Glycosaminoglycan synthesis in explants derived from bleomycin-treated fibrotic hamster lungs

Glycosaminoglycan synthesis was studied in explant cultures of hamster lungs 15 and 45 days following intratracheal administration of Bleomycin. At both time points, a statistically significant increase in 35S-sulfate incorporation into glycosaminoglycans was seen in the Bleomycin-treated explants compared with that of the controls. Furthermore, the percentage of label associated with dermatan sulfate was significantly higher in the treated explants than in controls at both 15 and 45 days. Conversely, the percentage of labeled heparin and/or heparan sulfate was significantly lower for the treated explants compared to controls at these times. These results indicate that glycosaminoglycan synthesis is altered from normal in this model of interstitial lung disease. Comparison of these data with previous measurements of glycosaminoglycan synthesis in another model of interstitial lung disease, induced by N-nitroso-N-methylurethane, reveals marked similarity in the changes from normal in 35S-labeling.     

Dynamics of proliferative activity of cultures of organotypic epithelial-mesenchymal recombinants from embryonic lungs of intact and urethane-receiving mice]

The dynamics of proliferative activity of cells was studied in the cultures of organotypic recombinants obtained from embryonic lung epithelium (E) and mesenchyma (M) of the intact and treated transplacentally by urethane mice (strain A). The labelling index (LI) of E and M in the aggregates obtained from treated embryonic lungs (EtMt) was significantly higher than LI in the aggregates from intact embryonic lungs (EiMi) in all days of cultivation (4-7-14). M from the treated embryonic lungs stimulated LI of the intact E (EiMt) but M from the intact embryonic lungs decreased LI of the treated E (EtMi).     

Morphogenetic characteristics of the aggregates formed in epithelial and mesenchymal recombinations of the lungs from intact and urethane-exposed mouse embryos

A study was made of the morphogenesis of organotypic aggregates obtained by epithelial mesenchymal recombinations from the lungs of embryonic mice, intact and treated with urethane. Normal growth and differentiation of organotypic structures were observed in long-term cultures of aggregates obtained by recombinations of the lung epithelium (E) and mesenchyma (M) from intact (i) embryonic mice (EiMi). Hyperplasia and squamous-cell metaplasia (with or without keratinization) of the epithelium were found in aggregates obtained from E and M of the treated mouse embryos (EtMt) and in aggregates obtained by recombinations of lung E and M from intact and treated embryos (EtMi, EiMt). The data obtained suggest that the alterations in epithelial mesenchymal interactions are of great significance for transplacental lung blastomogenesis and that the mesenchymal lung cells play an important part in mediation of the transplacental carcinogenous effects on epithelial target cells via subsequent epithelial mesenchymal tissue interactions.     

Hormonal regulation of beta-adrenergic receptors in fetal rabbit lung in organ culture

Explants of fetal rabbit lung were established on the 25th day of gestation. These were maintained in serum-free medium for periods up to 10 days. During this time, the cultures exhibited morphological changes typical of terminal lung differentiation. Morphological evidence was also obtained for synthesis and secretion of pulmonary surfactant in these explants. beta-Adrenergic receptors were identified in these lung explants. Exposure of the explants to 10(-7)M dexamethasone on the third day of culture resulted in a significant increase in the number of beta-adrenergic receptors in the tissue without a change in receptor affinity. The effect of dexamethasone in organ culture was dose-dependent, a maximum increase in receptor number being observed within 48 hours of incubation with a hormone concentration of 1 x 10(-7)M. Exposure of the explant tissue to 1 x 10(-7)M triiodothyronine resulted in no significant increase in the concentration of beta-adrenergic receptors and no change in receptor affinity. These results suggest that glucocorticoids may potentiate the effects of beta-adrenergic agents in the fetal lung by increasing the numbers of their receptors. The effects of triiodothyronine upon the fetal lung do not appear to be mediated by this mechanism.     

Effect of cortisol on the synthesis of lamellar body glycerophospholipids in fetal rabbit lung tissue in vitro

The effect of cortisol on the rate of choline incorporation into tissue phosphatidylcholine was investigated in lung explants from fetal rabbits of 19-28 days gestational age. The explants were incubated in medium with or without fetal calf serum for up to 7 days. When lung tissues were incubated in serum-free medium, a stimulatory effect of cortisol on tissue phosphatidylcholine synthesis was found in explants from 21-, 24-, 26- and 28-day fetal rabbits; a stimulatory effect of cortisol was observed in 19-day fetal lung explants only if fetal calf serum was present in the culture medium. To assess directly the effect of cortisol on the synthesis of lamellar body phosphatidylcholine, choline incorporation into phosphatidylcholine associated with a purified lamellar body fraction isolated from lung explants of 21- and 28-day fetal rabbits was also investigated. Cortisol caused a marked stimulation of synthesis and accumulation of lamellar body phosphatidylcholine in lung explants from both 21- and 28-day fetal rabbits. The magnitude of the stimulatory effect of cortisol on the rate of synthesis of lamellar body phosphatidylcholine was always greater than the effect of cortisol on the rate of choline incorporation into lipids of tissue homogenates. The relative rates of synthesis of lamellar body phosphatidylinositol and phosphatidylglycerol were also significantly altered in lung explants from 21- and 28-day fetal rabbits by cortisol treatment. Lamellar bodies that were formed initially in the fetal lung explants were enriched in phosphatidylcholine and phosphatidylinositol and had a relatively low phosphatidylglycerol content. With cortisol treatment there was a decrease in the relative rate of synthesis of lamellar body phosphatidylinositol and an increase in the relative rate of synthesis of phosphatidylglycerol. The stimulatory effect of cortisol on the synthesis of lamellar body phosphatidylcholine was observed at an earlier time-point of incubation than was the effect of cortisol on the relative rates of synthesis of lamellar body phosphatidylinositol and phosphatidylglycerol. The temporal sequence of the cortisol-induced changes in the synthesis of lamellar body glycerophospholipids, therefore, reflects that which occurs with maturation in vivo.     

Sensitivity of embryonal lungs of mice, rats and humans to exposure to nitrosomethylurea in organ cultures

With the direct action of nitrosomethylurea (NMU) in a concentration of 0.05 mg/ml on the organic cultures of the embryonic lung of mice of the A line, Wistar rats and man they developed a different degree of degenerative changes and hyperplastic epithelial proliferates. A toxic effect prevailed in the cultures at the initial experimental periods. The most sensitive to the toxic action of NMU was the lung tissue of rat embryos, and the least--of mice. The incidence of hyperplastic proliferates was, on the contrary, the greatest in the cultures of mouse lungs, and the least--of rat lungs. The sensitivity of the embryonic lungs of the man and rodents to the toxic action of NMU in repeated administration into the nutrient medium diminished during the cultivation. There was an increase of survival of the experimental cultures in comparison with the intact control.     

Localization and accumulation of fibronectin in rabbit fetal lung tissue

An interaction between mesenchyme and epithelium is required for the normal differentiation of fetal lung tissue. This morphogenic interaction may be mediated, in part, by changes in the composition and/or structure of the extracellular matrix. Therefore, we characterized the localization and accumulation of fibronectin, an extracellular-matrix component, during several stages of lung development in the rabbit fetus in vivo as well as in day-21 rabbit fetal lung explants maintained in vitro. Fibronectin was detected immunocytochemically in the basement-membrane zone beneath the epithelial ducts in lung tissue obtained from rabbit fetuses at 19 and 21 days of gestation. In fetal lung tissue obtained at these early stages of lung development, mesenchymal cells were stained only at their periphery. Immunostaining for connective-tissue fibronectin increased greatly between days 24 and 31 of gestation. A similar increase in the intensity of immunostaining for connective-tissue fibronectin was observed in rabbit fetal lung explants that had been maintained in vitro for 7 days. The concentration of fibronectin in fetal lung tissue obtained at different days of gestation was determined using a specific enzyme-linked immunoadsorbent assay (ELISA) and was found to increase from 1.7 ng/micrograms protein in fetal lung tissue obtained at day 19 of gestation to 7.3 ng/micrograms protein in fetal lung tissue obtained at day 24 of gestation. The levels of fetal lung fibronectin then remained relatively constant through to day 31 of gestation. A similar increase in fibronectin concentration was observed in day-21 fetal lung explants maintained in vitro for 7 days.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activities of enzymes of phospholipid and fatty acid synthesis in fetal and adult rat type II pneumocytes

Although differentiated fetal and adult type II pneumocytes are ultrastructurally similar, it is not known whether there are metabolic differences between them. We measured the activities of selected enzymes of phospholipid and fatty acid synthesis in fetal and adult rat type II cells, in late gestation fetal rat lung explants and in intact lung from rat fetuses of comparable gestational age. The activity of 1-acylglycerophosphocholine acyltransferase was significantly greater in adult type II cells than in fetal type II cells, fetal explants or intact fetal lung. The activity of CDP diacylglycerol:glycerol-3-phosphate 3-phosphatidyltransferase was similar in fetal and adult type II cells, but significantly lower in explants and intact fetal lung. There was a significant positive correlation between the percentage of alveolar epithelial cells in the cultures and tissue studied and CDP diacylglycerol:glycerol-3-phosphate 3-phosphatidyltransferase activity. This suggests that the previously reported correlation between phosphatidylglycerol synthesis and the percentage of alveolar epithelial cells in various lung culture systems may be related to the activity of this enzyme. Phosphatidylglycerol synthesis and CDP diacylglycerol:glycerol-3-phosphate 3-phosphatidyltransferase activity may be metabolic markers of type II cells, whereas the acyltransferase activity may be an indicator of type II cell maturation.     

Ultrastructural study of Pneumocystis carinii in explant cultures of rabbit lung and in cultures with and without feeder cells

Pneumocystis carinii-parasitized lung explants were obtained from corticoid-treated rabbits and maintained in vitro. Twenty-one days after the beginning of explant cultures, the ultrastructural morphology of trophozoite, precyst and cyst forms was normal as compared to the in vivo ultrastructure of P. carinii from infected rabbit. However, after the 36th day, only altered forms of P. carinii were observed. Lung tissue showed only minor alterations. Intracytoplasmic lamellar inclusions were observed in type 2 alveolar cells from which they were released. While the total number of parasites increased approximately 4-fold from day 0 to day 41, trophozoite counts increased approximately 6 times. Pneumocystis cells from inocula and supernates of cultures with and without Vero cells showed important ultrastructural alterations.     

Proteoglycan synthesis in organ cultures from regions of bovine tendon subjected to different mechanical forces.

Synthesis of proteoglycans by morphologically and chemically distinct regions of bovine flexor tendon was investigated in explant cultures. Proximal regions of the flexor tendon which experience only tensile forces and have low contents of proteoglycans initially exhibited relatively low rates of proteoglycan synthesis but high rates of collagen synthesis. The predominant proteoglycan produced by all proximal explants was of small hydrodynamic size and appeared similar to that extracted from proximal tissue. In contrast, explants derived from the distal tendon region, which experiences frictional and compressive forces in addition to tensile forces, and has a high content of proteoglycans, showed relatively high initial rates of proteoglycan synthesis and lower rates of collagen synthesis. These distal explants produced primarily large proteoglycans on the first day in culture. Turnover of newly synthesized proteoglycans was not detectable in proximal tissue, and was low in distal tissue. Loss of unlabelled proteoglycan from proximal and distal explants was not detected during the 12 days of culture. These observations suggest that the increase in specific types of proteoglycans in regions of tendon subjected to frictional and compressive forces is the result of elevated synthesis rates in this tissue. Two alterations in proteoglycan synthesis occurred during the 12-day culture period. (1) The rate of proteoglycan synthesis by all explants increased with time in culture. (2) The proportion of small proteoglycans synthesized by distal explants increased from 32% of the total proteoglycan produced on day 1, to 80% of that produced on day 12. Explants from proximal tendon continued to produce only small proteoglycans throughout the 12 days in culture. This switch in proteoglycan phenotype, resulting in decreased synthesis of large proteoglycans by the distal tissue, may be due to a lack of compressive forces on the cultured explants.     

Elastin synthesis during perinatal lung development in the rat

The rate of soluble elastin synthesis was estimated in lung explants from rats of differing ages to better define periods in lung development important to the deposition of lung elastin. Lungs from rat pups at days 1, 3, 7, 9, 12, 15, and 21 post-parturition and from adult rats were incubated in a defined medium containing L-[3H]valine. Following incubation, labelled soluble elastin (tropoelastin) was separated from other soluble proteins by coacervation and electrophoresis on polyacrylamide gels containing sodium dodecyl sulfate. The tropoelastin synthetic rate was then estimated after correcting for differences in recovery of radioactivity as tropoelastin and lung tissue L-[3H]valine specific activity. Maximal rates of elastin synthesis were observed in lung explants from 7-12-day-old rats. The rate of elastin synthesis during this period was 5-8-times the rate observed in adult rat lung (expressed per g of fresh lung) and represented approx. 2% of the total protein synthesis. Moreover, the values derived from lung explant culture for elastin synthesis were consistent with values for lung elastin deposition in the perinatal rat (5-10 micrograms elastin/h per g lung).     

Changes in the Syntheses of DNA,RNA and Protein during Early Somatic Embrogenesis in Sainfoin(Onobrychis viciaefolia Sccp.)

Hypocotylar explants of Onobrychis viciaefolia Scop. were cultured on LS basal medium supplemented with 1 mg/l BA and 1 mg/l KT. After two weeks of culture, calli were initiated on the surface of sections. Light-Yellow callus from .one of the explants was selected and proliferated on the medium above. Then it was transfered to LS medium with 1 mg/l BA to initiate somatic embryogenesis. The activity of RNA synthesis increased rapidly during the first two days. Of embryogenic culture and then decreased, but on the 5th day increased gradually. The activity of protein synthesis increased during the first three days and was the highest on the 3rd day. The activity of DNA synthesis had no mark change and emerged, a small peak on the 5th day. All the activities of syntheses of DNA, RNA and protein were higher on embryogenic culture than on nonembryogenic culture.     

红豆草体细胞胚胎发生早期DNA,RNA和蛋白质的合成动态变化

红豆草(Onebrychis viciaefolia Scop.)下胚轴切段在含有1mg/IBA、1mg/1 KT 的 LS培养基上培养,两周后产生愈伤组织,通过筛选、克隆得到大量的具有胚胎发生潜力的非胚性愈伤组织,当将其转移到含1mg/1BA 的 LS 培养基上后可诱导体细胞胚胎发生。应用放射性同位素液体闪烁技术测得在胚性培养的前2天,RNA 合成速度迅速上升,随后下降,第五天后又呈缓慢上升趋势,尔后平稳。蛋白质合成速度在胚性培养的第三天达到高峰,升高很快。而 DNA 合成速度变化平缓,只是在胚性培养的第五天出现一较小的峰。胚性培养过程的 DNA、RNA 蛋白质合成速度均高于非胚性培养。     

The effect of Solcoseryl on in-vitro cultured cells

Explants of peripherical nervous system (PNS), skin and ventriculus cordis from chick embryo were cultivated in Maximow chambers and the effect of Solcoseryl, Fa. Solco Basel AG, on some morphological parameters was tested. 1. The growth of tissue cultures is influenced by Solcoseryl in relation to concentration and time of application. The index of area in cultures of PNS and cor increased within the first days. By long time application up to 6 days in vitro the index of area decreased and the index was the same than in controls. Explants of skin showed no essential stimulation of growth. 2. The number of cells per unit of culture in the outgrowth of PNS, cor and skin was different influenced. The density of cells in cultures of PNS and skin decreased (signif. difference). In explants of heart we could not observe a difference between the inside and outside of the outgrowth. An influence of Solcoseryl on the degree of migration is discussed. 3. The area of cell nuclei from heartcells was observed. The area decreased under the influence of Solcoseryl. The difference is significant. 4. The mitotic index of heart cells increased by application of Solcoseryl within the first 2 and 3 days in vitro. 5. The number of nucleoli per nucleus of heart cells under experimental conditions increased significant. It is discussed, Solcoseryl influenced in vitro metabolic processes in suitable systems; stimulation of cell proliferation and migration and rns-synthesis was observed within the first days of cultivation. In-vitro-systems are important objects and they are suitable for tests of pharmaca in vitro.     

The ontogeny of the mouse estrogen receptor: the pelvic region

The appearance of estrogen receptors was examined during the course of fetal and neonatal development in the pelvic region of the mouse; 3H-diethylstilbestrol (DES) was administered via the maternal circulation to developing mice on days 4, 7, 10, 13, 14, 15, and 17 of gestation or to neonates on the day of birth. Localization of the ligand was monitored autoradiographically. The earliest appearance of estrogen receptors occurred in the mesenchyme around the genital ducts on day 13 of pregnancy. On subsequent days, estrogen-concentrating cells appeared in certain mammary-gland cells, connective-tissue strands, in perichondrium associated with specific developing bones, skin, interstitial tissue of the testis, in a sheath of cells surrounding the colon, and in the urethra. The significance of cells containing estrogen receptors in these locations is discussed in reference to a transplacental action of estrogens and the clinical ramifications of DES.     

Insulin and transferrin restore important cellular functions of human fetal kidney in serum-free organ culture.

A model previously developed in our laboratory to culture human fetal kidneys in serum-free chemically defined medium was used to evaluate the direct influence of potential regulators on nephrogenesis. The aim of the present work was to verify the effects of insulin and transferrin, two hormones considered as essential in other serum-free culture systems. Explants of renal cortex from human fetuses (15-21 weeks) were cultured for 2 and 5 days in serum-free Leibovitz's L-15 medium (37 degrees C, 95% air - 5% CO2). The addition of transferrin (5 micrograms/mL) had no effect, but insulin (30, 60, and 125 mU/mL) increased DNA and protein syntheses in a dose-dependent manner. The influence of insulin (125 mU/mL) was potentiated by the addition of transferrin and the combination of the two stimulated DNA synthesis by threefold on day 2 when compared with controls and by sixfold on day 5 of culture. After 5 days, synthesis was restored to values observed at day 0. Transferrin did not modify the insulin effect on protein synthesis, since the latter was already maximally stimulated as early as day 2 of culture and at levels well above that of uncultured explants (day 0). The activities of four hydrolases considered as markers of brush border differentiation were not importantly changed by any of the hormones, supplemented alone or in combination. The results indicate that proliferation rather than differentiation is the parameter mostly influenced by these two hormones. The combination of insulin plus transferrin restores cellular functions of human fetal kidney explants cultured in serum-free medium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Urethane interaction with nucleic acids and proteins from non-malignant fast growing tissues

The interaction of urethane metabolite(s) with macromolecules in tissues of pregnant and partially hepatectomized mice was studied. Pregnant mice were treated with tritiated urethane on the 17th day of pregnancy. Partially hepatectomized mice were studied at 72 h and 198 h after the operation. Operated mice were injected with tritiated urethane, 6 h before killing. It was observed that the specific radioactivity of lung DNA was highest in pregnant mice whereas in partially hepatectomized mice the specific radioactivities of lung DNA and regenerating liver DNA were comparable. It was also observed that binding to macromolecules was greatest at 72 h when the highest mitotic activity in regenerating liver occurs.     

Macromolecular synthesis in organ cultures of neonatal rat lung

Organ cultures of newborn rat lungs synthesize and accumulate DNA, RNA, collagen and noncollagenous proteins almost at a linear rate for at least 5 days. During this period the synthesis of collagen consistently exceeds the synthesis of noncollagenous proteins in a pattern similar to neonatal lung growth in vivo. Although some morphological characteristics of lung architecture are distorted after culture, fundamental structural similarities to lungs growing in intact animals are retained. When these cultures are maintained in atmospheres rich in oxygen, increased collagen synthesis is observed, a response similar to that of lungs in intact animals exposed to high oxygen concentrations in vivo. Our studies suggest that lung organ cultures may be a suitable system for investigating the biochemical aspects of lung tissue-environmental interaction.     

Urethane and N-nitrosodiethylamine are mutagenic for the Syrian hamster fetus

Urethane and N-nitrosodiethylamine are soluble environmental carcinogens that initiate tumors transplacentally, but have a mixed history of effectiveness in mutagenesis assays in vitro or in vivo with adult rodents. To test for their transplacental mutagenicity, Syrian hamster fetuses at 12 days in gestation were exposed transplacentally to urethane or N-nitrosodiethylamine at 0.5 or 1.0 mM/kg. The fetal cells were isolated on day 13 of gestation and tested for diphtheria toxin resistance as a mutation marker. Both compounds were significantly mutagenic, at both doses, causing 6- to 20-fold increases in mutations compared with controls. Compared with N-nitrosodiethylamine, urethane was somewhat more effective as a mutagen with a more marked dose-response. These results are consistent with mutagenesis as part of the mechanism of transplacental carcinogenicity of urethane and N-nitrosodiethylamine.     

Proliferation and cytotoxicity of thymus lymphocytes in vitro]

Proliferative and cytollytical activity of lymphocytes was compared in lymphocyte alloimmunization of the spleen and intact thymus. The count of live cells and DNA-synthesizing cells in the thymocyte monoculture was 10--15-fold, and in mixed thymus cell culture--about 5-fold lower than the corresponding amounts of spleen cells. The index of immune thymocyte stimulation was several times greater than that of the immune cells of the spleen. The cytotoxicity peak was observed on the 4th--5th day of stimulation when the cytolytic activity of the immune thymocytes approached the action of the immune cells of the spleen. Low DNA synthesis and a marked cytotoxic activity of immune thymocytes signified that stimulation of the thymus cells in vitro permitted to obtain cell population with a high content of cytolytic T-lymphocytes.