Vesicular Appearance Research Articles

Sodium channel activity of lobster nerve membrane treated with purified phospholipase A 2

Sodium channel activity was determined by measuring the veratridine-tetrodotoxin-sensitive sodium influx in reconstituted vesicles prepared from lobster nerve membrane and soybean lipids. The sodium channel activity was abolished by treatment of membranes, prior to reconstitution, with purified phospholipase A 2. When the treatment with phospholipase A 2 was carried out in the presence of bovine serum albumin the channel activity was fully preserved. Electron microscopy revealed that the normal vesicular appearance of the membranes was changed to an amorphous mass by treatment of the membranes with enzyme alone. A population of preserved vesicular structures was observed when bovine serum albumin was present during the enzyme treatment. Analysis of the membrane components indicate that there is no relationship between phospholipid composition and sodium channel activity.

The monocyte-macrophage system in granulomatous inflammation.

Granulomas may be immunologically induced or non-immunologically induced. In immunologically induced granulomas cells of the monocyte-macrophage series take on the appearance of epitheloid cells. Ultrastructurally epithelioid cells may have a secretory appearance with much rough endoplasmic reticulum or take on highly degenerate vesicular appearance. Other epithelioid cells look like activated macrophages. Secretory epithelioid cells may be found associated with acute local inflammation as in borderline tuberculoid leprosy in reaction, the lepromin reaction, following injection of BCG vaccine and in experimental zirconium granulomas. In these situations there may also be strong histological and biochemical evidence of increased fibroblast activity and collagen synthesis. It is suggested that these cells are actively secreting a fibroblast-activating factor. Epithelioid cells may lose their Fc receptors, undifferentiated macrophages in lepromatous leprosy can lose their C3 receptors. It is suggested that in a number of situations granuloma formation may be associated with complement activation through the alternative pathway as in the case of mycobacterial granulomas. Toxic granulomas produced by metals may be caused by C3 being split by plasmin after conversion from plasminogen by activation of the Hageman factor.

Dual function during development of rat sympathetic neurones in culture.

Many sympathetic principal neurones of the superior cervical ganglion of the newborn rat are known to be plastic with respect to the choice between norepinephrine (NE) and acetylcholine (ACh) as transmitter; when the neurones are dissociated and placed in culture, a majority of them can be shifted from an initial, immature, adrenergic state to a cholinergic state by co-culture with a variety of non-neuronal cells or by medium conditioned by such cells. To study this transition it has been helpful to grow single neurones, each in a microculture which also contains cardiac myocytes. The transmitter status of a neurone can be assayed by recording its effect on the myocytes (adrenergic excitation, cholinergic inhibition or dual function); then a fine structural assay of the neurone based on the appearance of the synaptic vesicles can be made and correlated directly with the physiology. In this paper we report the following findings on principal neurones developing in such microcultures. (i) During the transition period, a majority of the neurones were dual in function and in vesicular appearance. (ii) The physiological effects and vesicular appearance varied from mainly adrenergic to mainly cholinergic. (iii) In preliminary attempts to follow the transition by recording at least twice from the same microculture, partial transitions were observed, always in the direction adrenergic-to-cholinergic. (iv) The transitions were not synchronous or fixed in time course even in pairs of neurones grown side by side in the same microculture.

Separation and properties of the cytoplasmic and outer membranes of vegetative cells of Myxococcus xanthus.

We have developed methods for separating the cytoplasmic and outer membranes of vegetative cells of Myxococcus xanthus. The total membrane fraction from ethylenediaminetetraacetic acid-lysozyme-treated cells was resolved into three major fractions by isopycnic density centrifugation. Between 85 and 90% of the succinate dehydrogenase and cyanide-sensitive reduced nicotinamide adenine dinucleotide oxidase activity was found in the first (I) fraction (rho = 1.221 g/ml) and 80% of the membrane-associated 2-keto-3-deoxyoctonate was found in the third (III) fraction (rho = 1.166 g/ml). The middle (II) fraction (rho = 1.185 g/ml) appeared to be a hybrid membrane fraction and contained roughly 10 to 20% of the activity of the enzyme markers and 2-keto-3-deoxyoctonate. No significant amounts of deoxyribonucleic acid or ribonucleic acid were present in the three isolated fractions, although 26% of the total cellular deoxyribonucleic acid and 3% of the total ribonucleic acid were recovered with the total membrane fraction. Phosphatidylethanolamine made up the bulk (60 to 70%) of the phospholipids in the membrane fractions. However, virtually all of the phosphatidylserine and cardiolipin were found in fraction I. Fraction III appeared to contain elevated amounts of lysophospholipids and contained almost three times the amount of total phospholipid as compared with fraction I. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis resolved approximately 40 polypeptides in the total membrane fraction. Two-thirds of these polypeptides were enriched in fraction I, and the remainder was enriched in fraction III. Fraction II contained a banding pattern similar to the total membrane fraction. Electron microscopy revealed that vegetative cells of M. xanthus possessed an envelope similar to that of other gram-negative bacteria; however, the vesicular appearance of the isolated membranes was somewhat different from those reported for Escherichia coli and Salmonella typhimurium. The atypically low bouyant density of the outer membrane of M. xanthus is discussed with regard to the high phospholipid content of the outer membrane.

Morphogenesis by dissociated immature rat testicular cells in primary culture

Dissociated cells from immature (15-day) rat testes maintained in primary culture were shown to undergo morphogenesis. Dissociated cells (1-4 X 10(6)) were cultured in 2 ml medium NCTC-135 containing 10% rat serum at 32.5 degrees C under an air; CO2 atmosphere (95%:5%). During the initial culture period (1-3 days) the dissociated cells attached to the culture dish and formed a monolayer. After 4-5 days of culture, aggregates of cells formed at discrete foci within the monolayer; these structures averaged 50 micrometer in diameter and numbered 3-4 X 10(3)/dish. Within 5-6 days of culture some of these aggregates (2-3 X 10(2)) released their contact with the substratum. Histological examination of released aggregates indicated a more regular, tissue-like organization with increasing time in culture. Aggregates at earlier time periods (4-5 days) exhibited concentric rings of cell while by day 6 some aggregates exhibited a vesicular appearance with a single layer of columnar epithelium surrounding a fluid-filled lumen. The type of structure formed varied with plating density; at densities less than 2 X 10(6)/dish the structures were spheroid while at greater densities tubes were formed. Time-lapse observations indicated that spheres formed by movements of cells into an aggregating center while tubes formed by a rolling from the edge of the monolayer. The cells comprising the vesicle epithelium appeared to be predominantly one cell type as determined by light microscopy. These results therefore indicate that dissociated testicular cells have the ability to reform tissue-like associations in culture.

Fractionation of membrane vesicles: I. A separation method for different populations of membrane vesicles of thymocytes by affinity chromatography on Con A-Sepharose

Affinity chromatography, a separation technique which makes use of biospecific properties, is well established for the separation of molecules in solution. We applied this method to the subfractionation of biomembranes. Using a microsomal fraction mainly consisting of plasma membrane from rabbit or calf thymocytes, 20–40% of the protein adhered specifically to concanavalin A-Sepharose, whereas the majority of the membrane vesicles were recovered from the effluent. The adherence involved the binding of Con A to membranes, as addition of the hapten sugar α-methyl-mannoside completely prevented separation. The fractions which bound to Con A-Sepharose could be eluted by combining mechanical forces with the addition of α-methyl-mannoside. All fractions exhibited the same vesicular appearance and were identical with respect to the phospholipid cholesterol ratio. The method proved to be highly reproducible and it offers a possible way for the subfractionation of membranes according to their biospecific structure.

Distribution of Acid Phosphatase in Chick Hensen's Node

Acid phosphatase distribution and yolk drop ultrastructure in Hensen's node of chick blastoderm at Hamburger-Hamilton stages 3 and 4 are described. At stage 3 large deposits of the reaction product are localised in type-A yolk drops, where signs of intensive degradation are seen. It seems that during this degradation lipid droplets are being formed in the degrading yolk drops. Partly digested yolk drops with a vesicular appearance are extruded from the cells, especially in deeper layers of the node. The phosphatase reaction product is also distributed into intercellular spaces. At stage 4 the cells of the node are more vacuolated and contain acid phosphatase and electron-dense yolk drops in lesser amounts. The possible physiological role of acid phosphatase in Hensen's node during gastrulation is discussed.

Some studies on the metabolism of phospholipids in plasma membranes from rat liver

1. 1. Rat liver plasma membranes were isolated by rate-isopycnic zonal centrifugation. A method is described for the size 14 zonal rotor. The isolated membranes had an isopycnic banding density of 41.2% sucrose (w/w). On the basis of studies with eleven marker enzymes and electron microscopy, at least 90% of the fraction consisted of plasma membranes. The electron micrographs showed a predominantly vesicular appearance with few junctional complexes. 2. 2. Plasma membranes exhibited virtually no CDP-choline: 1, 2-diacyl- sn-glycerol cholinephosphotransferase activity and thus appear incapable of significant in vitro synthesis of phosphatidylcholine by the cytidine nucleotide pathway. 3. 3. Phosphatidylglycerol was the major product of sn-glycerol-3-phosphate esterification by plasma membranes in the presence of CDP-diglyceride at pH 7.5. The product was identified by thin-layer chromatography using pure phosphatidylglycerol as a reference compound, and by paper chromatography of the products obtained by hydrolysis with phospholipase D. 4. 4. Incubation of plasma membranes with labeled exogenous phospholipids in the presence of Ca 2+ resulted in the formation of fatty acids and monoacylphosphoglycerides. Analyses of the reaction products indicated that the phospholipase A mainly attacks the 2-position of the phosphoglyceride molecule. Plasma membrane phospholipase A also acted on labeled endogenous phosphatidylethanolamine in the presence of Ca 2+. 5. 5. A partial characterization of the phospholipase indicated that it required Ca 2+ for optimal activity, was insensitive to N-ethylmaleimide and deoxycholate, and was relatively heat stable. Its pH optimum was 8.0. The following was the preferred order of hydrolysis: phosphatidylethanolamine > phosphatidylglycerol > > > phosphatidylcholine. 6. 6. Lysophospholipase was not detected in rat liver plasma membranes under conditions in which maximal activity of this enzyme was observed in rat liver 100 000 × g supernatant.

Native and reformed Mycoplasma laidlawii membranes compared by freeze-etching

1. 1. The ultrastructure of native and reformed Mycoplasma laidlawii membranes was compared by the freeze-etching technique. 2. 2. Reformed membranes were prepared by dialysis of M. laidlawii membrane material solubilized in sodium dodecyl sulfate against buffer containing 5 or 20 mM Mg 2+. 3. 3. Both the convex and concave fracture faces of freeze-cleaved native M. laidlawii membranes showed large areas studded with numerous globular particles, approx. 75–150 Å in diameter. 4. 4. The reformed membranes produced with 5 mM Mg 2+ appeared mostly as long membranous sheets, while those produced with 20 mM Mg 2+ usually had a vesicular appearance. 5. 5. The fracture faces of the reformed membranes were relatively smooth and did not contain the globular particles seen in cleaved native membranes. 6. 6. This finding indicates that the ultrastructural organization of the reformed membranes differs from that of native M. laidlawii membranes.

Ameliorating effect of Allium Sativum on high-fat diet induced fatty liver in albino rats.

Objective:To assess the hepatoprotective effect provided by fresh garlic on fatty liver induced by high-fat diet.Methods:This experimental study was carried out at BMSI, JPMC from October to November 2008. Thirty adult albino rats, 200-240 gram weight, were divided into three groups. Group A received control diet, Group B received high-fat diet (20 mg butter/100 gm diet) and Group C received high-fat diet with fresh garlic (20 mg butter with 6 gm fresh garlic/100 gm diet). The groups were further divided on the basis of duration of treatment, four weeks and eight weeks respectively. The rats were sacrificed, liver removed, weighed and relative liver weight calculated. Hepatic tissue was processed and tissue slides stained with haematoxylin and eosin.Results:There was significant increase in relative liver weight in group B animals as compared to the control animals, which decreased significantly in group C. Haematoxylin and eosin stained sections revealed ballooned hepatocytes having vesicular appearance with pyknotic nuclei in high-fat group which were preserved to a great extent in group C animals.Conclusion:This study has shown that use of fresh garlic along with high-fat diet prevents its damaging effects on liver to a great extent.

THE CYTOLOGICAL EFFECTS OF PODOPHYLLIN AND PODOPHYLLOTOXIN ON THE FERTILIZED EGGS OF CHAETOPTERUS

1. Fertilized eggs of the polychaete annelid, Chaetopterus pergamentaceus, were treated with various dilutions of podophyllin and podophyllotoxin, beginning five minutes after insemination. Observations were made on the living eggs and on whole-mount cytological preparations made from samples fixed at various intervals during development. Dosages ranging from 0.1 to 0.00001 mg./ml. were tested.2. Beginning as soon as 60 seconds after the beginning of treatment with moderate to high dosages (0.1 to 0.005 mg./ml.) of podophyllin, the asters of the egg maturation figure began to fade, followed by disappearance of the spindle by about three minutes after the initiation of treatment. The nine egg chromosomes remained in the ring configuration (characteristic of the first maturation division in this form) for approximately two to five minutes longer (depending on the exact dosage), after which they were gradually enclosed in a membrane-surrounded area which appears to have been derived from the spindle substance. By this time they had taken on a somewhat vesicular appearance. The sperm chromosomes were often similarly affected by the podophyllin, although the onset of these effects was usually somewhat slower than for the eggs. Both egg and sperm chromosomes remained contained in this membrane for approximately an hour, after which they were released to lie loose in the egg cytoplasm. No further development occurred at these dosages.3. After treatments with podophyllin concentrations of 0.00005 to 0.00001 mg./ml., a semblance of normal development ensued, although the resulting larvae were usually abnormal. In egg-samples fixed 24 minutes after the beginning of treatment, there was occasional evidence of reduction in size of the asters; by 47-57 minutes after the initiation of treatment, multipolar figures were observed, as well as two entirely separate complex metaphases, often in uncleaved cytoplasm. There was frequently karyokinesis without accompanying cytokinesis. When cleavage continued to occur, it was often retarded and/or abnormal.4. Shape changes ("pear" and polar lobe stages) characteristic of the normal development of Chaetopterus eggs were observed, even after treatment with high dosages which destroyed the achromatic figure. Pseudo-polar bodies, usually without chromatin, were also noted, as well as giant polar bodies.5. The effects of podophyllotoxin were qualitatively much like those of podophyllin, but the dosage required to produce them was very much lower (0.001 mg./ml. for podophyllotoxin vs. 0.01 mg./ml. for podophyllin to completely block division, for example).6. Even short durations of exposure to either podophyllin or podophyllotoxin (10-20 minutes, as compared with the 60-minute duration routinely used in the other experiments) were sufficient to induce marked abnormality with moderate dosages.7. The effects of mitomycin C, N-dichloroacetyl DL serine (sodium) and quercetin were also tested. None of these agents was effective in producing cytological abnormality in Chaetopterus eggs, at the concentrations tested.

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.

A nuclear body in meiosis of Bromus

1. A structure which is tentatively called a “nuclear body” has been observed in microsporocytes in a large number of species of Bromus. It was found in diploid and polyploid species and in species with complements of large (L-)chromosomes, medium (M-)chromosomes or L- and M-chromosomes. These species were collected in North and South America and Europe. It is suggested that the nuclear body occurs regularly in species of the genus Bromus. 2. The nuclear body was first visible at pachytene. It was generally single. In some individuals it disappeared at the end of prophase; in others it was present during later stages of meiosis, in some instances as late as the quartet. The nuclear body was not observed when the quartet cells had separated. Before it disappeared the nuclear body underwent a sudden or gradual diminution. It decreased in size, or became faint-staining and faded away; or it decreased in size and faded. The nuclear body generally disappeared at the end of prophase in the diploid and tetraploid species and was present at metaphase-I and later stages in higher polyploid species. 3. When the nuclear body was present in later stages of meiosis it behaved as follows: It was extruded from the spindle at metaphase-I and frequently came to lie in the polar region of the cell; it was not included in either nucleus at telophase-I. A single nuclear body was present in one dyad cell; it lay outside the spindle at metaphase-II, and was included in one of the cells of the quartet. 4. A very great range in the size of the nuclear body was found in early stages of meiosis. Some variation in size was found within individuals and among individuals of the same species. Present data indicate some relationship between chromosome number and size of the nuclear body. The largest nuclear bodies were found in duodecaploid and octoploid and the smallest in diploid species; however an exact correspondence was not observed. 5. The nuclear body is extremely variable in shape. It has a vesicular appearance; there is great variation in the number, size and staining of the vesicles. The appearance of the nuclear body may vary within and among anthers and individuals. 6. The nuclear body is not associated with the nucleolus at pachytene, nor with the nucleolar organizing regions of specific bivalents. The nuclear body is often touching a bivalent or attached by fine strands to a bivalent during prophase. However, there appears to be no specifically differentiated region of any bivalent with which it is generally associated. The nuclear body may be associated with terminal or interstitial regions of bivalents which differ in size or chromomere pattern. It was more frequently associated with bivalents in some individuals than others. 7. The nuclear body was not found in mitotic divisions in shoot or root tips and thus appears to be related specifically to the meiotic divisions. It may arise as an exudate from some chromosomes, or it may be extruded from the nucleolar material; it seems less likely to arise from the karyolymph. It may be composed of some materials discarded in preparation for meiosis or during its earliest stages, which are perhaps related to concurrent changes in chromosome organization. 8. Observations are considered which suggest that a structure similar to the nuclear body observed in Bromus may be present in other plant genera.

An integrated morphological and biochemical study of a purified preparation of the succinate and DPNH oxidase system.

Electron micrographs of a purified succinate and DPNH oxidase system prepared from heart muscle reveal that it has a vesicular appearance and is membranous in nature. In keeping with its vesicular appearance is the fact that light scattering by this preparation shows marked changes as the molarity of the suspending medium is altered. Treatment of this preparation with 0.5 per cent deoxycholate solutions removes a large part of the lipide material, which comprises almost half of the dry weight of the preparation. The residue, which still contains the "core" of the cytochrome electron transmitter system, as shown by spectroscopic and enzymatic experiments, is still structured and is membranous in morphological appearance. It is concluded that the enzyme preparation is largely composed of fragmented mitochondrial membranes, and some of the consequences of the localization of the succinate and DPNH oxidase systems in or on these membranes are discussed.