Glucocorticoid enhances surfactant proteolipid Phe and pVal synthesis and RNA in fetal lung

Two newly described surfactant proteolipids (SPL), Phe and pVal, are produced by proteolytic processing of distinct precursors of Mr = 40,000 and 22,000, respectively. These proteins are structurally related and intimately associated with surfactant phospholipids. We now demonstrate the expression of both SPL(Phe) and SPL(pVal) in explants of human fetal lung from 16-24 weeks of gestation. Content, synthesis, and mRNA for the proteolipids were low prior to organ culture of fetal lung. Induction of synthesis of the proteolipids occurred rapidly in explant culture in the absence of exogenous hormones and was enhanced by addition of dexamethasone. Increased synthesis of the proteolipids was detected by enzyme-linked immunosorbent assay and by [35S]methionine incorporation into the glycosylated Mr = 40,000-43,000 SPL (Phe) precursor. The response to dexamethasone occurred rapidly and contrasted with effects of dexamethasone on the expression of surfactant-associated protein- (SAP) 35, a distinct surfactant glycoprotein. 8-Br-cAMP did not significantly increase proteolipid content but markedly increased synthesis of SAP-35 in identical cultures. Increased proteolipid content was associated with increased mRNA for each protein as determined by the Northern blot analysis. Proteolipid RNA was also increased by 8-Br-cAMP, however, not to the extent observed with the glucocorticoid. Immunohistochemical analysis of fetal lung with anti-proteolipid antiserum confirmed that the dexamethasone-enhanced synthesis of the proteins by Type II epithelial cells. The time and hormone dependence of the regulation of expression of both SPL(Phe) and SPL(pVal) precursors were distinct from that of SAP-35. Expression of the surfactant proteolipids increased during explant culture of human fetal lung and was further enhanced by glucocorticoid. Developmental and hormonal regulation of the surfactant proteolipids may be important factors in surfactant function at birth.     

Differential effects of epidermal growth factor and transforming growth factor-beta on synthesis of Mr = 35,000 surfactant-associated protein in fetal lung

Differentiation of pulmonary Type II epithelial cells in late gestation is associated with the synthesis of pulmonary surfactant required for adaptation to air breathing at birth. In the present work, induction of synthesis of a Type II epithelial cell protein, surfactant-associated glycoprotein of Mr = 35,000 (SAP-35) was studied in human fetal lung tissue obtained at 15-24 weeks of gestation. SAP-35 content increased during organ culture in the absence of exogenous hormones. Epidermal growth factor or triiodothyronine stimulated the induction of SAP-35 synthesis during culture. Stimulation by epidermal growth factor (EGF) was detected as early as 2 days and persisted for up to 5 days in culture. Response to EGF was dose-dependent (0.01-10 ng/ml) and was associated with enhanced incorporation of [35S]methionine into immunoprecipitable SAP-35. Increased SAP-35 synthesis was associated with increased SAP-35 RNA as assessed by Northern blot and hybridization assays with human SAP-35 cDNA. Effects of EGF were comparable to the induction of SAP-35 synthesis by 8-bromo-cAMP. In contrast to the stimulatory effect of EGF and triiodothyronine, SAP-35 content was inhibited by transforming growth factor-beta. Both the stimulatory and inhibitory effects of these agents on SAP-35 content were associated with concomitant changes in SAP-35 synthesis. These findings demonstrate multihormonal control of SAP-35 expression and strongly implicate both EGF and transforming growth factor-beta in the regulation of surfactant apoprotein synthesis.     

Synthesis of surfactant-associated protein, 35,000 daltons, in fetal lung

The major human pulmonary surfactant-associated protein of 35,000 daltons (Da) (SAP-35), consists of a group of related proteins of 27,000-36,000 Da, with isoelectric points ranging from pH 4.6 to 5.2. SAP-35 precursors were identified by immunoprecipitation of protein products of in vitro translation of normal adult human poly(A)+ mRNA with human SAP-35 antiserum. The translation products nearly comigrated with the most basic components of alveolar SAP-35 (mol mass = 24,500-27,000 Da). Processing of the primary translation products by canine pancreatic microsomal membranes increased their apparent molecular weight to 29,000-30,000-Da forms, which were sensitive to endoglycosidase F, suggesting the addition of asparagine-linked oligosaccharides to the molecules. A smaller protein of 24,500 Da was generated during treatment with canine microsomal membranes likely representing cleavage of a signal peptide. SAP-35 was not detected in explants of [35S]methionine-labeled fetal lung (20-24 wk gestation) after 1 day of culture or immunoprecipitates of in vitro translated poly(A)+ mRNA isolated from fetal human lung. However, after 3-5 days of organ culture, synthesis of SAP-35 was readily detected by immunoprecipitation of [35S] methionine-labeled tissue. Fully sialylated (neuraminidase-sensitive forms) comigrated with fully glycosylated SAP-35 isolated from human surfactant. High mannose (endoglycosidase H-sensitive precursors) were also synthesized by the organ cultures and were distinct from the secreted form in surfactant. Synthesis of surfactant-associated SAP-35 and its precursors was induced in association with morphological maturation of the type II epithelial cell during organ culture of human fetal lung.     

Synthesis of surfactant components by cultured type II cells from human lung

We examined the effect of monolayer culture on surfactant phospholipids and proteins of type II cells isolated from human adult and fetal lung. Type II cells were prepared from cultured explants of fetal lung (16-24 weeks gestation) and from adult surgical specimens. Cells were maintained for up to 6 days on plastic tissue culture dishes. Although incorporation of [methyl-3H]choline into phosphatidylcholine (PC) by fetal cells was similar on day 1 and day 5 of culture, saturation of PC fell from 35 to 26%. In addition, there was decreased distribution of labeled acetate into PC, whereas distribution into other phospholipids increased or did not change. The decrease in saturation of newly synthesized PC was not altered by triiodothyronine (T3) and dexamethasone treatment or by culture as mixed type II cell/fibroblast monolayers. The content of surfactant protein SP-A (28-36 kDa) in fetal cells, as measured by ELISA and immunofluorescence microscopy, rose during the first day and then fell to undetectable levels by the fifth. Synthesis of SP-A, as measured by [35S]methionine labeling and immunoprecipitation, was detectable on day 1 but not thereafter. Levels of mRNAs for SP-A and for the two lipophilic surfactant proteins SP-B (18 kDa) and SP-C (5 kDa) fell with half-times of maximally 24 h. In contrast, total protein synthesis measured by [35S]methionine incorporation increased and then plateaued. In adult cells, the content of SP-A and its mRNA decreased during culture, with time-courses similar to those for fetal cells. We conclude that in monolayer culture on plastic culture dishes, human type II cells lose their ability to synthesize both phospholipids and proteins of surfactant. The control of type II cell differentiation under these conditions appears to be at a pretranslational level.     

Differential regulation of hCG alpha and beta subunit mRNAs in JEG-3 choriocarcinoma cells by 8-bromo-cAMP

The coordinate regulation of human chorionic gonadotropin (hCG) subunit synthesis by JEG-3 choriocarcinoma cells was studied at the pretranslational level. The responses of the hCG alpha and beta mRNAs were measured during stimulation with the potent cAMP analog 8-bromo-cAMP (8-Br-cAMP) using 32P-labeled hCG alpha and beta cDNA probes. The hCG alpha mRNA (850 bases) and beta mRNA (1050 bases) from JEG-3 cells were identical in size to that of their respective mRNAs from placenta, by Northern blot analysis. After 48 h of stimulation with 2 mM 8-Br-cAMP, production of immunoreactive alpha and beta subunits increased 25- and 52-fold, respectively; corresponding levels of the alpha and beta mRNAs increased 36- and 43-fold, respectively, in a dot blot hybridization assay. Total cellular protein, DNA content, and messenger RNA pools were not altered by treatment with 8-Br-cAMP. The temporal coordination of the expression of the hCG alpha- and beta-subunit genes was examined by comparing the time course of stimulation of the respective mRNAs and the production of immunoreactive subunits. The kinetic responses of the alpha and beta mRNAs differed: the increase in hCG alpha mRNA preceded the increase in hCG beta mRNA, while levels of free alpha subunit and intact hCG increased in parallel with the increase in beta mRNA. hCG alpha mRNA levels increased rapidly between 8 and 24 h after the addition of 8-Br-cAMP, and approached a plateau by 48 h. The levels of hCG beta mRNA increased steadily throughout the 8-48 h period. These results demonstrate that the cAMP analog 8-Br-cAMP differentially regulates hCG subunit biosynthesis in JEG-3 cells at a pretranslational level, and that the stimulation by 8-Br-cAMP in this system appears to be relatively selective for hCG subunits.     

Regulation of activin beta A mRNA level by cAMP.

We demonstrated the presence of five species of the activin beta A mRNA in human placenta and one major RNA associated with two minor RNAs of the activin in the fetal membrane. We investigated the effect of 8-bromo-cAMP (8-Br-cAMP) on accumulation of activin beta A subunit mRNA in human fibrosarcoma HT1080 cells. Although low levels of the activin mRNA were detectable in the untreated cells, the one main RNA species was predominantly accumulated by 8-Br-cAMP. We propose that generation of multiple activin mRNAs in the fetal membrane and cAMP-treated HT1080 cells is presumably due to a cell-specific alternative polyadenylation.     

Regulation of cytochrome P-450c by glucocorticoids and polycyclic aromatic hydrocarbons in cultured fetal rat hepatocytes

The actions of polycyclic aromatic hydrocarbons and glucocorticoids to regulate the synthesis of cytochrome P-450c (the major isozyme induced by polycyclic aromatic hydrocarbons) were investigated in fetal rat hepatocytes maintained in primary monolayer culture. Treatment of hepatocytes in culture with 1,2-benzanthracene resulted in a 50-fold increase in 7-ethoxycoumarin O-deethylase activity. The level of P-450c increased in the cells in a time-dependent fashion as determined by immunoelectrophoretic analysis. The inductive effect of BA was potentiated approximately 1.6- to 2.3-fold when 1 microM dexamethasone was included in the culture medium. However, dexamethasone alone had little or no effect on the induction of P-450c. The rate of synthesis of P-450c was examined by immunoisolation of the specific isozyme from total cellular proteins radiolabeled with [35S]methionine and from the protein products formed during in vitro translation of the isolated mRNA. In addition, the amount of mRNA specific for cytochrome P-450c was determined by Northern blot analysis of RNA extracted from cultured cells. The changes in the rates of synthesis and mRNA levels were found to parallel the changes in enzyme activity. The concentration of dexamethasone required to cause a half-maximal increase in P-450c content in the presence of 1,2-benzanthracene was between 10(-8) and 10(-7) M. It is concluded that glucocorticoids act synergistically with polycyclic aromatic hydrocarbons to increase the levels of P-450c expressed in the fetal rat liver, and that this action is likely mediated by the classical type II glucocorticoid receptor.     

Differential effects of ACTH or 8-Br-cAMP on growth and replication in a functional adrenal tumor cell line

The effects of ACTH and 8-Br-cAMP on growth and replication of a functional mouse adrenal tumor cell line (Y-1) were investigated. ACTH and 8-Br-cAMP both inhibited DNA synthesis and replication when added to randomly growing cell cultures. ACTH addition and serum deprivation each arrested cells in G₁; an additional point of arrest in G₂ occurred with 8-Br-cAMP. Cells whose growth was arrested in G₁ by ACTH had a significantly larger volume and protein and RNA content compared to cells arrested in G₁ by serum deprivation. When ACTH or 8-Br-cAMP was added with serum to cells arrested by serum deprivation, the wave of DNA synthesis and cell division seen with serum was abolished. ACTH and 8-Br-cAMP had no effect on the serum-induced increases in protein and RNA content, rates of leucine incorporation into protein and uridine incorporation into RNA, and RNA polymerase I activity observed in cells during the pre-replicative period. Partial inhibition of the serum-induced increase in uridine transport occurred. ACTH and cAMP do not appear to inhibit replication by generalized negative pleiotypic effects but rather to inhibit the initiation of DNA synthesis more specifically. The ACTH-arrested Y-1 cell resembles an in vivo hypertrophied adrenal cortical cell.     

Developmental regulation of DUOX1 expression and function in human fetal lung epithelial cells

The purpose of this study was to determine the expression and cellular functions of the epithelial NADPH oxidase DUOX1 during alveolar type II cell development. When human fetal lung cells (gestational age 11-22 wk) were cultured to confluency on permeable filters, exposure of cells to a hormone mixture (dexamethasone, 8-Br-cAMP, and IBMX, together referred to as DCI) resulted in differentiation of cells into a mature type II phenotype as assessed by expression of lamellar bodies, surfactant proteins, and transepithelial electrical parameters. After 6 days in culture in presence of DCI, transepithelial resistance (2,616 +/- 529 Omega.cm(2)) and potential (-8.5 +/- 0.6 mV) indicated epithelial polarization. At the same time, treatment with DCI significantly increased the mRNA expression of DUOX1 ( approximately 21-fold), its maturation factor DUOXA1 ( approximately 12-fold), as well as DUOX protein ( approximately 12-fold), which was localized near the apical cell pole in confluent cultures. For comparison, in fetal lung specimens, DUOX protein was not detectable at up to 27 wk of gestational age but was strongly upregulated after 32 wk. Function of DUOX1 was assessed by measuring H(2)O(2) and acid production. Rates of H(2)O(2) production were increased by DCI treatment and blocked by small interfering RNA directed against DUOX1 or by diphenylene iodonium. DCI-treated cultures also showed increased intracellular acid production and acid release into the mucosal medium, and acid production was largely blocked by knockdown of DUOX1 mRNA. These data establish the regulated expression of DUOX1 during alveolar maturation, and indicate DUOX1 in alveolar H(2)O(2) and acid secretion by differentiated type II cells.     

Surfactant protein of molecular weight 28,000-36,000 in cultured human fetal lung: cellular localization and effect of dexamethasone

We have examined the effect of explant culture and hormones on the major surfactant associated protein of Mr 28,000-36,000 (SP 28-36) in human fetal lung. Explants of 16- to 23-week gestation lung were maintained for up to 5 days in culture. Polyclonal antibodies raised to SP 28-36 purified from alveolar proteinosis lung lavage were used in immunofluorescence experiments (n = 11). There was no specific fluorescence seen in frozen sections of preculture tissue. In explants cultured without serum or hormones, fluorescence was seen in most epithelial cells lining potential airspaces. In cultures treated with 10 nM dexamethasone and 2 nM T3 much brighter fluorescence was seen in virtually all epithelial cells. Immunofluorescence studies on cell monolayers prepared from explants confirmed that SP 28-36 is found in the cytoplasm of type II cells but not in fibroblasts. The pattern of fluorescence was consistent with the presence of SP 28-36 on rough endoplasmic reticulum. SP 28-36 mRNA was measured in isolated cell populations using a 32P-labeled cDNA probe. mRNA levels were manyfold higher in type II cell preparations (purity 78-92%) than in fibroblasts (purity 81-97%). A competitive enzyme linked assay was developed to quantify SP 28-36. The SP 28-36 content of five lungs before culture (17-23 weeks) was less than 0.02 microgram/mg DNA. During explant culture without hormones the SP 28-36 content increased exponentially. Exposure to dexamethasone accelerated the increase in SP 28-36 content. T3, alone or in the presence of dexamethasone, did not influence SP 28-36 content. We conclude that SP 28-36 content is very low in human fetal lung before 24 weeks gestation. Explant culture and treatment with dexamethasone synchronize development of type II cells from epithelial precursors, and induce synthesis of SP 28-36 in type II cells. These findings provide evidence of concomitant regulation by glucocorticoids of the phospholipid synthetic enzymes and the major protein of pulmonary surfactant.     

Direct evidence that the protein kinase catalytic subunit mediates the effects of cAMP on tyrosine aminotransferase synthesis

The effect of purified beef heart cAMP-dependent protein kinase catalytic subunit on tyrosine aminotransferase activity in intact cultured rat H35 hepatoma cells was directly tested by micro-injection using human red blood cell ghosts as vehicles. Although the micro-injection procedure itself produced temporary fluctuations in protein synthesis and in tyrosine aminotransferase activity in H35 cells, after a recovery period of 8-12 h, these parameters returned to normal in parallel with restoration of full inducibility of the aminotransferase by both 8-Br-cAMP and dexamethasone. Eight to sixteen hours after fusion of H35 cells with unloaded ghosts, ghosts loaded with bovine serum albumin or mock-loaded with the partially purified protein kinase catalytic subunit, no significant change in the activity of the aminotransferase was detected. In contrast, fusion with ghosts loaded with the catalytic subunit at concentrations between 0.1-2 mg/ml caused reproducible 2-3-fold increases in enzyme activity. Homogeneous preparations of the catalytic subunit exhibited even greater potency as an inducer. The effect was both time- and concentration-dependent and was abolished by inactivation of the catalytic subunit with N-ethylmaleimide prior to loading. The partially purified inhibitor of protein kinase from beef heart, while not affecting basal tyrosine aminotransferase activity, selectively inhibited the ability of 8-Br-cAMP but not that of dexamethasone to stimulate the activity of this enzyme. In addition, micro-injection of the pure regulatory subunit of the kinase blocked the response of the aminotransferase to low concentrations of 8-Br-cAMP. These results provide strong support for the proposition that the catalytic subunit of protein kinase mediates the effects of cAMP on the synthesis of tyrosine aminotransferase.     

The development of surfactant synthesis in fetal rabbit lung organ culture exhibits a sex dimorphism

Sex differences in amniotic fluid and lung lavage surfactant have been found. Although these studies suggest that augmented fetal surfactant synthesis occurs earlier in the female fetus, there is little direct evidence for a sex difference in fetal surfactant synthesis. We studied the synthesis of surfactant by evaluating the appearance of labelled phospholipids in lamellar bodies recovered from sex-specific organ culture of fetal rabbit lungs. Furthermore, we studied the ability of dexamethasone to stimulate surfactant synthesis in male and female fetal lungs. Organ culture was begun on day 21 of gestation. After 5 days the incorporation of [1,3-14C]glycerol into phosphatidylcholine (PC), disaturated phosphatidylcholine, phosphatidylinositol (PI), and phosphatidylglycerol was studied. Female lungs in organ culture synthesized more disaturated PC per milligram protein than male lungs. In the presence of dexamethasone (10(-8) M) and dihydrotestosterone (10(-8) M) an increased synthesis was noted in the female cultures of PC (270%), disaturated PC (234%), PI (281%), and phosphatidylglycerol (754%). No significant increase in the synthesis of PC or disaturated PC was observed in the male cultures. However in the male cultures smaller increases in the synthesis of PI (193%) and of phosphatidylglycerol (360%) were observed. Overall, dexamethasone stimulated synthesis in females but not in males such that significant differences in the synthesis of all phospholipids were found in the presence of 10(-8) M dexamethasone. These studies show that the synthesis of surfactant in the fetal lung is sexually dimorphic, as is the ability of dexamethasone to regulate synthesis. An understanding of the mechanism which causes these differences may provide important insight into the control of the developmental clock which regulates the orderly progression of development.     

Factors affecting cytotrophoblast cell viability and differentiation: Evidence of a link between syncytialisation and apoptosis

A relationship between cytotrophoblast differentiation (syncytialisation) and apoptosis is hypothesised to exist, but has not been clearly determined. To address this, we explored the effects of cAMP, an inducer of syncytialisation, on human choriocarcinoma cell differentiation and viability under three different culture conditions related to diverse survival status: no serum, 10% fetal calf serum or 10% charcoal-stripped fetal calf serum. 8-Br-cAMP increased BeWo cell viability in culture media without serum, but viability was decreased in a dose- and time-dependent manner when serum was present. The appearance of apoptotic nuclei fragments were only observed when BeWo cells were cultured in media containing serum combined with 8-Br-cAMP treatment. In addition, the ratio of FasL to Fas expression following treatment with 8-Br-cAMP increased by 20-fold in 10% charcoal-stripped fetal calf serum media and 65-fold 10% fetal calf serum media, and activation of caspase-3 also required media with serum. The markers of syncytialisation (syncytin 1 expression and human chorionic gonadotropin secretion) were induced significantly by 8-Br-cAMP, and were higher in 10% fetal calf serum media than in 10% charcoal-stripped fetal calf serum media, than in the absence of serum. Syncytia formation was stimulated by 8-Br-cAMP and this required serum in the media. We now show that factors contained within serum are necessary for cAMP-stimulated cytotrophoblast differentiation, that syncytialisation involves apoptotic events, and that a lack of serum based factors could switch the cellular program away from differentiation.     

Regulation of the synthesis of the major surfactant apoprotein in fetal rabbit lung tissue

Antibodies directed against the major apoprotein of rabbit lung surfactant, a 29-36-kDa glycoprotein, were used to study changes in the levels of translatable surfactant apoprotein mRNA in rabbit lung tissue during development, as well as the effects of cortisol and cyclic AMP analogues on the levels of surfactant apoprotein and its mRNA in fetal rabbit lung tissue in organ culture. The major surfactant apoprotein and its mRNA were undetectable in lung tissues of 21-day gestational age fetal rabbits. Translatable mRNA specific for the major surfactant apoprotein was first detectable in lung tissues of 26-day fetuses, increased 25-fold on day 28, reached peak levels at day 31, and declined after birth. Incubation of 21-day fetal rabbit lung explants with cortisol in serum-free medium resulted in an increase in the specific content of the 29-36-kDa apoprotein. Cyclic AMP analogues and forskolin, an activator of adenylate cyclase, also caused a marked increase in the accumulation of surfactant apoprotein. When fetal lung explants were incubated with cortisol and dibutyryl cyclic AMP in combination, the specific content of the surfactant apoprotein was increased to levels greater than that of explants treated with either cortisol or dibutyryl cyclic AMP alone. These effects of dibutyryl cyclic AMP and cortisol on surfactant apoprotein accumulation were associated with comparable changes in the levels of translatable surfactant apoprotein mRNA. Thus, we have shown for the first time that the induction of pulmonary surfactant apoprotein synthesis during differentiation in vitro and in vivo is associated with an increase in the level of translatable mRNA and that cortisol and cyclic AMP increase both the accumulation of the major surfactant apoprotein and the corresponding mRNA in fetal rabbit lung tissue in vitro.     

Fetal and neonatal rat pancreas in organ culture: age-related effects of corticosterone on the development of the islet cells

Fetal (18 days postcoitum) and neonatal (3-day) pancreatic explants were grown in organ culture with or without supplementation with corticosterone (0.1 micrograms/ml). After 0, 4, and 8 days of culture, the specific hormone-positive, islet cell volumes were determined by the use of immunocytochemical and morphometric methods. The insulin, glucagon, and somatostatin contents of the explants were estimated by radioimmunoassays. In the fetal explants, all of the islet cell populations increased in volume and the content of each of the hormones increased over an 8-day period of culture. Supplementation with corticosterone resulted in a restriction of the increases of the alpha and delta cell volumes and in the somatostatin content of the explants. In the neonatal explants, the volumes of the alpha and delta cells and the glucagon and somatostatin contents decreased over a 4-day culture period. The presence of corticosterone in the culture medium preserved these cells and their hormone content. Co-culture of 18-day fetal and 3-day neonatal pancreata in control medium for 8 days resulted in a significant decrease in the content of all three of the islet hormones in the fetal explants. These results suggest that a substance harmful to the islet cells is released from the degenerating acinar cells. Thus, the effects of the steroid on the islets may be mediated through its effects on the acinar tissue.     

Insulin and prolactin synergize to induce translation of human serum albumin in the mammary gland of transgenic mice

A dramatic uncoupling of the expression of chimaeric -lactoglobulin (BLG)/human serum albumin (HSA) gene constructs at the RNA and protein levels was observed in cultured mammary explants of virgin transgenic mice. Upon explantation, both HSA RNA and protein were expressed at high levels. However, when the explants were grown in hormone-free medium, HSA RNA continued to accumulate, whereas the synthesis of the corresponding protein was dependent on the presence of insulin and prolactin with a minor contribution of hydrocortisone. The untranslated HSA RNA was indistinguishable from its translatable counterpart in its mobility on agarose gels, was transported normally from the nucleus to the cytoplasm and was translated efficiently in rabbit reticulocyte lysate. In the presence of cycloheximide, HSA RNA rapidly disappeared, suggesting a dependency on ongoing protein synthesis. Its estimated half-life of 5--6 h in hormone-free medium increased significantly in the presence o f insulin, hydrocortisone and prolactin and was comparable to that of -casein RNA. The uncoupling of the expression of the BLG/HSA transgenes at the RNA and protein levels was also confirmed by in situ hybridization and immunohystochemistry on sections from virgin mammary explants. HSA synthesis was initiated within 13 h of the addition of insulin and prolactin in explants that had accumulated untranslated HSA RNA and was fourfold higher than that observed with insulin alone. Addition of hydrocortisone contributed to an additional 20% in HSA synthesis. We believe this is the first demonstration of translational control of exogenous milk protein gene expression in the mammary gland of transgenic animals     

In vitro chondrogenesis and cell viability

Anterior somites cultured with (NSA) or without (SA) notochord, and posterior somites cultured with (NSP) or without notochord (SP) were compared with respect to changes in their DNA content, their potential to synthesize the active sulfate principle phosphoadenosine phosphosulfate (PAPS), and their ability to accumulate ³⁵S-sulfate.Chondrogenesis was observed in the NSA, NSP, and SP explants, but was rarely noted in the SA explants. A decrease in DNA content during the initial 48 hr of culture was common to all explants. After this initial decrease, DNA content increased most in those explants forming cartilage. The synthesis of PAPS by cell-free extracts of each type of somite explant also decreased during the initial period of culture. Only extracts of those explants undergoing chondrogenesis showed increases in PAPS synthesis with continued culture. Each type of somite explant accumulated ³⁵S-sulfate into chondroitin sulfate during the first hours of culture. The non-chondrogenic SA explants accumulated little ³⁵S-sulfate during the period of culture. At varying times after 24 hr the chondrifying explants (NSA, SP, and NSP) initiated an increased rate of accumulation of ³⁵S-sulfate.Cartilage nodules, increases in DNA content, PAPS synthesis and ³⁵S-sulfate accumulation occurred within the same 24 hr period, during the 2nd day in NSP explants, the 3rd day in NSA explants, and between the 3rd and 4th day for SP explants. A hypothesis of in vitro somite chondrogenesis based on differential cell viability is presented.     

Effect of cortisol on human fetal lung in organ culture

Human fetal lung tissue obtained during the second trimester was cultured as organ culture with or without cortisol. The effect of cortisol on the phospholipid metabolism, as related to the appearance of osmiophilic lamellar bodies and the localisation of newly incorporated choline, was studied. In cortisol-treated explants, the concentration of saturated lecithins and the incorporation of (Me-3H)-choline into saturated lecithins increases significantly concomitantly with an increased number of osmiophilic lamellar bodies. The labelled choline is predominantly associated with these bodies. The obtained results indicate that cortisol accelerates the synthesis of pulmonary surfactant in the human fetal lung as early as in the second trimester.     

Epidermal growth factor (EGF) influences DNA synthesis in human fetal kidneys maturing in serum-free organ culture

Human fetal kidney explants (13-17 weeks of gestation) were maintained in serum-free organ culture. The influence of epidermal growth factor (EGF) was determined after 2 and 5 days by evaluating DNA and protein synthesis as well as the activities of five brush border hydrolases. During the studied period the overall morphology was preserved and the analysed parameters remained constant. Only DNA synthesis decreased after 2 days. The addition of EGF to the medium did not change any of the cell activities, except DNA synthesis. In fact, the incorporation of [3H]thymidine was significantly stimulated by 105% in 5-day explants cultured in the presence of the growth factor. These results indicate that EGF directly influences proliferation but not maturation of brush border enzymes in fetal human kidneys in culture.