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Biomedical subjects

J Floros

Publications and source records attributed to J Floros.

At least 91 records · Page 5Linked to original sources
PubMed1986

Characterization of bovine surfactant for infants with respiratory distress syndrome.

Exogenous surfactant treatment of surfactant-deficient disease states is now under study in a number of centers, using a variety of surfactant preparations. We have chosen one preparation because of its current and potential clinical usefulness, and we have characterized it using selected tests and assays that we thought would be necessary (although not necessarily sufficient) to justify extended clinical use. We found its lipid composition to resemble that of other surfactants derived from lung mince. There is little variation among several batches with regard to lipid composition or surface tension-lowering capability. Morphologic heterogeneity occurs in individual samples of pelleted material studied by electron microscopy. Arterial oxygenation is improved when the material is administered to animals depleted of surfactant. A low molecular weight protein was identified that reacted with antibody that specifically binds nonserum surfactant proteins in a number of animal species (including human and cow). The characteristics of this surfactant preparation should be useful for comparison as newer and simpler products become available.

Animals↗
PubMed1985

Glucocorticoids affect the synthesis of pulmonary fibroblast-pneumonocyte factor at a pretranslational level.

Glucocorticoids accelerate fetal lung maturation by acting on the fetal lung fibroblast to induce the synthesis of fibroblast-pneumonocyte factor which in turn stimulates pulmonary surfactant synthesis by the alveolar type II cell. We have studied the site of glucocorticoid regulation of fibroblast-pneumonocyte factor synthesis in primary cultures of fetal rat lung fibroblasts. Conditioned media from fetal rat lung fibroblasts exposed to cortisol stimulate [Me-3H]choline incorporation into saturated phosphatidylcholine by primary cultures of fetal rat lung alveolar type II cells. This effect is blocked by the presence of actinomycin D during the first, but not the second, 24 h of incubation of the fibroblasts with cortisol. Cycloheximide blocks this effect if present during either the first or second 24 h of incubation. We fractionated mRNA from fetal rat lung fibroblasts incubated in the presence or absence of dexamethasone and observed that cell-free translation products from a fraction of approximately 500 bases possess biological activity in the bioassay. Such activity is only present in cell-free translation products of mRNA isolated from fibroblasts treated with dexamethasone. These results suggest that glucocorticoids act at a pretranslational level to induce production of fibroblast-pneumonocyte factor and that the primary translation products are biologically active.

Animals↗
PubMed1985

Biosynthesis and in vitro translation of the major surfactant-associated protein from human lung.

We have characterized a 32,000-36,000-dalton sialoglycoprotein group that is an integral component of the lipoprotein complex called pulmonary surfactant. Our results from the cell-free translation of human lung RNA show that this protein consists of two similarly-sized precursor components of about 29,000-31,000 daltons. Tunicamycin treatment of the lung tissue prevents formation of the normal protein and results in the accumulation of these precursor components which are also seen under normal conditions in very small amounts. Although in vitro translation in the presence of dog pancreatic microsomes suggests that a cleavable signal peptide sequence is present in these precursor molecules, it does not appear that this cleavage occurs in vivo.

Electrophoresis, Polyacrylamide Gel↗
PubMed1981

Characterization and biological activity of cloned simian virus 40 DNA fragments.

The biological activity of fragments of the SV40 genome was determined by manual microinjection of the fragments into the nuclei of mammalian cells. Fragments of the SV40 A gene (that codes for the T antigens) were obtained either directly by digestion with restriction endonucleases or after cloning into plasmid pBR322. Three different biological activities were studied: expression of T antigen, induction of cell DNA synthesis, and, in a few cases, reactivation of repressed ribosomal RNA genes. By using a number of fragments with deletions in the various portions of the SV40 A gene, we have been able to conclude that: 1) the sequences from 0.65 to 0.51 map units are not needed for the induction of cell DNA synthesis; 2) the sequences from 0.42 to 0.17 map units are not needed for the induction of cell DNA synthesis; and 3) the critical sequences for the induction of cell DNA synthesis, 0.51 to 0.42 map units, are different from those necessary for the reactivation of repressed ribosomal RNA genes (0.39-0.33 map units). These results indicate that the information for these two fundamental processes of cell proliferation resides in two separate and distinct domains of the SV40 A gene.

Animals↗
PubMed1980

Reactivation of G0 nuclei by S-phase cells.

tsAF8 cells are a temperature sensitive (ts) mutant of BHK that arrest in G1 at the nonpermissive temperature or after serum deprivation. G0 tsAF8 were fused by polyethylene glycol with other G0 tsAF8 cells, and the fusion products were incubated at the nonpermissive temperature. The homokaryons were incapable of entering S phase under these conditions. However, when S phase tsAF8 cells were fused with G0 tsAF8 cells, both nuclei in the homokaryons entered S phase, even when the fusion products were incubated at the nonpermissive temperature. In addition S phase tsAF8 cells, if fused with chick erythrocytes, can reactivate the chick nucleus, even if the heterokaryons are placed at nonpermissive temperature. Therefore S phase information of tsAF8 can induce DNA synthesis, after fusion, in mammalian G0 AF8 cells and chick erythrocytes even when the homokaryons or heterokaryons are incubated at the nonpermissive temperature.

Animals↗
PubMed1978

Stimulated DNA synthesis in frog nuclei by cytoplasmic extracts of temperature-sensitive mammalian cells.

Cytoplasmic extracts of proliferating cells stimulate DNA synthesis in isolated nuclei of Xenopus laevis liver. When tested by the same assay, cytoplasmic extracts of resting cells are completely inactive. When cytoplasmic extracts are prepared from cell cycle-specific temperature-sensitive mutants arrestd in the G1 phase of the cell cycle by the nonpermissive temperature, they also fail to stimulate DNA synthesis in frog nuclei. The results indicate that, to stimulate DNA synthesis in isolated frog nuclei, essentially all information of G1 cells must be present.

Animals↗
PubMed1978

Characterization of ts13 cells a temperature-sensitive mutant of the G1 phase of the cell cycle.

ts 13 cells are a temperature-sensitive (ts) mutant of BHK cells that are known to arrest in G1 when shifted to the nonpermissive temperature. We have determined the entry into S of ts13 cells in five different growth conditions, namely: 1) quiescent, sparse cultures stimulated to proliferate by serum. 2) Quiescent, dense cultures stimulated by serum. 3) Quiescent, sparse cultures stimulated by trypsinization and replating. 4) Quiescent, dense cultures stimulated by trypsinization and replating. 5) Mitotic cells collected by mitotic detachment. For each different growth condition we have also determined the execution point of the mutant function, i.e. the time at which a shift-up to the nonpermissive temperature no longer prevents the entry of cells into S. The median time of entry into S and the execution point varied in different growth conditions, but the distance between the median execution point and the median time of entry into S was remarkably constant, i.e. 3.2 hr. In addition we have fused ts 13 cells cells with chick erythrocytes and studied the ability of ts13 cells in heterokaryon formation to induce DNA synthesis in chick nuclei. Although ts13 cells can induce DNA synthesis in chick nuclei at the permissive temperature, they fail to do so when fused and stimulated at the nonpermissive temperature of 39.5 degrees C.

Animals↗
PubMedDate not supplied

Inhibition of lung maturation by monoclonal antibodies against fibroblast-pneumonocyte factor.

Fetal lung maturation, especially the onset of surfactant formation by alveolar type II cells, seems to be regulated by endogenous fetal glucocorticoids. Recent studies suggest that glucocorticoids do not act directly on the type II cell but rather on the lung mesenchyme. In response to glucocorticoids, the mesenchyme produces and secretes a polypeptide, fibroblast-pneumonocyte factor, which in turn stimulates surfactant synthesis by the alveolar type II cell. We report here the generation of hybridomas secreting monoclonal antibodies to rat lung fibroblast-pneumonocyte factor. Two monoclonal antibodies studied in detail reduced the cortisol-stimulated synthesis of saturated phosphatidylcholine in organotypic cultures of fetal rat lung cells and blocked the stimulatory effect of fibroblast-pneumonocyte factor in type II cells from these cultures. When embryonic chicks were injected on day 15 of incubation with either monoclonal antibody, they showed on days 20 and 21 biochemical evidence of delayed lung maturation as compared with controls. These effects were organospecific. Our observations support a physiological role for fibroblast-pneumonocyte factor in prenatal lung maturation.

Animals↗
PubMedDate not supplied

Induction of AP-1 binding to intron 1 of SP-A1 and SP-A2 is implicated in the phorbol ester inhibition of human SP-A promoter activity.

A deletional analysis of the SP-A1 promoter in NCI-H441 cells was performed to identify potential cis-acting elements involved in phorbol ester-mediated repression of human SP-A transcription. The phorbol ester TPA reduced SP-A1 and SP-A2 promoter activity to approximately 35% to 45% compared to that of control cells. The inhibitory effect of TPA was significantly reduced upon removal of the region +64/+394 relative to the SP-A1 transcription start site. Using NCI-H441 nuclear proteins, electromobility shift assay analysis showed that the intron region +309/+329 of SP-A1 and the corresponding region of SP-A2 formed sequence-specific DNA/protein complexes that were induced by TPA exposure. The region +318/+324 of SP-A1 contains sequences similar to a consensus AP-1 binding site, TGACTGA (TCACTGA for SP-A2), which when mutated (TGAGAGT) prevented the formation of the TPA-induced DNA/protein complex. The TPA-induced complex was supershifted in the presence of antibody against the Jun family of proteins, but not the Fos family of proteins. These results suggest that the binding of AP-1 or an AP-1--like factor to the first intron of SP-A1 and SP-A2 may be involved in the phorbol ester inhibition of human SP-A gene expression.

Binding Sites↗
PubMedDate not supplied

Glucose decreases steady state mRNA content of hydrophobic surfactant proteins B and C in fetal rat lung explants.

The streptozotocin-induced diabetic (STZ-DB) rat model is associated with fetal hyperglycemia, but with low to normal plasma insulin concentration. Because surfactant protein (SP) mRNA content in fetal rat lung is decreased in STZ-DB pregnancy, we investigated the effect of increasing concentrations of glucose on SP gene expression in lung organ cultures. SP mRNA content (SP-A, SP-B, SP-C) was assessed by Northern blot analysis in fetal day 20 lung explants (term = 22 days) cultured for 44 hours in medium containing 10, 25, 50, or 100 mM glucose. Our findings were (1) No consistent alteration in SP-A mRNA content was observed at different glucose concentrations (P > .05); (2) SP-B and SP-C mRNA content were reduced in a dose-dependent manner when glucose concentration was increased from 10 mM to 100 mM. The mRNA content, compared to 10 mM glucose, decreased to 50-60% at 25 mM glucose, to 20-25% at 50 mM glucose, and to lower than 10% at 100 mM glucose (P < .01). These findings indicate that the decrease in SP-B and SP-C mRNA in fetuses of STZ-DB rats may be, in part, due to a direct effect of hyperglycemia, whereas the decrease in SP-A mRNA content in STZ-DB rats appears to be due to other effects of diabetes in pregnancy.

Animals↗
PubMedDate not supplied

The utility of postmortem lung for RNA studies; variability and correlation of the expression of surfactant proteins in human lung.

Postmortem human lung tissue was evaluated for its utility in studies of the mRNAs for the surfactant proteins. Data obtained from different analytical procedures indicated that surfactant protein mRNAs are quite stable in these tissues with a half-life of 10 to 12 h. These analyses revealed no major regional differences in the mRNA levels for the surfactant protein A (SP-A) and surfactant protein B (SP-B) although small differences were present in the levels for the surfactant protein C (SP-C). Analysis of adult surgical lung specimens indicated that there is greater individual variation in the mRNA levels for SP-A and SP-B compared to SP-C among individuals. Furthermore, in a given individual the level of SP-A mRNA correlated well with that of SP-B, whereas the level of SP-C mRNA did not correlate with either that of SP-A or SP-B.

Cadaver↗
PubMedDate not supplied

Localization of pulmonary surfactant proteins using immunohistochemistry and tissue in situ hybridization.

Surfactant, a complex mixture of lipids and proteins, is produced by the type II alveolar epithelial cells. Numerous studies have localized surfactant protein A (SP-A) to type II cells of the lung, and recent studies have shown that the type II cells in the human lung are also the site of synthesis of SP-B, one of the hydrophobic surfactant proteins. There have been conflicting reports about additional sites of SP-A production. We have studied the localization of the mRNAs for SP-A, SP-B, and SP-C in the rat and for SP-C in the human lung by tissue in situ hybridization using cRNA probes. The mRNAs for all three rat surfactant proteins and for human SP-C were found in type II alveolar epithelial cells. In addition, the mRNAs for rat SP-A and SP-B were found in nonciliated bronchiolar cells. SP-C mRNA was not detectable in the bronchiolar cells of both rat and human lung tissue. Immunohistochemical studies in the rat lung with antisera to SP-A and SP-B confirmed the presence of the protein in cell types where the mRNA was found, as well as in some alveolar macrophages. Alveolar macrophages in both rat and human lung tissue were negative for all mRNAs. Further studies are required to ascertain whether there are differences in the processing, function, and regulation of these proteins in the different cell types that produce them.

Animals↗
PubMedDate not supplied

Conservation analysis of rat and human SP-A gene identifies 5' flanking sequences of rat SP-A that bind rat lung nuclear proteins.

As an initial step toward understanding regulation of tissue-specific expression of SP-A, 5' flanking sequences of the rat SP-A gene and human SP-A I gene were cloned, sequenced, and compared using dot matrix analysis. Two regions were identified, each with a considerable degree of homology between the two species. One region was proximal to the TATAA box, at position -225/-17 in rats and -226/-36 in humans, and the other at position -1115/-1026 in rats and -938/-851 in humans. Studies in rats revealed the specific binding of rat lung nuclear proteins to each of the conserved 5' flanking regions identified in rat SP-A. Binding studies using the rat proximal (rPPS) or distal (rDPS) promoter segments, or overlapping fragments of these segments, with rat nuclear extracts detected the presence of a number (1-4) of lung-specific DNA/protein complexes. When nuclear proteins from liver, a nonexpressing tissue, were used the binding profile of certain nuclear proteins differed from that of the lung. These studies, taken together, suggest that sequences within identified conserved DNA segments in the 5' flanking region of the rat SP-A gene contribute to its tissue-specific expression in rats.

Animals↗