Glycoprotein biosynthesis by organ cultures of hamster trachea

Conditions have been developed for maintaining hamster tracheas in organ culture for at least 10 days. Secreted glycoproteins labelled with [¹⁴C]glucosamine and [³H]fucose were isolated from the spent medium and digested with papain, and the digest was fractionated on DEAE-Sephadex by stepwise elution with NaCl. The fractions eluted by 0.1 and 0.2 M NaCl and some of the products eluted with 0.4 M NaCl were shown to be derived from epithelial glycoproteins. Glycosaminoglycans were eluted by 0.4 M and by 1.25 M NaCl. Glycopeptides isolated from the epithelium by homogenization, ethanol precipitation and papain digestion, and defined as “intracellular”, gave a very similar profile on DEAE-Sephadex. The 0.1 M glycopeptide peak was the major fraction of epithelial origin from both secreted and “intracellular” material; it labelled extensively with both glucosamine and fucose and had a molecular weight of approx. 5000 (as judged by its elution from Sephadex G-75). This fraction was purified further by chromatography on Sephadex G-75 and DEAE-Sephadex; its amino acid and carbohydrate compositions were determined.     

Structure and metabolism of glycoproteins and glycosaminoglycans secreted by organ cultures of rabbit trachea.

1. When cultured in medium 199 in an atmosphere of O2+CO2 (95:5) rabbit tracheal explants retained their viability for up to 21 days. 2. The explants secreted into the culture medium three electrophoretically separable components which were eluted in the non-retarded fraction from Sephadex G-200. 3. Digestion with papain and fractionation with a LiCl gradient on DEAE-cellulose resulted in the separation of these components, which were identified as a sialic acid-rich glycoprotein of epithelial origin, and chondroitin sulphate and hyaluronic acid from sub-epithelial cartilage and connective tissue. 4. Incorporation of radioactive precursors ( D-[U-14-C]glucose, D-[1-14-C]glucosamine, D-[1-14-C]galactose and Na2-35SO4) into these secreted macromolecules was indicative of their active biosynthesis by the tracheal tissue.     

Intracellular levels of adenosine triphosphate in hamster trachea organ cultures exposed toMycoplasma pneumoniae cells or membranes

Summary The amount of adenosine triphosphate (ATP) in hamster trachea organ cultures was determined with a technique based on light emission from a luciferin/luciferase/ATP reaction. The amount of ATP, expressed as ng per mg dry weight, was consistent in tracheal explants prepared from various animals and changed negligibly when explants were cultivated in vitro for several days. The amount of ATP was related directly to cellular activity and integrity in the epithelium since inactivation by heat or freeze-thaw rapidly depleted measurable ATP, and ciliary activity and ATP content were related directly. When tracheal explants were infected with 10⁵ to 10⁷ CFU of virulentMycoplasma pneumoniae cells, both ciliary activity and ATP content in the tissue dropped dramatically after approximately 5 to 8 days (up to 85% and 60% decreases, respectively). Exposure of explants to 50 to 200 μg per ml of purifiedM. pneumoniae membranes also caused significant decreases in ciliary activity and ATP. When explants were infected with attenuated or nonvirulent mycoplasmas, ciliary activity was only slightly decreased, while ATP values often rose slightly. The technology associated with the determination of ATP levels in tracheal explants should prove useful as a new, objective, analytical approach to cell viability in organ cultures. This investigation was supported in part by the National Institutes of Health (PHS Grant AI 12559), by a Biomedical Sciences Support Grant made to the University of Illinois School of Life Sciences, and by the University Research Board.     

Intracellular levels of adenosine triphosphate in hamster trachea organ cultures exposed to Mycoplasma pneumoniae cells or membranes

The amount of adenosine triphosphate (ATP) in hamster trachea organ cultures was determined with a technique based on light emission from a luciferin/luciferase/ATP reaction. The amount of ATP, expressed as ng per mg dry weight, was consistent in tracheal explants prepared from various animals and changed negligibly when explants were cultivated in vitro for several days. The amount of ATP was related directly to cellular activity and integrity in the epithelium since inactivation by heat or freeze-thaw rapidly depleted measurable ATP, and ciliary activity and ATP content were related directly. When tracheal explants were infected with 10(5) to 10(7) CFU of virulent Mycoplasma pneumoniae cells, both ciliary activity and ATP content in the tissue dropped dramatically after approximately 5 to 8 days (up to 85% and 60% decreases, respectively). Exposure of explants to 50 to 200 microgram per ml of purified M. pneumoniae membranes also caused significant decreases in ciliary activity and ATP. When explants were infected with attenuated or nonvirulent mycoplasmas, ciliary activity was only slightly decreased, while ATP values often rose slightly. The technology associated with the determination of ATP levels in tracheal explants should prove useful as a new, objective, analytical approach to cell viability in organ cultures.     

Functional and ultrastructural effects of nontypeable haemophilus influenzae in a hamster trachea organ culture system

Summary A hamster trachea organ culture system was utilized to evaluate quantitatively the effects of a strain of nontypeableHaemophilus influenzae (NTHI) and culture supernatants of the same strain on ciliary activity. Tracheal explants were maintained in organ culture for 96 to 144 h and ciliary activity was observed daily with an inverted microscope. Explants continuously exposed to a strain of NTHI had a progressive decline in ciliary activity which was significantly lower than uninfected controls evaluated concomitantly by 48 h of exposure and thereafter. Histologic studies revealed a progressive degeneration of mucosal cells and exfoliation of ciliated cells. Scanning electron microscopy showed little adherence of NTHI to the mucosal surface. Sterile broth cultures of NTHI and supernatants of organ cultures infected with the same NTHI strain had no adverse effect on ciliary activity. Infected tracheal explants treated with ampicillin 24, 48, or 72 h after continuous bacterial challenge had no significant decline in ciliary activity compared to controls. The lack of adherence and the histologic changes observed when hamster trachea cultures were infected with NTHI suggested a toxin might mediate the damage observed. Broth and organ culture supernatants, however, produced no damage. Therefore, further studies are needed to determine the role, if any, of a toxin in the production of damage to hamster tracheal explants by NTHI. This work was supported by a Merit Review grant from the Veterans Administration and by Grant AI-19641 from the National Institute of Allergy and Infectious Diseases.     

Incorporation of fucose and glucosamine into cell bound and medium released macromolecules.

(1) Determinations were carried out on the incorporation of fucose-6-(3H) and glucosamine-6-(3H) into trichloracetic acid insoluble macromolecules which remained bound to the cells or were released into the medium of chick embryo muscle cell cultures. The radioactivity determined in the medium was corrected for unspecific binding of label to components of the medium. (2) During an incorporation period of six hours the incorporation per microgram DNA with fucose as label into cell bound macromolecules is about twice as high as the incorporation into macromolecules released into medium. With glucosamine about twice as much is incorporated into medium released into the cell bound macromolecules. (3) The incorporation per microgram DNA increased during a culture period of three days but the increase ceases at different times during this culture period when determined with fucose or glucosamine or for cell bound and medium released material. (4) An increase in cell density increases the incorporation per DNA of fucose and to a much slighter extent that of glucosamine. Reduction of cell density by addition of cytosine arabinoside to the medium does not increase the incorporation per microgram DNA. (5) The effect of changes of fibroblast/myoblast ratios on the incorporation of fucose and glucosamine were examined. No significant effect was observed for a ratio of 10-30% fibroblasts when control cultures or cultures after cell sedimentation were maintained in complete medium. Marked changes were observed after culture in medium without protein components. Under these conditions an increase in the fibroblast/myoblast ratios were observed as well as an increase in the incorporation of label into medium released and a decrease into cell bound macromolecules.     

Differential release of prostaglandins by organ cultures of human fetal trachea and lung

Summary Human fetal lung at 16–19 weeks gestation has a partially differentiated epithelium, and in organ culture, distal airsacs dilate and the epithelium autodifferentiates to type I and II pneumatocytes, processes regulated by endogenous prostaglandin PGE₂. Human fetal trachea, at the same gestation, has a terminally differentiated mucociliary epithelium but after 4–6 d in organ culture, develops squamous metaplasia. Tracheal cultures restricted to 3 d have normal phase-contrast and light microscopy appearances and immunohistochemical reactivities (epithelium: cytokeratin 7,8,18; glutathione S-transferase pi-isozyme; epithelial membrane antigen and mesenchyme; desmin; vimentin). In human fetal trachea organ cultures, the predominant prostaglandins released are 6-keto-PGF_1α, PGF_2α, and PGE₂, a pattern similar to that previously described for human adult trachea and lung. In fetal lung cultures, 13,14-dihydro-15-keto-PGF_2α is the major prostaglandin released with lesser amounts of 13,14-dihydro-15-keto-PFG_2α, PGF_2α, PGE₂, and 6-keto-PGF_1α. Human fetal lungin vitro has the competence to self-differentiate, as early as 12 weeks gestation and presence of high levels in fetal lung of the inactive metabolite 13,14-dihydro-15-keto-PGE₂ relative to PGE₂ suggests that active prostaglandin catabolism may be one of the mechanisms to retard this stage of maturationin vivo by limiting PGE₂ availability. Surprisingly, the profile of prostaglandins released from fetal lung organ culture does not change to that of a mature lung with terminal differentiation of the epithelium, and this may indicate differences in the expression of key prostaglandin-metabolizing enzymes in developing human fetal lung in culture and within utero ontogeny.     

Incorporation of ammonium into intracellular UDP-activated N-acetylhexosamines and into carbohydrate structures in glycoproteins.

The negative effects of ammonia on animal cells, especially in vitro cultures, are well known, but the mechanism of how ammonia inhibits cell growth and influences the glycosylation of proteins is not completely understood. We investigated the ammonium action on the synthesis of the intracellular UDP-N-acetylhexos- amines (UDPGNAc), which are precursors of glycosylation as well as on N-linked oligosaccharides of a recombinant human IL-2 mutant variant model glycoprotein expressed in BHK-21 cells under defined and controlled culture conditions in a continuously perfused bioreactor. The examinations were based on our previous observations that increased ammonia concentrations in the medium lead to the intracellular formation and accumulation of UDPGNAc (Ryll et al., 1994). The kinetics of formation of the UDPGNAc pool after adding ammonia and its reconstitution to normal conditions are shown. To study the pathway leading to the intracellular increase of UDPGNAc, the uptake and incorporation of 15NH4+ was confirmed by the detection of 15N in UDP-N-acetylglucosamine (UDP-GlcNAc). UDP-GlcNAc was purified using high pH anion-exchange chromatography with pulsed amperometric detection and analyzed by GC/MS. The proportion of UDP-GlcNAc containing 15N was approximately 60% and corresponds quantitatively to the increased intracellular concentration of UDP-GlcNAc. In order to confirm the direct influence of ammonia on protein glycosylation, the human IL-2 mutant glycoprotein variant IL-Mu6, bearing a novel N-glycosylation site, has been produced under defined protein-free medium conditions in the presence of 15NH4Cl. IL-Mu6 glycoprotein was purified and N-glycans released were analyzed by matrix-assisted laser desorption ionization time of flight mass spectroscopy. Maximally 60-80% of N-acetylated sugars in N-glycan structures contained 15N indicating that ammonium is used as a building block during synthesis of the carbohydrate structures expressed from in vitro cultivated mammalian cells.     

Studies on bone matrix glycoproteins. Incorporation of [1-14C]glucosamine and plasma [14C]glycoprotein into rabbit cortical bone

The radioactively labelled constituents present in bone matrix were compared 12 days after injection of either [(14)C]glucosamine or plasma [(14)C]glycoprotein. Both precursors are utilized in the synthesis of organic matrix by bone tissue. Cortical bone from animals injected with [(14)C]glucosamine contains radioactivity derived from glucosamine and plasma glycoproteins and all glycoprotein fractions are labelled. Plasma [(14)C]glycoprotein labels the less acidic glycoproteins to a greater extent than the more acidic components. An antibody has been raised against the less-acidic-glycoprotein fraction of bone. The latter contains a glycoprotein of alpha-mobility that appears to be concentrated specifically in bone tissue and which is present also in plasma. This alpha-glycoprotein accounts for a large proportion of the components labelled and retained in bone matrix after [(14)C]glucosamine injection.     

Biosynthesis of respiratory-tract mucins. Incorporation of radioactive precursors into glycoproteins by canine tracheal explants in vitro

1. Canine tracheal explants, cultured in medium 199, actively incorporated radioactive precursors into secreted macromolecules in vitro. 2. Puromycin, 6-diazo-5-oxo-l-norleucine and ouabain markedly inhibited the incorporation of these precursors. 3. Exogenous glucosamine at concentrations above 20mm caused a greater than 50% inhibition of the incorporation of l-[G-(3)H]fucose and l-[U-(14)C]serine. 4. Carbohydrate content of the purified secretions was approximately 50% and consisted principally of galactose, N-acetylglucosamine, N-acetylgalactosamine, fucose and sialic acids. 5. Chromatography on DEAE-cellulose and Bio-Gel A-150m and equilibrium density-gradient centrifugation in a CsCl gradient confirmed the presence of mucous glycoproteins. 6. Electrophoresis on 1% agarose gels gave profiles that were identical with canine respiratory mucus obtained in vivo. 7. These results support the utility of the explant system for studies of respiratory secretions.     

Incorporation of N-fluoroacetyl-d-glucosamine into hyaluronate by rabbit tracheal explants in organ culture

1. Incubation of rabbit tracheal explants with N-[(3)H]acetyl-d-glucosamine and N-acetyl-d-[1-(14)C]glucosamine led to labelling of a number of soluble macromolecular products separable from the medium, after papain digestion, by ion-exchange chromatography. 2. With N-acetyl-d-[1-(14)C]glucosamine in the incubation medium, a neutral glycoprotein, two acidic glycoprotein fractions, hyaluronic acid and a glycosaminoglycan fraction were obtained and all were radioactively labelled. Similar labelling occurred with N-fluoroacetyl-d-[1-(14)C]glucosamine or N-fluoro[(3)H]acetylglucosamine as precursor. 3. Maximal labelling was obtained at 96h after incubation of cultures. N-Fluoroacetyl-glucosamine under these conditions was incorporated into hyaluronate less efficiently than N-acetylglucosamine. 4. With N-fluoroacetyl-d-[1-(14)C]glucosamine as precursor, a hyaluronate component was separated that on enzymic degradation by glycosidases (hyaluronidase, beta-glucuronidase and N-acetyl-beta-hexosaminidase) yielded a (14)C-labelled oligosaccharide fraction together with N-acetyl-d-[1-(14)C]glucosamine and N-fluoroacetyl-d-[1-(14)C]glucosamine, consistent with some exchange of N-acetyl groups having occurred. 5. The results on enzymic degradation of labelled macromolecules by glycosidases suggest that the presence of incorporated N-fluoroacetyl side chains may render the hyaluronate analogue more resistant to hyaluronidase.     

Incorporation of fluorescently labeled contractile proteins into freshly isolated living adult cardiac myocytes.

When fluorescently labeled contractile proteins are injected into embryonic muscle cells, they become incorporated into the cells' myofibrils. In order to determine if this exchange of proteins is unique to the embryonic stage of development, we isolated adult cardiac myocytes and microinjected them with fluorescently labeled actin, myosin light chains, alpha-actinin, and vinculin. Each of these proteins was incorporated into the adult cardiomyocytes and was colocalized with the cells' native proteins, despite the fact that the labeled proteins were prepared from noncardiac tissues. Within 10 min of injection, alpha-actinin was incorporated into Z-bands surrounding the site of injection. Similarly, 30 sec after injection, actin was incorporated into the entire I-bands at the site of injection. Following a 3-h incubation, increased actin fluorescence was noted at the intercalated disc. Vinculin exchange was seen in the intercalated discs, as well as in the Z-bands throughout the cells. Myosin light chains required 4-6 h after injection to become incorporated into the A-bands of the adult muscle. Nonspecific proteins, such as fluorescent BSA, showed no association with the myofibrils or the former intercalated discs. When adult cells were maintained in culture for 10 days, they retain the ability to incorporate these contractile proteins into their myofibrils. T-tubules and the sarcoplasmic reticulum could be detected in periodic arrays in the freshly isolated cells using the membrane dye WW781 and DiOC6[3], respectively. In conclusion, the myofibrils in adult, as in embryonic, muscle cells are dynamic structures, permitting isoform transitions without dismantling of the myofibrils.     

Incorporation of exogenous fatty acids into phospholipids by cultured hamster fibroblasts. Effect of SV40 transformation

In situ incorporation of two saturated (palmitic, 16:0; stearic, 18:0) and three unsaturated fatty acids (oleic, 18:1; linoleic, 18:2; arachidonic, 20:4) into the four major phospholipids, sphingomyelin, PC, PI and PE, was followed. Transformed cells incorporated unsaturated fatty acids more rapidly, whereas no significant differences were found concerning saturated fatty acids. In vitro determination of phospholipid acylation showed that incorporation of coenzyme A-activated forms of two saturated fatty acids (16:0 and 18:0) and one unsaturated fatty acid (18:1) into phospholipids was increased in transformed cells. Comparison of results obtained in situ and in vitro strongly suggests that incorporation of fatty acids into phospholipids in cultured cells is not limited by acyltransferase activities.