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PubMed1973

The optical and electron microscopic determination of pulmonary asbestos fibre concentration and its relation to the human pathological reaction.

The quantitative extraction of asbestos fibres from asbestotic lung by alkali digestion has been refined by maceration of the tissue without prior drying, the minimum use of centrifugation, and the adoption of phase contrast microscopy. Preliminary experiments suggested that, using this technique, asbestos fibre counts were accurate to within at least +/- 20% and in most instances to within +/- 10%. The method was used to assess asbestos concentrations in lung tissue showing various degrees and forms of fibrosis. The results, as determined by light microscopy, indicated that uncoated fibres generally outnumbered coated fibres. In mild and moderate asbestosis there was a progressive increase in concentration of asbestos fibres, both coated and uncoated, with increasing severity of fibrosis, whereas in severe asbestosis no correlation existed between the fibre concentration and the form or the extent of the pathological reaction. It is suggested that the severe fibrosis results from the supervention of non-specific inflammatory processes. Asbestos fibre diameter distributions, gauged by electron microscopy, were fairly constant irrespective of the degree of fibrosis. Optically visible fibres constituted between 12 and 30% of the total, so that an optical count may be said to give an approximate indication of the total asbestos concentration and, so far as asbestosis is concerned, may well serve for comparative purposes. The relation between asbestos and neoplasia will, however, require identification and quantitation of particular types of the mineral by microanalytical techniques.

Asbestos↗

PubMed1966

A fiber apparatus in the nucleus of the yeast cell.

The structure and mode of division of the nucleus of budding yeast cells have been studied by phase-contrast microscopy during life and by ordinary microscopy after Helly fixation. The components of the nucleus were differentially stained by the Feulgen procedure, with Giemsa solution after hydrolysis, and with iron alum haematoxylin. New information was obtained in cells fixed in Helly's by directly staining them with 0.005% acid fuchsin in 1% acetic acid in water. Electron micrographs have been made of sections of cells that were first fixed with 3% glutaraldehyde, then divested of their walls with snail juice, and postfixed with osmium tetroxide. Light and electron microscopy have given concordant information about the organization of the yeast nucleus. A peripheral segment of the nucleus is occupied by relatively dense matter (the "peripheral cluster" of Mundkur) which is Feulgen negative. The greater part of the nucleus is filled with fine-grained Feulgen-positive matter of low density in which chromosomes could not be identified. Chromosomes become visible in this region under the light microscope at meiosis. In the chromatin lies a short fiber with strong affinity for acid fuchsin. The nucleus divides by elongation and constriction, and during this process the fiber becomes long and thin. Electron microscopy has resolved it into a bundle of dark-edged 150 to 180 A filaments which extends between "centriolar plaques" that are attached to the nuclear envelope.

Cell Nucleus↗

PubMed1990

A band of F-actin containing podosomes is involved in bone resorption by osteoclasts.

Isolated rabbit osteoclasts cultured on devitalized thin bone slices excavate resorption lacunae that can be visualized with brightfield or phase-contrast microscopy. Superimposition of the brightfield images of such resorption lacunae and the fluorescence images of the corresponding osteoclasts after fixation and staining with rhodamine-conjugated phalloidin revealed that a bright fluorescent band of F-actin-containing podosomes precisely outlined the resorption lacunae in stationary osteoclasts. When the resorption lacunae were being extended laterally, the clearly delineated band of podosomes corresponded to the advancing edge of the resorbing osteoclast and the most recently excavated part of the lacunae. Reshaping and reorganization of the bright bands preceded development of the lateral boundary of the lacunae. Podosomes forming these bands were highly dynamic, changed in size and location, and appeared and disappeared continuously. Their lifespan varied between 2 and 12 min. Similar bands were also seen in vivo in bone-resorbing osteoclasts on the endocranial surface of growing calvariae. Podosomes disappeared in osteoclasts treated with calcitonin, resulting in the disruption of the fluorescent bands. Our results suggest that podosomes are an essential part of the resorption apparatus of osteoclasts.

Actins↗

PubMed1996

Axenic culture of reptilian Blastocystis isolates in monophasic medium and speciation by karyotypic typing.

The growth of axenic reptilian isolates of Blastocystis in Iscove's modified Dulbecco's medium (IMDM) was studied and the morphology of the parasite was examined by phase-contrast microscopy. The chromosomal patterns of these reptilian isolates of Blastocystis were examined by pulsed-field gel electrophoresis (PFGE) and compared with those of B. hominis and B. lapemi, a sea snake Blastocystis. IMDM with 10% horse serum supported excellent growth of the reptilian Blastocystis isolates. The parasites from all the isolates were predominantly vacuolar, but multivacuolar and amoeboid forms were also seen. Amoeboid forms with rather elongate pseudopodia were also observed. There were some differences in size, morphology, and growth characteristics in the different reptilian isolates. The karyotypic patterns of the Blastocystis isolates from tortoise, iguana, and python were distinctly different from one another and from those obtained with B. hominis and B. lapemi. On the basis of the above-mentioned differences in chromosomal patterns, the tortoise, iguana, and python isolates are described as new species, viz., B. geocheloni sp. nov. from Geochelone carbonaria (red-footed tortoise), B. cycluri sp. nov. from Cyclura cornuta (rhino iguana), and B. pythoni sp. nov. from Python reticulatus (reticulated python).

Animals↗

PubMed1983

Morphological and electrophysiological aspects of dissociated cultures of rat CNS.

Rat central nervous system has been cultured up to 6 weeks after complete dissociation. Maturation of different cell types has been followed in the quasi monolayer by phase contrast microscopy. Dorsal root ganglion (DRG) neurones usually differed from central nervous system (CNS) neurones by their spherical shape accompanied by only one or two processes, exact identification of cell types, however, was usually only possible by combining morphology with electrophysiology. Scanning electron-microscopy revealed a more extensive arborization of neurites and a higher number of presumed synaptic structures in cultures after 2 weeks of culturing. Layers of ependymal cells were also found. The different cell types were further identified by determining their membrane properties. Glial cells had higher resting membrane potentials (-56 +/- 9.7 mV) than CNS neurones (-49 +/- 10.2 mV), while the membrane potential of DRG neurones lay in-between the two (-53 +/- 1.7 mV). The sequence for input resistance was: DRG neurones (30 +/- 9.3 M omega) greater than CNS neurones (18 +/- 10.5 M omega) greater than glial cells (9.3 +/- 5.2 M omega). In CNS neurones the input resistance is correlated with the membrane potential, which is not the case for glial cells. Action potentials of DRG neurones exhibited delayed repolarisation increasing the spike duration to three times that of CNS neurones.

Action Potentials↗

PubMed1975

Inhibition of multiplication of Toxoplasma gondii by human monocytes exposed to T-lymphocyte products.

The multiplication of Toxoplasma gondii was quantitated in human monocytes in vitro by phase-contrast microscopy. Toxoplasma multiplication was identical in monocytes from subjects byt was significantly inhibited in cells from both sources if the monocytes were preincubated with immune lymphocytes and toxoplasma monocytes were preincubated with immune lymphocytes and toxoplasma antigen. Supernates prepared from toxoplasma-immune lymphocytes incubated with toxoplasma antigen were also effective in inducing in monocytes the capacity to inhibit toxoplasma multiplication. Supernative acitivty was evident after lymphocytes and antigen were incubated for as little as 15 min. The instruction of monocytes was also repid and reversible. Monocytes were fully induced to inhibit toxoplasma multiplication after a 2 h exposure to an active supernate, but they lost their inhibitory capacity on culture in vitro for 48 h in the absece of immune cells or their products. The lymphocytes particupating in the monocyte induction were identified as t cells. The in vitro stimulation of monocytes appeared to exhibit some specificity, since no inhibition of toxopreotein derivative and lymphocytes from tuberculin-positive subjects, concanavalin a-stimulated lymphocytes, or their supermates. Supernates which induced monocytes to inhibit toxoplasma multiplication did not influence parasite growth in HeLa cells.

Adult↗

PubMed1998

Retroviral vector-mediated gene transfer into keratocytes: in vitro effects of polybrene and protamine sulfate.

BACKGROUND: To determine the potential of somatic gene transfer as a novel technique for modulating corneal wound healing on a cellular level, the successful transduction of human keratocytes should be ascertained in vitro. In addition, the ability of different polycations to increase the transduction efficiency and their antiproliferative and cytotoxic effects should be assessed. METHODS: To test transduction efficiency (X-Gal staining), cultured human keratocytes were incubated for 2 h with a retroviral vector bearing the beta-galactosidase gene, with and without the addition of polybrene or protamine sulfate. To test the antiproliferative and cytotoxic effects, cultured human keratocytes were incubated with various concentrations of polybrene and protamine sulfate (0.08 to 800 micrograms/ml) for 2, 24 and 72 h, and evaluations were performed by means of an XTT-based colorimetric assay and phase-contrast microscopy. RESULTS: Human keratocytes in vitro were transduced successfully with the beta-galactosidase gene (3.5 +/- 1.0%). Transduction efficiency was significantly (P < or = 0.01) improved by addition of a polycation (from 12.3 +/- 1.7% to 18.6 +/- 2.3%), but there was no significant difference between the effects of polybrene and those of protamine sulfate. Both drugs induced a highly significant dose-dependent inhibition of proliferation (P < 0.001). ID50 ranged from 11 to 22 micrograms/ml with polybrene and from 15 to 244 micrograms/ml with protamine sulfate. Only with doses of 80 and 800 micrograms/ml did protamine sulfate produce less antiproliferative effects than polybrene (P < or = 0.04). The lowest concentrations induced no morphological signs of cytotoxicity, whereas these signs were mild at 8 micrograms/ml and moderate to severe at the highest concentrations. CONCLUSIONS: Both polybrene and protamine sulfate can significantly improve the in vitro efficiency of successful retroviral vector-mediated gene transfer into keratocytes. Mild cytotoxic and moderate antiproliferative effects are to be expected in cultured keratocytes with a standard transduction procedure (8 micrograms/ml for 2 h).

Cell Division↗

PubMed2003

Human keratocytes cultured on amniotic membrane stroma preserve morphology and express keratocan.

PURPOSE: To develop a new method of expanding human corneal keratocytes in serum while maintaining their characteristic morphology and keratocan expression. METHODS: Human keratocytes were isolated from central corneal buttons by digestion in 1 mg/mL of collagenase A in DMEM and seeded on plastic or the stromal matrix of human amniotic membrane (AM) in DMEM with different concentrations of FBS. On confluence, cells on AM were continuously subcultured for six passages on AM or plastic. In parallel, cells cultured on plastic at passages 3 and 11 were reseeded on AM. Cellular morphology and cell-cell networks were assessed by phase-contrast microscopy and a cell viability assay, respectively. Expression of keratocan was determined by RT-PCR and Western blot analysis. RESULTS: Trephined stroma yielded 91,600 +/- 26,300 cells (ranging from 67,000 to 128,000 cells per corneal button). Twenty-four hours after seeding, cells appeared dendritic on AM, even in 10% FBS but fibroblastic on plastic. Such a difference in morphology correlated with expression of keratocan assessed by RT-PCR and Western blot, which was high and continued at least to passage 6 on AM, even in 10% FBS, but was rapidly lost each time when cells on AM were passaged on plastic. Fibroblasts continuously cultured on plastic to passages 3 and 11 did not reverse their morphology or synthesize keratocan when reseeded on plastic in 1% FBS or on AM. CONCLUSIONS: Human keratocytes maintain their characteristic morphology and keratocan expression when subcultured on AM stromal matrix even in the presence of high serum concentrations. This method can be used to engineer a new corneal stroma.

Amnion↗

PubMed1993

Expression of type I and type III procollagen by lens epithelial cells.

PURPOSE: To identify the types of procollagen that are synthesized by cultured bovine lens epithelial cells and to determine what types of post-translational modifications are involved in procollagen biosynthesis and what patterns of procollagen gene expression occur in these cells. METHODS: The epithelial nature of the cultured cells was confirmed with transmission electron and phase contrast microscopy. To label collagen, primary or secondary monolayer cultures were pulsed with [3H] proline for various periods of time. Procollagens were identified by immunoprecipitation with specific antibodies, pepsin digestion, and electrophoretic separation on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Procollagen messenger RNA was identified by Northern blot analysis using specific DNA probes. RESULTS: In the normal lens epithelium, cells are attached to each other by tight junctions punctuated with desmosomes. After the proteins were labeled, they were immunoprecipitated with antibodies specific to collagen precursors and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to further confirm their identification as types I and III procollagen or processed forms of these proteins. The secretion of procollagen was inhibited by treatment with 2,2'-dipyridyl and bands of underhydroxylated procollagen were observed in the cell layer after electrophoresis. The sizes of messenger RNA observed were 6.9 kb and 5.4 kb for type alpha 1(I), 5.0 kb for type alpha 2(I), and 5.3 kb for the type III transcripts. CONCLUSIONS: This is the first report that lens epithelial cells express fibrillar procollagen genes, making the lens capsule a unique ocular basement membrane.

Animals↗

PubMed1995

The effect of periodontal parameters on the subgingival microbiota around implants.

The study aimed to examine the relationship between the subgingival flora around implants and their periodontal parameters. Plaque samples from 561 implants (279 patients) were analyzed by means of differential phase contrast microscopy and compared with the sample site's probing depth, bleeding tendency on probing, and plaque and gingivitis indices. If possible, one implant with deep and one with shallow pockets were selected within the same patient. The impact of the intraoral exposure time on the microbial composition around the implants was examined cross-sectionally, with the same group of patients. Only tendencies can be detected by the latter, and no concrete conclusions can be drawn. From the clinical parameters, increased probing depth was found to detrimentally increase the proportion of spirochetes and motile organisms, whereas the other parameters were found to be of minor importance. For partially edentulous patients only, there was a tendency for increased proportions of spirochetes and motile organisms the longer the intraoral exposure time. These observations emphasize the importance of the periodontal health of the remaining teeth (as a reservoir of pathogenic microorganisms) in partial edentulous patients rehabilitated by means of implants and indicate the importance of shallow pockets around implants (flap trimming when aesthetics and phonetics allow).

Bacteria↗

PubMed1991

Increase of lipid fluidity and suppression of proliferation resulting from liposome uptake by human keratinocytes in vitro.

The in vitro effects of liposomes on HaCaT human keratinocytes were studied with regard to their uptake, lipid fluidity and proliferation of the cells. Oligolamellar liposomes, prepared from soya bean phospholipids, had a mean size of 150 mm and consisted predominantly of phosphatidylcholine (83%) and phosphatidylethanolamine (10%) and the fatty acids comprised mainly linoleic acid (66%) or other unsaturated fatty acids. After 6 and 24 h of incubation with 1 and 0.1% w/v of liposomal lipids, phase-contrast microscopy revealed marked cytoplasmic vacuolization of the cells. Keratinocytes treated with the liposomes contained aggregations of multilaminated lipid material without delimiting cell membranes. The cellular lipid fluidity (reciprocal of diphenylhexatriene fluorescence polarization P-value) correlated with liposomal concentration and incubation time. A significant elevation of lipid fluidity (P less than 0.05) was observed with 1 and 0.1% liposomes after 1 h of incubation (81.8 +/- 4.7 and 95.7 +/- 1.2% of control P value) and for 0.01% liposomes after 3 h (96.2 +/- 1.5%). Maximum fluidity occurred after 48 h of exposure to 1% liposomes (42.1 +/- 3.1%). Exposure to liposomal lipids for 24 and 48 h resulted in suppressed cell proliferation with 50% inhibition concentrations (IC50), being 0.06% for incorporation of [3H]-thymidine. 0.08% for [14C]-amino-acid incorporation and greater than 1% for protein content per well after 24 h of exposure. The cells were able to proliferate and lipid fluidity returned to normal within 7 days following discontinuation of incubation with liposomal lipids.

Cell Division↗

PubMed1992

Cytotoxicity of ophthalmic preservatives on human corneal epithelium.

Because the corneal epithelium invariably encounters the full concentration of the preservative that is contained in multi-dose topical ophthalmic preparations, we investigated the cytotoxicity of several of these agents by using a sensitive model of human corneal epithelial cells in vitro. Primary cultures of epithelial cells were prepared from freshly enucleated globes. At confluence, all experimental cultures received a single dose of preservative at the concentration present in marketed formulations. The serum in the culture medium simulated the possible neutralizing effect of proteins present in the tear film in vivo. The cells were observed continuously by phase-contrast microscopy and time-lapse videomicrography for 24 hrs. Benzalkonium chloride at a concentration of 0.01% and chlorobutanol at 0.5% caused immediate cell retraction, as well as cessation of normal cytokinesis, cell movement, and mitotic activity; the epithelial cells degenerated within 2 hrs and 8 hrs, respectively. Cultures treated with chlorobutanol developed conspicuous blebs on the cell surface after 3 to 5 hrs of exposure. Thimerosal (0.001%) caused cell retraction, cessation of mitotic activity, and total cell destruction within 9 hrs. Sorbic acid (0.1% and 0.2%) greatly reduced cell movement and suppressed mitotic activity, but no cell death occurred. At concentrations of 50 ppm and 30 ppm, H2O2 instantaneously caused a marked retraction of the cells, followed by cessation of cytokinesis, cell movement, and mitosis. Retraction and death of the epithelial cells occurred within 12-24 hrs after exposure to 1 ppm H2O2 in serum-free medium. Polyquaternium ammonium chloride (0.001%) and polyaminopropyl biguanide (0.00005%) had no discernible effects on cytokinetic movement or on the mitotic activity of the epithelial cells. We relate our findings in vitro to those reported in vivo and discuss the mechanism of cytotoxicity of the various preservatives.

Cell Death↗

PubMed1975

Ultrastructural effects of lysozymes on the cell wall of Caryophanon latum.

When Caryophanon latum was exposed to egg white lysozyme in isotonic sucrose and observed by phase-contrast microscopy, protoplasts emerged along the length of the trichomes, apparently at sites corresponding to cross septa. Electron microscopy of sections revealed that this enzyme initially attacked the core of the septal peptidoglycan and delamination of septa resulted. The inner densely staining layer of the lateral and polar wall (considered to contain peptidoglycan as the major component) remained intact except for destruction at the advancing tip of partial septa; protoplasts or cell debris could escape from the gaps formed at developing septa. Treatment of intact trichomes with pronase, a lipase - phospholipase C mixture, EDTA, glutaraldehyde, or heat, before exposure to egg white lysozyme did not alter this pattern nor did it render the remaining peptidoglycan more susceptible to attack. The wall material external to the peptidoglycan was solubilized by pronase. The peptidoglycan remaining after lysozyme treatment was not morphologically changed by treatment with pronase. Lysozyme derived from Chalaropsis hydrolyzed incomplete septa initially, while the lateral and polar wall and complete septa were degraded later. Therefore, it is most probable that the inner dense layer does contain the peptidoglycan component and that some biochemical maturation distinguishes the substrate for these enzymes in the lateral wall and septa.

Bacteria↗

PubMed1996

Effects of triiodothyronine on morphology, growth behavior, and the actin cytoskeleton in mouse osteoblastic cells (MC3T3-E1).

We investigated the effects of thyroid hormone treatment on morphology, growth behaviour, and cytoskeletal structures of long-term cultured MC3T3-E1 cells. Morphological investigations were carried out on native cells by phase contrast microscopy and on epon-embedded semithin sections. The area covered by the cell and matrix layers (tissue-like area), percent extracellular matrix, average height of tissue-like area, and length and height of single cells were measured histomorphometrically on the cross sections. F-actin was analyzed histochemically and quantitated after fluorochrome-labeled phalloidin staining using confocal microscopy and fluorometry. Significant differences between control and T3-treated cells were found after confluency, but not in subconfluent cultures. Control cells continued to proliferate forming multilayers, and produced increasing amounts of extracellular matrix. In contrast, T3-treated cells stopped to proliferate forming two cell layers at the maximum. These cells were flattened, distinctly enlarged, and polygonal in shape. Histochemical staining for F-actin revealed three different staining patterns, depending on the position of the cell within the multilayer of control cultures. Basal cells contained a large number of thick stress fibers in parallel arrangement. Intermediate cells exhibited only a few thick actin filament bundles located at the outermost periphery. The superficial cells were characterized by a large number of thin, parallel-oriented microfilament bundles extending across the entire cytoplasm. The actin pattern of T3-treated cells resembled that of the basal cell layer of the control cells. The amount of F-actin increased with the prolonged T3 treatment. We conclude from these data that the known specific cellular responses to T3 treatment are accompanied by significant morphological alterations indicating pivotal effects of thyroid hormones on osteoblastic differentiation.

3T3 Cells↗

PubMed1989

Quantitative analysis of cell motility and chemotaxis in Dictyostelium discoideum by using an image processing system and a novel chemotaxis chamber providing stationary chemical gradients.

An image processing system was programmed to automatically track and digitize the movement of amebae under phase-contrast microscopy. The amebae moved in a novel chemotaxis chamber designed to provide stable linear attractant gradients in a thin agarose gel. The gradients were established by pumping attractant and buffer solutions through semipermeable hollow fibers embedded in the agarose gel. Gradients were established within 30 min and shown to be stable for at least a further 90 min. By using this system it is possible to collect detailed data on the movement of large numbers of individual amebae in defined attractant gradients. We used the system to study motility and chemotaxis by a score of Dictyostelium discoideum wild-type and mutant strains, including "streamer" mutants which are generally regarded as being altered in chemotaxis. None of the mutants were altered in chemotaxis in the optimal cAMP gradient of 25 nM/mm, with a midpoint of 25 nM. The dependence of chemotaxis on cAMP concentration, gradient steepness, and temporal changes in the gradient were investigated. We also analyzed the relationship between turning behavior and the direction of travel during chemotaxis in stable gradients. The results suggest that during chemotaxis D. discoideum amebae spatially integrate information about local increases in cAMP concentration at various points on the cell surface.

Cell Movement↗

PubMed2003

Human lens epithelial cell damage and stimulation of their secretion of chemical mediators by benzalkonium chloride rather than latanoprost and timolol.

OBJECTIVE: To investigate the effects of latanoprost, timolol maleate, and benzalkonium chloride on cell damage and induction of the secretion of chemical mediators of stress and wound healing by human lens epithelial cells in culture. METHODS: Cells from a human lens epithelial cell line (SRA01/04) were cultured in Dulbecco minimum essential medium supplemented with 5% fetal bovine serum. The amounts of latanoprost (50 micro g/mL), timolol maleate (5 mg/mL), or benzalkonium chloride (200 micro g/mL) used in eyedrops, and x10 to x1000 dilutions thereof, were added to the medium. After 7 days' culture, cell morphological changes were assessed using phase-contrast microscopy, and cell-free culture supernatants were collected for prostaglandin E2 (PGE2), interleukin 1alpha (IL-1alpha), and interleukin 6 (IL-6) iodine I 125 radioimmunoassay, enzyme-linked immunosorbent assay, and chemiluminescent enzyme immunoassay, respectively. RESULTS: All cells that were cultured with the concentrations of latanoprost, timolol, or benzalkonium chloride used in eyedrops detached from the culture dish and died within 3 days. At a x10 dilution of latanoprost or timolol or a x100 dilution of benzalkonium chloride, no proliferation or elongation of the cells was observed. Secretions of PGE2, IL-1alpha, and IL-6 at x10 dilutions of latanoprost or timolol were 3 to 77 times higher than in controls, whereas they were 190 to 305 times higher at a x180 dilution of benzalkonium chloride. The amounts of these soluble mediators in culture supernatants depended on the dose of latanoprost, timolol, or benzalkonium chloride added. CONCLUSION: Our results indicate that benzalkonium chloride, used as the preservative in eyedrops containing latanoprost or timolol, is the agent most damaging to lens epithelial cells and most strongly stimulates the expression of soluble chemical mediators in these cells.

Antihypertensive Agents↗

PubMed1988

Condensation of DNA in situ in metaphase chromosomes induced by intercalating ligands and its relationship to chromosome banding.

Interactions of certain intercalating cationic ligands with nucleic acids result in the formation of products that undergo condensation and agglomeration; this transition in solution can be monitored by light-scatter measurements. In the present study, using such intercalators as the antitumor drug mitoxantrone or fluorochromes acridine orange and quinacrine, we induced condensation of DNA in situ in Chinese hamster chromosomes. The in situ products scattered light and could be detected by darkfield- or phase-contrast microscopy. In the darkfield the complexes had a characteristic granular appearance and often generated a banding pattern on the chromosomes. In contrast, condensation of DNA in situ by the nonintercalating polyvalent cations (Co3+, spermine4+), while enhancing the chromosome's image contrast, did not produce the granular products or the banding. The condensation of free DNA, single or double stranded, natural or synthetic, the latter of various base composition and configuration, was also measured in solution. The condensation in solution and in situ was observed at similar concentrations of the respective ligands. The intercalating dye ethidium bromide, which did not condense DNA in solutions of moderate and high ionic strength, also did not generate the granular products or banding on chromosomes. The data also show that both base composition and configuration are important factors in determining the sensitivity of DNA to condensation by particular intercalating ligands. The studies suggest that the phenomenon of DNA condensation by intercalating dyes, which shows a high degree of specificity with respect to primary and secondary structures of DNA, may be associated with mechanisms of chromosome banding induced by the intercalating thiazine dyes in Giemsa staining or by quinacrine. Observation of chromosome banding based on light-scatter detection in darkfield microscopy allows the study of interactions between DNA and the ligands that neither fluoresce nor generate colored products. This principle of chromosome "counter-staining" can be explored by flow cytometry.

Acridine Orange↗

PubMed1984

Immunoreactive insulin from mouse brain cells in culture and whole rat brain.

Foetal mouse brain cells were cultured as described previously [Sotelo, Gibbs, Gajdusek, Toh & Wurth (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 653-657] without added insulin and without foetal calf serum after 12 days in culture. Examination by phase-contrast microscopy showed that these modifications did not appear to affect growth and development of the cells adversely. Silver impregnation of the cultures and indirect immunofluorescence following reaction with tetanus toxin showed that a high proportion of the cells resembled neurones. Analysis of concentrated culture medium by radioimmunoassay and high-pressure liquid chromatography (h.p.l.c.) revealed that the cells produced two main forms of immunoreactive insulin which differed from authentic pancreatic insulin in retention time. Immunoreactive somatostatin was also produced in culture and this was resolved into at least three forms by h.p.l.c. Immunoreactive insulin was also extracted from whole rat brain by using two published procedures. The method of Havrankova, Schmechel, Roth & Brownstein [Proc. Natl. Acad. Sci. U.S.A. (1978) 75, 5737-5741] consistently gave greater yields of insulin than did that of Eng & Yalow [Diabetes (1980) 29, 105-109] and the concentration was about three times that of plasma. The extracted insulin was further characterized by h.p.l.c. in each case and was found to behave like authentic pancreatic insulin. The production of insulin and somatostatin by foetal mouse brain cells in culture suggests that they may be a useful model system for studies of neuropeptide biosynthesis.

Animals↗