids were 61 per cent of the total vs 76 per cent of the total for myelin sphingomyelin. The non-identity of axonal and myelin sphingolipid fatty acids substantiates the belief that they are intrinsic axonal constituents. These findings do not rule out the possibility of a close metabolic relationship between the sphingolipids of the axon and its myelin sheath.     

Growth mechanism of myelin figures of phosphatidylcholine

The initial growth process of myelin figures, rod-like lyotropic liquid-crystalline structures, formed by phosphatidylcholine in water, ethylene glycol or glycerin, is suggested to be diffusion-limited with an apparent diffusion coefficient D of approx. 10(-6) cm2/s. D can be expressed by the sum of two processes. One is considered to describe the diffusion of an aggregate of phosphatidylcholine molecules and the other mainly to describe a lateral diffusion in the bilayer membranes which constitute myelin figures.