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J Floros

Publications and source records attributed to J Floros.

At least 19 recordsLinked to original sources
PubMed1995

Translation in vivo of 5' untranslated-region splice variants of human surfactant protein-A.

Transcripts of human SP-A genes, SP-A1 and SP-A2, undergo alternative splicing of 5' untranslated-region exons. We reverse-transcribed and amplified free cytoplasmic and polysome-bound RNA and showed that (a) all splice variants of both genes are translated in vivo, (b) the relative translatability of splice variants can differ among individuals, and (c) the relative levels of different SP-A splice variants differ among individuals.

Base Sequence↗
PubMed1995

Dinucleotide repeats in the human surfactant protein-B gene and respiratory-distress syndrome.

Pulmonary surfactant, a lipoprotein complex, is essential for normal lung function, and deficiency of surfactant can result in respiratory-distress syndrome (RDS) in the prematurely born infant. Some studies have pointed towards a genetic contribution to the aetiology of RDS. Because the surfactant protein B (SP-B) is important for optimal surfactant function and because it is involved in the pathogenesis of pulmonary disease, we investigated the genetic variability of the SP-B gene in individuals with and without RDS. We identified a 2.5 kb BamHI polymorphism and studied its location, nature and frequency. We localized this polymorphism in the first half of intron 4 and found that it is derived by gain or loss in the number of copies of a motif that consists of two elements, a 20 bp conserved sequence and a variable number of CA dinucleotides. Variability in the number of motifs resulting from either deletion (in 55.3% of the cases with the variation) or insertion (44.7%) of motifs was observed in genomic DNAs from unrelated individuals. Analysis of 219 genomic DNAs from infants with (n = 82) and without (n = 137) RDS showed that this insertion/deletion appears with significantly higher frequency in the RDS population (29.3 as against 16.8%, P < 0.05).

Base Sequence↗
PubMed1995

Cloning, characterization, and development expression of a rat lung alveolar type I cell gene in embryonic endodermal and neural derivatives.

We report here the identification and characterization of a novel gene, T1 alpha, expressed in high abundance in adult rat lung, fetal lung, and early fetal brain. T1 alpha was identified by a monoclonal antibody previously shown to be specific for an antigen expressed by alveolar epithelial type I cells. The cDNA for T1 alpha is 1.85 kb and identifies a single mRNA species of the same size on Northern blots of adult rat lung. The longest open reading frame of the cDNA is 498 bases which would encode a protein of approximately 18 kDa. The protein has a putative membrane spanning domain near the C-terminus but lacks consensus sequences for N-glycosylation. Northern blots and RT-PCR show high expression of T1 alpha in adult lung, with marginally detectable expression in adult brain, intestine, and kidney. RT-PCR analysis shows expression of T1 alpha in freshly isolated type I cells (50-60% purity) but not in highly purified type II cells or other lung cells. We believe therefore that T1 alpha is primarily if not uniquely expressed in alveolar type I cells in the adult rat. Polyclonal antisera against a 16-amino-acid peptide identified in the deduced sequence reacts with the apical membranes of adult type I cells in lung tissue sections but does not label other cell types. The above antiserum as well as the original monoclonal antibody recognize a single approximately 18-kDa protein derived from bacterial expression of a construct containing the T1 alpha open reading frame. By RT-PCR T1 alpha is detected in rat lung from Day 13.5 onward, but is detected by in situ hybridization earlier in lung, brain and neural derivatives, and foregut. Expression is down-regulated in all but lung tissues as development proceeds.

Amino Acid Sequence↗
PubMed1995

Human SP-A: then and now.

In the short span of ten years, our understanding of human surfactant-associated protein A (SP-A) has advanced rapidly at both the level of the protein and the level of the gene. In the period 1984-1988, the protein was biochemically characterized and two SP-A precursors were identified. The molecular characterization was begun with the publication of an SP-A genomic sequence and sequences of two SP-A cDNAs, suggesting the presence of two SP-A genes. In the period 1991-1992, an SP-A pseudogene, a second SP-A genomic sequence, and an SP-A allelic variant were described. Since that time, a picture of increasing complexity has emerged from studies of the two SP-A genes. This complexity includes alternative splicing of 5' untranslated exons, allelic variants of both SP-A genes, and sequence heterogeneity within the 3' untranslated region. The challenge for the future will be to discover the physiological significance of the genetic complexity of human SP-A.

Alleles↗
PubMed1995

5' splicing and allelic variants of the human pulmonary surfactant protein A genes.

Human pulmonary surfactant protein A (SP-A) is encoded by two genes, SP-A1 and SP-A2. Reports from our laboratory and other investigations have shown heterogeneity in both genes within three regions (the 5' untranslated [5' UT], the coding, and the 3' untranslated [3' UT] regions). To more fully examine the variability in these regions and characterize the transcription start site in each gene, we used primer extension and 5' RACE to clone and then sequence cDNA clones from two individuals. These cDNAs extended from the transcription start site to approximately 40% of the 3' UT segment. The in vitro translatability of selected cDNAs was also tested. After analysis of our data, we found that: (1) the 5' UT of SP-A genes contains four (A, B, C, D for SP-A1) or three (A, B, D for SP-A2) untranslated exons, three of which (A, B, D) vary in length, and one of which (C) is new; (2) these exons are alternatively spliced and the major splice patterns as well as their relative frequency vary between the two genes (the major pattern for SP-A1 is AD'[81%] and the major patterns for SP-A2 are ABD [44%] and ABD'[49%]); (3) the SP-A1 gene uses three transcription start sites with equal frequency, whereas the SP-A2 gene uses only one; (4) splicing variability occurs among alleles and among individuals; (5) three previously undescribed alleles exist for the SP-A1 gene (6A2, 6A3, 6A4) and two for the SP-A2 gene (1A1, 1A2); and (6) a core group of 10 invariant nucleotides and four invariant amino acids can be used to discriminate between SP-A1 and SP-A2 alleles.

Alleles↗
PubMed1995

Surfactant protein B in human fetal lung: developmental and glucocorticoid regulation.

Pulmonary surfactant protein B (SP-B) enhances phospholipid film formation in vitro and is essential for normal surfactant function in vivo. We examined human fetal lung before and during explant culture for content and cellular localization of SP-B mRNA and protein. SP-B mRNA was low in preculture specimens (18-20 wk) but hybridization signal increased over epithelial cells during culture and was enhanced by dexamethasone treatment (10 nM). SP-B immunofluorescence was very low in preculture specimens, increased during culture, and was uniformly intense in epithelial cells of dexamethasone-treated tissue. With a newly developed immunoassay, SP-B protein was undetectable in preculture lung (< 2% of adult), appeared during culture (26% of adult), and was further increased approximately 3-fold by dexamethasone treatment (86% of adult); lung tissue of two newborn infants contained 7-9-fold more SP-B than is found in the adult. Using Western blot with enhanced chemiluminescence, mature SP-B was undetectable in 16-wk specimens but was present in 19-24-wk preculture tissue at 0.2-2.9% of the adult level. By comparison, SP-B mRNA content is 14 and 50% of adult level in 19- and 24-wk lung tissue, respectively; levels increase 3-fold during culture and a further 3-fold with dexamethasone. Based on these observed differences between mRNA and protein content, we conclude that basal SP-B gene expression in epithelial cells of human fetal lung is regulated primarily at the level of translation or protein stability, whereas glucocorticoids act transcriptionally. We speculate that SP-B protein accumulates only as type II cells differentiate and acquire lamellar bodies for processing and storage of SP-B.

Adult↗
PubMed1995

Dexamethasone enhances surfactant protein gene expression in streptozotocin-induced immature rat lungs.

Because surfactant protein (SP) mRNA levels in rat fetuses are increased by maternal dexamethasone (dex) treatment and decreased in streptozotocin-induced diabetic (STZ-DB) pregnancy, we investigated the in vivo effects of dex on SP gene expression in STZ-DB pregnancy. The mRNA levels of SP (SP-A, SP-B, SP-C) were assessed in d 18 and 20 fetuses by Northern blot analysis, and nuclear run-on assays were performed with lung nuclei from d 20 fetuses (term = 22 d). Our findings indicate: 1) dex causes a greater increase in SP-A and SP-B mRNA levels in d 18 (12-16-fold) compared with day 20 (4-6-fold) fetuses (p < 0.05) in normal and STZ-DB pregnancy; 2) a 2-3-fold increase in SP-C mRNA levels was observed in response to dex in d 18 and 20 fetuses; 3) the increase in transcription of SP-A and SP-B in d 20 fetuses after dex is 68 and 60%, respectively, of the increase in their mRNA levels whereas in STZ-DB, the decrease in transcription compared with mRNA levels is 3.67-fold for SP-A and 2.42 fold SP-B; and 4) changes in SP-C transcription in either in vivo model, dex-treated or STZ-DB, correspond well with changes in mRNA levels. Together, these findings indicate that dex can enhance SP expression in STZ-DB immature lungs and support differential regulation of fetal SP genes in the models studied.

Animals↗
PubMed1994

Steel factor affects SCL expression during normal erythroid differentiation.

Steel factor is one of the growth factors that controls the proliferation and differentiation of hematopoietic cells and SCL, also known as Tcl-5 or Tal-1, is a transcription factor involved in erythropoiesis. In this report, we studied the role of SCL in the proliferation of human peripheral blood burst-forming unit-erythroid (BFU-E) and the effects of Steel factor on SCL expression in proliferating erythroid cells. BFU-E-derived colonies increase progressively in size, as determined by cell number, from day 7 to day 14 of culture, with the greatest increase in colony size (10-fold expansion) occurring between day 7 and day 10. SCL protein levels in BFU-E-derived cells were highest in day 7 cells and decreased progressively from day 7 to day 14 of culture, suggesting an association of SCL with erythroid proliferation. In contrast, SCL mRNA levels did not decrease significantly between day 7 and day 14 cells, suggesting that posttranscriptional mechanisms are largely responsible for the decrease in SCL protein observed. The role of SCL in Steel factor-induced erythroid proliferation was then examined. In BFU-E-derived colonies cultured with Steel factor, colony size was significantly increased compared to control. In day 7 and day 10 erythroid precursors cultured with Steel factor, SCL protein was increased significantly compared to control. The increase in SCL protein levels in early erythroid precursors stimulated with Steel factor suggests one mechanism through which Steel factor may enhance normal erythroid proliferation. SCL mRNA levels assessed by Northern blot in day 7 cells did not increase significantly in response to Steel factor stimulation, suggesting that posttranscriptional mechanisms may also be important in the increase in SCL protein observed in response to Steel.

Base Sequence↗
PubMed1994

Human SP-A: genotype and single-strand conformation polymorphism analysis.

We have previously characterized two surfactant protein A (SP-A) cDNAs termed 1A and 6A, as well as a 6A allelic variant termed 6A1. These sequences are quite heterogeneous at the 3' untranslated region (3'UT). Differences between 6A and 6A1 alleles include an 11-bp insertion/deletion 407 bases downstream from the start of the translation termination codon and a base pair polymorphism (C or G) in exon 1 (position 1,193; White, Damm, Miller, Spratt, Schilling, Hawgood, Benson, and Cordell. Nature Lond. 317: 361-363, 1985). The 11-bp (GCCCACTGCCT) segment is present in 6A1 and absent in 6A. The 6A/6A genotype, in a small number of specimens, showed a trend toward a higher frequency in the black Nigerian population compared with Caucasians. In this report, we examine the frequency of the 6A genotype in a larger number of samples from Caucasians and black Nigerians as well as the meiotic stability of the 3'UT heterogeneity. Slot-blot analysis and allele-specific oligonucleotide probes have confirmed that the 6A/6A genotype is more frequent in the Nigerian population. Single-strand conformation polymorphisms in the 3'UT appear to be stably inherited by members of a three-generation family, suggesting that these nucleotide variants represent natural polymorphisms in the population.

Base Sequence↗
PubMed1993

Immunogold localization of SP-A in lungs of infants dying from respiratory distress syndrome.

Prematurely born infants can develop the neonatal respiratory distress syndrome (RDS) because of a deficiency of pulmonary surfactant. This lipoprotein complex synthesized by type II pneumocytes has different ultrastructural forms--intra- and extracellular lamellar bodies, which within the alveoli are transformed into tubular myelin, and this in turn gives rise to the surface monolayer, the functionally active form of surfactant. We have previously shown that at autopsy RDS lungs lack tubular myelin and have decreased immunoreactivity for antisera to surfactant protein A (SP-A), an important component of tubular myelin. Therefore, we proposed a role for SP-A in the conversion of lamellar bodies to tubular myelin and in the pathogenesis of RDS. To explore this possibility further, we compared in 14 RDS and 14 control lungs the distribution of SP-A in ultrathin sections, using affinity-purified rabbit anti-human-SP-A IgG and goat anti-rabbit IgG-conjugated with 10 nm colloidal gold particles. In controls, gold label was present in lamellar bodies, endoplasmic reticulum, on the cytoplasmic membrane of type II cells, and on lamellar bodies and tubular myelin either within alveoli or macrophages. In RDS lungs, reduced label was present in the same intracellular compartments and organelles, except in tubular myelin, which is absent. It is postulated that if SP-A is indeed necessary for the conversion of lamellar bodies to tubular myelin, in RDS either there is a deficiency of adequate amounts of functional SP-A or some other important component of surfactant is missing.

Extracellular Space↗
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↗