Biomedical subjects

B R Stripp

Publications and source records attributed to B R Stripp.

34 records · Page 2Linked to original sources

PubMed1996

Altered surfactant function and structure in SP-A gene targeted mice.

The surfactant protein A (SP-A) gene was disrupted by homologous recombination in embryonic stem cells that were used to generate homozygous SP-A-deficient mice. SP-A mRNA and protein were not detectable in the lungs of SP-A(-/-) mice, and perinatal survival of SP-A(-/-) mice was not altered compared with wild-type mice. Lung morphology, surfactant proteins B-D, lung tissue, alveolar phospholipid pool sizes and composition, and lung compliance in SP-A(-/-) mice were unaltered. At the highest concentration tested, surfactant from SP-A(-/-) mice produced the same surface tension as (+/+) mice. At lower concentrations, minimum surface tensions were higher for SP-A(-/-) mice. At the ultrastructural level, type II cell morphology was the same in SP-A(+/+) and (-/-) mice. While alveolar phospholipid pool sizes were unperturbed, tubular myelin figures were decreased in the lungs of SP-A(-/-) mice. A null mutation of the murine SP-A gene interferes with the formation of tubular myelin without detectably altering postnatal survival or pulmonary function.

Animals↗

PubMed1996

Clara cell secretory protein: a determinant of PCB bioaccumulation in mammals.

Clara cell secretory protein (CCSP) is a product of nonciliated cells of the conducting airway epithelium. The normal physiological function of CCSP is unknown. However, the ability of CCSP to bind small lipophilic molecules, such as steroid hormones and certain pollutants, has led to speculation that this protein may mediate the biological accumulation of potentially harmful polychlorinated biphenyl (PCB) metabolites within the lung. To investigate the contribution of CCSP in the in vivo accumulation of methylsulfonyl-PCB, a line of mice was established that were homozygous for a null allele of the CCSP gene. CCSP-deficient mice were healthy and fertile, with no gross physiological or pathological abnormalities Parenteral challenge with the PCB metabolite 4-methylsulfonyl-2,2',4',5,5'-pentachlorobiphenyl (MeSO2-PCB) demonstrated that CCSP-deficient mice no longer accumulate this class of pollutants within lung and kidney tissues. These data demonstrate that CCSP is the determinant for MeSO2-PCB accumulation within mice and support the notion that bioconcentration of MeSO2-PCB pollutants occurs at sites of CCSP localization, such as the respiratory and reproductive tracts of humans.

Animals↗

PubMed1995

Targeted disruption of the surfactant protein B gene disrupts surfactant homeostasis, causing respiratory failure in newborn mice.

Surfactant protein B (SP-B) is an 8.7-kDa, hydrophobic protein that enhances the spreading and stability of surfactant phospholipids in the alveolus. To further assess the role of SP-B in lung function, the SP-B gene was disrupted by homologous recombination in murine mouse embryonic stem cells. Mice with a single mutated SP-B allele (+/-) were unaffected, whereas homozygous SP-B -/- offspring died of respiratory failure immediately after birth. Lungs of SP-B -/- mice developed normally but remained atelectatic in spite of postnatal respiratory efforts. SP-B protein and mRNA were undetectable and tubular myelin figures were lacking in SP-B -/- mice. Type II cells of SP-B -/- mice contained no fully formed lamellar bodies. While the abundance of SP-A and SP-C mRNAs was not altered, an aberrant form of pro-SP-C, 8.5 kDa, was detected, and fully processed SP-C peptide was markedly decreased in lung homogenates of SP-B -/- mice. Ablation of the SP-B gene disrupts the routing, storage, and function of surfactant phospholipids and proteins, causing respiratory failure at birth.

Animals↗

PubMed1995

Plasticity of airway cell proliferation and gene expression after acute naphthalene injury.

The goal of this study was to determine the temporal and spatial sequence of events that accompany lung injury and repair after parenteral administration of the Clara cell-specific cytotoxicant, naphthalene. Changes in airway epithelial cells were evaluated by measuring alterations in the expression of markers for differentiated Clara cells (CYPIIF and Clara cell 10-kDa secretory protein, CC10), distal airway/alveolar type II cells (surfactant protein B; SP-B) and for cycling/proliferating cells (cyclin dependent kinase 1;CDK1). Naphthalene-induced Clara cell cytotoxicity resulted in the exfoliation of epithelial cells containing CC10 protein. This was accompanied by a dramatic reduction in the abundance of mRNA for CC10 and CYPIIF. Large numbers of CDK1 mRNA-positive cells were identified in and around bronchioles and terminal bronchioles 48 h after treatment. This cellular proliferation resulted in the population of airways by immature epithelial cells lacking normal levels of CC10 mRNA but overexpressing SP-B mRNA. Seventy-two hours after naphthalene treatment a reduction in CDK1 mRNA-positive cells was noted within bronchioles and terminal bronchioles at all locations, with the exception of airway bifurcations. At airway bifurcations CDK1 mRNA appeared to be more abundant at the 72-h time point than at 48 h. Comparison of these sections with serial sections probed for CC10 mRNA demonstrated a correlation between the expression of CDK1 and CC10 mRNA at bifurcations. Temporal increases in the abundance of CC10 mRNA observed at later time points were largely accounted for by the processive maturation of newly repopulated cells neighboring bifurcations in bronchioles. These studies identify spatially distinct populations of cells that act in concert to repopulate naphthalene-injured airways and support the notion that branch point cells play an important role in the maturation of newly regenerated airway epithelial cells after acute injury.

Animals↗

PubMed1994

Utilization of modified surfactant-associated protein B for delivery of DNA to airway cells in culture.

Pulmonary surfactant lines the airway epithelium and creates a potential barrier to successful transfection of the epithelium in vivo. Based on the functional properties of pulmonary surfactant protein B (SP-B) and the fact that this protein is neither toxic nor immunogenic in the airway, we hypothesized that SP-B could be modified to deliver DNA to airway cells. We have modified native bovine SP-B by the covalent linkage of poly(lysine) (average molecular mass of 3.3 or 10 kDa) to the N terminus of SP-B and formed complexes between a test plasmid and the modified SP-B. Transfection efficiency was determined by transfection of pulmonary adenocarcinoma cells (H441) in culture with the test plasmid pCPA-RSV followed by measurement of activity of the reporter gene encoding chloramphenicol acetyltransferase (CAT). Transfections were performed with DNA.protein complexes using poly(lysine)10kDa-SP-B ([Lys]10kDa-SP-B) or poly(lysine)3.3kDa-SP-B ([Lys]3.3kDa-SP-B), and results were compared with transfections using unmodified poly(lysine).DNA, unmodified SP-B.DNA, or DNA only. For [Lys]10kDa-SP-B.pCPA-RSV preparations, CAT activity was readily detectable above the background of [Lys]3.3kDa-SP-B or unmodified SP-B. The SP-B-poly(lysine) conjugates were effective over a broad range of protein-to-DNA molar ratios, although they were optimal at approximately 500:1-1000:1. Transfection efficiency varied with the tested cell line but was not specific to airway cells. Addition of replication-defective adenovirus to the [Lys]10kDa-SP-B.pCPA-RSV complex enhanced CAT activity about 30-fold with respect to that produced by the [Lys]10kDa-SP-B.pCPA-RSV complex alone. This increase suggests routing of the adenoviral.[Lys]10kDa-SP-B.pCPA-RSV complex through an endosomal pathway. Effects of covalent modification on the secondary structure of SP-B were examined by Fourier transform infrared spectrometry (FTIR). Results of FTIR indicated that the conformation of [Lys]10kDa-SP-B was comprised primarily of alpha-helical structure compared with a predominantly aggregated structure of unmodified poly(lysine). We conclude that poly(lysine) conjugates of SP-B effectively deliver DNA in vitro and may have utility as DNA delivery vehicles to the airway in vivo.

Adenoviridae↗

PubMed1994

Structure and regulation of the murine Clara cell secretory protein gene.

Clara cell secretory protein (CC10 or CCSP) is an abundant component of airway secretions and has the ability to bind small hydrophobic molecules. Genomic clones were isolated for the murine gene coding for Clara cell secretory protein. Nucleotide sequence analysis revealed that the gene was composed of three exons that span 4316 bp. Organization of the murine CCSP gene was very similar to that of the rabbit gene that codes for the biochemically related uteroglobin protein. Messenger RNAs for both genes were coded for by three exons, counterparts of which encode similar structural and functional protein domains. Transcriptional regulatory elements in the 5' flanking DNA were conserved between species, as were the functional properties of these elements when characterized in assays of promoter function. These data support the notion that Clara cell secretory protein genes from the rat and mouse, and the uteroglobin gene in rabbit, represent interspecies homologues.

Amino Acid Sequence↗

PubMed1994

Airway epithelial cell expression of interleukin-6 in transgenic mice. Uncoupling of airway inflammation and bronchial hyperreactivity.

We produced transgenic mice which overexpress human IL-6 in the airway epithelial cells. Transgenic mice develop a mononuclear cell infiltrate adjacent to large and mid-sized airways. Immunohistochemistry reveals these cells to be predominantly CD4+ cells, MHC class II+ cells, and B220+ cells. Transgenic mice and nontransgenic mice had similar baseline respiratory system resistance (0.47 +/- 0.06 vs 0.43 +/- 0.04 cmH2O/ml per s at 9 wk of age, P = NS and 0.45 +/- 0.07 vs 0.43 +/- 0.09 cmH2O/ml per s at 17 wk of age, P = NS). Transgenic mice, however, required a significantly higher log dose of methacholine to produce a 100% increase in respiratory system resistance as compared with non-transgenic littermates (1.34 +/- 0.24 vs 0.34 +/- 0.05 mg/ml, P < or = 0.01). We conclude that the expression of human IL-6 in the airways of transgenic mice results in a CD4+, MHC class II+, B220+ lymphocytic infiltrate surrounding large and mid-sized airways that does not alter basal respiratory resistance, but does diminish airway reactivity to methacholine. These findings demonstrate an uncoupling of IL-6-induced airway lymphocytic inflammation and airway hyperresponsiveness and suggest that some forms of airway inflammation may serve to restore altered airway physiology.

Airway Resistance↗

PubMed1994

Regulation of gene expression in the lung.

Lung gene expression is regulated by a complex interaction of autocrine, paracrine, and endocrine factors. With cloning of lung-specific genes, the mechanisms of transcriptional control of lung gene expression are beginning to be deciphered. This review focuses on expression are beginning to be deciphered. This review focuses on recent research that identifies cis-active sequences and trans-active proteins interacting to control expression of lung-specific genes. Delineating the mode of gene regulation is important in beginning to understand the mechanisms underlying the cellular differentiation required for fetal development of lung cells and recovery of lung cells from acute and chronic injury.

Acute Disease↗

PubMed1993

The lung-specific CC10 gene is regulated by transcription factors from the AP-1, octamer, and hepatocyte nuclear factor 3 families.

We have shown that a large fragment (-2339 to +57) from the rat CC10 gene directed lung-specific expression of a reporter construct in transgenic animals. Upon transfection, a smaller fragment (-165 to +57) supported reporter gene expression exclusively in the Clara cell-like NCI-H441 cell line, suggesting that a Clara cell-specific transcriptional element resided on this fragment (B. R. Stripp, P. L. Sawaya, D. S. Luse, K. A. Wikenheiser, S. E. Wert, J. A. Huffman, D. L. Lattier, G. Singh, S. L. Katyal, and J. A. Whitsett, J. Biol. Chem. 267:14703-14712, 1992). The interactions of nuclear proteins with a particular segment of the CC10 promoter which extends from 79 to 128 bp upstream of the CC10 transcription initiation site (CC10 region I) have now been studied. This sequence can stimulate both in vitro transcription in H441 nuclear extract and transient expression of reporter constructs in H441 cells. Electrophoretic mobility shift assays using extracts from H441, HeLa, rat liver, and fetal sheep lung cells were used to demonstrate that members of the AP-1, octamer, and HNF-3 families bind to CC10 region I. Transcription factors from H441 cells which are capable of binding to CC10 region I are either absent in HeLa, rat liver, and fetal sheep lung extracts or enriched in H441 extracts relative to extracts from non-Clara cells.

Animals↗

PubMed1992

cis-acting elements that confer lung epithelial cell expression of the CC10 gene.

To define cis-acting genetic elements responsible for cell-specific transcriptional regulation of the CC10 gene, DNA sequences spanning nucleotides -2338 to +49 of the rat CC10 gene were linked to a reporter gene coding for chloramphenicol acetyltransferase (CAT). In transient expression assays, CC10 sequences were capable of restricting CAT expression to a human lung adenocarcinoma cell line similar to pulmonary Clara cells. Transgenic mice harboring the hybrid RtCC10-CAT construct expressed high levels of CAT activity specifically within protein extracts of lung and trachea. Transcripts for the CAT reporter gene colocalized with those for the endogenous murine CC10 gene within the airways of transgenic mice. Functional analysis of deletion mutants identified stimulatory, inhibitory, and cell type-specific transcriptional regulatory elements. The results of gel retention and DNaseI protection assays suggest that a transcriptional stimulatory region located between -320 and -175, and a cell type-specific regulatory element located between -175 and +49, result from a series of protein-DNA interactions occurring at -220 to -205 and -128 to -86, respectively. Lung epithelial specific transcriptional regulatory elements described herein will be useful for expression of chimeric genes within epithelial cells lining the trachea, bronchi, and bronchioles of mice.

Adenocarcinoma↗

PubMed1992

Human cystic fibrosis transmembrane conductance regulator directed to respiratory epithelial cells of transgenic mice.

Human cystic fibrosis transmembrane conductance regulator (CFTR) was expressed in transgenic mice under the control of transcriptional elements derived from the human surfactant protein C (SP-C) gene. The hCFTR mRNA was expressed in lungs and testes: in the lung, we found hCFTR mRNA in bronchiolar and alveolar epithelial cells, and CFTR protein in respiratory epithelial cells. While the level of expression of hCFTR mRNA varied, hCFTR mRNA and protein were detected in pulmonary epithelial cells of several lines. Lung weight, morphology, somatic growth and reproductive capacity were not altered by expression hCFTR in lung and testes of the transgenics. Our findings suggest that hCFTR can be safely transferred to lung epithelial cells for CF therapy.

Animals↗

PubMed1990

Strategies for analysis of gene expression: pulmonary surfactant proteins.

Gene transcription is regulated by the formation of protein-DNA complexes that influence the rate of specific initiation of transcription by RNA polymerase. Recent experimental advances allowing the identification of cis regulatory sequences that specify the binding of trans acting protein factors have made significant contributions to our understanding of the mechanistic complexities of transcriptional regulation. These methodologies have prompted the use of similar strategies to elucidate transcriptional control mechanisms involved in the tissue specific and developmental regulation of pulmonary surfactant protein gene expression. The purpose of this review is to describe various methodologies by which molecular biologists identify and subsequently assay regions of nucleic acids presumed to be integral in gene regulation at the level of transcription. It is well established that genes encoding surfactant proteins are subject to regulation by hormones, cytokines, and a variety of biologically active reagents. Perhaps future studies utilizing molecular tools outlined in this review will be valuable in identification of DNA sequences and protein factors required for the regulation of lung surfactant genes.

Animals↗

PubMed1988

Nucleotide sequence of the envelope protein genes of a highly virulent, neurotropic strain of Newcastle disease virus.

The envelope glycoproteins of Newcastle disease virus (NDV), hemagglutinin-neuraminidase (HN) and fusion (F) proteins, play important roles in determining the host immune response and the virulence of that particular virus strain. The complete nucleotide sequence of the HN and F genes of a highly neurovirulent strain of NDV (Texas G. B., 1948) was determined in an effort to study the molecular basis of this strain's neurotropic properties. Comparison of the predicted amino acid sequences for the HN and F among the American NDV strains revealed that the Texas G. B. and Beaudette C envelope genes are closely related to each other and are less closely related to the avirulent B1 Hitchner strain. We have found 11 amino acid changes in the predicted HN protein between the Beaudette C and Texas G. B. strain but only 2 conservative amino acid changes (amino acids 11 and 197) in the F protein between these two strains. Although the virulence of NDV strains has been related to sequences at the cleavage site of F0, the property of neurovirulence cannot depend solely upon these sequences because there are no sequence differences between the Beaudette C and Texas G. B. strains. We suggest that the neurovirulence phenotype could be due to the molecular properties of the HN protein; however, we cannot exclude the possibility that the two conservative amino acid differences between the two F proteins could also play a role in determining the phenotypic differences between these two virus strains.

Amino Acid Sequence↗

PubMedDate not supplied

Clara cell secretory protein-deficient mice differ from wild-type mice in inflammatory chemokine expression to oxygen and ozone, but not to endotoxin.

The in vivo function of Clara cell secretory protein (CCSP) is unknown. Biologic and biochemical properties associated with CCSP have led to speculation that it participates in pulmonary inflammatory control. Our earlier studies have demonstrated that CCSP-deficient mice are more sensitive to either hyperoxia or ozone toxicity and show altered oxidant-induced pulmonary proinflammatory responses. In this study we test the hypothesis that altered chemokine responses seen in CCSP-/- mice following oxidant stress are a direct consequence of altered immunoregulation associated with CCSP deficiency. To test this hypothesis we utilized three distinct models of inducing pulmonary toxicity: hyperoxia and ozone (O3), which cause epithelial cell injury, and endotoxin, which causes pulmonary inflammation independent of direct epithelial cell injury. Wild-type (WT) or CCSP-/- strain 129 mice were exposed to O3 at 1.0 ppm for 24 hours, oxygen (O2) > 99% for 68 hours or inhalation of 0.0575 microgram endotoxin per mouse for 10 minutes and examined 6 hours postexposure. Mice displayed increased sensitivity to O3, as demonstrated by increased abundance of mRNAs encoding Eotaxin, macrophage inflammatory protein (MIP)-1 alpha, and MIP-2, after 4 hours of exposure, whereas WT mice were unaltered from controls. Increased sensitivity to hyperoxia was also observed, as demonstrated by increased abundance of mRNAs encoding Eotaxin, MIP-1 alpha, MIP-1 beta, MIP-2, and interferon-gamma inducible (IP)-10 after 68 hours of exposure, whereas WT mice were unaltered from controls. In contrast, WT and CCSP-/- mice responded identically 6 hours postinhalation of 0.0575 microgram lipopolysaccharide (LPS) per mouse. PMN response was 63% and 64% in WT and CCSP-/- mice, respectively. Messenger RNAs encoding Eotaxin, MIP-1 alpha, MIP-1 beta, MIP-2, IP-10, and MCP-1 were increased identically. We conclude that CCSP does not participate in regulation of the endotoxin-elicited pulmonary inflammatory response. Identical inflammatory and chemokine responses of CCSP-/- and WT mice in response to a nonepithelial toxic agent (endotoxin) suggest that altered inflammatory control observed between WT and CCSP-/- mice following O2 and O3 exposure is not the result of altered immunoregulation.

Animals↗

PubMedDate not supplied

Inflammatory and antioxidant gene expression in C57BL/6J mice after lethal and sublethal ozone exposures.

Ozone (O3) is a highly reactive and toxic oxidant pollutant. The objective of this study is to compare cytokine, chemokine, and metallothionein (Mt) changes elicited by lethal and sublethal exposure to ozone in a genetically sensitive strain of mice. Eight-week-old C57BL/6J mice were exposed to 0.3 ppm ozone for 0, 24, or 96 hours; 1.0 ppm ozone for 0, 1, 2, or 4 hours; or 2.5 ppm ozone for 0, 2, 4, or 24 hours. After 24 hours of exposure to 0.3 ppm ozone, increases in mRNA abundance were detected for messages encoding eotaxin, macrophage inflammatory protein (MIP)-1 alpha, and MIP-2. These increases persisted through 96 hours of exposure. At this time point messages encoding lymphotactin (Ltn) and metallothionein were also increased. After 4 hours of 1.0 ppm ozone exposure, increases in mRNA abundance were detected for messages encoding eotaxin, MIP-1 alpha, MIP-2, and interleukin (IL)-6. Mt mRNA abundance was increased after 1 hour of exposure and persisted through 4 hours, although the magnitude of the alterations increased. After 2 hours of 2.5 ppm ozone exposure, increases were detected for messages encoding eotaxin, MIP-1 alpha, MIP-2, IL-6, and Mt. These increases persisted through 4 hours of exposure. Lung weights of mice exposed to 2.5 ppm ozone for 24 hours were approximately 2 times greater than air-exposed mice. At this dose lethality occurred by 36 hours. Increased mRNAs for eotaxin, MIP-1 alpha, MIP-2, and Mt were to a higher magnitude than were detected after 2 and 4 hours of exposure. Messages encoding IL-12, IL-10, interferon (IFN)-gamma, IL-1 alpha, IL-1 beta, and IL-1Ra were unaltered at all time points and doses examined. Our results demonstrate dose- and time-dependent changes in chemokine, cytokine, and Mt mRNA abundance and that early acute changes may be predictive of subacute and chronic responses to ozone.

Animals↗

PubMedDate not supplied

Inflammatory and epithelial responses in mouse strains that differ in sensitivity to hyperoxic injury.

The pulmonary response to various toxicants including bleomycin, ozone, ionizing radiation, and hyperoxia is highly variable among mouse strains. The current study tests the hypothesis that at a similar stage of injury, regardless of strain, expression of inflammatory cytokine and epithelial marker genes would be similar, indicating a common pathway of injury progression. Three strains of mice, C57B1/6J, 129/J, and C3H/HeJ, ranging from sensitive to resistant, were exposed to > 95% O2 for varying times. Ribonuclease protection was used to quantify changes in cytokine mRNA. Despite differences in the kinetics, each strain demonstrated similar hyperoxia-induced changes in the abundance of interleukin (IL)-6, IL-1 beta, IL-3, and tumor neucrosis factor (TNF)-alpha. For each strain, death was accompanied by similar increases in cytokine mRNAs above steady-state control levels. Other inflammatory cytokines, including IL-1 alpha, IL-4, and interferon (IFN)-gamma, were unaltered in all strains at all times. In situ hybridization analysis of the epithelial markers, surfactant protein B (SPB), and clara cell secretory protein (CCSP) at the time of proinflammatory induction showed a similar pattern of expression in all strains. Increased SPB was detected in bronchiolar epithelium, while the number of type II cells expressing this message declined. Both the number of cells expressing CCSP as well as abundance per cell declined. These results suggest that although differences in acute sensitivity to hyperoxia exist between mouse strains, once initiated, acute epithelial cell injury and associated inflammatory changes follow the same pattern in all strains.

Animals↗