Immunohistochemical localization of a low molecular weight surfactant-associated protein in human lung

We used an antiserum to a hydrophobic 6 KD surfactant-associated protein to localize this protein in human lung tissue. This antiserum does not crossreact with the 35 KD surfactant-associated protein. By light microscopy using the indirect immunoperoxidase technique, the protein appears to be localized within Type II alveolar epithelial cells. Staining is also detectable in alveolar macrophages and occasionally within the lumina of alveoli and bronchioles. No staining was detected within the alveolar septa or in association with blood vessels. An identical distribution is seen for the 35 KD surfactant-associated protein using an antiserum specific for that protein.     

Autoradiographic localization of specific [3H]dexamethasone binding in fetal lung

The cellular and subcellular localization of specific [3H]dexamethasone binding was examined in fetal mouse lung at various stages of development and in human fetal lung at 8 weeks of gestation using a rapid in vitro steroid incubation technique followed by thaw-mount autoradiography. Competition studies with unlabeled steroids demonstrate the specificity of [3H]dexamethasone labeling, and indicate that fetal lung mesenchyme is a primary glucocorticoid target during lung development. Quantitative binding studies, involving incubation of intact tissue with competing ligand and subsequent subcellular fractionation, show this to be specific, nuclear binding characteristic of glucocorticoid receptors. Autoradiographs of [3H]dexamethasone binding in lung tissue at early stages of development demonstrate that the mesenchyme directly adjacent to the more proximal portions of the bronchiolar network is heavily labeled. In contrast, the epithelium which will later differentiate into bronchi and bronchioles, is relatively unlabeled. Distal portions of the growing epithelium, destined to become alveolar ducts and alveoli, do show nuclear localization of [3H]dexamethasone. Because of the known importance of the mesenchyme in controlling lung development and the ability of glucocorticoids to stimulate lung development, these results suggest that many of the growth-promoting effects of glucocorticoids may be mediated through the mesenchyme. In addition, by utilizing a technique which allows the simultaneous examination of extracellular matrix components and [3H]dexamethasone binding, a relationship is observed between extensive mesenchymal [3H]dexamethasone binding and extensive extracellular matrix accumulation. Since glucocorticoids stimulate the synthesis of many extracellular matrix components, these results suggest a role for these hormones in affecting mesenchymal-epithelial interactions during lung morphogenesis.     

The effect of dexamethasone on transferrin secretion by cultured fetal rat hepatocytes

Cultured fetal rat hepatocytes derived from 12, 15 and 19-day gestation rats are capable of secreting transferrin. When dexamethasone is added to the medium an increased secretion rate is observed. The changes in secretion rates in control as well as dexamethasone-treated cells during culture have been shown to correlate with the level of mRNA coding for transferrin. Immunocytochemical experiments show that initially all hepatocytes contain transferrin which is localized in the lumina of the perinuclear space, rough endoplasmic reticulum and in the saccules and vesicles of the Golgi apparatus. During culture, particularly in control cells, the intensity of labelling varies from cell to cell. In addition, adjacent cells are observed to label more intensely in different intracellular organelles.     

Collagen glucosyl- and galactosyltransferases of cultured human fetal lung fibroblasts

Collagen-galactosyltransferase and collagen-glucosyltransferase activities have been studied in cultured human fetal lung WI-38 and IMR-90 diploid fibroblasts. These enzymes functioned in concert to synthesize glucosylgalactosylhydroxylysine units as found naturally in collagens, basement membranes, and certain serum glycoproteins. The transferases used UDP-Gal and UDP-Glc as glycose donors, collagens and collagen-derived peptides or glycopeptides as glycose acceptors, and worked best in the presence of manganese as a required divalent cation. Two pH optima, between pH 6 and 6.5 and between pH 7.5 and 8, were noted for each type of transferase, and these optima, particularly in the case of glucosyltransferase, were evident regardless of size of acceptor employed in the assay. About 35% of the total activity of each enzyme was found in the soluble fractions of cell homogenates, and, of the particulate fraction activities, about 50% could be released by mild sonication or by treatment with Triton X-100. Assessment of transferase activities as a function of cellular aging in culture revealed that significant decreases in enzyme levels occurred as the cell approached senescence (late Phase II), and these effects were reversed when cells attained senescence (Phase III). Addition of ascorbic acid to young cultures, under conditions known to increase endogenous collagenpeptide hydroxylation, caused no effects on the activities of the glycosyltransferases toward exogenous acceptors. These results suggested that the activities of collagen-hydroxylases and glycosyltransferase might not be coordinately regulated, and that, regardless of the hydroxylation events, glycosylation of the peptide might be limited to a specific fraction of hydroxylysine residues during the post-translational modification of collagen.     

Surfactant strengthens the inhibitory effect of C-reactive protein on human lung macrophage cytokine release

In this study we investigated the effect of acute-phase levels of C-reactive protein (CRP) on cytokine production by pulmonary macrophages in the presence or absence of pulmonary surfactant. Both human alveolar and interstitial macrophages as well as human surfactant were obtained from multiple organ donor lungs. Precultured macrophages were stimulated with LPS alone or together with IFN-gamma in the presence or absence of CRP, surfactant, and combinations. Releases of TNF-alpha and of IL-1beta to the medium were determined. We found that CRP could modulate lung inflammation in humans by decreasing the production of proinflammatory cytokines by both alveolar and interstitial macrophages stimulated with LPS alone or together with IFN-gamma. The potential interaction between CRP and surfactant phospholipids did not overcome the effect of either CRP or surfactant on TNF-alpha and IL-1beta release by lung macrophages. On the contrary, CRP and pulmonary surfactant together had a greater inhibitory effect than either alone on the release of proinflammatory cytokines by lung macrophages.     

Stage-dependent processing and localization of a Plasmodium falciparum protein of 130,000 molecular weight

A Plasmodium falciparum protein of 130,000 molecular weight (m.w.) has been identified, cloned in Escherichia coli, and completely sequenced (Kochan et al. 1986). The protein appeared to bind to soluble glycophorin, a host erythrocyte surface protein. In the present study, extracts of parasites from different intraerythrocytic stages were immunoblotted with antibodies, raised against a 30,000 m.w. fusion protein corresponding to the 3' end of the 130,000 m.w. protein. It was demonstrated that the protein is synthesized at the trophozoite stage, accumulates at the schizont stage, and is processed at the merozoite stage to a triplet of three polypeptides. The processed proteins are present in the culture supernatant at the time of merozoite burst from the red cell. Immunofluorescent staining of the parasite at different intracellular stages indicates that the protein is localized on the parasite at the trophozoite stage. At late trophozoite stage, it appears to be transported to the erythrocyte cytoplasm, where it is present in small vesicles or inclusions. In mature schizonts the protein accumulates around the plasma membrane of the erythrocyte. At the segmenter stage, just prior to merozoite release, it appears also to surround the intracellular merozoite, as well as the erythrocyte plasma membrane. The soluble 130,000 m.w. protein binds to erythrocytes but binds significantly greater to erythrocyte membranes, suggesting it binds to an internal domain of glycophorin rather than the domain exposed on the surface. The 130,000 m.w. protein is present in 11 different geographic isolates of P. falciparum from diverse geographic origins. Its molecular weight is similar in all isolates.     

Induction of gamma-glutamyl transferase by dexamethasone in cultured fetal rat hepatocytes

Hepatocytes isolated as a relatively pure population from normal fetal rats were maintained in primary monolayer culture for 4-10 days. Hepatocytes exhibited a small increase in basal gamma-glutamyl transferase (GGT) activity over time. Exposure to dexamethasone (10(-6) mol/l) elicited a rise in GGT activity after a lag of 24 h. The presence of the steroid was necessary to maintain induction, and its removal resulted in reversal of induction. The maximal response was 2- to 3-fold, 72 h after exposure to the steroid. After this maximal response, a gradual decay in enzyme activity occurred, despite the continuous presence of the hormone. Actinomycin D or cycloheximide given prior to/or simultaneously with the steroid prevented the induction, thus suggesting that both RNA and protein biosynthesis are necessary for induction to occur.     

Refolding of a high molecular weight protein: salt effect on collapse

Small-angle neutron scattering experiments were performed on dilute solutions of a high molecular weight protein (fibronectin, M = 580 kg/mol) in four cases: native conditions; unfolded state obtained by a denaturing agent (urea); and two badly refolded (or collapsed) states obtained by progressive elimination of the denaturing agent in salt-containing or salt-free solutions. Our main result is concerned by the conformation of the protein as the attempt for refolding is driven with or without salt. In salt-containing solution, we observe unambiguously that the protein chain collapses at large length scales but still obeys to a Gaussian statistics at short length scales. In other words, the globule embodies a large quantity of solvent compared to the compact situation. In salt-free solutions, the badly refolded protein is not globular but displays both a coil-like and an open conformation at large length scales and a local high density area. This behavior is discussed with respect to the scaling theories for polymers and polyampholytes.     

Novel compounds lowering the cellular isoform of the human prion protein in cultured human cells

Purpose: Previous studies showed that lowering PrP^C concomitantly reduced PrP^Sc in the brains of mice inoculated with prions. We aimed to develop assays that measure PrP^C on the surface of human T98G glioblastoma and IMR32 neuroblastoma cells. Using these assays, we sought to identify chemical hits, confirmed hits, and scaffolds that potently lowered PrP^C levels in human brains cells, without lethality, and that could achieve drug concentrations in the brain after oral or intraperitoneal dosing in mice. Methods: We utilized HTS ELISA assays to identify small molecules that lower PrP^C levels by ⩾30% on the cell surface of human glioblastoma (T98G) and neuroblastoma (IMR32) cells. Results: From 44,578 diverse chemical compounds tested, 138 hits were identified by single point confirmation (SPC) representing 7 chemical scaffolds in T98G cells, and 114 SPC hits representing 6 scaffolds found in IMR32 cells. When the confirmed SPC hits were combined with structurally related analogs, >300 compounds (representing 6 distinct chemical scaffolds) were tested for dose–response (EC₅₀) in both cell lines, only studies in T98G cells identified compounds that reduced PrP^C without killing the cells. EC₅₀ values from 32 hits ranged from 65 nM to 4.1 μM. Twenty-eight were evaluated in vivo in pharmacokinetic studies after a single 10 mg/kg oral or intraperitoneal dose in mice. Our results showed brain concentrations as high as 16.2 μM, but only after intraperitoneal dosing. Conclusions: Our studies identified leads for future studies to determine which compounds might lower PrP^C levels in rodent brain, and provide the basis of a therapeutic for fatal disorders caused by PrP prions.     

Lamellar bodies of cultured human fetal lung: content of surfactant protein A (SP-A), surface film formation and structural transformation in vitro

Lamellar bodies were isolated from dexamethasone and T3-treated explant cultures of human fetal lung, using sucrose density-gradient centrifugation. We examined their content of surfactant apoprotein A (SP-A), and their ability to form surface films and to undergo structural transformation in vitro. SP-A measured by ELISA composed less than 2% of total protein within lamellar bodies; this represented, as a minimum estimate, a 2-12-fold enrichment over homogenate. One- and two-dimensional gel electrophoresis also suggested that SP-A was a minor protein component of lamellar bodies. Adsorption of lamellar bodies to an air/water interface was moderately rapid, but accelerated dramatically upon addition of exogenous SP-A in ratios of 1:2-16 (SP-A:phospholipid, w/w). Similar adsorption patterns were seen for lamellar bodies from fresh adult rat and rabbit lung. Lamellar bodies incubated under conditions that promote formation of tubular myelin underwent structural rearrangement only in the presence of exogenous SP-A, with extensive formation of multilamellate whorls of lipid bilayers (but no classical tubular myelin lattices). We conclude that lamellar bodies are enriched in SP-A, but have insufficient content of SP-A for structural transformation to tubular myelin and rapid surface film formation in vitro.     

Intracellular localization of the high molecular weight microtubule accessory protein by indirect immunofluorescence

Microtubule accessory proteins were isolated from porcine brain microtubules by phosphocellulose chromatography, and the high molecular weight protein (HMW protein), purified from this microtubule-associated fraction by electrophoretic elution from SDS gels, was used to raise antisera in rabbits. In agarose double diffusion tests, the antiserum obtained forms precipitin lines with purified HMW protein but not with tau protein or tubulin. When rat glial cells (strain C6) are examined by indirect immunofluorescence, this serum specifically stains a colchicine-sensitive filamentous cytoplasmic network in interphase cells, a network indistinguishable from that seen when cells are treated with antitubulin serum. In dividing cells, specific staining of the mitotic spindle and the stem body is observed with the antiserum to HMW protein. These studies indicate that HMW protein, like tau protein, is associated with microtubules in intact cells.     

Purification of normal cellular prion protein from human platelets and the formation of a high molecular weight prion protein complex following platelet activation

A method for the extraction and purification of PrP(C), in its native monomeric form, from outdated human platelet concentrates is described. Both calcium ionophore platelet activation and lysis in Triton X-100 were evaluated as methods for the extraction of soluble platelet PrP(C) in its monomeric form. Following platelet activation, the majority of released PrP(C) was detected as a disulphide linked high molecular weight complex, which under reducing conditions could be separated into what appear to be stable non-disulphide linked PrP dimers or PrP covalently linked to another as yet unidentified protein. This phenomenon appears to be unique to activation since only monomeric PrP(C) was detected following lysis of resting platelets. Subsequently, PrP(C) was purified from the Triton X-100 lysate by sequential cation ion exchange and Cu2+ affinity chromatography. From 10 L of outdated platelet concentrate, we were able to recover 1.29 mg PrP(C) at a purity of 92%.     

The effect of human urogastrone on lung phospholipids in fetal rabbits

Previous in vivo studies have demonstrated that mouse epidermal growth factor (EGF) can enhance fetal lung maturation. We have examined the effect of urogastrone, the human equivalent of mouse EGF and a related growth factor, on the phospholipid profile of fetal rabbit lung lavage and its action on fetal rabbit Type II pneumocytes in culture. Urogastrone (1 or 8 micrograms) given i.p. to fetal rabbits on day 25 of gestation resulted in increased total phospholipid, phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine contents, increased phosphatidylinositol and phosphatidylethanolamine as a proportion of phospholipid and decreased sphingomyelin as a proportion of phospholipid in lung lavages on day 28. These changes were unaccompanied by alterations in body weight or lung weight, DNA or protein concentrations. Urogastrone (16 micrograms) resulted in increased fetal deaths. Phospholipid profiles on day 27 were unchanged after fetal administration of urogastrone (1 microgram) on day 25. Urogastrone (0.01 and 0.1 ng/ml) added to fetal rabbit Type II pneumocytes in culture for 24 h enhanced the incorporation of radiolabelled choline and thymidine into phosphatidylcholine and DNA respectively. These findings indicate that human urogastrone can alter the phospholipid composition of the rabbit lung in a similar manner to that which occurs during maturation of the lung surfactant system in late pregnancy. This effect can be achieved, at least in part, by a direct action on Type II pneumocytes.     

Choice of expression vector alters the localization of a human cellular protein

The fusion of synthetic epitopes with proteins of interest is an important tool in the identification and characterization of recombinant proteins. Several mammalian expression vectors are commercially available containing unique identification tags or epitopes. These vectors offer a great advantage to researchers, as highly specific antibodies and purification resins against these specific epitopes are readily available. The tags facilitate immunologic assays and the purification of the recombinant proteins. The fusion of these epitopes with the recombinant proteins is not expected to alter the behavior of the protein of interest. In this report, we demonstrate that the mere expression of a cellular protein, hVIP/mov34, which we earlier identified as a cellular HIV-1 Vpr ligand, in two different vectors clearly altered its localization pattern in HeLa cells. Specifically, cloning of hVIP/mov34 in pcDNA3/HisA resulted in its nuclear localization, whereas the expression of this gene from a TOPO cloning expression vector, pcDNA3.1/V5/His, resulted in cytoplasmic expression. The native staining pattern of hVIP/mov34 using polyclonal antisera raised against hVIP/mov34 demonstrated cytoplasmic staining. During cloning, other leader sequences intended for targeting this protein into a cytoplasmic or a nuclear location were not fused to the actual ORF of this protein. Also, the amino acid sequence of the fusion region arising from cloning of hVIP/mov34 in both vectors does not match any reported NLS sequences. These results indicate that the choice of the expression vectors, as well as the position of synthetic epitopes, can significantly alter the behavior and the biology of recombinant proteins. This result suggests the need for a careful examination of these features when characterizing a newly identified protein.     

Induction of surfactant protein in fetal lung. Effects of cAMP and dexamethasone on SAP-35 RNA and synthesis

Synthesis of surfactant-associated glycoprotein of Mr = 30,000-35,000 (SAP-35) was induced in explant culture of human fetal lung obtained from 8 to 24 weeks of gestation. SAP-35 synthesis and content increased markedly during 1-5 days in organ culture in association with the morphologic maturation of Type II epithelial cells and the appearance of lamellar bodies. [35S] Methionine labeling of the explants and subsequent immunoprecipitation of SAP-35 demonstrated distinct high-mannose precursors and sialylated SAP-35 forms as early in culture as SAP-35 synthesis was detectable. The increase in SAP-35 synthesis was associated with increased SAP-35 RNA of 2.1 kilobases as assessed by hybridization assay with [32P]cDNA specific for human SAP-35. Specific SAP-35 RNA increased during organ culture and both SAP-35 content and SAP-35 RNA increased in the absence of exogenous hormones in 2% carbon-stripped fetal calf serum. SAP-35 content and synthesis was stimulated by 8-Br-cAMP. Addition of 100 microM 8-Br-cAMP, enhanced both the concentration of SAP-35 protein and the SAP-35 RNA as assessed by hybridization assay. In contrast, treatment of the explants with dexamethasone was associated with decreased SAP-35 protein synthesis, SAP-35 content, and decreased SAP-35 RNA levels compared to untreated explants. Inhibition by dexamethasone occurred at all gestational ages tested, was dose-dependent, and detectable within 24-48 h during organ culture. Dexamethasone significantly inhibited both basal and cAMP-induced SAP-35 synthesis. Induction of pulmonary surfactant protein (SAP-35) synthesis during organ culture of human fetal lung was associated with increased SAP-35 RNA. SAP-35 synthesis and SAP-35 RNA were inhibited by dexamethasone and enhanced cAMP.     

Surfactant proteins A (SP-A) and D (SP-D): Levels in human amniotic fluid and localization in the fetal membranes

Surfactant proteins A (SP-A) and D (SP-D) are major proteins, in the lung, which are composed of collagenous and globular domains. They show an overall similarity to the serum complement protein C1q, which is involved in the initiation of antibody-dependent defence mechanisms. Both SP-A and SP-D were detected, immunochemically, in amniotic fluid as early as 26 weeks gestation and, as expected, SP-A levels rose sharply from 32 weeks towards term. By contrast, SP-D levels in the same samples rose only moderately. Immunochemistry of paraffin sections of fetal membranes, revealed the presence of both SP-A and SP-D in the amniotic epithelium and chorio-decidual layers. SP-A and SP-D are both lectins and therefore they may play a role in the antibody-independent recogniton and clearance of pathogens in the amniotic fluid.     

Low molecular weight fibroblast collagen: structure, secretion, and differential expression as a function of fetal and cellular age

A unique low molecular weight collagen that was highly resistant to proteolytic degradation was originally isolated from fetal calf ligamentum nuchae fibroblasts and hence termed FCL-1 [Sage, H., Mecham, R., Johnson, C., & Bornstein, P. (1983) J. Cell Biol. 97, 1933-1938]. The differential expression of this protein was studied as a function both of fetal (donor) age and of subcultivation in vitro. Concomitant isolation, subculture, and metabolic radiolabeling experiments performed on cell strains from fetal calf ligament (FCL) and fetal bovine skin (FBS) representing different gestational ages (85-270 days in utero) showed that (a) FCL-1 was synthesized preferentially by fibroblasts from younger animals and (b) expression of FCL-1 diminished as a function of increased passage in culture. Levels of FCL-1, measured as percent of total radiolabeled culture medium protein that precipitated in a concentration range of 20-50% ammonium sulfate, ranged from 22% in FCL 85 cells to 7.7% in FCL 270 (term) cells. FBS fibroblasts at passages 6-10 secreted from 13% to 6% FCL-1, respectively. When cells from an 85-day fetal ligament were allowed to accumulate copious extracellular matrix in vitro, the production of FCL-1 was increased to 32%. FCL-1 was not immunoreactive with polyclonal antibodies directed toward most of the sequences of the interstitial type I and type III procollagens. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the apparent molecular weight of FCL-1 was 13 000 (on the basis of collagen peptide standards) and approximately 30 000 (on the basis of globular protein standards). Incubation with bacterial collagenase produced a stable cleavage product of Mr 8000 (by collagen standards) or 17 000 (by globular standards). In contrast, pepsin removed a small peptide of approximately 1000-2000 in molecular weight from FCL-1, and a gradual but progressive proteolysis of the collagen was observed over a period of 1-6 h. Pulse-chase studies revealed a secretion time of approximately 60 min for FCL-1, without the appearance of any processed, intermediate forms. These studies confirm that FCL-1 represents a novel member of the collagen gene family that manifests differential expression as a function of development.     

Airway branching patterns and cytodifferentiation in cultured fetal hamster lung

Intact fetal hamster lungs were taken for culture on gestational day 12, when only lobar bronchi and primary bronchioles are established and the epithelial cells are undifferentiated. Explants were maintained on Transwell collagen membranes for 2 and 4 days in BGJb medium alone, with 5% FBS, or with the following additives: insulin, transferrin, hydrocortisone, cholera toxin, EGF, and vitamin A. Development of the respiratory tree was affected differently by each medium formulation. BGJb medium with 5% FBS permitted near normal branching of airways and presumptive alveoli. In contrast, BGJb medium alone permitted only limited branching of these structures. BGJb medium with additives permitted branching but markedly altered normal development. The differentiation of endocrine and secretory cells was monitored by immunolabeling for serotonin and calcitonin gene-related peptide, and Clara cell protein, respectively. Ciliated cells were identified by morphology. All medium formulations supported the timely differentiation of endocrine, secretory, and ciliated cells. The ultimate goal of our studies is to characterize factors that influence airway branching and cytodifferentiation during fetal lung development. This study showed that near normal airway branching and cytodifferentiation were supported in vitro by BGJb medium with 5% FBS. Although cytodifferentiation occurred with the two other formulations, airway development was impaired.