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J Floros

Publications and source records attributed to J Floros.

At least 73 records · Page 4Linked to original sources
PubMed1991

Hormonal effects on the surfactant protein B (SP-B) mRNA in cultured fetal rat lung.

Glucocorticoids, triiodothyronine (T3), and cyclic adenosine monophosphate (cAMP) have been shown previously to modulate phosphatidylcholine and surfactant protein A (SP-A) synthesis in fetal rat lung explant cultures. In this report, we have examined the hormonal regulation of the rat surfactant protein B (SP-B) mRNA to determine whether SP-B expression is coordinately regulated with the surfactant phospholipids or with SP-A. Dexamethasone (1 to 200 nM) and cAMP (200 microM) had a stimulatory effect on SP-B mRNA levels, whereas T3 tended to inhibit the accumulation of SP-B mRNA. In combination experiments, treatment with dibutyryl-cAMP (200 microM) and dexamethasone (100 nM) resulted in about a 22-fold increase, whereas dexamethasone or dibutyryl-cAMP alone produced 18- and 2-fold increases, respectively. When the cAMP analogue 8-bromo-cAMP (200 microM) was used in combination with dexamethasone, there was no significant difference between the combined effect and that of dexamethasone alone. T3 treatment, however, resulted in a significant reduction of the dexamethasone-induced stimulation from about a 22-fold to a 14-fold increase. Tissue in situ hybridization showed that dexamethasone stimulated the levels of SP-B mRNA in cells from both the alveolar and bronchiolar epithelium. These data indicate that there are differences in the hormonal regulation of the components of surfactant, suggesting that they are independently regulated.

Animals↗
PubMed1990

Regulation of surfactant protein A mRNA by hormones and butyrate in cultured fetal rat lung.

We have previously shown that dexamethasone, triiodothyronine (T3) and dibutyryl adenosine 3',5'-cyclic monophosphate (cAMP) stimulate phosphatidylcholine (PC) synthesis in fetal rat lung explants in culture. There are also additive interactions between these agents with regard to PC synthesis. In this study we examined the regulation of surfactant protein A (SP-A) mRNA in fetal rat lung in culture. Dexamethasone increased SP-A mRNA in the explants in a dose-dependent fashion (1-200 nM), but T3 did not. Whereas 8-bromo-cAMP increased SP-A mRNA, a decrease was observed with dibutyryl cAMP. These findings support the view that at least some of the genes involved in the synthesis of the various components of surfactant are independently regulated. Since we observed differences in the effects of a cAMP analogue which contained butyrate and one that did not, explants were then cultured with Na butyrate, a known regulator of gene expression. A significant decrease in SP-A mRNA was observed at mM concentrations. Exposure of the explants to alpha-aminobutyric acid, a butyric acid analogue which is elevated in the blood of infants of diabetic mothers, resulted in a significant decrease in SP-A mRNA at a concentration 1/25 of that required for Na butyrate. This observation raises the question of whether the decreased SP-A levels reported in fetuses of diabetic mothers may, at least in part, be related to this metabolite.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1989

Postnatal stimulation of rat surfactant protein A synthesis by dexamethasone.

The effects of postnatal dexamethasone treatment in vivo on the synthesis of surfactant protein A (SP-A) were examined at the protein and RNA levels. Rats ranging from 1 day old to adult were injected with 200 micrograms of dexamethasone/kg body wt or with vehicle alone and were killed 24 h after injection. One portion of the lung was metabolically labeled with [35S]methionine, the proteins immunoprecipitated using an antiserum to SP-A, and analyzed electrophoretically. Both newly synthesized intracellular and secreted SP-A levels were increased by dexamethasone, reaching averages of 2.3 and 4.5 times control values, respectively. Another portion of the lung tissue was used for RNA analysis. SP-A mRNA levels were also elevated an average of 1.4 times control values by hormone treatment. Dose-response experiments using 16-day-old pups showed that both total SP-A, as measured by enzyme-linked immunosorbent assay, and total SP-A mRNA levels were elevated with dexamethasone treatment, reaching maximal stimulation at 2 mg. We conclude that postnatal dexamethasone treatment in vivo results in increased levels of both newly synthesized SP-A and SP-A mRNA, suggesting that pretranslational events may in part contribute to this process.

Animals↗
PubMed1989

Initiation of fetal rat lung phospholipid and surfactant-associated protein A mRNA synthesis.

To determine whether the initiation of fetal lung surfactant phospholipid production and the activation of the gene for the 35-kD surfactant-associated protein are dependent on circulating corticosteroids, we cultured dexamethasone-responsive explants of 15- to 17-d fetal rat lung in medium with 1% FCS (controls), charcoal-stripped 1% FCS, or a variety of glucocorticoid antagonists. The steroid antagonist RU 486 almost completely abolished specific cytoplasmic and nuclear dexamethasone binding in the explants but had no glucocorticoid-agonist activity. There was a significant increase in disaturated phosphatidylcholine synthesis during 7 d in culture in control explants (78%) and in those cultured with Charcoal-stripped serum (83%), RU 486 (82%), or the other glucocorticoid antagonists--clotrimazole, cortexelone, and 11-ketoprogesterone. Specific mRNA for surfactant-associated protein A was not detectable in preculture 17-d lung tissue, but accumulated to the same extent in cultures with or without RU 486 in the medium. These findings support the view that expression of the genes responsible for the synthesis of the various components of surfactant is not induced by glucocorticoids, but by signals contained within the lung tissue itself. The role of circulating hormones is later acceleration and modulation of surfactant production.

Animals↗
PubMed1989

Expression of the 35kDa and low molecular weight surfactant-associated proteins in the lungs of infants dying with respiratory distress syndrome.

Newborn respiratory distress syndrome (RDS) results from a deficiency of pulmonary surfactant. Surfactant has three ultrastructural forms: lamellar bodies, which, when secreted from Type II pneumocytes, transform into tubular myelin; tubular myelin in turn gives rise to the phospholipid monolayer at the air-fluid interface in the alveolus that constitutes functional surfactant. It has been shown previously that the lungs of infants dying from RDS lacked tubular myelin despite the presence of abundant lamellar bodies, whereas the lungs of control infants dying from other causes had both tubular myelin and lamellar bodies. An abnormality in the conversion of lamellar bodies to tubular myelin in RDS was proposed as a possible explanation for this finding. To evaluate the role of surfactant proteins (SPs) in this conversion, the authors re-examined the lungs of 11 RDS infants and 10 control infants for reactivity with antisera to high and low molecular weight SPs. In control infants, abundant intense staining with antisera to both types of SPs was found, but in the RDS lungs, staining was weaker than that in controls and less intense for high molecular weight compared to low molecular weight SPs. In lungs from patients with RDS, although staining increased with increasing gestational and post-natal ages, the intensity was less than control levels at all ages. The correlation of deficiency of SPs in RDS with lack of tubular myelin suggests that SPs may be involved in the conversion of normal lamellar bodies to tubular myelin and that the deficiency of SPs could explain the persistent respiratory distress in the presence of surfactant phospholipid synthesis.

Anti-Infective Agents↗
PubMed1988

Proline hydroxylation alters the electrophoretic mobility of pulmonary surfactant-associated protein A.

Studies from several laboratories involving amino acid analysis and sequencing of the Mr 35,000 pulmonary surfactant-associated proteins (SP-A) have detected hydroxyproline residues. These residues are present in a region with a collagen-like sequence that has been revealed by direct amino acid sequencing and from the deduced amino acid sequence of the cDNA clones coding for SP-A. We treated human lung tissue with tunicamycin to block N-glycosylation and with 2,2-dipyridyl to inhibit the hydroxylation of proline residues. The SP-A synthesized under these conditions showed a shift in apparent molecular weight to 27,000 and 29,000 compared to 29,000 and 31,000 for SP-A synthesized in the presence of tunicamycin alone. Dipyridyl treatment alone caused an alteration in electrophoretic mobility similar to that seen with tunicamycin, although this was more difficult to evaluate since changes in molecular weight due to glycosylation occurred under these conditions. These results indicate that proline hydroxylation in the collagen-like portion of SP-A decreases its electrophoretic mobility.

2,2'-Dipyridyl↗
PubMed1988

Localization of surfactant protein synthesis in human lung by in situ hybridization.

In order to investigate the sites of synthesis of the pulmonary surfactant-associated proteins, we performed tissue in situ hybridization. We used frozen sections of human lung tissue and 35S-UTP-labeled cRNA probes to localize mRNAs for the 35 kDa surfactant-associated protein (PSP-A) and for the precursor of one of the hydrophobic, low molecular weight surfactant-associated proteins (PSP-B). We found that PSP-A mRNA is present only in the alveolar epithelial type II cells with alveolar macrophages, bronchiolar epithelium, and other cells of the interstitutium being negative. PSP-B mRNA is present in both alveolar type II cells and in some cells of the bronchiolar epithelium. Macrophages and other cells were negative. The data in this report demonstrate that: (1) type II pneumonocytes are capable of synthesizing both PSP-A and PSP-B, (2) some cells of the human bronchiolar epithelium contain PSP-B mRNA but not PSP-A, and (3) human alveolar macrophages do not synthesize either PSP-A or PSP-B.

Humans↗
PubMed1988

Surfactant-associated proteins in tracheal aspirates of infants with respiratory distress syndrome after surfactant therapy.

We have developed enzyme-linked immunoassays (ELISAs) that measure major proteins that are associated with pulmonary surfactant. Using these ELISAs, we tested sequential tracheal aspirates from infants severely ill with respiratory distress syndrome (RDS) who had been treated either with exogenous surfactant or with placebo within 8 h of birth. On average, we found low concentrations of surfactant proteins in tracheal aspirates on Day 1 of life, with increases evident by Day 3. The surfactant used in this study (TA surfactant) contains only the low molecular weight (6 kDa) surfactant proteins and not the 35 kDa surfactant protein. As we expected, those who were treated with TA surfactant more frequently had detectable concentrations of low molecular weight surfactant protein on the second day of life when compared with control infants. No differences were evident in the concentrations of surfactant proteins between the 2 groups by Day 3, nor were differences evident between the 2 groups evident for 35 kDa surfactant protein during the first 3 days of life. Increased low molecular weight surfactant proteins in tracheal aspirates 1 to 2 days after surfactant therapy may occur either because of persistence of exogenous surfactant proteins and/or enhanced surfactant protein production. Comparisons with measurements from other groups of patients with RDS confirm that absence of both surfactant proteins reflects alveolar surfactant deficiency.

Enzyme-Linked Immunosorbent Assay↗
PubMed1987

Dihydrotestosterone blocks fetal lung fibroblast-pneumonocyte factor at a pretranslational level.

Fibroblast pneumonocyte factor (FPF) synthesis by fetal rat lung fibroblasts is augmented during gestation in the presence of cortisol. The control and cortisol-augmented levels of FPF production, as determined by FPF ability to stimulate saturated phosphotidylcholine synthesis by lung epithelial Type II cells, is delayed during development in fibroblasts derived from male fetuses as compared to those derived from female fetuses. The mechanism by which this delay occurs has been addressed. Pregnant rats treated in vivo with dihydrotestosterone (DHT) showed decreased FPF activity from control or cortisol-treated fibroblasts derived from 20-day-old male or female fetuses. In vitro translated proteins of size-fractionated lung RNA from 19-day-old fibroblasts that were pretreated with DHT in vitro showed decreased FPF activity compared to nontreated samples. This decreased FPF activity was present even if the DHT-pretreated cells were stimulated with cortisol prior to RNA preparation. Using a mouse model of testicular feminization that contains no receptors for androgens showed no change in the cortisol augmented FPF activity when the fibroblasts were pretreated with DHT. These data taken together suggest that the delayed FPF production of male-derived lung fibroblasts is a physiologic process which requires androgen receptors, and the mechanism by which androgens inhibit FPF production appears to affect events occurring mainly at a pretranslational level.

Animals↗
PubMed1987

Isolation of a cDNA clone encoding a high molecular weight precursor to a 6-kDa pulmonary surfactant-associated protein.

Mammalian surfactant is an incompletely defined mixture of lipids and associated proteins of molecular mass 35,000 Da and approximately 6,000 Da. Surfactant preparations which are highly effective in treating respiratory distress syndrome in premature infants lack the 35-kDa proteins, but contain the 6-kDa proteins. We isolated and partially sequenced one of these low molecular weight proteins from the lung lavage material of an alveolar proteinosis patient. Oligonucleotides deduced from the sequence were used as probes to isolate a human cDNA clone. The clone codes for a 42-kDa protein which contains the sequence of the 6-kDa protein. Messenger RNA coding for the 42-kDa protein was identified in human lung RNA by in vitro translation and immunoprecipitation of the translation products with an antiserum against purified bovine surfactant 6-kDa proteins. Immunoprecipitation of the 42-kDa primary translation product is inhibited by the presence of the bovine 6-kDa protein. These observations suggest a precursor-product relationship of the 42-kDa protein to one of the 6-kDa proteins.

Amino Acid Sequence↗
PubMed1987

The 35 kd pulmonary surfactant-associated protein is encoded on chromosome 10.

The genomic components identified by each of two closely related cDNA clones for the major 35 kilodalton non-serum surfactant-associated proteins (PSP-A) were shown to derive from human chromosome 10 by Southern blot analysis of DNAs from human-rodent somatic cell hybrids. By in situ hybridization to human metaphase chromosomes, the cDNA probes were localized to the region 10q21-q24.

Animals↗
PubMed1986

Post-translational modification of the major human surfactant-associated proteins.

The major protein in human pulmonary surfactant is a sialoglycoprotein of 32-36 kDa (PSP-A) that has been shown by translation of lung mRNA in vitro to be derived from precursor molecules of 29-31 kDa [Floros, Phelps & Jaeusch (1985). J. Biol. Chem. 260, 495-500]. We show here that two-dimensional gel patterns of PSP-A similar to that of the primary translation products are obtained by incorporation of [35S]methionine in the presence of tunicamycin or by N-glycanase digestion of the 32-36 kDa group. Additional gel patterns are also observed in which the isoelectric-point heterogeneity is similar to that of either tunicamycin-treated tissue or primary translation products, but with higher molecular masses. The gel patterns showing higher-molecular-mass components are obtained when terminal sialic acid addition is prevented by the incubation of lung tissue with monensin or when terminal sialic acids are digested from the fully processed protein with neuraminidase. The 32-36 kDa forms have been shown to contain [14C]mannose. Pulse-chase experiments indicate that the acidic isoforms in the protein group arise from basic isoforms that are detectable within 10 min.

Electrophoresis, Polyacrylamide Gel↗
PubMed1986

Isolation and characterization of cDNA clones for the 35-kDa pulmonary surfactant-associated protein.

A group of 35,000-dalton sialoglycoproteins is the major non-serum protein component of pulmonary surfactant. Tryptic fragments of these proteins were sequenced, and oligonucleotide probes were synthesized based on the amino acid sequences. A human lung cDNA library was then screened using the oligonucleotide probes, and clones coding for these proteins were identified and characterized. By in vitro transcription-translation experiments we have associated individual clones with particular proteins. The data suggest that co-translational modifications of two primary translation products account for many of the isoforms observed by two-dimensional gel electrophoresis in the precursors of 35,000-dalton sialoglycoproteins.

Amino Acid Sequence↗
PubMed1986

Primary translation products, biosynthesis, and tissue specificity of the major surfactant protein in rat.

Rat lung tissue was labeled with [35S]methionine and the major surfactant-associated proteins immunoprecipitated using a specific antiserum. The protein pattern obtained was very similar to that seen in rat bronchoalveolar lavage. Rat lung mRNA was subsequently translated in an in vitro rabbit reticulocyte system, and surfactant-associated protein-related polypeptides were immunoprecipitated. A 26-kDa polypeptide was identified and characterized as follows. (a) Unlabeled surfactant proteins added to the immunoprecipitation mixture completely inhibited its immunoprecipitation. (b) Two-dimensional gel electrophoresis of the 26-kDa protein resolved it into 3 isoforms. (c) Inclusion of dog pancreatic microsomes in the translation mixture resulted in the formation of two distinct higher molecular weight groups of isoforms, suggesting that the 26-kDa protein is destined to become a glycoprotein. Immunoprecipitation of [35S]methionine-labeled rat lung tissue proteins after tunicamycin treatment resulted in 3 isoforms, identical to the ones seen in the primary translation products. In addition, expression of the surfactant proteins appears specific to the lung.

Animals↗