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1.
Sreedhara Sangadala U. Ramadas Bhat Joseph Mendicino 《Molecular and cellular biochemistry》1993,126(1):37-47
The structures of high molecular weight sulfated oligosaccharide chains in mucins purified from the sputum of a patient with cystic fibrosis and blood group H determinant were established. Reduced oligosaccharides released by treatment with alkaline borohydride were separated by ion exchange chromatography on DEAE-Agarose and a fraction containing multisulfated chains was further purified by lectin affinity chromatography to completely remove small amounts of sialylated chains. A major sulfated oligosaccharide fraction containing chains with an average of 160 to 200 sugar residues was isolated by gel filtration on BioGel P-10 columns and individual subfractions were characterized by methylation analysis, periodate oxidation and sequential glycosidase digestion before and after desulfation. Carbohydrate analysis yielded Fuc, Gal and GldNAc in a ratio of 1:2:2.1 and only one galactosaminitol residue for every 160-to 200 sugar residues. The average molecular weight of oligosaccharide chains in these fractions was between 27,000 and 40,000 daltons. Structural analysis showed that these high molecular weight chains contained varying amounts of the repeating unit shown in the following oligosaccharide. Only one in about every 10 repeating units contained sulfate esters.Several shorter chains which contain 2 to 3 sulfate esters were also isolated from this multisulfated oligosaccharide fraction. The structures proposed for these oligosaccharides indicate that they are lower molecular weight chains with the same general structure as those found in the high molecular weight sulfated oligosaccharides.
Taken collectively, the results of these studies show that a major sulfated oligosaccharide fraction in resporatory mucin purified from the mucus of patients with cystic fibrosis contains high molecular weight branched chains that consist of a repeating oligosaccharide sequence with sulfate linked to the 6 positions of galactose and possibly GlcNAc residues in the side chains. 相似文献
2.
D. P. Chora L. Reddy S. K. Gupta L. Wan P. A. Mathieu R. L. Shoemaker J. S. Rhim 《In vitro cellular & developmental biology. Animal》1994,30(8):539-546
Summary Cystic fibrosis (CF) involves abnormalities in mucus production and secretion of the airway. Studies of the regulation of
airway mucin production and secretion has been difficult due to the lack of in vitro models of the airway epithelial cells
which express functional differentiation. Because the majority of the mucin in the airway is apparently produced by the submucosal
glands, we have focused our attention on the development of cell culture models of human airway submucosal glands. This report
describes the propagation of CF airway submucosal gland epithelial cells which continue to express mucin production. The CF
bronchus was obtained from a 31-yr-old patient who received a double lung transplant. The glands were dissected out and primary
cultures prepared by the explant/outgrowth procedure. The cells were immortalized by infection with Adl2-SV40 hybrid virus.
The cultures are maintained in serum-free keratinocyte basal medium supplemented with insulin (5μg/ml), hydrocortisone (0.5μg/ml), epidermal growth factor (10 ng/ml), bovine pituitary extract (25μg/ml), and antibiotics. Cultures were passaged using 0.125% trypsin in Ca+2 and Mg+2-free Hanks’, balanced salt solution. Polymerase chain reaction (PCR) analysis demonstrated that the cells were homozygous
for the ΔF508 mutation. Morphologic observations showed that the cells were epithelial and were interconnected by sparsely
distributed desmosomes. Their cytoplasm contained secretory-type structures including abundant Golgi, rough endoplasmic reticulum,
and secretory vesicles. Immunofluorescent studies determined that all cells were positive for cytokeratins, mucin glycoconjugates,
and cystic fibrosis transmembrane conductance regulator. The cultures secreted substantial amounts of mucin glycoproteins
and expressed the MUC-2 mucin gene. Patch clamp experiments revealed that the cells expressed defective Cl− channels which were not activated by Forskolin. 相似文献
3.
Edward DeBuysscher John Kennedy Joseph Mendicino 《In vitro cellular & developmental biology. Plant》1984,20(5):433-446
Summary Mucus-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.
This investigation was supported by United States Public Health Service Grant HL 20868 from the National Heart, Lung and Blood
Institute and AM 28187 from the National Institute of Arthritis, Diabetes and Digestive and Kidney Diseases. 相似文献
4.
Long-term culture of normal and cystic fibrosis epithelial cells grown under serum-free conditions 总被引:8,自引:0,他引:8
Dieter C. Gruenert Carol B. Basbaum Jonathan H. Widdicombe 《In vitro cellular & developmental biology. Plant》1990,26(4):411-418
Summary The understanding of pathways associated with differentiated function in human epithelial cells has been enhanced by the development
of methods for the short-term culture of human epithelial cells. In general these methods involve the use of serum. The subculture
and maintenance of epithelial cells in long-term culture has been more problematic. A serum-free medium developed for human
bronchial epithelial cells was slightly modified and found to be useful for the subculture and long-term maintenance of not
only bronchial epithelial cells, but also tracheal, nasal polyp, and sweat gland epithelial cells from either normal or cystic
fibrosis individuals. The cells maintained epithelial-specific characteristics after multiple subcultures. Monolayers of epithelial
cells showed junctional complex formation, the presence of keratin, and micro villi. Functional studies with Ussing chambers
showed short circuit current (Isc) responses to isoproterenol, bradykinin, or calcium ionophore (A23187) in subcultured tracheal and bronchial cells.
This work is supported by grants HL41928 and DK39619 (DCG), HL24136 (CBB), and HL42368 (JHW and DCG) from the National Institutes
of Health, Bethesda, MD. 相似文献
5.
Peggy Wallace John R. Kennedy Joseph Mendicino 《In vitro cellular & developmental biology. Animal》1994,30(3):168-180
Summary The morphologic and functional properties of explant out-growth cells and epithelial cells isolated from swine trachea epithelium
by proteolysis were examined. A mixed population of ciliated, serous, and basal cells, obtained from out-growths, from proteolysis
of trachea epithelium, and from unattached explants in organ culture, all yielded cell cultures that were composed almost
entirely of mucus-secreting cells. When the cells were grown in primary or secondary culture on a modified collagen matrix
in supplemented HAM:DMEM (1:1) medium they expressed a mucus-secreting phenotype with numerous mucus granules at various stages
of maturation and incorporated [3H]GlcN and35SO4 into secreted mucin glycoproteins. Results obtained in these studies suggest that extensive transdifferentiation of ciliated
and serous cells to mucus-secreting cells occurs after the release and during subsequent attachment and culture. Ciliated
cells containing mucus granules were seen in various stages of cilia resorption. Basal cells containing mucus granules were
also frequently observed. The number of mucus-secreting cells and the synthesis of mucin glycoproteins increased dramatically
with time of attachment and culture, whereas cell proliferation, population doubling time of 72 h, and incorporation of [3H]thymidine into DNA increased much more slowly. The number of mucus-secreting cells correlated closely with the level of
secretion of mucin glycoproteins. Taken collectively, these studies help to elucidate the transdifferentiation process, which
dramatically increases the number of mucus-secreting cells after disruption and release of epithelial cells from swine tracheobronchial
epithelium. A similar mechanism involving disruption of the extracellular matrix may be involved in the stimulation of hypersecretion
of mucus and mucin glycoproteins by chemical and infections irritants. 相似文献
6.
Sreedhara Sangadala Dooha Kim John M. Brewer Joseph Mendicino 《Molecular and cellular biochemistry》1991,102(1):71-93
Summary The oligosaccharide chains in human and swine trachea and Cowper's gland mucin glycoproteins were completely removed in order to examine the subunit structure and properties of the polypeptide chains of these glycoproteins. The carbohydrate, which constitutes more than 70% of these glycoproteins, was removed by two treatments with trifluoromethanesulfonic acid for 3 h at 3° and periodate oxidation by a modified Smith degradation. All of the sialic acid, fucose, galactose, N-acetylglucosamine and N-acetylgalactosamine present in these glycoproteins was removed by these procedures.The deglycosylated polypeptide chains were purified and characterized. The size of the monomeric forms of all three polypeptide chains were very similar. Data obtained by gel filtration, release of amino acids during hydrolysis with carboxypeptidase B and gel electrophoresis in the presence of 0.1% dodecyl sulfate showed that a major fraction from each of the three mucin glycoproteins had a molecular size of about 67 kDa. All of the deglycosylated chains had a tendency to aggregate. Digestion with carboxypeptidases showed that human and swine trachea mucin glycoproteins had identical carboxyl terminal sequences, -Val-Ala-Phe-Tyr-Leu-Lys-Arg-COOH. Cowper's gland mucin glycoprotein had a similar carboxyl terminal sequence, -Val-Ala-Tyr-Leu-Phe-Arg-Arg-COOH. The yield of amino acids after long periods of hydrolysis with carboxypeptidases showed that at least 85% of the polypeptide chains in each of the deglycosylated preparations have these sequences. These results suggested that the polypeptide chains in these deglycosylated mucin glycoprotein preparations were relatively homogeneous.The deglycosylated polypeptide chains as well as the intact mucin glycoproteins had blocked amino terminii. The purified polypeptide chains were digested with trypsin-TCPK, and S. aureus V8 protease and the resulting peptides were isolated by gel electrophoresis in the presence of 0.1% dodecyl sulfate and by HPLC. Two partial amino acid sequences from swine trachea mucin glycoprotein, two partial sequences from human trachea mucin glycoprotein and three partial sequences from Cowper's gland mucin glycoprotein were determined. The partial amino acid sequences of the peptides isolated from swine trachea mucin glycoprotein showed more than 70% sequence homology to a repeating sequence present in porcine submaxillary mucin glycoprotein. Five to eight immunoprecipitable bands with sizes ranging from about 40 kDa to 46 kDa were seen when the polypeptide chains were digested with S. aureus V8 protease. All of the bands had blocked amino terminii and differed by a constant molecular weight of about 1.5 kDa. These data suggest that the polypeptides were formed by cleavage of glutamic acid residues present at regular intervals in the chains of all three mucin glycoproteins. These large immunoreactive peptides were formed by the removal of smaller peptides from the carboxyl terminal end of the deglycosylated mucin glycoprotein chains. Taken collectively, these findings indicate that the polypeptide chains in these mucin glycoproteins are very similar in subunit structure and that there is a high degree of homology between their polypeptide chains. 相似文献
7.
Cindy Lloyd John R. Kennedy Joseph Mendicino 《In vitro cellular & developmental biology. Plant》1984,20(5):416-432
Summary Swine 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, 10−5
M, it decreased the secretion of mucin glycoproteins. Vitamin A, at a concentration of 10−9
M, 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.
This investigation was supported by U.S. Public Health Service Grants HL 20868, HL 24688, and HL 24718 from the National Heart,
Lung and Blood Institute, Bethesda, MD, and AM 28187 from the National Institute of Arthritis, Diabetes and Digestive and
Kidney Diseases, Bethesda, MD. 相似文献
8.
Altered terminal glycosylation, with increased fucosylation and decreased sialylation is a hallmark of the cystic fibrosis (CF) glycosylation phenotype. Oligosaccharides purified from the surface membrane glycoconjugates of CF airway epithelial cells have the Lewis x, selectin ligand in terminal positions. This review is focused on the investigations of the glycoconjugates of the CF airway epithelial cell surface. Two of the major bacterial pathogens in CF, Pseudomonas aeruginosa and Haemophilus influenzae, have binding proteins which recognize fucose in -1,3 linkage and asialoglycoconjugates. Therefore, consideration has been given to the possibility that the altered terminal glycosylation of airway epithelial glycoproteins in CF contributes to both the chronic infection and the robust, but ineffective, inflammatory response in the CF lung. Since the glycosylation phenotype of CF airway epithelial cells have been modulated by the expression of wtCFTR, the hypotheses which have been proposed to relate altered function of CFTR to the regulation of the glycosyltransferases are discussed. Understanding the effects of mutant CFTR on glycosylation may provide further insight into the regulation of glycoconjugate processing as well as new approaches to the therapy of CF. 相似文献
9.
Rhim AD Kothari VA Park PJ Mulberg AE Glick MC Scanlin TF 《Glycoconjugate journal》2000,17(6):385-391
Cystic fibrosis (CF) has a characteristic glycosylation phenotype usually expressed as a decreased ratio of sialic acid to fucose. The glycosylation phenotype was found in CF/T1 airway epithelial cells (F508/F508). When these cells were transfected and were expressing high amounts of wtCFTR, as detected by Western blot analysis and in situ hybridization, the cell membrane glycoconjugates had an increased sialic acid content and decreased fucosyl residues in 1,3/4 linkage to antennary N[emsp4 ]-acetyl glucosamine (Fuc1,3/4GlcNAc). After the expression of wtCFTR decreased, the amount of sialic acid and Fuc1,3/4GlcNAc returned to levels shown by the parent CF cells. Sialic acid was measured by chemical analysis and Fuc1,3/4GlcNAc was detected with a specific 1,3/4 fucosidase. CF and non-CF airway cells in primary culture also had a similar reciprocal relationship between fucosylation and sialylation. It is possible that the glycosylation phenotype is involved in the pathogenesis of CF lung disease by facilitating bacterial colonization and leukocyte recruitment. 相似文献
10.
Verhaeghe C Tabruyn SP Oury C Bours V Griffioen AW 《Biochemical and biophysical research communications》2007,356(3):745-749
Cystic fibrosis is a common genetic disorder characterized by a severe lung inflammation and fibrosis leading to the patient's death. Enhanced angiogenesis in cystic fibrosis (CF) tissue has been suggested, probably caused by the process of inflammation, as similarly described in asthma and chronic bronchitis. The present study demonstrates an intrinsic pro-angiogenic status of cystic fibrosis airway epithelial cells. Microarray experiments showed that CF airway epithelial cells expressed several angiogenic factors such as VEGF-A, VEGF-C, bFGF, and PLGF at higher levels than control cells. These data were confirmed by real-time quantitative PCR and, at the protein level, by ELISA. Conditioned media of these cystic fibrosis cells were able to induce proliferation, migration and sprouting of cultured primary endothelial cells. This report describes for the first time that cystic fibrosis epithelial cells have an intrinsic angiogenic activity. Since excess of angiogenesis is correlated with more severe pulmonary disease, our results could lead to the development of new therapeutic applications. 相似文献
11.
Altered O-glycosylation and sulfation of airway mucins associated with cystic fibrosis 总被引:6,自引:0,他引:6
Cystic fibrosis (CF) is the most lethal genetic disorder in Caucasians and is characterized by the production of excessive amounts of viscous mucus secretions in the airways of patients, leading to airway obstruction, chronic bacterial infections, and respiratory failure. Previous studies indicate that CF-derived airway mucins are glycosylated and sulfated differently compared with mucins from nondiseased (ND) individuals. To address unresolved questions about mucin glycosylation and sulfation, we examined O-glycan structures in mucins purified from mucus secretions of two CF donors versus two ND donors. All mucins contained galactose (Gal), N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), fucose (Fuc), and sialic acid (Neu5Ac). However, CF mucins had higher sugar content and more O-glycans compared with ND mucins. Both ND and CF mucins contained GlcNAc-6-sulfate (GlcNAc-6-Sul), Gal-6-Sul, and Gal-3-Sul, but CF mucins had higher amounts of the 6-sulfated species. O-glycans were released from CF and ND mucins and derivatized with 2-aminobenzamide (2-AB), separated by ion exchange chromatography, and quantified by fluorescence. There was nearly a two-fold increase in sulfation and sialylation in CF compared with ND mucin. High performance liquid chromatography (HPLC) profiles of glycans showed differences between the two CF samples compared with the two ND samples. Glycan compositions were defined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Unexpectedly, 260 compositional types of O-glycans were identified, and CF mucins contained a higher proportion of sialylated and sulfated O-glycans compared with ND mucins. These profound structural differences in mucin glycosylation in CF patients may contribute to inflammatory responses and increased pathogenesis by Pseudomonas aeruginosa. 相似文献
12.
Schulz BL Sloane AJ Robinson LJ Prasad SS Lindner RA Robinson M Bye PT Nielson DW Harry JL Packer NH Karlsson NG 《Glycobiology》2007,17(7):698-712
Cystic fibrosis (CF) is characterized by chronic lung infection and inflammation, with periods of acute exacerbation causing severe and irreversible lung tissue damage. We used protein and glycosylation analysis of high-molecular mass proteins in saline-induced sputum from CF adults with and without an acute exacerbation, CF children with stable disease and preserved lung function, and healthy non-CF adult and child controls to identify potential biomarkers of lung condition. While the main high-molecular mass proteins in the sputum from all subjects were the mucins MUC5B and MUC5AC, these appeared degraded in CF adults with an exacerbation. The glycosylation of these mucins also showed reduced sulfation, increased sialylation, and reduced fucosylation in CF adults compared with controls. Despite improvements in pulmonary function after hospitalization, these differences remained. Two CF children showed glycoprotein profiles similar to those of CF adults with exacerbations and also presented with pulmonary flares shortly after sampling, while the remaining CF children had profiles indistinguishable from those of healthy non-CF controls. Sputum mucin glycosylation and degradation are therefore not inherently different in CF, and may also be useful predictive biomarkers of lung condition. 相似文献
13.
Katja Sobczak Andrei Segal Nadine Bangel‐Ruland Judith Semmler Willy Van Driessche Hermann Lindemann Ralf Heermann Wolf‐Michael Weber 《The journal of gene medicine》2009,11(9):813-823
Background
Cystic fibrosis (CF) respiratory epithelia are characterized by a defect Cl? secretion and an increased Na+ absorption through epithelial Na+ channels (ENaC). The present study aimed to find an effective inhibitor of human ENaC with respect to replacing amiloride therapy for CF patients. Therefore, we developed specific antisense oligonucleotides (AON) that efficiently suppress Na+ hyperabsorption by inhibiting the expression of the α‐ENaC subunit.Methods
We heterologously expressed ENaC in oocytes of Xenopus laevis for mass screening of AON. Additionally, primary cultures of human nasal epithelia were transfected with AON and were used for Ussing chamber experiments, as well as biochemical and fluorescence optical analyses.Results
Screening of several AON by co‐injection or sequential microinjection of AON and ENaC mRNA in X. laevis oocytes led to a sustained decrease in amiloride‐sensitive current and conductance. Using primary cultures of human nasal epithelia, we show that AON effectively suppress amiloride‐sensitive Na+ absorption mediated by ENaC in CF and non‐CF tissues. In western blot experiments, it could be shown that the amount of ENaC protein is effectively reduced after AON transfection.Conclusions
Our data comprise an initial step towards a preclinical test with AON to reduce Na+ hyperabsorption in CF epithelia. Copyright © 2009 John Wiley & Sons, Ltd.14.
Altered glycosylation is a phenotypic characteristic of cystic fibrosis (CF), and some of the alterations are summarized. The lungs are the site of the lethal pathology of the disease. Therefore, two of the characteristics were examined in CF and non-CF immortalized airway epithelial cell lines (AEC). The activity of -l-fucosidase was elevated (280%) in CF AEC when compared with non-CF AEC, whereas the activities of the other lysosomal enzymes which were examined were similar in both cell types. -l-Fucosidase activity was transiently increased in the non-CF cells after treatment with Brefeldin A (BFA) for 6 h. Thus BFA caused the normal cells to express a phenotypic characteristic of CF. Glycopeptides from the CF and non-CF AECs metabolically labeled withl-[3H]fucose were examined for binding to lentil lectin-Sepharose. A higher percentage of CF glycopeptides bound to lentil lectin, 43% compared with 23% for non-CF control. In addition a higher percentage of CF glycopeptides were bound tightly to lentil lectin and required 0.2M -methylmannoside to be eluted. This species of tightly bound glycopeptides increased dramatically to 77% from 46% when the CF AEC were treated with BFA. In contrast, the non-CF cell glycopeptides had a minor decrease in tightly bound glycopeptides to 26% from 33% after BFA treatment. Thus, the CF AEC showed fucosylation alteration observed previously for other CF cells and tissue. 相似文献
15.
Cystic fibrosis is a disease for which a number of Phase I clinical trials of gene therapy have been initiated. Several factors account for the high level of interest in a gene therapy approach to this disease. CF is the most common lethal inherited disease in Caucasian populations. The lung, the organ that is predominantly responsible for the morbidity and mortality in CF patients, is accessible by a non-invasive method, the inhalation of aerosols. The vectors employed in the Phase I trials have included recombinant adenoviruses, adeno-associated viruses and cationic lipids. While there have been some positive results, the success of the vectors until now has been limited by either immunogenicity or low efficiency. A more fundamental obstacle has been the absence of appropriate receptors on the apical surface of airway epithelial cells. Molecular conjugates with carbohydrate substitution to provide targeting offer several potential advantages. Lactosylated polylysine in which 40% of the lysines have been substituted with lactose has been shown to provide a high efficiency of transfection in primary cultures of CF airway epithelial cells. Other important features include a relatively low immunogenicity and cytotoxicity. Most importantly, the lactosylated polylysine was demonstrated to give nuclear localization in CF airway epithelial cells. Until now, most non-viral vectors did not have the capability to provide nuclear localization. These unique qualities provided by the lactosylation of non-viral vectors, such as polylysine may help to advance the development of molecular conjugates sufficiently to warrant their use in future clinical trials for the gene therapy of inherited diseases of the lung. 相似文献
16.
Protein profiles of cultured cystic fibrosis (CF) lung epithelial cells were analyzed by two-dimensional gel electrophoresis and mass spectrometry (MS). The analysis gave rise to a protein map over the pI range of 4-7, and a molecular weight range of ca. 100-10 kDa. The map contains 194 identified proteins, which were detectable by silver stain. All silver stained features were identified by matrix-assisted laser desorption/ionization-time of flight MS of tryptic peptides. Some proteins were found to be represented by multiple features on the 2-D gel. Among the high abundance proteins identified were sets of proteins associated with inflammation, including the classical NFkappaB, p65 (RelA) and NFkappaB, p65 (RelB). We suggest that this composite atlas of the high abundance CF lung epithelial proteome will serve as a reference database for future studies of candidate CF drugs, validating different approaches to CFTR gene therapy, and analogous investigations of other types of human lung disorders. 相似文献
17.
Heparins/heparan sulfates modulate the function of proteins and cell membranes in numerous biological systems including normal and disease processes in humans. Heparin has been used for many years as an anticoagulant, and anticoagulant heparin-mimetics were developed several decades ago by chemical sulfation of non-mammalian polysaccharides, e.g., an antithrombotic sulfated xylan. This pharmaceutical, which comprises a mixture of sulfated oligoxylans, also mimics most other biological actions of natural heparins in vitro, including inhibition of the human immunodeficiency virus, but the molecular basis for these actions has been unclear. Here, numerous Components of the sulfated oligoxylan mixture were isolated and when bioassayed in the case of anti-HIV-1 infectivity revealed that a structural specificity underlines the capacity of sulfated xylan to inhibit HIV-1, rather than a non-specific mechanism. Components were isolated by chromatographic fractionation through Bio-Gel P10 in 0.5M ammonium bicarbonate. This fractionation revealed an elution range associated with apparent molecular weights of 22 000 to <1500 relative to standard heparin and heparan sulfates and newly prepared sulfated oligosaccharide standards. Components were characterized by metachromatic absorption spectroscopy, ultracentrifugation, GlcA analysis, and potency against HIV-1 infectivity, both in the tetrazolium cytotoxicity assay and in syncytium-forming assays, in CD4-lymphocytes. Structural specificity was indicated by the differential potencies exhibited by the Components: Highest activity (cytotoxicity) was exhibited by Components in the chromatographic region 5500 in mass (50% effective (inhibitory) concentration=0.5–0.7 g ml–1 in the first fractionation series, and 0.1–0.5 g ml–1 in a second series). The potency declined sharply below 5400 in mass, but with an exception; a second structure exhibiting relatively high potency eluted among low-mass oligosaccharides which had an average size of a nonomer. Components displayed differential potencies also against the syncytium-forming infectivity of HIV-1. The high potency against syncytium-formation was retained by Components down to a minimum size of about 4500 in mass, smaller than the 5400 required above. One in ten of the 1,4-linked xyloses in the native xylan are substituted with a monomeric 1,2 DGlcA branch. We have speculated that pharmaceutical actions of sulfated xylan might be related to structures involving the -D linked substituents and this was examined using a space-filling model of a sulfated octaxylan and by analyses of Components for GlcA content. Understanding structure/function relations in the heparin-like actions of these agents would be of general significance for the careful examination of their potential clinical usefulness in many human processes modulated by heparins, including AIDS. 相似文献
18.
The molecular and biochemical basis of cystic fibrosis 总被引:5,自引:0,他引:5
19.
Selective isolation and culture of a proliferating epithelial cell population from the hamster trachea 总被引:7,自引:0,他引:7
William E. Goldman Joel B. Baseman 《In vitro cellular & developmental biology. Plant》1980,16(4):313-319
Summary A reliable cell isolation technique was developed to allow the cultivation of cells from the hamster respiratory tract. Repeated
thermolysin treatments and gradient centrifugation yielded a cell culture completely free from contamination by fibroblasts.
Viable cells could be isolated from as little tissue as a single hamster trachea, but in vitro proliferation occurred only
if the hamster was less than 4 months of age. The cultured cells could be repeatedly passaged and subcultured for weeks by
employing normal tissue culture techniques. Morphologically, the monolayers appeared to be a homogeneous population of epithelial
cells, and successful cloning of freshly isolated single cells resulted in apparently identical cultures. The epithelial origin
of these cells was also suggested by continued growth in minimum essential medium withd-valine substituted forl-valine. The relative ease with which this cell type can be isolated, cultured, and manipulated in vitro should encourage
its application as a model of the respiratory epithelium.
This research was supported by Public Health Service Grant P50-HL 19171 and Research Career Development Award 1-K04-AI 00178
to J. B. B. 相似文献
20.
特发性肺纤维化(IPF)是一种严重影响肺通气与换气功能的下呼吸道慢性疾病,其发病机理目前尚不明确,表现为异常的间质炎症和纤维化,以及肺泡结构的破坏。而Ⅱ型肺泡上皮细胞(ATⅡ)作为维持肺结构和功能的关键细胞,在肺部纤维化的发生和发展中极其重要。在IPF中,各种原因所致的ATⅡ的受损和衰老凋亡,可能是纤维化发生的是始动因素。而在这之后,关于临时基质的形成、成纤维细胞的聚集、激活以及间质-上皮转化的过程,异常的ATⅡ也参与其中,并发挥着重要的作用。 相似文献