A mechanism of airway injury in an epithelial model of mucociliary clearance

We studied the action of sodium metabisulphite on mucociliary transport in a frog palate epithelial injury model, hypothesizing that it may be useful for the study of mechanisms of airway injury. Sodium metabisulphite (MB) releases SO₂ on contact with water. SO₂ is a pollutant in automobile fumes and may play a role in the exacerbation of airway disease symptoms. We first investigated its effect on mucociliary clearance. MB 10^-1 M, increased mucociliary clearance time (MCT) by 254.5 ± 57.3% of control values, (p < 0.001, n = 7). MB 10^-4 and 10^-2 M did not interfere with mucus clearance time compared to control values. In MB-treated frog palates, MCT did not return to control values after one hour (control, 97.3 ± 6.3% vs. MB, 140.9 ± 46.3%, p < 0.001, n = 7). Scanning EM images of epithelial tissue were morphometrically analyzed and showed a 25 ± 12% loss of ciliated cells in MB palates compared to controls with an intact ciliary blanket. Intact cells or groups of ciliated cells were found in scanning EM micrographs of mucus from MB-treated palates. This was associated with increased matrix metalloproteinase (MMP-9) activity in epithelial tissue and mucus. We suggest that the loss of ciliated cells as a result of MMP-9 activation prevented full recovery of MCT after MB 10^-1 M. The mechanism of action may be on epithelial cell-cell or cell-matrix attachments leading to cell loss and a disruption of MCT. Further studies are warranted to determine whether this is an inflammatory mediated response or the result of a direct action on epithelial cells and what role this mechanism may play in the progression to chronic airway diseases with impaired mucociliary clearance.     

In vivo clearance of surfactant lipids during acute pulmonary inflammation.

We studied the action of sodium metabisulphite on mucociliary transport in a frog palate epithelial injury model, hypothesizing that it may be useful for the study of mechanisms of airway injury. Sodium metabisulphite (MB) releases SO₂ on contact with water. SO₂ is a pollutant in automobile fumes and may play a role in the exacerbation of airway disease symptoms. We first investigated its effect on mucociliary clearance. MB 10^-1 M, increased mucociliary clearance time (MCT) by 254.5 ± 57.3% of control values, (p < 0.001, n = 7). MB 10^-4 and 10^-2 M did not interfere with mucus clearance time compared to control values. In MB-treated frog palates, MCT did not return to control values after one hour (control, 97.3 ± 6.3% vs. MB, 140.9 ± 46.3%, p < 0.001, n = 7). Scanning EM images of epithelial tissue were morphometrically analyzed and showed a 25 ± 12% loss of ciliated cells in MB palates compared to controls with an intact ciliary blanket. Intact cells or groups of ciliated cells were found in scanning EM micrographs of mucus from MB-treated palates. This was associated with increased matrix metalloproteinase (MMP-9) activity in epithelial tissue and mucus. We suggest that the loss of ciliated cells as a result of MMP-9 activation prevented full recovery of MCT after MB 10^-1 M. The mechanism of action may be on epithelial cell-cell or cell-matrix attachments leading to cell loss and a disruption of MCT. Further studies are warranted to determine whether this is an inflammatory mediated response or the result of a direct action on epithelial cells and what role this mechanism may play in the progression to chronic airway diseases with impaired mucociliary clearance.     

Influence of rheological properties of human bronchial secretions on the ciliary beat frequency

We studied the relations between the mucociliary beat frequency (MF) measured photometrically on the depleted frog palate and the rheological properties of sputum collected in patients with chronic obstructive pulmonary disease. MF was lower (p less than 0.001) with sputa (11.3 +/- 3.3 Hz) than with frog mucus (16.9 +/- 3.3 Hz) used as controls. The relative transport rate (Tr) of sputa was closely correlated (r = 0.81, p less than 0.001) to the relative MF. Significant correlations were observed between MF on the one hand and viscosity (r = -0.68, p less than 0.01), elastic modulus (r = -0.70, p less than 0.01) and spinability (r = +0.49, p less than 0.05) on the other. These results suggest that abnormalities in the rheological properties of bronchial secretions may impair the mucociliary transport rate by first decreasing the ciliary beat frequency.     

Mucociliary clearance in chronic sinusitis: related human nasal clearance and in vitro bullfrog palate clearance

Nasal mucociliary clearance was measured in both healthy subjects and patients with chronic sinusitis using saccharin granule technique. Nasal mucociliary transit time (ST) was significantly slower in the patients with chronic sinusitis compared with that in controls (p less than 0.005). Nasal mucus collected from each nasal cavity was used for in vitro bullfrog palate clearance studies and compared to the in vivo nasal ST. Mucociliary clearance rate (MTR) on frog palate was 12.5 +/- 2.5 mm/min in the mucus from control subjects, 6.1 +/- 1.5 mm/min in the mucus from the patients. The difference was statistically significant (p less than 0.005). The MTR on frog palate in the patients whose nasal ST was within normal range was significantly slower than that in controls (p less than 0.005), but not significantly different from that in the patients whose nasal ST was over the normal range. These results suggest that the nasal mucous properties which decreased the mucociliary clearance on frog palate did not contribute to the mucociliary clearance of the patients who had a normal one. No significant correlation existed between MTR on frog palate and nasal ST in both control and chronic sinusitis. In chronic sinusitis patients, decelerated nasal ST was recovered significantly by normal saline nebulization compared with the value before the nebulization (p less than 0.01). None of the significant change of ST was observed in control before and after the nebulization.     

Infection of ciliated cells by human parainfluenza virus type 3 in an in vitro model of human airway epithelium

We constructed a human recombinant parainfluenza virus type 3 (rPIV3) that expresses enhanced green fluorescent protein (GFP) and used this virus, rgPIV3, to characterize PIV3 infection of an established in vitro model of human pseudostratified mucociliary airway epithelium (HAE). The apical surface of HAE was highly susceptible to rgPIV3 infection, whereas only occasional cells were infected when virus was applied to the basolateral surface. Infection involved exclusively ciliated epithelial cells. There was little evidence of virus-mediated cytopathology and no spread of the virus beyond the ciliated cell types. Infection of ciliated cells by rgPIV3 was sensitive to a neuraminidase specific for alpha2-6-linked sialic acid residues, but not to a neuraminidase that cleaves alpha2-3- and alpha2-8-linked sialic acid residues. This provided evidence that rgPIV3 utilizes alpha2-6-linked sialic acid residues for initiating infection, a specificity also described for human influenza viruses. The PIV3 fusion (F) glycoprotein was trafficked exclusively to the apical surface of ciliated cells, which also was the site of release of progeny virus. F glycoprotein localized predominately to the membranes of the cilial shafts, suggesting that progeny viruses may bud from cilia per se. The polarized trafficking of F glycoprotein to the apical surface also likely restricts its interaction with neighboring cells and could account for the observed lack of cell-cell fusion. HAE derived from cystic fibrosis patients was not more susceptible to rgPIV3 infection but did exhibit limited spread of virus due to impaired movement of lumenal secretions due to compromised function of the cilia.     

Functional activity of ciliated outgrowths from cultured human nasal and tracheal epithelia

Primary cultures of respiratory epithelium were produced as outgrowths from human fetal and adult tracheal and nasal polyp explants. Video recordings of the epithelial cell outgrowths were carried out after 5 days of culture and the ciliary beating frequency was analyzed by using a video technique. Uniform fields of differentiated ciliated cells were observed near the edge of the explant. In the transition region of the outgrowth from the explant to the outgrowth periphery, isolated ciliated cells were present, as well as cells with fused cilia. The ciliary beating frequency of the outgrowth of well-differentiated ciliated cells (13.5 +/- 1.4 Hz) was significantly higher (p less than 0.001) than the beating frequency of both the explant (11.9 +/- 0.7 Hz) and the ciliated cells with fused cilia (9.8 +/- 1.7 Hz). The same differentiation stages and functional activities were observed in the outgrowth cultures, whatever their origin. These in vitro models are comparable with each other and therefore could be useful for studying the ciliogenesis and functional activity of the human respiratory epithelium.     

Mucociliary transport determined by in vivo microdialysis in the airways of normal and CF mice

We report a novel method to measure mucociliary transport (MCT) in both the upper and lower airways of normal and CF mice. The in vivo microdialysis technique involves placing a small quantity of dye on the airway surface and a microdialysis probe a defined distance from the site of dye deposition. The dye is transported toward the probe by ciliary transport and, upon reaching the microdialysis probe, diffuses across the dialysis membrane and is collected in the dialysate leaving the probe. The rate of MCT is calculated from the length of time from dye deposition to recovery. The rate of tracheal MCT in normal mice was 2.2 +/- 0.45 (SE) mm/min (n = 6), a value similar to that in reports using other techniques. MCT in CF mice was not different (2.3 +/- 0.29, n = 6), consistent with previous observations suggesting that tracheal ion transport properties are not different between CF and normal mice. The rate of MCT in the nasal cavity of normal mice was slower than in the trachea (1.3 +/- 0.26, n = 4). MCT in the CF mouse nasal cavity (1.4 +/- 0.31, n = 8), a region in which the CF mouse exhibits bioelectric properties similar to the human CF patient, was, again, not different from the normal mouse, perhaps reflecting copious gland secretion offsetting Na(+) and liquid hyperabsorption. In conclusion, we have developed a versatile, simple in vivo method to measure MCT in both upper and lower airways of mice and larger animals.     

Presence of gamma-aminobutyric acid in embryonic palates of AJ and SWV mouse strains

The presence of gamma-aminobutyric acid (GABA) in the embryonic palate was sought as a criterion for its role in regulating palate development. GABA was measured by a gas chromatographic-mass spectrometric (GC-MS) method using the heptafluorobutyryl (HFB)-cyclohexyl-GABA derivative, which gave the necessary sensitivity and specificity to measure low levels of GABA in the presence of contaminating substances. GABA was measured in dissected embryonic palates at various times of development in the AJ mouse strain. GABA levels were lower in day 14 AJ palates (0.19 +/- 0.01 nmol/mg protein) than at days 13 (0.28 +/- 0.03) and 15 (0.30 +/- 0.04). Comparable levels were observed in fore- and hindlimbs at day 14, whereas levels were lower in embryonic tongue and higher, as was expected, in embryonic brain. To confirm the presence of GABA in the palate, it was analyzed in growing palate mesenchymal cells in primary and secondary cultures as well as in serum-free medium. In addition, GABA levels were compared in the SWV mouse strain; this strain exhibits a more efficient active uptake mechanism and diazepam produces a higher frequency of cleft palate in this strain than in AJ. SWV contained one and one-half to three times higher concentrations of GABA in excised palates and cultured palate cells than the AJ strain. Furthermore, when GABA levels in skin fibroblasts of the two strains were measured, SWV cells contained 2.7-fold greater GABA than AJ cells. The present results provide additional evidence for the role of GABA in palate development.     

Effect of estrogen on the metabolism of cortisol and cortisone in the baboon fetus at midgestation

To determine whether the metabolism of cortisol (F) and cortisone (E) in the baboon fetus is regulated by estrogen, fetal interconversion of F/E was measured at midgestation after an experimental increase in placental estradiol (E2) production. Six baboons (Papio anubis) received increasing numbers of androstenedione implants (50 mg) inserted s.c. at 8-day intervals between Days 70 and 100 of gestation (term = Day 184) to elevate the production of estrogen; controls (N = 8) received no treatment. On Day 100 of gestation, each animal was anesthetized with ketamine:halothane/nitrous oxide, the fetus was exteriorized and [3H] F/[14C] E was infused via a fetal femoral vein for 70 min. Blood samples were then obtained from the contralateral fetal femoral vein, the umbilical vein/artery, and a maternal saphenous vein. After purification of F and E, the metabolic clearance rate (MCR), peripheral interconversion, and placental extraction of F and E were calculated. Maternal serum E2 concentrations (ng/ml; mean +/- SE) between Days 80 and 100 of gestation were greater (p less than 0.01) in androstenedione-treated baboons (2.2 +/- 0.2) than in untreated controls (1.2 +/- 0.1). Although the MCR of F was similar in control (5.2 +/- 0.3 1/day) and treated (7.7 +/- 1.0 1/day) animals, the MCR of E (13.5 +/- 2.0 1/day) was increased (25.8 +/- 2.5 1/day; p less than 0.05) by androstenedione treatment. Placental extraction of F (59 +/- 9%) was lower (p less than 0.01) than that of E (82 +/- 5%) in untreated baboons and was not affected by androstenedione treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of atypical antipsychotics on visceral fat distribution in first episode, drug-naive patients with schizophrenia

The aim of this study was to determine the location of antipsychotic-induced weight gain in drug na?ve, first episode patients with schizophrenia. Various fatness and fat distribution parameters (by Computerized Tomography scanning and anthropometry) and 1600 hr plasma cortisol were measured in 19 (15 men and 4 women) subjects with schizophrenia (mean age = 31.0 years; mean body mass index [BMI] = 24.6 kg/m2) and an equal number of age- and sex- matched controls (mean age = 32.6 yr; mean BMI = 23.0 kg/m2). Patients were then given either olanzapine or risperidone. Sixteen patients were re-tested following a treatment period lasting approximately 6 months. Patients with schizophrenia, had significantly more intra-abdominal fat [IAF] (116.8 +/- 20.2 cm2 vs. 38.0 +/- 4.8 cm2, respectively; t = 3.80, df = 18, p < 0.0001) and had higher levels of plasma cortisol (360.2 +/- 49.6 nmol/L vs. 192.7 +/- 19.7 nmol/L, respectively; t = 3.13, df = 18, p < 0.003) than appropriately matched control subjects. Treatment with atypical antipsychotics did not result in a significant increase in IAF (116.8 +/- 20.2 cm2 vs. 131.7 +/- 20.9 cm2; p = NS) though visceral fat stores still remained significantly higher than those seen in controls (38.0 +/- 4.8 cm2) (F = 9.34; df = 2, 51; p < 0.0003). However, plasma levels of cortisol did significantly decrease (360.2 +/- 49.6 nmol/L +/- vs. 316.2 +/- 48.4 nmol/L; p < 0.05). Pre-treatment levels of IAF did not differ between those who received risperidone and those who were given olanzapine (123.0 +/- 35.9 cm2 vs. 113.1 +/- 15.7 cm2, respectively; t = 0.20, df = 16, p < 0.84). The increase in IAF did not differ between those given risperidone and those who received olanzapine (26.9 +/- 12.1 cm2 vs. 18.24 +/- 11.44 cm2, respectively; t = 0.50, df = 16, p < 0.63). Patients with drug na?ve, first episode, schizophrenia have higher levels of visceral fats stores as compared to matched control subjects. Treatment with atypical antipsychotics does not result in a significant increase in IAF distribution.     

Development and ciliation of the palate in two frogs, Bombina and Xenopus; a comparative study

The morphological changes that occur during metamorphosis in the palates of two types of anuran larvae (a discoglossid, Bombina orientalis, and a pipid, Xenopus laevis) are compared. In B. orientalis the structural changes are accompanied by the ciliation of the palate epithelium. Ciliation begins in the anterior region of the palate and continues in a posterior direction throughout metamorphosis. By contrast, the palate of X. laevis never becomes ciliated during its development. Instead, two ciliated grooves develop between the choanae (nasal openings) and the esophageal opening. The grooves transport mucus and trapped objects out of the internal nares and toward the esophagus. These grooves are compared to similar structures on the palate of adult B. orientalis. The timing and pattern of ciliogenesis during metamorphosis in each of these anurans is also described relative to well-established staging series for external frog development. We show that the onset and location of ciliogenesis are consistent and predictable in these anurans and, therefore, make the frog palate an excellent system for the study of ciliogenesis.     

Identification of novel ciliogenesis factors using a new in vivo model for mucociliary epithelial development

Mucociliary epithelia are essential for homeostasis of many organs and consist of mucus-secreting goblet cells and ciliated cells. Here, we present the ciliated epidermis of Xenopus embryos as a facile model system for in vivo molecular studies of mucociliary epithelial development. Using an in situ hybridization-based approach, we identified numerous genes expressed differentially in mucus-secreting cells or in ciliated cells. Focusing on genes expressed in ciliated cells, we have identified new candidate ciliogenesis factors, including several not present in the current ciliome. We find that TTC25-GFP is localized to the base of cilia and to ciliary axonemes, and disruption of TTC25 function disrupts ciliogenesis. Mig12-GFP localizes very strongly to the base of cilia and confocal imaging of this construct allows for simple visualization of the planar polarity of basal bodies that underlies polarized ciliary beating. Knockdown of Mig12 disrupts ciliogenesis. Finally, we show that ciliogenesis factors identified in the Xenopus epidermis are required in the midline to facilitate neural tube closure. These results provide further evidence of a requirement for cilia in neural tube morphogenesis and suggest that genes identified in the Xenopus epidermis play broad roles in ciliogenesis. The suites of genes identified here will provide a foundation for future studies, and may also contribute to our understanding of pathological changes in mucociliary epithelia that accompany diseases such as asthma.     

The ciliary rootlet maintains long-term stability of sensory cilia

The striated ciliary rootlet is a prominent cytoskeleton originating from basal bodies of ciliated cells. Although a familiar structure in cell biology, its function has remained unresolved. In this study, we carried out targeted disruption in mice of the gene for rootletin, a component of the rootlet. In the mutant, ciliated cells are devoid of rootlets. Phototransduction and ciliary beating in sensory and motile cilia initially exhibit no apparent functional deficits. However, photoreceptors degenerate over time, and mutant lungs appear prone to pathological changes consistent with insufficient mucociliary clearance. Further analyses revealed a striking fragility at the ciliary base in photoreceptors lacking rootlets. In vitro assays suggest that the rootlet is among the least dynamic of all cytoskeletons and interacts with actin filaments. Thus, a primary function of the rootlet is to provide structural support for the cilium. Inasmuch as photoreceptors elaborate an exceptionally enlarged sensory cilium, they are especially dependent on the rootlet for structural integrity and long-term survival.     

Newt lungs: a versatile system for the study of mucociliary transport

Lungs of the newt, Taricha granulosa, provide a versatile system for studying mucociliary transport. The mucociliary epithelium is restricted to a ventral strip of epithelium which extends the entire length of the lung. The transport process can be studied in the isolated lung or in a variety of functional components derived from it. These components include: (1) isolated sheets of the mucociliary epithelium, (2) flattened epithelial sheets grown in long term primary culture, (3) dissociated ciliated and mucous cells, (4) functional, demembranated ciliary tufts, and (5) isolated populations of discrete, demembranated axonemes. The latter two models can be reactivated with MgATP and made to beat at frequencies comparable to those measured in intact cells. Featuring large cells (25-30 microns in diameter) and cilia (12-13 microns in length) in comparison to other mucus-transporting systems, coupled with the ability to perform 'physiological' experiments at ambient temperatures, newt lungs afford a convenient system to study mechanisms involved in the control of ciliary beat frequency, waveform and coordination at several levels of organization.     

Mucus and the mare: how little we know

Uterine infections are a major cause of infertility, but the role of mucus in equine uterine defense is not well understood. Mucociliary currents play an important role in protecting mucous membranes, including the upper and lower respiratory tracts of mammals, and are required for feeding and oxygenation of many aquatic invertebrates. Although phagocytosis has long been considered the first line of uterine defense in the mare, there are concerns about its efficacy in the uterine lumen. Additional local defenses, such as mucociliary currents, have therefore been proposed. The uterine epithelium exhibits alternating mucus-secreting and ciliated cells supporting a mucopolysaccharide blanket, features shared with mucociliary membranes throughout the animal kingdom. Gross uterine anatomy, such as continuity of uterine and cervical folds, may indicate adaptations to mucociliary clearance. In addition, ciliated cells obtained in uterine lavages often display motility. Disruptions of mucociliary clearance play major roles in pathogenesis of mucosal infections in humans, including pneumonia, chronic sinusitis, and otitis media. Establishing drainage is a major goal of therapy in treatment of chronic sinusitis, hastening return of mucociliary function. Similar disruptions may occur in equine uterine infections, associated with accumulations of uterine fluid, loss of endometrial folds, and cervical trauma. Possible clinical implications of mucociliary clearance in the mare are discussed, however the role of mucociliary clearance in the mare remains speculative.     

Association of cytochrome P450 genes polymorphisms (CYP1A1 and CYP1A2) with the development of chronic obstructive pulmonary disease in Bashkortostan

To assess the role that polymorphisms of cytochrome P450 genes play in genetic predisposition to chronic obstructive pulmonary disease (COPD), the allele and genotype distributions of CYPIA1 (2455 A/G, 3801T/C) and CYP1A2 (-2464T/delT, -163C/A) genes were studied in Tatar and Russian COPD patients and in cases of healthy individuals (Russian, Tatar and Bashkir), residents of Bashkortostan. It was shown that the CYP1A1 and CYP1A2 genes haplotypes frequency distribution patterns do not differed between Tatars and Russians ethnic groups (chi2 = 0.973, df = 3, p = 1.00 and chi2 = 1.546, df = 3, p = 0.92, respectively). Analysis of the the CYP1A1 and CYP1A2 genes haplotypes revealed statistically significant differences in the haplotypes frequency distributions between Bashkirs versus Russians and Tatars (chi2 = 12.328, df= 3,p = 0.008; chi2 = 9.218, df=3, p = 0.034, respectively for CYP1A1 gene and (chi2 = 18.779, df=3, p = 0.0001, chi = 14.326, df=3, p = 0.003, respectively for CYP1A2 gene). The (-2467)delT allele and CYP1A2*1D haplotype of CYPIA2 gene was associated with higher risk of COPD in Tatar ethnic group (OR = 1.83, 95% CI 1.24-2.71, chi2 = 9.48, p = 0.003 and chi2 = 9.733, p = 0.0027, Pcor = 0.008; OR = 3.908, 95% CI 1.56-10.19, respectively). On the other hand the CYP1A2*1A haplotype had protective effect (chi2 = 6.319, p = 0.0127, Pcor = 0.038; OR = 0.6012, 95% CI 0.402-0.898). But at the same time we did not find any differences in the genotypes and haplotypes frequency distributions of the CYP1A2 gene within the patients and healthy groups in Russian ethnic group. We also did not find any association of CYP1A1 gene with COPD in ethnic groups of Bashkortostan.     

Impact of Cardiopulmonary Bypass on Respiratory Mucociliary Function in an Experimental Porcine Model

Background

The impact of cardiac surgery using cardiopulmonary bypass (CPB) on the respiratory mucociliary function is unknown. This study evaluated the effects of CPB and interruption of mechanical ventilation on the respiratory mucociliary system.

Methods

Twenty-two pigs were randomly assigned to the control (n = 10) or CPB group (n = 12). After the induction of anesthesia, a tracheostomy was performed, and tracheal tissue samples were excised (T0) from both groups. All animals underwent thoracotomy. In the CPB group, an aorto-bicaval CPB was installed and maintained for 90 minutes. During the CPB, mechanical ventilation was interrupted, and the tracheal tube was disconnected. A second tracheal tissue sample was obtained 180 minutes after the tracheostomy (T180). Mucus samples were collected from the trachea using a bronchoscope at T0, T90 and T180. Ciliary beat frequency (CBF) and in situ mucociliary transport (MCT) were studied in ex vivo tracheal epithelium. Mucus viscosity (MV) was assessed using a cone-plate viscometer. Qualitative tracheal histological analysis was performed at T180 tissue samples.

Results

CBF decreased in the CPB group (13.1 ± 1.9 Hz vs. 11.1 ± 2.1 Hz, p < 0.05) but not in the control group (13.1 ± 1 Hz vs. 13 ± 2.9 Hz). At T90, viscosity was increased in the CPB group compared to the control (p < 0.05). No significant differences were observed in in situ MCT. Tracheal histology in the CPB group showed areas of ciliated epithelium loss, submucosal edema and infiltration of inflammatory cells.

Conclusion

CPB acutely contributed to alterations in tracheal mucocilliary function.     

Calcium control of ciliary arrest in mussel gill cells.

After several hours in 20 mM sodium phosphate and 40 mM KCI (pH 7.4) or similar simple solutions, ciliated cells exfoliate en masse from stripped gill epithelium of freshwater mussels, e.g., Elliptio complanatus. Three types of ciliated cells--lateral (L), laterofrontal (LF), and frontal (F)--can be distiniguished and counted separately in the suspensions. About one-half of the cells of each type remain motile. Motility is unaffected by addition of 10(-5) M A23187 or 10(-2) M Ca+2 added separately, but when ionophore and Ca+2 are added together, ciliary beat is largely arrested. Treatment of the cells with Triton X-100 (Rohm & Haas Co., Philadelphia, Pa.) results in a total loss of motility as the ciliary membrane becomes disrupted. Such models can be reactivated by addition of ATP and Mg+2. All ciliated cell types are reactivated to about the same extent. At least 80% of the activity of the untreated preparation returns. Ca+2-EGTA buffers added to the reactivating solutions permit titration of free Ca+2 concentration vs. percent motility. Activity is unchanged for all cell types at Ca+2 less than 10(-7) M; at 10(-6) Ca+2, L cilia of all cell types are arrested differentially, whereas at Ca+2 greater than 10(-4) M most cilia of all cell types are arrested. We conclude: (a) that increasing cytoplasmic Ca+2 is directly responsible for ciliary arrest, (b) that the readily reversible physiological arrest response of the L cilia in the intact gill is caused by a rise in free Ca+2 in narrow limits from ca. 5 x 10(-7) M to ca. 8 x 10(-7) M, and (c) that the site which is sensitive to Ca+2 is part of the ciliary axoneme or the basal apparatus.     

Mhp107 is a member of the multifunctional adhesin family of Mycoplasma hyopneumoniae

Mycoplasma hyopneumoniae is the causative pathogen of porcine enzootic pneumonia, an economically significant disease that disrupts the mucociliary escalator in the swine respiratory tract. Expression of Mhp107, a P97 paralog encoded by the gene mhp107, was confirmed using ESI-MS/MS. To investigate the function of Mhp107, three recombinant proteins, F1(Mhp107), F2(Mhp107), and F3(Mhp107), spanning the N-terminal, central, and C-terminal regions of Mhp107 were constructed. Colonization of swine by M. hyopneumoniae requires adherence of the bacterium to ciliated cells of the respiratory tract. Recent studies have identified a number of M. hyopneumoniae adhesins that bind heparin, fibronectin, and plasminogen. F1(Mhp107) was found to bind porcine heparin (K(D) ～90 nM) in a dose-dependent and saturable manner, whereas F3(Mhp107) bound fibronectin (K(D) ～180 nM) at physiologically relevant concentrations. F1(Mhp107) also bound porcine plasminogen (K(D) = 24 nM) in a dose-dependent and physiologically relevant manner. Microspheres coated with F3(Mhp107) mediate adherence to porcine kidney epithelial-like (PK15) cells, and all three recombinant proteins (F1(Mhp107)-F3(Mhp107)) bound swine respiratory cilia. Together, these findings indicate that Mhp107 is a member of the multifunctional M. hyopneumoniae adhesin family of surface proteins and contributes to both adherence to the host and pathogenesis.