Molecular characterization of centriole assembly in ciliated epithelial cells

Ciliated epithelial cells have the unique ability to generate hundreds of centrioles during differentiation. We used centrosomal proteins as molecular markers in cultured mouse tracheal epithelial cells to understand this process. Most centrosomal proteins were up-regulated early in ciliogenesis, initially appearing in cytoplasmic foci and then incorporated into centrioles. Three candidate proteins were further characterized. The centrosomal component SAS-6 localized to basal bodies and the proximal region of the ciliary axoneme, and depletion of SAS-6 prevented centriole assembly. The intraflagellar transport component polaris localized to nascent centrioles before incorporation into cilia, and depletion of polaris blocked axoneme formation. The centriolar satellite component PCM-1 colocalized with centrosomal components in cytoplasmic granules surrounding nascent centrioles. Interfering with PCM-1 reduced the amount of centrosomal proteins at basal bodies but did not prevent centriole assembly. This system will help determine the mechanism of centriole formation in mammalian cells and how the limitation on centriole duplication is overcome in ciliated epithelial cells.     

Depletion of serum-derived exosomes aggravates heat stress-induced damage of bovine mammary epithelial cells

Molecular Biology Reports - Exosomes are involved in intercellular communication, affecting many physiological and pathological process. The present study evaluated the effects of serum exosomes on...     

Effect of inducers of lipid peroxidation on the behavior of ciliated epithelial cells

Television microscope and original image treatment system were used for monitoring and recording the ciliary activity (beat frequency) of gill ciliated epithelia of the mussel Mytilus edulis (Bivalvia) and of the rat tracheal ciliated epithelia in response to the following prooxidants: H₂O₂, Fe⁺², Fe⁺² + ascorbic acid and NADP-H + ADP + Fe⁺². Mussel ciliated cells proved to be more sensitive to the influence of the prooxidants than rat cells. The reactions of ciliated epithelial cells of mollusks and rats to the inducers of lipid peroxidation were not similar to behavioral responses of these cells under the action of low-dose ionizing radiation.     

Equine bronchial epithelial cells differentiate into ciliated and mucus producing cells in vitro

We describe a method for creating differentiated equine bronchial epithelial cell cultures that can be used for in vitro studies including airway disease mechanisms and pathogen–host interactions. Our method is based on the culturing of human tracheobronchial epithelial cells at an air–liquid interface (ALI) in specific serum-free, hormone-supplemented medium. Bronchial epithelial cells are isolated and grown on T-Clear® insert membranes. Within 2 to 3 wk, cells differentiate into ciliated and mucus producing cells as demonstrated by confocal and electron microscopy. Furthermore, the demonstration of the two major gel-forming mucin species, Muc5ac and Muc5b, in our bronchial epithelial cell culture system validates this method for studies of respiratory tract disease of the horse.     

Mechanical sensitivity and cell coupling in the ciliated epithelial cells ofMytilus edulis gill

Summary Transmission electron microscopy has not provided strong evidence for gap junctions inMytilus edulis gill tissue, in spite of extensive physiological evidence for coupled ciliary arrest in lateral cells and coupled activation in abfrontal cells. To investigate the kinds and relative distribution of cell junctions and also to determine whether ciliary membrane particle differences exist in these two types of oppositely mechanically sensitive cells, we analyzed the structure of these and two other ciliated cell types (frontal and laterofrontal) by freeze-fracture replication. Gap junctions occur in all four ciliated cell types, but they are relatively small and of variable morphology, often consisting of elongate, winding complexes of membrane particles. Statistically, such structures rarely would be recognized as gap junctions in thin sections. Gap junctions appear to be most abundant between the highly coupled abfrontal cells, minimal between laterofrontal cells, and not evident in the epithelial cells that separate coupled ciliated cell types. The ciliary necklaces of the mechanically activated abfrontal cilia are typically 4- or 5-stranded while those of the remaining three cell types are mainly 3-stranded. In developing gill tips, ciliated cells have abundant gap junctions and newly formed cilia have a full complement of necklace particles. Nascent lateral cilia are not mechanically sensitive, indicating that the acquisition of mechanosensitivity does not correlate with the presence of ciliary necklace or other membrane particles. Lateral and laterofrontal cells become sensitive to neurotransmitters soon after the appearance of the latter during development, but mechanosensitivity of both lateral and abfrontal cells arises substantially later.     

Effects of 1.8 GHz radiofrequency field on DNA damage and expression of heat shock protein 70 in human lens epithelial cells

To investigate the DNA damage, expression of heat shock protein 70 (Hsp70) and cell proliferation of human lens epithelial cells (hLEC) after exposure to the 1.8 GHz radiofrequency field (RF) of a global system for mobile communications (GSM). An Xc-1800 RF exposure system was used to employ a GSM signal at 1.8 GHz (217 Hz amplitude-modulated) with the output power in the specific absorption rate (SAR) of 1, 2 and 3 W/kg. After 2 h exposure to RF, the DNA damage of hLEC was accessed by comet assay at five different incubation times: 0, 30, 60, 120 and 240 min, respectively. Western blot and RT-PCR were used to determine the expression of Hsp70 in hLECs after RF exposure. The proliferation rate of cells was evaluated by bromodeoxyuridine incorporation on days 0, 1 and 4 after exposure. The results show that the difference of DNA-breaks between the exposed and sham-exposed (control) groups induced by 1 and 2 W/kg irradiation were not significant at any incubation time point (P > 0.05). The DNA damage caused by 3 W/kg irradiation was significantly increased at the times of 0 and 30 min after exposure (P < 0.05), a phenomenon that could not be seen at the time points of 60, 120 or 240 min (P > 0.05). Detectable mRNA as well as protein expression of Hsp70 was found in all groups. Exposure at SARs of 2 and 3 W/kg for 2 h exhibited significantly increased Hsp70 protein expression (P < 0.05), while no change in Hsp70 mRNA expression could be found in any of the groups (P > 0.05). No difference of the cell proliferation rate between the sham-exposed and exposed cells was found at any exposure dose tested (P > 0.05). The results indicate that exposure to non-thermal dosages of RF for wireless communications can induce no or repairable DNA damage and the increased Hsp70 protein expression in hLECs occurred without change in the cell proliferation rate. The non-thermal stress response of Hsp70 protein increase to RF exposure might be involved in protecting hLEC from DNA damage and maintaining the cellular capacity for proliferation.     

The distribution of cationized ferritin receptors on ciliated epithelial cells of rat trachea

The interaction of tracheal cilia with the biphasic mucus layer covering the surface of the mammalian respiratory tract may be influenced by many cell surface coat components including those having an overall negative charge. In order to assess the distribution of ciliary anionic sites, cationized ferritin (CF) was used to label the surface of rat tracheal epithelium. If pieces of trachea were fixed with 3% glutaraldehyde and treated with CF at low (L) (0.08 mg/ml), medium (M) (0.32 mg/ml PBS), or high (H) (0.64 mg/ml PBS) concentrations, the label was distributed evenly over the entire external surface of the ciliary membrane at all concentrations. Unfixed tracheal tissue was also treated with L, M, and H CF for 1 or 5 min at 4 degrees C in order to minimize lateral redistribution of CF receptors. To ensure accessibility of the cell surface to CF the samples were agitated thoroughly during exposure. Exposure for 1 min to L, M, and H CF resulted in a light binding of ferritin particles on all portions of the ciliary membrane with occasional areas of multilayered binding distributed randomly on the ciliary shaft. When unfixed trachea was treated with CF for 5 min at 4 degrees C, CF binding was similar except heavier and more uniform. In no instance was there any preferential binding of CF to the ciliary tips at any of the concentrations used. Moreover, as indicated by the CF binding pattern at L concentrations, high density negative charges are present over almost the entire surface of the cilium. These results suggest that, unlike the ciliary membrane of other organs such as oviduct, negatively charged cell surface coat molecules are present on all areas of the ciliary membrane of rat tracheal epithelia.     

Myosin at the apical pole of ciliated epithelial cells as revealed by a monoclonal antibody

A monoclonal antibody (CC-212), obtained in a fusion experiment in which basal bodies from quail oviduct were used as immunogen, has been shown to label the apical pole of ciliated cells and to react with a 200-kD protein. This monoclonal antibody was demonstrated to be an anti-myosin from smooth muscle or from nonmuscular cells using the following criteria: On Western blots it reacted with the myosin heavy chains from gizzard and platelet extracts and from cultured cell line extracts, but did not react with striated muscle myosin heavy chains. By immunofluorescence it decorated the stress fibers of well-spread cells with a characteristic striated pattern, while it did not react with myotubes containing organized myofibrils. On native ciliated cells as well as on Triton-extracted ciliated cortices from quail oviduct, this monoclonal antibody decorated the apical pole with a stronger labeling of the periphery of the apical area. Ultrastructural localization was attempted using the immunogold technique on the same preparation. Myosin was associated with a filamentous material present between striated rootlets and the proximal extremities of the basal bodies. No labeling of the basal body itself or of axoneme was observed.     

The development of ciliated and mucus cells from basal cells in hamster tracheal epithelial cell cultures

Hamster tracheal epithelia consist of three cell types: ciliated, mucus and basal cells. Autoradiographic data from several studies suggest that either basal or non-ciliated columnar cells may serve as stem cells for regeneration of lost or damaged ciliated and mucus cells. The objective of the present study was to examine the role of basal cells in the formation of ciliated and mucus cells in hamster tracheal epithelial (HTE) cell cultures via tritiated thymidine ([3H]-TdR) autoradiography. When 3 day cultures were pulsed with [3H]-TdR for 6 hr and incubated for 2 additional days in non-radioactive media (5 day total) label was present in the nuclei of basal and columnar epithelial cells suggesting that the labeled columnar cells may be derived from basal cells. However, the morphological reorganization occurring during this 2 day interval may create difficulties in this interpretation. Since these morphological changes are minimal during the 6 day to 8 day in vitro period, 6 day HTE cultures were pulsed with [3H]-TdR for 6 hr and incubated for 2 additional days in non-radioactive media (8 day total), and examined to further study the fate of labeled basal cells during this period. Analysis of these 8 day cultures revealed that labeled nuclei were present in both basal cells and adjacent ciliated and mucus cells. These results do not exclude the possibility of non-basal cell origin of ciliated and mucus cells in other systems but suggest that, at least in HTE cultures, undifferentiated basal cells have the ability to develop into ciliated and mucus cells.     

Caveolin-3 and eNOS colocalize and interact in ciliated airway epithelial cells in the rat

In ciliated airway epithelial cells endothelial nitric oxide synthase as well as several other membrane bound proteins are located in the apical cell pole. To date, mechanisms that serve to target and to keep these proteins in this region are unknown. Endothelial nitric oxide synthase is known to target to caveolae by interaction with caveolin-1 or caveolin-3. Since caveolin-1 is found only in a subpopulation of ciliated cells at the basolateral cell membrane, we examined if caveolin-3 could be responsible for the apical localization of endothelial nitric oxide synthase in ciliated cells. We used real-time RT-PCR, laser-assisted microdissection, Western blotting and double-labeling immunohistochemistry to examine the presence of caveolin-3 in the airway epithelium of the rat. Indeed, we found caveolin-3-mRNA as well as protein in ciliated cells throughout the trachea and the bronchial tree. Caveolin-3-immunoreactivity was confined to the apical region and was colocalized with endothelial nitric oxide synthase and the high affinity choline transporter in a compartment distinct from the plasma membrane at the light microscopic level. No caveolae were found in the apical plasma membrane of ciliated cells but a tubulovesicular network was present in the apical region that reached up to the basal bodies of the cilia and was in close contact with mitochondria. Co-immunoprecipitation of caveolin-3 with endothelial nitric oxide synthase verified that both proteins interact in airway ciliated cells. These findings indicate that caveolin-3 is responsible to keep endothelial nitric oxide synthase in a membrane compartment in the apical region of ciliated cells.     

Appearance and disappearance of motile ciliated epithelial cells in sputum during acute respiratory infection

Motile ciliated cells were observed in sputum of a patient during two attacks of acute respiratory disease (ARD-1 and ARD-2). These cells appeared in the sputum on the fifth day of the disease in ARD-1 and on the third day in ARD-2. The period of massive desquamation of ciliated cells from the respiratory tract surface was 3 h in ARD-1 and no more than 1 h in ARD-2. Desquamation of ciliated cells was assumed to be due to the simultaneous influence of two factors: viruses and some unknown proinflammatory agents. Either factor alone, be it respiratory virus infection or inflammatory mediators (chemokines, neutrophils, or reactive oxygen species), is insufficient to cause desquamation. Desquamation of ciliated cells ceases simultaneously with the disappearance of viruses. Desquamated cells are supposed to contain virus particles and desquamation is considered to be one of the host defense mechanisms against viruses.Translated from Fiziologiya Cheloveka, Vol. 31, No. 1, 2005, pp. 137–140.Original Russian Text Copyright © 2005 by Fedotova, Fedotov.     

Patterns of protein synthesis in various cells after extreme heat shock

The analysis of proteins synthesized in rat thymocytes and mouse teratocarcinoma PCC-4 Aza 1 and myeloma Sp2/0 cells after 1 h of treatment at 42 or 44 degrees C was carried out. Shock at 42 degrees C reduced the total synthetic rate of proteins in all three cell lines and induced "classical" heat-shock protein with a mass of 70 kDa (hsp 70). Heat shock at 44 degrees C resulted in almost complete inhibition of protein synthesis; only a small amount of hsp 70 was synthesized. Meanwhile a new 48-kDa polypeptide (pI = 7.5) was found in the cells exposed to severe heat shock. This protein was compared by peptide mapping with other known polypeptides of the same size: heat-shock protein from chicken embryo cells and mitogen-stimulated polypeptide from human lymphoid cells. The peptide maps were not identical. It was also shown that after a shock at 44 degrees C teratocarcinoma cells were able to accumulate anomalous amounts of hsp 70 despite hsp 70 synthesis inhibition. The data show that reaction of various cells to extreme heat shock depends heavily on cell type.     

Repair of x-ray damage in aging WI-38 cells

Rate of strand rejoining and the ability to perform repair replication were determined in young ad old X-irradiated WI-38 cells. No differences in either process were apparent and we conclude that reduced efficiency in one or both of them is not responsible for _{in vitro} aging of human cells.     

Isolation, in silico characterization and chromosomal localization of a group of cDNAs from ciliated epithelial cells after in vitro ciliogenesis

Background

Immotile cilia syndrome (ICS) or primary ciliary dyskinesia (PCD) is an autosomal recessive disorder in humans in which the beating of cilia and sperm flagella is impaired. Ciliated epithelial cell linings are present in many tissues. To understand ciliary assembly and motility, it is important to isolate those genes involved in the process.

Results

Total RNA was isolated from cultured ciliated nasal epithelial cells after in vitro ciliogenesis and expressed sequenced tags (ESTs) were generated. The functions and locations of 63 of these ESTs were derived by BLAST from two public databases. These ESTs are grouped into various classes. One group has high homology not only with the mitochondrial genome but also with one or more chromosomal DNAs, suggesting that very similar genes, or genes with very similar domains, are expressed from both mitochondrial and nuclear DNA. A second class comprises genes with complete homology with part of a known gene, suggesting that they are the same genes. A third group has partial homology with domains of known genes. A fourth group, constituting 33% of the ESTs characterized, has no significant homology with any gene or EST in the database.

Conclusions

We have shown that sufficient information about the location of ESTs could be derived electronically from the recently completed human genome sequences. This strategy of EST localization should be significantly useful for mapping and identification of new genes in the forthcoming human genome sequences with the vast number of ESTs in the dbEST database.     

Endogenous calcitonin gene-related peptide protects human alveolar epithelial cells through protein kinase Cepsilon and heat shock protein

The intracellular mechanisms of ischemic preconditioning (PC) in preventing lung dysfunction following transplantation, shock, and trauma remain poorly understood. Previously, we have shown that alveolar epithelial cells secrete calcitonin gene-related peptide (CGRP) under inflammatory stress. Using a hypoxia/reoxygenation (H/R) and PC model, we found that CGRP was also secreted from human type II alveolar epithelial cells (A549) after PC. The locally released CGRP interacted with its receptor on the membrane of A549 cells and elicited downstream signals mediating the PC effect, because hCGRP(8-37), a specific CGRP receptor antagonist, attenuated the protective effect of PC. Pre-inhibition of CGRP protein synthesis by small interfering RNA exacerbated (but overexpression of the CGRP gene ameliorated) H/R-induced cell death, which supports the autocrine effect of CGRP on A549 cells. Exogenous bioactive CGRP mimicked the beneficial effect of PC and up-regulated the expression of heat shock protein 70 (HSP70), which might act as the end effector to maintain cell viability. These effects were sensitive to hCGRP(8-37), calphostin C (a protein kinase C (PKC) inhibitor), and 5-hydroxydecanoic acid (a mitochondrial K(+)(ATP) channel blocker) but were insensitive to protein kinase A blockers. Moreover, CGRP induced the membrane translocation of PKCepsilon. PKCV1-2 (a cell-permeable inhibitory peptide of PKCepsilon) effectively abolished CGRP-induced HSP70 expression and cell protection. Therefore, PC induces CGRP secretion from human alveolar epithelial cells, and the locally released CGRP acts back on these cells, protecting them from H/R injury. The post-receptor signaling of CGRP is through PKCepsilon-dependent expression of HSP70.