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PubMed1997

Pathogenicity of cilia-associated respiratory (CAR) bacillus isolates for F344, LEW, and SD rats.

We conducted experiments to test whether rats of F344, LEW, and SD strains differ in susceptibility to mycoplasma-free isolates of cilia-associated respiratory (CAR) bacillus, whether Mycoplasma pulmonis can affect expression of CAR bacillus disease, and whether isolates of CAR bacillus differ in virulence for rats. In the first experiment, 24 rats of each strain were inoculated intranasally with 10(7) bacilli of CAR bacillus X1428D/AS, and 24 rats of each strain were inoculated with sterile medium (controls). Eight weeks later, eight inoculated rats and eight control rats of each strain were euthanatized, eight inoculated and eight control rats were given 10(6.5) colony-forming units of M. pulmonis X1428D, and eight inoculated rats and eight control rats were sham inoculated. Four rats of each group were euthanatized 4 or 8 weeks after the second inoculation. Severity of lesions in nasal passages, middle ear, trachea, and lungs was assessed by scoring. Rats of all three strains given CAR bacillus had typical lesions of similar severity; M. pulmonis X1428D was avirulent and did not exacerbate CAR bacillus disease. In the second experiment, groups of eight rats of F344 and SD strains were given 10(5) or 10(7) CAR bacillus X1328E, X1428D/AS, or X2450D and euthanatized 8 or 16 weeks later. Isolates X1428D/AS and X2450D caused similar lesions in rats of both strains and at both doses, but CAR bacillus X1328E was avirulent. Rats of the tested strains are similarly susceptible to CAR bacillus disease, but CAR bacillus isolates differ in virulence.

Animals↗
PubMed1999

Isolation of cilia-associated respiratory (CAR) bacillus from pigs and calves and experimental infection of gnotobiotic pigs and rodents.

Filamentous, gram-negative bacteria morphologically similar to cilia-associated respiratory (CAR) bacillus of rodents and rabbits were isolated from the tracheas of 5 pigs and 4 calves. All pigs but none of the calves had histologic lesions of chronic tracheitis. In silver-stained histologic sections, CAR bacilli were adhered to the tracheal epithelium of each pig but were not found in the calves. Like CAR bacillus of rats, the bacteria displayed gliding motility and grew only in cell culture or cell culture medium supplemented with fetal serum. Initially, all isolates were contaminated by Mycoplasma spp. This contamination was eliminated from 4 pig isolates by limiting dilutions, and mycoplasma-free isolates were used to intranasally inoculate gnotobiotic pigs and CAR bacillus-free mice and rats and to immunize guinea pigs. The gnotobiotic pigs remained healthy, and when they were necropsied 4 and 7 weeks after infection no macroscopic or microscopic lesions were found in the respiratory tract. However, CAR bacillus was isolated at both times from the nasal cavities and tracheas of inoculated pigs, and the ciliated tracheal epithelium of infected pigs necropsied 7 weeks after infection was colonized by low numbers of CAR bacillus-like bacteria. The rats and mice remained healthy through week 12 postinoculation, and evidence of short- or long-term colonization was not detected by histologic examination or culture. When used as primary antibody for immunohistochemical staining, sera from guinea pigs immunized with pig CAR bacillus specifically stained CAR bacilli colonizing the respiratory epithelium of naturally infected pigs, whereas sera collected prior to immunization failed to react with the bacteria. These results indicate that CAR bacilli are unlikely to be primary pathogens of pigs or cattle and that rodents do not act as reservoirs.

Animals↗
PubMed1979

Numerical density of convex, nonbranching organelles in anisotropically oriented cells. Cilia in tangential chondrocytes.

A stereological expression relating the mean number of organelle profiles per cell profile, Np, to the mean number of organelles per cell, Nc, is first derived and then tested for cilia in tangenital articular chondrocytes where Nc is known to be 1.0. Nc derived stereologically for this organelle-cell model is 1.19. This close approximation of the estimate to a known value provides empirical evidence of the usefulness of the derived stereological expression.

Animals↗
PubMed2005

Caught Nek-ing: cilia and centrioles.

The Nek family of cell-cycle kinases is widely represented in eukaryotes and includes numerous proteins that were described only recently and remain poorly characterized. Comparing Neks in the context of clades allows us to examine the question of whether microbial eukaryotic Neks, although not strictly orthologs of their vertebrate counterparts, can provide clues to ancestral functions that might be retained in the vertebrate Neks. Relatives of the Nek2/NIMA proteins play important roles at the G2-M transition in nuclear envelope breakdown and centromere separation. Nek6, Nek7 and Nek9 also seem to regulate mitosis. By contrast, Nek1 and Nek8 have been linked with polycystic kidney disease. Results of statistical analysis indicate that the family coevolved with centrioles that function as both microtubule-organizing centers and the basal bodies of cilia. This evolutionary perspective, taken together with functional studies of microbial Neks, provides new insights into the cellular roles of the proteins and disease with which some of them have been linked.

Animals↗
PubMed1997

HSP70 and HSP90 homologs are associated with tubulin in hetero-oligomeric complexes, cilia and the cortex of Tetrahymena.

We show in the present study that homologs of hsp90 and hsp70 are induced by heat shocks in Tetrahymena and appear to form a high molecular mass complex (approximately 700 kDa) with tubulin. Three members of the hsp70 family (hsp72, 73, and 78) and one member of the hsp90 family (hsp82) have been identified by immunological or by a combination of immunological and sequencing methods. The known components of the 700 kDa complex and the conditions under which it can be recovered suggest that it may be an induced protective assemblage rather than a normal processing intermediate. Immunoblotting and immunofluorescence studies suggest further that large amounts of hsp73 and lesser amounts of hsp82 are associated with mature microtubules in both cilia and the cortex in this cell type. Some site-specific localizations of the identified heat shock proteins were also noted in non-microtubular components of the cell cortex.

Animals↗
PubMed1984

Attachment of the cap to the central microtubules of Tetrahymena cilia.

The central microtubule cap is bound to the ends of the two central microtubules in Tetrahymena thermophila cilia by plug-shaped structures similar in appearance to the distal filament plugs attached to the ends of the A-microtubules. The caps have been separated from the microtubules and are composed of a bead, two plates, and two peg-like plugs to which the microtubules are attached. The structure of the cap is discussed in relation to the directionality of microtubule assembly in vivo.

Animals↗
PubMed2003

Photolysis of caged calcium in cilia induces ciliary reversal in Paramecium caudatum.

Intracellular Ca(2+) concentration controls both the pattern and frequency of ciliary and flagellar beating in eukaryotes. In Paramecium, it is widely accepted that the reversal of the direction of ciliary beating (ciliary reversal) is induced by an increase in intra-ciliary Ca(2+) levels. Despite this, the Ca(2+)-sensitive region of the cilium that initiates ciliary reversal has not been clearly identified. We injected caged calcium into living P. caudatum cells and applied ultraviolet (UV) light to portions of the injected cells to raise artificially the intracellular Ca(2+) level ([Ca(2+)](i)). UV application to the upper ciliary region above the basal body induced ciliary reversal in injected cells. Furthermore, UV application to the tips of cilia induced weak ciliary reversal. Larger areas of photolysis in the cilium gave rise to greater angles of ciliary reversal. These results strongly suggest that the Ca(2+)-sensitive region for ciliary reversal is distributed all over the cilium, above the basal body.

Animals↗
PubMed1988

A cation channel for K+ and Ca2+ from Tetrahymena cilia in planar lipid bilayers.

The single-channel properties for monovalent and divalent cations of a voltage-independent cation channel from Tetrahymena cilia were studied in planar lipid bilayers. The single-channel conductance reached a maximum value as the K+ concentration was increased in symmetrical solutions of K+. The concentration dependence of the conductance was approximated to a simple saturation curve (a single-ion channel model) with an apparent Michaelis constant of 16.3 mM and a maximum conductance of 354 pS. Divalent cations (Ca2+, Ba2+, Sr2+, and Mg2+) also permeated this channel. The sequence of permeability determined by zero current potentials at high ionic concentrations was Ba2+ greater than or equal to K+ greater than or equal to Sr2+ greater than Mg2+ greater than Ca2+. Single-channel conductances for Ca2+ were nearly constant (13.9 pS-20.5 pS) in the concentrations between 0.5 mM and 50 mM Ca-gluconate. In the experiments with mixed solutions of K+ and Ca2+, a maximum conductance of Ca2+ (gamma Camax) and an apparent Michaelis constant of Ca2+ (K Cam) were obtained by assuming a simple competitive relation between the cations. Gamma Camax and K Cam were 14.0 pS and 0.160 mM, respectively. Single-channel conductances in mixed solutions were well-fitted to this competitive model supporting that this cation channel behaves as a single-ion channel. This channel had relatively high-affinity Ca2+-binding sites.

Animals↗
PubMed2005

Pseudomonas aeruginosa lectins I and II and their interaction with human airway cilia.

The bacterium Pseudomonas aeruginosa (PA) produces two carbohydrate binding lectins, designated PA lectin-I and lectin-II (PA-IL, PA-IIL). Both lectins are used by the bacterium to adhere to the glycocalyx of mammalian cells. In addition, the lectins immobilize ciliary beat. The kinetics of ciliary beat inhibition by each individual lectin have been analysed; however, their joint action on cilia has not been reported. Here we demonstrate that PA-IL and PA-IIL inhibit ciliary beat in a similar time-dependent manner. If applied simultaneously, ciliary beat inhibition after five hours of incubation was weaker than if each lectin was applied separately. Thus it can be hypothesized that the lectins compete for the same binding site(s) of the glycocalyx. Sugar inhibition experiments demonstrate that D-galactose and L-fucose inhibit both lectins, although clear preferences of D-galactose for PA-IL and of L-fucose for PA-IIL exist. These interactions have to be kept in mind when designing sugar-based therapies.

Adhesins, Bacterial↗
PubMed1987

Serodiagnosis of cilia-associated respiratory bacillus infection by the indirect immunofluorescence assay technique.

Antibody to cilia-associated respiratory (CAR) bacillus was detected by the indirect immunofluorescence assay (IFA) technique using tracheal sections of infected mice as antigen in serum samples collected from rats infected naturally and experimentally. Nine of 23 cases of natural infection were positive in IFA antibody, with titres ranging from 1:10 to 1:80, and all these antibody-positive cases were also histologically positive. The remaining 14 cases were negative in both IFA antibody and histological diagnosis, even though some of them were infected with Sendai virus and Mycoplasma pulmonis. In the experimental infection, serum samples collected from 18 rats on days 4, 7, 14, 21, 28 and 56 post-inoculation (PI) (three rats for each point) and examined for IFA antibody revealed that seroconversion occurred in one rat on day 14 PI and in three rats on day 21 PI. Antibody titres of 1:80 to 1:160 remained to the termination of the experiment. The IFA technique was useful for the diagnosis of CAR bacillus infection except in the early stage of the infection.

Animals↗
PubMed1989

Pathology of rats intranasally inoculated with the cilia-associated respiratory bacillus.

Five-week-old Wistar/Ms rats were inoculated intranasally with a lung homogenate containing a strain of cilia-associated respiratory (CAR) bacillus and were examined on days 4, 7, 14, 21, 28 and 56 postinoculation (PI). Some rats showed clinical signs with wheezing and considerable body weight loss from day 21 PI. Gross lesions, including enlargement of lungs with focal atelectasis, bronchiectasis and emphysema, were observed from day 21 PI. Histologically, round cell infiltration was first present in the lamina propria of the nasal respiratory mucosa on day 7 PI. From day 14 PI, colonization of the CAR bacillus (4-8 micron in length), associated with round cell infiltration in the lamina propria and the peripheral regions, was observed in the ciliated mucosa of the bronchioles, bronchi, trachea and nasal cavities. Generally, the lesions progressed and expanded from upper to lower airways with time. Sporadic mucopurulent bronchopneumonia was observed from day 21 PI in some rats. The CAR bacilli (0.2-0.25 micron in diameter) were also demonstrated electron-microscopically in the ciliated epithelium of the intrapulmonary airways. The CAR bacillus antigen was demonstrated on the ciliated mucosa of the affected airways by the indirect immunofluorescence assay technique. Microbiological examination revealed that the rats used in this study were free from other known respiratory pathogens throughout the experimental period. Thus, it is suggested that the CAR bacillus alone can produce a murine respiratory disease. Fourteen days were needed for pathological lesions to develop.

Animals↗
PubMed1989

Transmission experiments of cilia-associated respiratory bacillus in mice, rabbits and guineapigs.

Transmission experiments of cilia-associated respiratory (CAR) bacillus were performed in mice in order to clarify the principal route of the infection, and in rabbits and guineapigs in order to examine their susceptibility. Determination of the infection was evaluated serologically by the indirect immunofluorescence assay (IFA) technique and histologically by the presence of CAR bacillus in the airways. BALB/c mice were intranasally inoculated with the SMR strain of CAR bacillus. The IFA antibody to the bacteria in these mice rose to more than 1:160 at 4 weeks postinoculation (PI) and the mice were utilized as transmitters for the following experiments. One out of 15 uninfected mice kept in intracage contact with infected mice became infected from 4 weeks after contact. Incidence of contact infection increased thereafter. On the other hand, there was no evidence of infection in the uninfected mice housed in the separate cages from the cage in which infected mice were housed throughout the 12-week observation period. The primary method of CAR bacillus transmission seems to be direct contact with infected mice or fomites contaminated by infected mice; airborne transmission appears to be of little importance. Rabbits and guineapigs were also intranasally inoculated with the SMR strain of CAR bacillus. IFA antibodies were positively detected by 4 weeks PI, but no CAR bacillus nor histological changes relating to the infection were observed in the airways of either species. It is suggested that rat origin CAR bacillus can transmit to rabbits and guineapigs, and that the infection can spread to other species of rodents and rabbits.

Animals↗
PubMed2000

Sequence of 16S rRNA gene of rat-origin cilia-associated respiratory (CAR) bacillus SMR strain.

The 16S rRNA gene of the SMR strain of cilia-associated respiratory (CAR) bacillus, which was isolated from a spontaneously infected rat at our institute, was sequenced. Its 1,521 nucleotides were determined. On the basis of the results of the sequence analysis, the SMR strain was found to be most closely related to members of the Flavobacter/Flexibacter group. This sequence was compared with the previously determined 16S rRNA gene sequences (rat-origin: three; mouse-origin: one; rabbit-origin: one) of CAR bacillus isolates. The SMR strain showed the highest sequence similarity (99.9%) to the rat-origin CARB-NIH strain (Schoeb et al., 1993), and it was concluded that the strains are identical.

Animals↗
PubMed2005

Mechanism of signal amplification in the olfactory sensory cilia.

Molecular mechanisms underlying olfactory signal amplification were investigated by monitoring cAMP dynamics in the intact sensory cilia. We saw that [cAMP]i increased superlinearly with time during odorant stimuli for >1 s. This time course was remarkably different from that obtained with the rapid quench method previously applied to the in vitro preparation, in which [cAMP]i change has been reported to be transient. The superlinear increase of [cAMP]i was attributable to a gradual increase of cAMP production rate that was consistent with the thermodynamical interaction model between elemental molecules, as has been revealed on the rod photoreceptor cell. It thus seems likely that the fundamental mechanism for molecular interactions between olfactory transduction elements is similar to that of the rod. In olfaction, however, cAMP production was extremely small (approximately 200,000 molecules/s/cell at the maximum), in contrast to the cGMP hydrolysis in the rod (250,000 molecules/photon). The observed numbers indicate that the olfactory receptor cell has lower amplification at the enzymatic cascade. Seemingly, such low amplification is a disadvantage for the signal transduction, but this unique mechanism would be essential to reduce the loss of ATP that is broadly used for the activities of cells. Apparently, transduction by a smaller number of second-messenger formations would be achieved by the fine ciliary structure that has a high surface-volume ratio. In addition, it is speculated that this low amplification at their enzymatic processes may be the reason why the olfactory receptor cell has acquired high amplification at the final stage of transduction channels, using Ca2+ as a third messenger.

Adenylyl Cyclases↗
PubMed2005

Model of calcium oscillations due to negative feedback in olfactory cilia.

We present a mathematical model for calcium oscillations in the cilia of olfactory sensory neurons. The underlying mechanism is based on direct negative regulation of cyclic nucleotide-gated channels by calcium/calmodulin and does not require any autocatalysis such as calcium-induced calcium release. The model is in quantitative agreement with available experimental data, both with respect to oscillations and to fast adaptation. We give predictions for the ranges of parameters in which oscillations should be observable. Relevance of the model to calcium oscillations in other systems is discussed.

Animals↗
PubMed1995

Detection of cilia-associated respiratory bacillus in experimentally and naturally infected mice and rats by the polymerase chain reaction.

Cilia-associated respiratory (CAR) bacillus was detected by means of the reverse transcription (RT)-polymerase chain reaction (PCR), and the results were compared with those of indirect immunofluorescence test (IFAT) for the detection of the organism. In the experimental infections, 15 mice were in contact with mice previously inoculated with CAR bacillus. Three mice each were tested at days 3, 5, 7, 12 and 20 postexposure. On day 3 postexposure, CAR bacillus was detected in oral swab samples from all 3 mice by RT-PCR, but was not detected in any sampling sites from the mice by IFAT. Total numbers of positive samples from nasal, oral and tracheal swabs obtained through the test were 6/15, 14/15 and 8/15, respectively, by RT-PCR, and 2/15, 6/15 and 3/15, respectively by IFAT. For the detection of CAR bacillus in samples from 52 rats, 34 serum antibody negative rats by enzyme-linked immunosorbent assay were also negative by RT-PCR and IFAT except for one sample from the oral cavity, and all serum antibody positive rats were positive for the organism by RT-PCR but it could not be detected in five of them by IFAT. By means of RT-PCR, no differences in the positive rates depending on sampling sites were observed except in one rat. The RT-PCR was found to be a specific, highly sensitive and reliable procedure for detecting CAR bacillus in mice and rats. The oral cavity was the most suitable site for the diagnosis of the early stage of this infection by RT-PCR.

Animals↗
PubMed1985

Electron microscopy of fat-storing cells in liver diseases with special reference to cilia and cytoplasmic cholesterol crystals.

Two hundred and ten fat-storing cells in the liver of patients with liver diseases were examined electron microscopically, and 5 of them were found to project a single cilium from the perinuclear region to the space of Disse. Three cilia were in the space between a hepatocyte and their own cell body, and 2 were between a sinusoidal endothelial cell and their own cell body. In cross sections of the cilium, the axoneme consisted of 9 pairs of microtubules arranged in a cylinder and lacked a central pair of microtubules ("9 + 0" pattern). Arms and spokes were also absent in the axoneme. Therefore, the single cilium of fat-storing cells was considered to be immotile. Some fat-storing cells from a patients with alcoholic liver cirrhosis contained an angular electron lucid inclusion, which was identical to cholesterol ester crystals, suggesting the presence of disturbed cholesterol metabolism.

Cholesterol↗
PubMed2006

Formation of primary cilia in the renal epithelium is regulated by the von Hippel-Lindau tumor suppressor protein.

Growing evidence points to defects in the primary cilium as a critical mechanism underlying renal cyst development. Inactivation of the VHL gene is responsible for the autosomal dominant condition von Hippel-Lindau (VHL) disease and is implicated in most sporadic clear cell renal carcinomas. Manifestations of VHL disease include cysts in several organs, particularly in the kidney. Here it is shown that VHL inactivation is associated with abrogation of the primary cilium in renal cysts of patients with VHL disease and in VHL-defective cell lines. Complementation of VHL-defective clear cell renal carcinoma cell lines with wild-type VHL restored primary cilia. Moreover, it is shown that the effects of VHL on the primary cilium are mediated substantially via hypoxia-inducible factor. The effect of VHL status on the primary cilium provides a potential mechanism for renal cyst development in VHL disease and may help in the understanding of how VHL acts as a tumor suppressor.

Carcinoma, Renal Cell↗