John R. Kennedy

The study of ciliary frequencies with an optical spectrum analysis system

Abstract The cilia of rabbit tracheal epithelial cells beat at the rate of from 6 to 21 cycles/sec at physiological temperatures. The cilia of each cell show a frequency response which fluctuates within a frequency range specific to that cell. Cells from the same area of the trachea tend to fluctuate in the same frequency range. The frequency may be constant for several seconds or it may vary second-by-second in the range appropriate to the cell being measured. The frequency range can be altered in part by temperature. At 37 °C the frequency range may fluctuate by ± 3.5 beats/sec. As the temperature is decreased ciliary beat becomes more coherent and the frequency range decreases to ± 0.5 beats/sec at 5 °C. Frequencies are obtained with a photomultiplier system attached to a phase-contrast microscope. The mixed population of frequencies produced by the cilia passing through the transmitted light are sorted by Fast Fourier Transform analysis into discrete frequencies. With this system, frequencies of individual ciliated cells can be analysed in real time and for unlimited duration.

The primordial germ cells in early mouse embryos: Light and electron microscopic studies

Abstract The migrating primordial germ cells in mouse embryos aged between 8.5 and 11 days were identified under the light and electron microscopes by means of localizing alkaline phosphatase activity, and their ultrastructure was studied. Most of the migrating primordial germ cells were round with smooth contour; they were invariably in close contact with irregularly shaped surrounding cells. The ultrastructural characteristics of early primordial germ cells included: prominent nucleoli, cytoplasmic protrusions into the nuclei, Golgi complexes with alkaline phosphatase activity, disappearance of granular endoplasmic reticulum with increasing age of the embryos, dense cytoplasm with extremely abundant ribosomes, and cytoplasmic dense granules. The significance of these findings is discussed in relation to the origin and migration of mouse primordial germ cells.

The fine structure of the digestive system of daphnia pulex crustacea cladocera

The alimentary canal of Daphnia pulex consists of a tube-shaped foregut, a midgut (mesenteron) with an anterior pair of small diverticula, and a short hindgut. The foregut and hindgut are structurally similar. Each is formed by a low cuboidal epithelium 5 mum tall and lined with a chitinous intima. The midgut wall consists of a simple epithelium resting on a thick beaded basal lamina which is surrounded by a spiraling muscularis. Anteriorly the midgut cells are columnar in shape being 30 mum in height each having a basal nucleus, anteriorly concentrated mitochondria and in apical border of long thin microvilli. Posteriorly the midgut cells become progressively shorter so that in the posteriormost region of the midgut the cells are 5 mum tall and cuboidal in shape. The microvilli concomitantly become shorter and thicker. All mesenteron cells contain the usual cytoplasmic organelles. The paired digestive diverticula are simple evaginations of the midgut. The wall of each consists of a simple epithelium of cuboidal cells 25 mum in height, each with a brushed border of long thin microvilli. Enzyme secretion appears to be holocrine in mode and not confined to any one region of the mesenteron though definitely polarized anteriorly. The thin gut muscularis encircles the entire length of the midgut and caeca. Thick and thin filaments appear to be in a 6:1 ratio.

Stimulation of frog ciliated cells in culture by acetylcholine and substance P

1. Ciliary beat frequency in epithelial outgrowths from cultured explants of Rana pipiens palate changed markedly from second to second. 2. Acetylcholine (10(-8) to 10(-3) M) and substance P (1.35 x 10(-7) to 1.35 x 10(-5) M) increased and stabilized ciliary beat frequency. The effect of acetylcholine and part of the effect of substance P were blocked by atropine (10(-4) M). 3. Acetylcholine appears to act directly and substance P both directly and indirectly through the release of acetylcholine.

Cigarette Smoke: The Effect of Residue on Mitochondrial Structure

Cigarette smoke residue is ciliatoxic to Tetrahymena pyriformis. Principal sites of activity are the mitochondria of the cell. The internal membranes of the mitochondria are degraded with time, correlating well with loss of ciliary activity and cell death.

Synthesis of mucin glycoproteins by epithelial cells isolated from swine trachea by specific proteolysis

SummaryMucus-producing cells were isolated from swine trachea mucosa by a method that included enzymatic digestion of the epithelial surface with Dispase, a neutral protease fromBacillus polymyxa, and differential attachment of the washed cells to culture flasks coated with collagen. Epithelial cells were the major cell type isolated by these procedures. Ciliated cells that did not attach to the flasks were removed by decantation, and fibroblasts were destroyed by the bacterial protease. The isolated cells synthesized respiratory mucins and the rate of secretion was increased about threefold when tracheas were exposed to sulfur dioxide. The cultured cells incorporated both [35S]O4 and [I-14C]N-acetylglucosamine into secreted mucin glycoproteins. The secretion of glycoprotein increased for about 3 d until the cells became confluent, and then a constant rate was observed for a period of at least 7 d. This increase in the output of mucin glycoprotein during the initial 3 d of culture was accompanied by a corresponding increase in the number of mucus-producing cells in the flasks. The results obtained in these and subsequent studies suggest that the rate of formation of mucus-producing cells may be a rate limiting step in the regulation of mucin glycoprotein synthesis in tracheal epithelium.The chemical, physical, and immunological properties of the glycoprotein secreted by isolated tracheal epithelial cells were very similar to the mucin glycoprotein purified from washes of swine trachea epithelium. The purified mucin glycoproteins showed complete cross-reaction with antibodies to trachea mucin glycoprotein. They were eluted near the void volume during gel filtration on Sepharose CL-6B columns. The glycoprotein isolated from culture media under the standard assay conditions had nearly the same carbohydrate composition as samples purified from washes of trachea epithelium. Reduced oligosaccharides released by β-elimination with dilute alkaline borohydride showed similar elution profiles during chromatography on Bio Gel P-6 colums. Taken collectively, these results suggest that the isolated epithelial cells secreted mucin glycoproteins that were very similar to those synthesized by the intact trachea epithelium under standard incubation conditions.

The effects of the organophosphorous insecticides Dursban and Lorsban on the ciliated epithelium of the frog palate in vitro

The ciliotoxic potential of the organophosphorous insecticides Dursban™ and Lorsban™, their active ingredient, chlorpyrifos, and their carrier ingredients (Blanks) were assessed. Since chlorpyrifos inhibits acetylcholinesterase, the acetylcholine-innervated ciliated epithelial cultures of frog palate were used as the model. All compounds caused a decrease in frequency of ciliary beat over time. EC50 values followed the same order as the time to inhibition. The orders were Lorsban > Dursban > chlorpyrifos, and Lorsban > Dursban ∼ Lorsban Blank > Dursban Blank. Stimulation of ciliary beating occurred immediately after exposure to all compounds, followed by inhibition. Dursban, Lorsban, and both Blanks elicited stimulatory effects in the presence of atropine. Atropine only blocked the initial stimulatory response with chlorpyrifos. In addition to chlorpyrifos, some component(s) of the inert ingredients were initially stimulatory but ultimately inhibitory to ciliary beating in the frog palate model. All compounds caused mitochondrial damage, including swelling, disruption of cristae, and loss of matrix.

CYTOTOXIC EFFECTS OF THE HERBICIDE 3-AMINO-1,2,4-TRIAZOLE ON DAPHNIA PULEX (CRUSTACEA:CLADOCERA)

Zero to 24 hour old specimens of Daphnia were exposed to 10 ppm 3-amino-1,2,4-triazole at 24° C until immobilized. The most consistent alteration in cell structure was expressed by the mitochondria especially those of muscle fibers. Not all muscle cells within a single animal nor all mitochondria within a single cell were affected equally. The most common alteration observed was folding of the outer membrane. This was accompanied by organelle swelling and a reduction in electron density of the ground matrix and number of cristae. In addition other cytotoxic effects were observed. These included general tissue swelling, disarrangement of myofilaments and dissociation of membranes. Mobile 0-24 hr old daphnids which were exposed to the herbicide for up to 15 hr showed no cell or organelle damage regardless of length of exposure. Data from acute static experiments suggest that the time of immobility is closely related to molt for 0-24 hr juveniles.

The immediate effects of silicon carbide whiskers upon ciliated tracheal epithelium

Considering the relationship between toxicity of dust and particle geometry, as exhibited by asbestos, we have examined short-term biological effects of SiC whiskers (SiCW) in vitro and in vivo. Cultured explants of tracheal epithelium were exposed to a range of SiCW concentrations. There were no dramatic effects on ciliary function as measured by an optical spectrum analysis system that provided discrete ciliary frequencies. Particles were swept by ciliary activity into nonciliated regions where foci of extensive cell damage and death were observed with whiskers penetrating epithelial layers into the underlying tissues. Similar necrotic foci were observed in tracheae from rats exposed by intratracheal instillation to SiC whiskers in vivo.

Regulation of the synthesis of mucin glycoproteins in swine trachea explants

SummarySwine tracheal epithelium has been cultured as explants in a chemically defined medium for periods of up to 2 wk. The viability of the explants was shown by the preservation of the ultrastructural features of cells in the epithelial layer and by the active incorporation of radioactive glucosamine and sulfate into secreted mucin glycoproteins. The rate of secretion of mucin glycoprotein was about 0.035 mg per cm2 per d. After initial 24 h lag period was shown to be due to the equilibration of intracellular mucin glycoprotein pools with radioactive precursors. The rate of secretion of glycoprotein showed a linear dependence on the area of the explant, and maximal incorporation was observed at 200 μM glucosamine. A higher concentration of35SO4, 1000 μM, was required for maximal incorporation of the precursor. Insulin at 0.1 to 1 μg/ml increased the rate of secretion twofold, whereas 0.1 to 100 μg/ml of hydrocortisone and 0.1 to 100 μg/ml of epinephrine significantly decreased the rate of secretion. Vitamin A had little or no effect of normal trachea explants at low concentrations, and, at higher concentrations, 10−5M, it decreased the secretion of mucin glycoproteins. Vitamin A, at a concentration of 10−9M, increased the rate of synthesis of glycoprotein at least fourfold in trachea explants from vitamin A-deficient rats.Mucus secretions collected from the surface of swine trachea and from the culture medium of trachea explants were purified. The mucus was solubilized by reduction and carboxymethylation, and the high molecular weight mucin glycoproteins were purified by chromatography on Sepharose CL-6B columns under dissociating conditions in 2M guanidine HCl. The mucin glycoproteins purified from swine trachea and from the culture medium of trachea explants were virtually indistingushable. They showed the same properties when examined by gel electrophoresis and immunoprecipitation. The purified glycoproteins contained about 25% protein, and serine, threonine, and proline were the principal amino acids present. More than 80% of the carbohydride chains in both samples were released by treatment with alkaline borohydride. Nearly the same molar ratio ofN-acetylgalactosamine,N-acetylglucosamine, galactose, fucose, sulfate, and sialic acid was found in both preparations.