Membrane-bound Spaces Research Articles

Synthesis of Mn3O4 nanoparticles and their application to cancer cells

The Mn3O4 nanoparticles were synthesized by optimizing the precipitation system MnCl2/H2O2/NH4OH. The material thus obtained was microstructurally characterized using X-ray diffraction (XRD), Fourier transform infrared (FT-IR) spectroscopy and scanning electron microscopy (SEM). The Mn3O4 nanoparticles were applied to fibroblast and cancer cells. In the concentration range 25–100 μg/ml the particles showed low cytotoxic effects on L929 fibroblast cells, medium cytotoxic effects on FsaR fibrosarcoma cells and high cytotoxic effects on SCCVII carcinoma cells. N-Acetyl-L-cysteine (NAC) increased cytotoxicity of Mn3O4 nanoparticles, which suggested that reactive oxygen species (ROS) were not responsible for their selective cytotoxic effect. It was shown that the Mn3O4 nanoparticles entered the cancer cells and aggregated inside the membrane-bound spaces that were recognized as destroyed mitochondria.

Changes in features of degenerating primary sensory neurons with time after capsaicin treatment.

Capsaicin (50 mg/kg) was injected into new born mice and 5 and 12 h, and 1, 2, 3, and 5 days later, their lumbar dorsal root ganglia (DRG) with the nerve roots were fixed by immersion. The morphological changes which ensued with time after treatment were examined by light and electron microscopy. The findings were as follows: (a) rapid degeneration of certain smaller B-type neurons, indicating their prompt death, was seen 5 h after treatment. Later, accumulated neurofilaments appeared in larger B-type neurons. Fissures of the cytoplasm and cell fragmentation were also observed as particular features of degeneration. Finally, these degenerating neurons, destined to die, appeared as small round figures with a disorganized nucleus. Severely degenerated neurons were seen throughout the survival time after treatment, but seemed to be most numerous after 2-3 days. (b) Three days after treatment the Nissl substances of large A-type neurons appeared dispersed, forming ring-like bundles in the periphery of cells. Cytoplasmic rupture and large membrane-bound spaces with fine granular or fibrillar materials, indicating peripheral cytolysis, were also conspicuous. Some of these cells showed severe degeneration clearly leading to cell death. The A-type neurons began to degenerate later than the B-type neurons. (c) Satellite cells showed an increased amount of electron-opaque cytoplasm that contained large vacuoles and neuronal cell debris. Mitotic figures were increased in satellite cells 3 days after treatment. (d) Unmyelinated axons in the dorsal root of mice treated with capsaicin became enlarged with accumulation of neurofilaments, synaptic vesicles or various kinds of vesicles, multivesicular bodies and mitochondria. Numerous dense lamellar bodies appeared in the unmyelinted axons within DRG 3 days after treatment, but were scarcely seen in the dorsal roots. Degeneration of the myelinated fibers increased with time. Interestingly, capsaicin seemed to have both a direct and indirect action on DRG neurons: its direct action induced rapid degeneration of the smaller neurons, whereas its indirect action induced relatively slow degeneration of the larger neurons, causing chromatolytic changes similar to those induced by periphal nerve axotomy. The injury to DRG neurons due to the indirect action seemed to be induced retrogradely.

Molecular immunocytochemistry of the CuZn superoxide dismutase in rat hepatocytes.

The distribution of CuZn superoxide dismutase (SOD) molecules in subcellular organelles in rat liver hepatocytes was studied using integrated biochemical, stereological, and quantitative immunocytochemical techniques. A known concentration of purified CuZn SOD in 10% gelatin was embedded alongside the liver tissue for the calculation of CuZn SOD concentrations in hepatocyte organelles and total CuZn SOD in the rat liver. Most of the CuZn SOD was located in the cytoplasmic matrix (73.1%) and in the nucleus (11.9%) with concentrations of 1.36 and 0.71 mg/cm3, respectively. Lysosomes contained the highest concentration (5.81 mg/cm3). Only low concentrations were measured in mitochondria (0.21 mg/cm3). Membrane-bound spaces of rough endoplasmic reticulum (ER), smooth ER, and the Golgi system did not contain significant concentrations of the enzyme. By adding up the concentrations in all subcellular compartments, a total liver content of CuZn SOD was established from the immunocytochemical measurements (0.386 +/- 0.028 mg/gm liver) that agreed closely with those obtained by biochemical assays (0.380 +/- 0.058 mg/gm liver). The average distances between two CuZn SOD molecules can be calculated from enzyme concentrations. It was determined that CuZn SOD molecules in the cytoplasmic matrix and nucleus were 34 and 42 nm apart, respectively. In peroxisomes and mitochondria, average intermolecular distance increased to approximately 60 nm and increased to 136 nm in smooth ER. CuZn SOD is a relatively abundant protein in the cytosol of hepatocytes and its distribution overlaps with major sites of O2- production. The efficiency of protection CuZn SOD can provide to cytosolic proteins from attacks by superoxide anion depends on the rate of O2- production, distribution of CuZn SOD relative to cytosolic proteins, and the relative reaction rates between O2- with both cytosolic proteins and CuZn SOD. Future studies of these substrate-enzyme relationships in vivo can lead to a greater understanding of how cells handle oxidant stress.

Chapter 6 Protein Sorting in the Secretory Pathway

Publisher SummaryThis chapter focuses on protein sorting in the secretory pathway. From primary and secondary biosynthetic sites in the cytosol and mitochondrial matrix, respectively, proteins and lipids are distributed to more than 30 final destinations in membranes or membrane-bound spaces, where they carry out their programmed function. Molecular sorting is defined, in its most general sense, as the sum of the mechanisms that determine the distribution of a given molecule from its site of synthesis to its site of function in the cell. The final site of residence of a protein in a eukaryotic cell is determined by a combination of various factors, acting in concert: (1) site of synthesis, (2) sorting signals or zip codes, (3) signal recognition or decoding mechanisms, (4) cotranslational or posttranslational mechanisms for translocation across membranes, (5) specific fusion–fission interactions between intracellular vesicular compartments, and (6) restrictions to the lateral mobility in the plane of the bilayer. Improvements in cell fractionation, protein separation, and immune precipitation procedures in the past decade have made them possible. Very little is known about the mechanisms that mediate the localization and concentration of specific proteins and lipids within organelles. Various experimental model systems have become available for their study. The advent of recombinant DNA technology has shortened the time needed for obtaining the primary structure of proteins to a few months.

Occurrence of Nuclear Vacuoles in Meiotic Prophase Nuclei in Compositae

SUMMARYThe occurrence of nuclear vacuoles (membrane-bounded spaces) in the meiotic prophase nuclei of pollen mother cells in 23 species of Compositae was examined by electron microscopy. In nuclei of all examined species, nuclear vacuoles were recognized. These nuclear vacuoles were classified into four types by their size and appearance; 1) nuclear vacuoles which consist of many elongated tubes, 2) nuclear vacuoles which are large in size and few in number, 3) nuclear vacuoles which consist of elongated thin tubes with rather constant diameter, and 4) nuclear vacuoles which consist of few and thin elongated tubes. Nuclear vacuoles seemed to be a characteristic feature of each genus without any clear intergenus taxonomic relationship. Nuclear vacuoles were found not only in pollen mother cells but also in embryo sac mother cells of two examined species, suggesting that nuclear vacuoles are specific structures in meiocytes and that they are involved in the meiotic process though the nature of their content...

Capacity of enucleated amoebae to recognize and respond to heterologous cytoplasm.

When amoebae are injected with heterologous cytoplasm from another type of amoeba, cellular organelles are segregated and degenerate within membrane-bounded spaces in the host's cytoplasm. In the present study, the role of the nucleus in this response was tested by injecting heterologous cytoplasm from Pelomyxa carolinensis into enucleate and enucleate Amoeba discoides. Samples were prepared for ultrastructural study at intervals between 15 min and 2d after the operation. In enucleate recipients of heterologous cytoplasm, organelles and cellular debris were observed in vacuole-like spaces indistinguishable in morphology and frequency from those of nucleated recipients. When donor cytoplasm included thoria particles, the electron-dense tracer was found along with degenerating organelles in the vacuole-like structures, confirming the presence there of injected cytoplasm. changes of comparable magnitude were not observed when either nucleated or enucleated cells were injected with homologous cytoplasm. The results indicate that nuclear activity is not necessary for the ultrastructurally observable responses of amoebae to heterologous cytoplasm. It is suggested therefore that the cytoplasm possesses a mechanism for recognition of at least some types of heterologous organelles.

Fine structure of the modified sertoli cells in the terminal segment of the seminiferous tubules of the bull, ram and goat

The ultrastructure of the epithelial cells in the terminal segment of the seminiferous tubules was studied in the bull, ram and goat. These cells were characterized by membrane-bounded spaces and intercellular dilatations, and abundant microtubules and microfilaments. The presence of vacuolated nucleoli and typical inter-Sertoli cell junctions in the cells of the terminal segment confirms the concept that these cells are actually modified Sertoli cells. Ligation of the efferent ductules in the bull for 24 h had no harmful effect upon the blood-testis barrier in the terminal segment.

Intracellular distribution of lead in Tetrahymena during continuous exposure to the metal.

Lead acetate (0.1--0.2%) forms a precipitate with the organic growth medium. The Tetrahymena cells ingest this lead-containing precipitate and cell growth is resumed after a variable lag period. Ingested lead is observed as electron-dense material in food vacuoles. Soon after exposure, cytoplasmic lead (preserved with certain fixation only) is revealed as electron-dense particles in cilia and in a halo around digestive vacuoles. Later the lead particles pervade the entire cell but after the lag period they are confined to membrane-bound spaces. In dilute growth medium, high concentrations of lead inhibit food-vacuole formation and cell growth. Under these conditions lead is deposited in alveoli of the pellicle and is also found in autophagic vacuoles and other membrane-limited structures. The study has revealed that lead enters Tetrahymena through the membrane of digestive vacuoles and through the cell surface. The change in distribution of lead during the lag period indicates that a mechanism is activated for removal of lead into membrane-bound spaces. The final storage of lead seems to be in lysosomes.

Membrane-bounded spaces in the cytoplasm of normal pig hepatocytes

The appearance of light microscopically visible vacuoles in hepatocytes from pigs 1, 3, 5, and 8 weeks old are described both after immersion and perfusion fixation. The vacuoles could not be demonstrated in hepatocytes from 1-day-old pigs. Ultrastructurally the vacuoles are demonstrated as membrane-bounded spaces (MBS) in the cytoplasm, mostly situated in the vascular pole of the hepatocytes. Some of the MBS are shown to communicate with the space of Disse and, less frequently, with the intercellular space. Ultrastructurally the MBS are also demonstrated in hepatocytes from 1-day-old pigs, but they are few, small, and only localized in the vicinity of the microvilli. By elevating the perfusion pressure all MBS are dilated and the communications with the cell surface are widened. The MBS are interpreted as invaginations of the plasma membrane, which increase the surface of the cells and probably facilitate the exchange of nutrients and metabolic products.

The Nucleus in the Spores of Rhinosporangium seeberi

Rhinosporidiosis is a disease occurring usually in the tropics and is characterized by the production of nasal polyps supposedly caused by a fungus, Rhinosporangium seeberi, which was claimed after light microscopic studies to proliferate by “amitotic” division of the nucleus. The organism was studied electron microscopically and the spores were found to be bound by a non-collagenous wall enclosing a variety of vesicles, some containing smooth amorphous material, others coiled threads, and still others dark granules. Typically mitochondria, endoplasmic tubules, Golgi, and a membrane-bound nucleus were not seen although a few rudimentary membrane-bound spaces are present. These features as well as failure to culture the organism cast doubts about its taxonomic position, and it seemed the organism was essentially some nucleic acid complex organizing mucopolysaccharides in subepithelial zones in the nasal mucous membrane.

Ultrastructure of a cardiac myxoma.

A cardiac myxoma of the left atrium was studied by means of the electron microscope. A single cell type was seen, occurring predominantly as closely-apposed groups of 3 to 6 cells, and characterized by extensively anastomosing microvilli forming peripheral intracytoplasmic membrane-bound spaces (some of which may have been the result of cellular secretory activity), prominent linear and parallel roughsurfaced endoplasmic reticulum, variable electron-dense intracytoplasmic deposits, and poorly developed and sparse mitochondria and Golgi apparatuses. No fibroblasts, smooth muscle cells, or vascular channels lined by endothelial cells were present, although these have been reported in some of the earlier electron microscopic studies of cardiac myxomas. An endocardial subendothelial reserve cell origin of this tumor appears most likely.

Digestion and the distribution of acid phosphatase in Blepharisma.

Suspensions of Blepharisma intermedium were fed latex particles for 5 min and then were separated from the particles by filtration. Samples were fixed at intervals after separation and incubated to demonstrate acid phosphatase activity. They were subsequently embedded and sectioned for electron microscopy. During formation of the food vacuole, the vacuolar membrane is acid phosphatase-negative. Within 5 min, dumbbell-shaped acid phosphatase-positive bodies, possibly derived from the the acid phosphatase-positive Golgi apparatus, apparently fuse with the food vacuole and render it acid phosphatase-positive. A larger type of acid phosphatase-positive, vacuolated body may also fuse with the food vacuole at later stages. At about 20 min after formation, acid phosphatase-positive secondary pinocytotic vesicles pinch off from the food vacuoles and approach a separate system of membrane-bounded spaces. By 1 hr after formation, the food vacuole becomes acid phosphatase-negative, and the undigested latex particles are voided into the membrane-bounded spaces. The membrane-bounded spaces are closely associated with the food vacuole at all stages of digestion and are generally acid phosphatase-negative. Within the membrane-bounded spaces, dense, pleomorphic, granular bodies are found, in which are embedded mitochondria, paraglycogen granules, membrane-limited acid phosphatase-containing structures, and Golgi apparatuses. The granular bodies may serve as vehicles for the transport of organelles through the extensive, ramifying membrane-bounded spaces.

The fine structure of fibrillar components and plasma membrane contacts in esophageal myoepithelium of Ascaris lumbricoides (var. suum)

Esophageal myoepithelium of Ascaris consists of syncytially arranged cells penetrated by deep plasmalemmal infoldings. Myofibrillae are transversely oriented to the long axis of the esophagus as bundles of thin (35–75) A and thick (140–175) A myofilaments. Nonmyofibrillar regions of the cytoplasm contain mitochondria, endoplasmic reticulum, and bundles of tightly packed, filaments 75–100 A thick. The myoepithelial plasmalemma is deeply invaginated and forms extensive cavernous, membrane-bounded spaces within the cytoplasm. The invaginated plasmalemmae come into “close” junctional contact forming two, morphologically distinct types of contact in the myofibrillar regions. One is oriented transverse to the long axis of the myofibrils and is similar in morphology to intercalated disks of cardiac muscle. The other is oriented longitudinally with respect to the long axis of the myofibrils and is similar in morphology to “tight junctions” of electrotonic transmitting synapses and between some vertebrate smooth muscle cells. The significance of the above findings is discussed in terms of the function of the two types of fibrillar components and membrane contacts found in the myoepithelium.

Morphological alterations in mammalian neurons in vitro in response to hypertonic solutions

Neurons in cultures of central nervous tissue exhibited marked structural changes when exposed to hypertonic solutions. Cellular reactions were described in living neurons as well as after fixation and staining in preparations observed with both the light and electron microscope. The structures involved in these changes were mainly the nucleolus, the nucleus and the Nissl substance. In living neurons, observed with phase contrast optics, the nucleolus became invisible in hypertonic medium. This change occurred within a few seconds, and it was reversible when the cells were brought back to isotonic solutions. Fixation of the cells while exposed to hypertonic solution caused the nucleolus to reappear as a granular body. In stained preparations it appeared as a more irregular body in contrast to the smoothly outlined nucleolus in normal cells. In electron microscopic preparations of neurons which were fixed while exposed to hypertonic solutions the nucleolus was visible only as “nucleolar shadow”, overlaid by a few small irregular bodies of higher electron density than other nuclear contents. The nuclear membrane of living neurons exposed to hypertonic media lost much of its sharp definition and became rather hazy in outline. The nuclear diameter increased about 10% in hypertonic medium, and the nuclear space became somewhat denser when observed with the phase contrast microscope. In Nissl stained preparations the nuclear space was filled with many small granular or rod-shaped bodies in contrast to the clear vesicular appearance of the nuclei of untreated cells. In electron microscopic preparations the nuclear space exhibited a spotty appearance due to the presence of electron dense and light areas. In living neurons immersed in hypertonic solutions the Nissl substance showed a slight increase in phase density, especially after repeated changes between hypertonic and isotonic solutions. Sometimes a distinct striation in the Nissl substance appeared. In Nissl stained preparations there was no marked change observed in comparison with normal cells. However, in the electron microscope, the Nissl substance of hypertonically treated cells exhibited a marked structural change. The membrane-bound spaces of the endoplasmic reticulum assumed a rather precise orientation parallel to the cell membrane so that in extreme cases a concentric arrangement of endoplasmic cisternae was observed. The normal arrangement of ribosomal granules in rosettes and clusters became disturbed and the granules were more uniformly distributed. The cells as whole units showed a distinct shrinkage in hypertonic solution which may account for the more crowded appearance of various organelles such as mitochondria and Golgi complexes. There was also a marked increase in agranular reticulum profiles and small membrane bound vesicles in treated cells. Vacuoles appeared frequently in the cytoplasm of treated cells; they disappeared upon re-immersion in isotonic medium.

Phagocytosis in the chick blastoderm

Chick blastoderms were cultured in vitro by the New [5] technique and treated with either colloidal gold or carmine solution. Considerably larger amounts of foreign particles were phagocytosed by the edge cells than by other parts of the blastoderm. In an additional group of experiments it was shown that the edge cells phagocytosed particles only when they were actively migrating over the vitelline membrane. Electron micrographs showed that the particles were taken into membrane-bounded spaces.

Metabolic and ultrastructural changes induced in adipose tissue by insulin.

The addition in vitro of insulin to rat adipose tissue (epididymal) produces marked metabolic changes which may be followed by measurement of the net gas exchange of the tissue. Using this method to monitor the metabolic action of insulin, concomitant observations with the electron microscope on the tissue have been made. These reveal that pronounced morphological changes are induced by insulin. The plasma membranes of the adipose cells become invaginated at many sites to form minute finger-like indentations. Numerous tiny, membrane-bounded vesicles are also present and arranged in relationship to the plasma membrane in such a way as to suggest that their formation occurred when a recessed fold was pinched off. Deeper in the cytoplasm, especially in specimens that had been incubated a longer time, numerous large, smooth, membrane-limited vesicles are seen. Finally, in these incubated specimens the cytoplasmic matrix has lost much of its granular nature, small lipid droplets are frequently found in the cytoplasm and suggestive changes have occurred in mitochondria. In control specimens, incubated without insulin for identical periods of time, indentations and vesicles in the plasma membrane are sparse at best and no vesicles or membrane-bound spaces appear deeper in the cytoplasm. The metabolic and morphologic changes induced by insulin seem to be interdependent events. Both changes appear to be initiated rapidly and concomitantly in the tissue. Both processes are initiated by insulin at concentrations considered to be physiological, 0.004 microg. (100 microunits) per ml. Insulin treated with alkali fails to initiate either process. It is concluded that insulin initiates pinocytosis in rat adipose tissue and the possible significance of this process in the mode of action of insulin is discussed.

Electron microscopy of the Fallopian tube epithelium of rabbits in oestrus

The epithelium of the Fallopian tube of rabbits in oestrus has been found to contain secretory cells and ciliated cells. Occasionally lymphocytes are encountered. The secretory cells show balloon-like bulges which are filled with secretory granules. The secretory granules are homogeneous and sometimes possess a limiting membrane. They seem to be synthetized in membrane-bounded spaces, here called secretion spaces, which can be found anywhere in the cytoplasm. No proof of a relationship between the secretion spaces and the structures of the Golgi zone or the system of granulated cytomembranes (α-cytomembranes) can be given. α-Cytomembranes form a well-developed system of vesicles. Opaque particles occur in clusters in the cytoplasm in a higher degree in the secretory cells than in the ciliated cells. The ciliated cells possess cilia which show the usual structural pattern. The cytoplasm of the ciliated cells contains only a few α-cytomembranes as compared with the cytoplasm of the secretory cells. An α-cytomembrane surrounds the nucleus of both ciliated and secretory cells. The assumption that secretory cells can transform into ciliated cells or vice versa cannot be confirmed although in a few cases a single cilium has been found on a secretory cell.