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J Floros

Publications and source records attributed to J Floros.

At least 19 recordsLinked to original sources
PubMed1992

Surfactant protein A expression is delayed in fetuses of streptozotocin-treated rats.

The content and distribution of the 26-to 38-kDa surfactant protein (SP-A) and its mRNA were determined in fetuses of control and streptozotocin (STZ)-treated Sprague-Dawley rats using immunohistochemistry, RNA blotting, and in situ hybridization. Female rats were treated with 50 mg/kg STZ before mating, and the fetuses were killed at fetal days 18-21 or on neonatal days 1 and 2 (day of birth = end of day 22). SP-A was barely detectable on fetal day 18 in controls and easily detected by fetal day 21. In the STZ group, SP-A was decreased compared with controls at fetal days 18-21. However, by neonatal days 1-2, there were no significant differences in SP-A levels between groups. SP-A mRNA was detectable at fetal day 18 in controls, but it was decreased in the STZ group at day 18-21 (P less than 0.02) and differences were no longer detected by neonatal days 1-2. SP-A and SP-A mRNA accumulated with advancing gestational age in both groups until neonatal days 1-2. The differences in SP-A and SP-A mRNA levels in the two groups diminished with advancing age but remained significant at fetal day 21. These data suggest that STZ-induced diabetes interferes with normal expression of SP-A in the developing fetal lung.

Animals↗
PubMed1992

An allelic variant of the 6A gene for human surfactant protein A.

Two human surfactant protein A (SP-A) cDNA clones (1A and 6A) encoding human SP-A genes have previously been identified and characterized by our laboratory. The present study was undertaken to assess potential heterogeneity in the 6A locus among specimens from white adults and infants with respiratory distress syndrome (RDS) as well as black adults. The entire and/or a portion of the published SP-A gene was amplified from different genomic DNAs using the polymerase chain reaction (PCR), cloned, and sequenced. The data revealed the presence of a variant (6A1) of the 6A gene with characteristics of the 1A gene. The most notable changes were a one-base change in exon 1 and an 11-base insertion in the 3' untranslated region (3'UT). Both changes are found in 1A gene. Analysis of the reverse-transcribed (RT) PCR products from lung mRNAs using 6A specific oligos showed that the 6A1 variant is transcribed. The prevalence of each SP-A allele was then assessed in a group of 20 Nigerian Blacks and 43 Whites (26 infants with RDS and 17 adults). Although the data did not reach statistical significance, a trend was shown where the 6A/6A genotype appeared with higher frequency in Blacks.

Alleles↗
PubMed1992

Surfactant protein C: hormonal control of SP-C mRNA levels in vitro.

We have studied hormonal regulation of the surfactant protein C (SP-C) in fetal 18-dah rat lung explants. SP-C mRNA was detected in Northern blots with a specific rat SP-C cDNA probe and quantified by densitometry. Treatment of the explants with dexamethasone resulted in a dose-dependent increase of the SP-C mRNA level. Transcriptional assays have shown that the regulation of SP-C mRNA by dexamethasone involves a transcriptional step. Administration of the cAMP analogues, 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) or dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP), produced a dose-dependent increase of SP-C mRNA levels, with maximum stimulation observed at 200 microM. The thyroid hormone T3 had no effect on SP-C mRNA levels, whether administered alone or in combination with dexamethasone. Variation in the effects of the above hormones on three surfactant protein mRNAs, SP-A, SP-B and SP-C, indicates that the hormonal regulation of the surfactant proteins is a complex process and that each gene is, in part, differentially regulated.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1992

Delayed hydrophobic surfactant protein (SP-B, SP-C) expression in fetuses of streptozotocin-treated rats.

Tissues from fetuses and neonates of control and streptozotocin (STZ)-treated Sprague-Dawley rats were used to study the content and distribution of the hydrophobic surfactant protein B (SP-B) and the mRNAs for SP-B and SP-C using immunohistochemistry, RNA blotting, and tissue in situ hybridization. A dose of 50 mg/kg STZ was used to treat female rats before mating. The fetuses were sacrificed at fetal days 18 through 21 and neonates were obtained on neonatal days 1 and 2 (day of birth = end of day 22). At fetal day 18, SP-B was barely detectable by immunohistochemistry in control animals but the levels were progressively increased through gestation and easily detected by fetal day 21. At all fetal ages, SP-B was decreased in the STZ group compared with control animals. Both SP-B and SP-C mRNA were detectable at fetal day 18 in the control group and increased with advancing gestational age. In fetal lungs from the STZ group, SP-B and SP-C mRNA also showed an increase with advancing gestational age, but the levels were decreased compared with controls at fetal days 18, 20, and 21 (P less than 0.05). At fetal day 19, this difference did not achieve statistical significance. Differences between the two groups were no longer detected by neonatal days 1 and 2. The difference between the STZ and control groups, in both protein (SP-B) and mRNA (SP-B and SP-C), diminished with advancing fetal age but remained significant up to fetal day 21.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1991

Dexamethasone in vivo raises surfactant protein B mRNA in alveolar and bronchiolar epithelium.

Glucocorticoids enhance the rate of maturation of the fetal lung, in part, by increasing production of surfactant by type II cells. We have examined the effect of in vivo dexamethasone treatment on mRNA levels for the hydrophobic surfactant protein, SP-B, in rat lung and compared the data to similar measurements of SP-A mRNA. Rats of known gestational and postnatal ages were injected intraperitoneally with dexamethasone (2 mg/kg) and killed after 24 h. SP-B mRNA levels in the lungs were determined by RNA blotting using rat SP-A and SP-B cDNA probes. SP-B mRNA levels increased 5.2-fold when 163 pairs of animals of all ages were examined. SP-B mRNA increases in fetuses (107 pairs) and postnatal animals (56 pairs) were 8.4- and 2.1-fold, respectively (P less than 0.0001). The high levels of stimulation in fetal lungs were largely due to high increases on gestational days 18 and 19 when the control levels are very low. There were no detectable gender differences in the population as a whole. Tissue in situ hybridization showed that type II cells, as well as some bronchiolar cells, responded to dexamethasone treatment throughout life. The significance and the reasons for the presence and hormonal responsiveness of SP-B in the bronchiolar epithelium are intriguing and warrant further investigation.

Aging↗
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↗