Nontypable Haemophilus influenzae (NTHI) has become the predominant cause of both acute suppurative otitis media and chronic otitis media with effusion. It has now been well-demonstrated that both outer membrane proteins and restriction fragment analysis of the bacterial genomes of concomitant nasopharyngeal and middle ear effusion isolates of NTHI are identical. It is therefore of critical importance to understand the mechanisms whereby bacteria that are present in normal healthy children in small numbers become the predominant organism in the nasopharynx in otitis media. The studies presented here suggest that nontypable Haemophilus influenzae can effectively decrease ciliary function as measured by stroboscopic illumination of ciliary beat frequency on human adenoidal organ culture. This organism also produces significant histopathologic and ultrastructural damage to the epithelial cells and cilia of adenoid organ culture, demonstrated by both light microscopy and scanning electron microscopy. The data suggest the following hypothesis: nontypable Haemophilus influenzae can destroy mucociliary function and allow increased bacterial replication in the mucus overlying the nasopharyngeal mucosa. The mucociliary system of the eustachian tube may also be involved in a similar manner, thus allowing bacteria to enter the middle ear space via the eustachian tube.

Adenoids↗

PubMed1988

Isolation and reactivation of highly-coupled newt lung cilia.

The paired lungs of the newt, Taricha granulosa, are simple, unbranched sacs, 3.5-5.0 cm in length. The inner epithelium overlying the pulmonary vein is differentiated into a mucociliary tract that extends the entire length of the lung. Populations of single, demembranated ciliary axonemes, 12-13 micron in length, can be isolated by extracting whole lungs or primary cultures of the ciliated epithelium with Triton X-100. The motile capabilities of the isolated axonemes are the highest yet obtained for any ciliary model. When exposed to a suitable reactivating medium containing Mg2+ and ATP, nearly 100% of the axonemes become motile. Uniform reactivation of high quality requires short extraction times, minimization of mechanical damage, and strict adherence to optimal conditions throughout the extraction, storage, and reactivation procedures. Significant deviations from either pH 7.0 or 0.12 M salt can lead to a rapid, irreversible decrease in the beat frequency of reactivated axonemes. Both DTT and EDTA serve to stabilize their motility. The isolated axonemes beat at 29.5 Hz in the presence of 1.75 mM ATP at 21 degrees C, matching the beat frequencies measured for cultured cells at the same temperature. With 5 mM ATP, beat frequencies over 40 Hz are measured. Our results show that neither the plasma membrane, accessory structures, nor hydrodynamic coupling of cilia are required for this activity and imply that the lack of these factors is not responsible for the low motile capabilities of ciliary models isolated previously.

Adenosine Triphosphate↗

PubMed1985

Flow birefringence of microtubules and its relation to birefringence measurements in cells.

Understanding the molecular basis of mitotic movements in living cells will require correlative experiments on intact cells, cell models, purified tubulin, and perhaps other biopolymers. Birefringence is one assay that is useful in all of these experimental situations. Heretofore, studies of birefringence changes during mitosis have lacked a quantitative basis for interpretation in terms of microtubule number and packing density. One of the aims of this work was to establish that relationship. Purified calf brain tubulin was polymerized to equilibrium and oriented in the hydrodynamic field of a microcapillary flow birefringence apparatus. The relationship between birefringence and microtubule packing density was determined by a combination of optical, electron microscopic, and biochemical methods. The data correlate surprisingly well with those obtained by others from in vitro measurements on isolated mitotic spindles. Using the flow birefringence data, the sensitivity of polarizing microscopes for detecting microtubules was examined and found to depend on microtubule packing density, object thickness, and instrumental factors that limit both the detection and measurement of weakly birefringent objects. Because of the dependence of measurement sensitivity on object thickness, a method of measuring the thickness of microtubule bundles using the dispersion of birefringence was developed. This method is capable of measuring thickness to within two or three Airy diffraction units and does not require any assumptions regarding object symmetry.

Animals↗

PubMed1985

Isolation of newt lung ciliated cell models: characterization of motility and coordination thresholds.

Demembranated ciliated cell models are useful for studying mechanisms responsible for the regulation of ciliary coordination and waveform. This paper describes procedures for isolating ciliated cells from the newt, Taricha granulosa, by trypsin dissociation, their subsequent demembranation by Triton X-100, and their reactivation with MgATP to produce highly motile, coordinated, ciliated cell models. Reactivation of cell models with a high degree of mechanochemical coupling depended on avoiding mechanical damage and maintaining optimal conditions during all stages of isolation and reactivation. Highly motile models were prepared from cells incubated in trypsin, treated briefly with EDTA, separated by gentle agitation, and concentrated by centrifugation at low gravitational forces. Optimal demembranation and reactivation conditions were similar to those described previously for isolated newt lung axonemes. Under these conditions, nearly 100% of the models were reactivated when provided with MgATP and 90-95% beat with coordinated waves. The ciliary tufts beat at frequencies within the range measured in living cells and their reactivated motility was stable for at least 30 min at constant MgATP. These highly coupled models were used to show (1) that development of coordination in the ciliary tuft occurs at a higher substrate concentration range (10-25 microM) than that required to initiate motility per se (2-10 microM; (2) that outer dynein arms may not contribute to beat frequency at substrate concentrations below 35 microM; and (3) that vanadate has effects both on beat frequency and coordination of the tufts.

Animals↗

PubMed1985

Newt lung ciliated cell models: effect of MgATP on beat frequency and waveforms.

Highly coupled newt lung ciliated cell models were used to study the effects of MgATP concentration on ciliary beat frequency and waveform. Models were prepared from ciliated lung cells of the newt Taricha granulosa by trypsin dissociation of the epithelium, demembranation with Triton X-100, and reactivation with MgATP, as described previously [Weaver and Hard, 1985]. Beat frequencies were measured stroboscopically. Ciliary waveforms of reactivated models and intact mucociliary epithelial sheets were determined by single frame analysis of high-speed movies. Waveform parameters calculated included the durations of the effective and recovery strokes, the angular swings and angular velocities of the ciliary base and tip, the position of the bend along the ciliary shaft during the recovery stroke, the velocity of recovery stroke bend propagation, and the ratio of the duration of recovery stroke bend propagation to the duration of the recovery stroke itself. We found that beat frequency varied biphasically in response to MgATP at 21 degrees C, as shown previously for isolated, individual, newt lung axonemes. Apparent Fmax (maximum beat frequency) and Km values of 25 Hz and 0.14 mM, and 35 Hz and 0.47 mM, respectively, were obtained for each linear segment of the biphasic double reciprocal plot. Demembranation did not alter either ciliary waveform or the pattern of coordination. In this system, metachrony is antilaeoplectic and ciliary waveform appears to be regulated independent of beat frequency.

Animals↗

PubMed1983

Muciliary transport in newt lungs: the ultrastructure of the ciliary apparatus in isolated epithelial sheets and in functional triton-extracted models.

High voltage and conventional electron microscopy were used to investigate the ultrastructure of the ciliary apparatus in intact and in Triton-extracted, reactivated sheets of mucociliary epithelium isolated from newt lung. Each long (about 13 microns) ciliary axoneme terminates on a barrel-shaped basal body which is anchored in the apical cytoplasm by a variety of accessory structures. A basal foot is associated with the midpoint of each basal body and acts as a focal point for numerous microtubules (MTs). In many cases MTs can be seen to interconnect the feet of neighbouring basal bodies. Attached to the proximal end of each basal body and extending in a direction opposite the basal foot is a large 'ciliary root'. Each ciliary root is associated with a distinct bundle of 6-7 nm microfilaments which appear to stain with the specific F-actin probe NBD-phallacidin. A single 3-4 microns long striated rootlet inserts into each ciliary root and extends toward the cell nucleus through an extensive network of microfilaments. At the level of the basal plate 'Y-shaped' structures appear to connect each axonemal outer doublet MT to the plasma membrane. All of these ciliary accessory structures are present in the same relationship in Triton-extracted models. Their morphology and distribution indicates that they serve to anchor the cilia in the apical cytoplasm. In addition some of these structures appear to be responsible for maintaining the structural and functional integrity of the ciliary field in the demembranated and reactivated models.

Animals↗

PubMed1977

Phase-randomized laser illumination for microscopy.

A simple apparatus is described that phase randomizes the output of a continuous argon ion laser, so that it may be used as a source of high intensity, monochromatic light for microscopy. The phase-randomizing device can be used with any laser, polarized or unpolarized, and of any desired power output and wavelength, including dye lasers for spectral studies. The randomizing system can be adapted to any light microscope and any optical system including bright-field, phase-contrast, Nomarski differential-interference, dark-field, and split-beam interference systems such as the Jamin-Lebedeff System. It can be used for a variety of photometric and photographic studies. The 514-nm wavelength appears to be relatively harmless to a variety of cells.

Lasers↗

PubMed1977

Behaviour of kinetochore fibres in Haemanthus katherinae during anaphase movements of chromosomes.

A laser light source along with a new method of preparing endosperm cells of Haemanthus katherinae for differential interference contrast (DIC) microscopy has led to increased visibility of kinetochore fibres. Little information is available concerning the behaviour of these fibres during anaphase in living cells. In metaphase, kinetochore fibres are seen as distinct bundles of microtubules, here referred to as 'filaments', extending from the kinetochore to the 'diffuse' pole. They possess an apparent globular substructure which corresponds to the moving 'particles or states' described previously from ciné films. In early anaphase, the filaments of each kinetochore fibre lose their parallel orientation characteristic of metaphase and splay out so that the more peripheral filaments intermingle with those of other kinetochore fibres. This process begins at the poles and proceeds as a wave toward the kinetochores as chromosomal movement progresses. This behaviour has been examined in relation to a number of proposed models for the mechanism of chromosome movement and has been found to place some constraints on some models but to be consistent with any model that hypothesizes that chromosomes move as a consequence of cumulative cohesive lateral interactions of microtubules.

Anaphase↗