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PubMed1978

Light and electron microscopic histochemistry of the monoamines in the human foetal sympathetic ganglion in culture.

Sympathetic ganglia of 13 to 19-week-old human foetuses were cultured in small pieces with and without nerve growth factor for up to 5 weeks in vitro. The cultures were studied using phase-contrast, fluorescence and electron microscopy. Monoamines were demonstrated with the formaldehyde-induced fluorescence method, with and without pretreatment of the cultures with catecholamines or monoamine oxidase inhibitor. In the long-term cultures, primitive sympathetic cells, sympathicoblasts of types I and II, and young sympathetic neurons showed a fine structure identical to that described earlier in vivo. There were virtually no satellite or Schwann cells in the cultures. The neurons showed a considerable capacity to grow new nerve fibres in culture, even without nerve growth factor. Nerve terminals with accumulations of synaptic vesicles were regularly observed, occasionally in synapse-like contact with other nervous structures. Large granular vesicles were regularly found in the sympathicoblasts after glutaraldehyde-osmium tetroxide fixation. After permanganate fixation, dense-cored vesicles typical of adrenergic neurons were not seen, either in the perikarya, or in the processes, although it was possible to demonstrate specific fluorescence. No small intensity fluorescent (SIF) cells were observed. Variable formaldehyde-induced fluorescence was observed in the nerve cell perikarya and nerve fibres. The intensity of the fluorescence increased after treatment of the cultures with monoamine oxidase inhibitor and after incubation with catecholamines.

Axons↗
PubMed1979

Aggregation of microtubule initiation sites preceding neurite outgrowth in mouse neuroblastoma cells.

By examining microtubule regrowth using immunofluorescence with antibody to tubulin, we have studied the structure and intracellular localization of microtubule initiation sites in undifferentiated and differentiated mouse neuroblastoma cells. The undifferentiated cells are round and lack cell processes. They contain an average of 12 initiation sites per cell. Each of these sites, which are located near the cell nucleus, initiates the growth of several microtubules in a radial formation. In contrast to the undifferentiated cells, neuroblastoma cells stimulated to differentiate by serum deprivation are asymmetrical, containing one or two very long neurites. These cells have a single, large microtubule initiation center which can be visualized not only by immunofluorescence but by phase-contrast and differential interference microscopy as well. The initiation site measures 3-4 mu in diameter and is located in the cell body along a line defined by the neurite. During cell differentiation, the large initiation, the large initiation center seems to be formed by the aggregation of many smaller sites. This process procedes neurite extension by about 24 hr. The growth of microtubules from this center appears to be highly oriented, since most microtubules initially grow into the neurite processes rather than into the cell interior. Thus major changes in the structure and location of microtubule initiation sites occur during the differentiation of neuroblastoma cells. Similar changes are likely to be involved in alterations in the morphology of other cell types.

Animals↗
PubMed1976

The cytotoxic action of leucocidan from Pseudomonas aeruginosa on human polymorphonuclear leucocytes.

Human polymorphonuclear leucocytes treated in vitro with leucocidin from Pseudomonas aeruginosa underwent characteristic morphological alterations as shown by phase-contrast and scanning electron microscopy. Within a few minutes of exposure to leucocidin the granulocytes became round, and protoplasmic extrusions appeared on the cell membrane, were withdrawn again and put out at another point of the cell. The final stage of the leucocidin-treated leucocyte was an enlarged, rounded vesicle with apparently intact plasma membrane. Omission of calcium ions from the diluting buffer caused certain differences in the morphologic appearance of the damaged leucocytes.

Calcium↗
PubMed1977

Comparison of the effects of mecillinam and 6-aminopenicillanic acid on Proteus mirabilis, Escherichia coli, and Staphylococcus aureus.

Single strains of Proteus mirabilis, Escherichia coli, and Staphylococcus aureus were grown on filter membranes placed on agar containing concentration series of mecillinam (FL 1060), 6-aminopenicillanic acid (6-APA), or ampicillin. P. mirabilis and E. coli were also exposed to combinations of mecillinam or 6-APA with ampicillin. Colony-forming units were counted, and cells were examined by interference phase-contrast and transmission electron microscopy. Mecillinam and 6-APA were very effective in reducing the viability of the two gram-negative species, but they were less effective against S. aureus. Combinations of mecillinam and 6-APA with ampicillin acted synergistically against the gram-negative bacilli. When the antibiotics were presented consecutively, their effects on viability were usually no greater than the effects of the individual antibiotics acting alone. When P. mirabilis and E. coli were exposed to mecillinam alone or in combination with ampicillin, the cells became rounded. 6-APA alone or in combination with ampicillin produced elongated polymorphic cells in these species. The most unusual morphological effects were ultrastructural. Mecillinam and, to a lesser extent, 6-APA produced inward growth of numerous pairs of trilamellar membranous structures within the cells. It is possible that these membranes represent the growth initiation of aberrant cross walls. Both mecillinam and 6-APA produced multiple, thick cross walls in S. aureus.

Ampicillin↗
PubMed1976

Bacteriological study on effects of beta-lactam group antibiotics in high concentrations.

The growth and viability of Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa exposed to various concentrations of a number of beta-lactam group antibiotics were determined. In S. aureus, the bacteriolytic and bactericidal activity of these drugs was lower at very high drug concentrations than that occurring at low concentrations, but these phenomena were not observed in E. coli and P. aeruginosa. Under phase-contrast and scanning electron microscopy, S. aureus treated with high concentrations of beta-lactam group antibiotics revealed a lower frequency of bacteriolysis than at low drug concentrations, and similarly by transmission electron microscopy fewer cells were transformed into spheroplasts at high drug concentrations. However, swelling of the cell wall septum was seen in many cells. Spheroplast formation occurred with the highest frequency at drug levels near the minimum inhibitory concentration and became less frequent as drug concentrations were increased.

Anti-Bacterial Agents↗
PubMed1978

Cytopathogenicity of Mycoplasma hyopneumoniae in porcine tracheal ring and lung explant organ cultures alone and in combination with monolayer cultures of fetal lung fibroblasts.

Porcine tracheal rings and lung explants alone and in combination with monolayer cultures of porcine lung fibroblasts (PLF) were separately inoculated with virulent strains of Mycoplasma hyopneumoniae and incubated at various times. The preparations were observed by bright-field, phase-contrast, and scanning electron microscopy. In PLF cultures, the strains at initial concentrations of 10(1.3) colony-forming units/ml increased within 3 days to 10(6) colony-forming units/ml, showed progressive clustering on the cells, and caused some sloughing. Introduction of a tracheal ring or lung explant into these mycoplasma-infected PLF cultures caused the explant to lose its epithelial ciliary motility. Eventually parts or whole cells of the respective ciliated epithelium were lost. Without infected PLF monolayers, the explants inoculated with M. hyopneumoniae were less susceptible to infection. When uninfected explants were incubated for 18 days or kept in stock for 2 months, they did not show the above changes. With 5 h postinoculation, M. hyopneumoniae cultures became intimately associated with the PLF culture, but when epithelial cell sloughing occurred, the mycoplasmal cells became dependent on the introduction of a fresh PLF monolayer or a tracheal or lung explant for survival.

Culture Techniques↗
PubMed1977

Morphology and ultrastructure of Crenothrix polyspora Cohn.

Naturally grown cell material of Crenothrix polyspora from the well of a waterworks was studied by means of phase-contrast and Nomarski interference microscopy as well as by transmission electron microscopy. The material consisted of clusters of sheathed filaments up to 2 cm long. Propagation forms observed were nonmotile, spherical cells that arose by simple ("macrogonidia") or multiple ("microgonidia") septation of the filamental tips. Ultrastructural analysis revealed Crenothrix to be procaryotic and gram negative, with several layers of sheath material surrounding the filaments. On thin sections, individual cells had elaborate membrane systems in the form of lamellar stacks. They resembled thylakoids of photosynthetic bacteria. Spectrophotometric analysis gave no indication of photosynthetic pigments. The cells also contained large hexagonal bodies, rod-shaped fibrillar elements, and polyphosphate granules.

Bacteria↗
PubMed1977

Effect of naturally occurring apatites on growth and morphology of algae.

Crystals (30--100 micrometer) of selected naturally occurring apatite (Ca10 (PO4)6(OH, F)2) samples were added to P-free (less than 0.001 microgram/ml total P) Bristol's medium (1-1000 microgram/ml of apatite) as the sole source of ortho-PO43-. The media were inoculated with washed, non-axenic cells of three chlorophycean algal species cultivated under PO43--deficient conditions. Phase-contrast and scanning electron microscopy revealed that at low slurry densities (1-10 microgram/ml of apatite), Ankistrodesmus braunii (ATCC 2744) cells were morphologically distorted. At concentrations of 100 and 1000 microgram/ml of apatite, more than 85% of the cells had undergone autospore formation within 7--10 days of incubation at 20 degrees C. Most autospores formed failed to germinate under high nutrient conditions. Scenedesmus longus (No. 1236) formed colonies when cultivated in Bristol's medium but daughter cells displayed a Chodatella-like unicellular morphology when grown in apatite media. Test algal species (Chlamydomonas dysosmos, S. longus, A. braunii) showed a marked preference for growth on apatite crystals over non-nutritive surfaces. Unialgal and mixed-algal cultures produced an extensive matrix of extracellular fibrous material in response to growth on crystals at concentrations greater than 10 microgram/ml of apatite.

Apatites↗
PubMed1979

Marginal bands in camel erythrocytes.

The elliptical, anucleate erythrocytes of camels have been examined for the presence of marginal bands and their constituent microtubules. Lysis of erythrocytes under microtubule-stabilizing conditions readily revealed marginal bands in at least 3 % of the cells, as observed by phase-contrast and darkfield light microscopy. Microtubules plus a marginal band-encompassing network of material are visible in lysed cell whole mounts with transmission electron microscopy. Marginal band microtubules are also evident in electron micrographs of thin-sectioned camel erythrocytes identifiable as reticuloyctes on the basis of submaximal electron density (reduced haemoglobin iron content) and presence of polysomes. The results suggest that marginal bands may be involved in morphogenesis of camel erythrocytes but are not required for maintenance of their ellipticity after cells are fully differentiated.

Animals↗
PubMedDate not supplied

[Light microscopic and electron microscopic study of the cells of tissue cultures irradiated with a neodymium laser].

Damage of tissue culture cells (Hela, a human amnion, a primary rat liver culture) caused by the neodymium laser radiation was studied by time-lapse phase-contrast microfilming and electron microscopy. The tested cultures were shown to display different sensitivity and the degree of cell alteration within the same cultures varied considerably. A physical mechanism of cell damage is probably of thermal and shock-wave nature.

Amnion↗
PubMed1975

Tropomyosin antibody: the specific localization of tropomyosin in nonmuscle cells.

An antibody against purified chicken skeletal muscle tropomyosin is used in indirect immunofluorescence to visualize the localization of tropomyosin in a variety of nonmuscle cells. The antibody produces a fluorescent pattern which is very similar to that obtained with an actin-specific antibody. This pattern is composed of fluorescent fibers which are shown to be coincident with the fibers seen with phase-contrast optics. High resolution epifluorescent microscopy reveals that fibers stained with the actin antibody show a continuous fluorescence, while fibers reacted with the tropomyosin antibody show a periodic fluorescence. Measurements indicate that the lengths of the fluorescent segments are variable with an average of 1.2 mum while the spacing between segments is approximately 0.4 mum.

Actins↗
PubMed1992

Microinjection of smg/rap1/Krev-1 p21 into Swiss 3T3 cells induces DNA synthesis and morphological changes.

Microinjection of either Ki-rasVal-12 p21 or the GDP-bound form of Ki-ras p21 plus smg GDP dissociation stimulator (GDS), a stimulatory GDP/GTP exchange protein for Ki-ras p21, smg/rap1/Krev-1 p21, and rho p21, into quiescent Swiss 3T3 cells induced DNA synthesis irrespective of the presence or absence of insulin. The guanosine 5'-(3-O-thio)triphosphate (GTP gamma S)-bound form of smg p21B or the GDP-bound form of smg p21B plus smg GDS also induced DNA synthesis but only in the presence of insulin. Either the GDP-bound form of Ki-ras p21 or the same form of smg p21B alone was inactive, but smg GDS alone was slightly active only in the presence of insulin. The morphology of the cells was analyzed by scanning electron, phase-contrast, and confocal laser scanning microscopies. Ki-rasVal-12 p21 induced membrane ruffling irrespective of the presence or absence of insulin. The GTP gamma S-bound form of smg p21B showed the same effect only in the presence of insulin. Either the GDP-bound form of Ki-ras p21, the same form of smg p21B, or smg GDS alone was inactive. Upon microinjection of Ki-rasVal-12 p21, stress fibers markedly decreased and the cells became round and piled up. In contrast, upon microinjection of the GTP gamma S-bound form of smg p21B, stress fibers did not markedly decrease and the cells neither became round nor piled up. These results indicate that both ras p21 and smg p21 are mitogenic in Swiss 3T3 cells but that their actions are slightly different.

3T3 Cells↗
PubMed1992

Responses of the solitary olfactory receptor cell of the guinea pig.

Olfactory receptor cells were isolated from the nasal mucosa of the guinea pig, using enzymatic digestion and mechanical dissociation. Under a phase-contrast microscope, the cells can be readily distinguished from other tissues, because of morphological characteristics. The intracellular free calcium ion concentrations ([Ca2+]i) of these cells were determined using the Ca2+ sensitive dye fura-2 and digital imaging microscopy. In the presence of 1 microM ionomycin, calcium-ionophore, there was an irreversible increase in [Ca2+]i in all the cells. During prolonged stimulation by 10 mM n-Amyl acetate under standard conditions, 16 of the 40 isolated olfactory cells responded with [Ca2+]i. There was no evidence of any increase in [Ca2+]i in the olfactory receptor cells in nominally Ca2+ free solution and stimulated by amyl acetate. When 3 mM CaCl2 was added, the [Ca2+]i increased remarkably. When cells were stimulated with amyl acetate in a CaCl2-depleted extracellular fluid supplemented with 10 microM verapamil (an L-type voltage-dependent calcium channel antagonist), no change occurred in [Ca2+]i. The addition of CaCl2 to this fluid did not elevate [Ca2+]i, in any cell. This would suggest that the [Ca2+]i increase in the presence of amyl acetate may mainly depend on the influx of calcium from the extracellular space. Our finding that the influx of extracellular calcium into cells was suppressed by verapamil suggests that the calcium channel, which is opened by amyl acetate, is a voltage-dependent L-type Ca2+ channel.

Animals↗
PubMed1979

Microcinematographic demonstration of synchronous and asynchronous myoepithelial contractions in mouse submandibular gland rudiments in organotypic culture.

Time-lapse phase-contrast cinematography revealed contractile activity within mouse submandibular salivary gland rudiments in organotypic culture. Three types of contraction were distinguishable. In type I (voiding contractions), all portions of the gland contracted synchronously, and the active state ranged from 30 min to 2 hr. In type II (priming contractions), all portions of the gland contracted synchronously, but the active state was shorter, ranging from 4 to 10 min. In type III (churning contractions), isolated foci in lobules or secretory units throughout the gland contracted asynchronously and had very short active states of about 1 min. By electron microscopy, myoepithelial cells could first be demonstrated in submandibular glands developing either in vitro or in vivo, at 21 days postconception. Contractions in the cultured rudiments began as early as 18 days postconception. Since neither smooth nor striated muscle could be identified in these glands by electron microscopy, the contractions are believed to result from myoepithelial activity that apparently may begin before ultrastructural evidence of myoepithelial differentiation is clearly present. Although, for over a century, myoepithelium has been presumed to have a contractile function and indirect evidence has lent ample support to this presumption, the present study represents the first direct cinematographic demonstration and characterization of myoepithelial contractions, under conditions in vitro.

Animals↗
PubMed1978

Monolayer cultures of rat pituitary intermediate lobe: growth and fine structure of epithelial cells.

Rat hypophyseal intermediate lobe cells, partially dispersed by trypsin, were cultivated on an inert surface. The attaching capacity, the growth of the cultures and the fine structure of the epithelial cells developed during cultivation were studied. The majority of the cell groups attached to the inert surface within 72 h after explantation. Epithelial islands developed, with a layer consisting of fibroblast-like cells beginning to form at their periphery. By the end of the second week of cultivation the epithelial islets and fibroblast-like cells formed a monolayer. The epithelial cells survived for five weeks. The granular cytoplasm of the epithelial island cells could be well identified on the phase-contrast microcinematographic frames of the young cultures; both epithelial and fibroblast-like cells divided. The mitotic ability of both cell types could be confirmed by their tritiated-thymidine incorporation, too. In young cultures, the fine-structural features of epithelial cells were practically identical with those of the glandular cells in intact glands. In older cultures, the secretory granules in the epithelial cells were lower in number; the localization of the granules at the periphery was predominating.

Animals↗
PubMed1979

Morphology of dissociated hippocampal cultures from fetal mice.

Dissociated hippocampal cultures from fetal mice (13--18 days gestational age) can be maintained for up to two months in culture. Cells grow as either isolated neurons or in small neuronal aggregates. Neurons remain small with a soma diameter of 15--20 micrometer even in mature cultures and develop extensively branched processes during the first two weeks in culture. After this time, processes become more difficult to visualize with phase-contrast optics because of a tendency to grow within the underlying non-neuronal cells. However, the presence of processes has been proved by silver-staining which demonstrates an organizational complexity ranging from a loosely reticulated neuropil to fascicles containing many fibers. More detailed study of individual neuronal morphology was carried out in cells filled with the fluorescent dye, Lucifer Yellow CH, in conjunction with the intracellular recording of synaptic and action potentials from dye-containing micropipettes. Dye-filled cells show a well-developed branching morphology. Process specializations include spines, beading, and basket-like endings. Processes tend to emanate from one side of the soma, either originating at the cell body or from a single trunk. Commonly there are 2--4 orders of branching, but up to 6 orders can occur (counted centrifugally from the soma). Electron microscopy revealed synapses distributed predominantly on dendrites with a smaller number on somata. Dendritic spines are present and are contacted principally by asymmetric synaptic junctions. Symmetric synapses are relatively more common on somata and proximal dendrites.

Action Potentials↗
PubMed1992

Altered expression of E-cadherin in gastric cancer tissues and carcinomatous fluid.

Expression of E-cadherin in 21 patients with various histological types of gastric carcinomas was studied by immunoperoxidase staining. Intercellular boundaries of almost all cancer cells in well and moderately differentiated adenocarcinomas stained as deeply for E-cadherin as normal gastric mucosa. However, singly infiltrating cells of those histological types were poorly stained. In poorly differentiated adenocarcinomas, cancer cells forming clusters stained lightly and those infiltrating singly stained even less. In signet ring cell carcinomas, hardly any staining was observed. In each histological type, the staining patterns and intensity at different layers of the gastric wall, were essentially the same. Cancer cells from carcinomatous ascites of gastric adenocarcinomas and pancreatic adenocarcinomas, and those from pleural effusion of lung adenocarcinomas were also studied by immunofluorescence staining. Of 11 specimens, ten were negative and only one from a lung adenocarcinomas was positively stained. By phase-contrast microscopic observations, none of these cancer cells including those from the lung adenocarcinomas, formed obvious cell-cell contacts. Cell aggregation assays confirmed the above results. The molecular weight of E-cadherin of cancer cells of lung adenocarcinomas was less than intact E-cadherin as revealed by Western blot analysis. These results suggest that depressed expression and/or impaired function of E-cadherin in cancer cells, facilitates their liberation from primary sites to infiltrate freely into tissue or fluid.

Adenocarcinoma↗
PubMed1979

Human reticulocyte maturation and its relevance to erythropoietic stress.

A comparative study of circulating human reticulocytes by phase-contrast microscopic observation and NMB staining revealed two different forms of reticulocytes which could be clearly distinguished by their distinctive morphologic appearance. These were (1) a multilobular motile form and (2) a cup-shaped nonmotile form. Sequential studies of different populations in the circulation of newborns showed that the multilobular form was the youngest and the cup-shaped form the more mature reticulocyte. The multilobular form was found in the circulation only under conditions of erythropoietic stress and could not be detected in the circulation of hematologically normal individuals. The presence of these characteristic forms in peripheral blood can be used as a reliable indicator of transient or persistent erythropoietic stress.

Adult↗