Search PubMed⌕ Search

Biomedical subjects

D S Strayer

Publications and source records attributed to D S Strayer.

At least 55 records · Page 3Linked to original sources
PubMed1996

Regulation of p53 gene expression by a poxviral transcription factor.

Identification of regulators of p53 expression is a crucial step in understanding the diverse functions of p53 and its role in cellular homeostasis and responsiveness to insult. Several viral proteins inactivate p53 as a modulator of cell cycle progression and apoptosis. Here, we report that a unique leporipoxviral transcription factor greatly increases levels of p53 mRNA. C7, an early transcription factor from malignant rabbit fibroma virus (MV), is an important determinant of MV virulence. Its effects on cellular gene expression were studied both during MV infection and in isolation, with C7 DNA cloned into a pKC4 expression plasmid. In both settings, C7 caused increased p53 mRNA levels. The increased p53 mRNA reflected new transcription. C7-induced increased transcription was selective: mRNAs for some cellular genes increased but those for many other genes (e.g., Bc12) were unchanged. Immunoblot and immunohistochemical analysis of pKC7-transfected and MV-infected cells showed that increased transcription led to an increase in p53 protein. EMSA analysis suggested that C7 bound the human p53 promoter between -240 and -614 bp. These studies document the direct effects of a viral transcription factor on cellular gene expression, specifically that it upregulates p53 transcription.

Animals↗
PubMed1996

Receptor-mediated regulation of pulmonary surfactant secretion.

Surfactant protein A (SP-A) regulates surfactant secretion via an SP-A specific type II cell membrane receptor (SPAR). We report here that two anti-SPAR monoclonal antibodies can modulate the secretory inhibition caused by SP-A. A2C and A2R are rat monoclonal antibodies raised independently and recognize a 32-kDa protein on rat alveolar type II cell membranes. Immunocytochemical studies show that these antibodies bind to isolated type II cells. Scatchard analysis confirms that SP-A binds alveolar type II cells through a single affinity receptor and shows that A2C and A2R recognize that same receptor. Both antibodies inhibit the binding of 125I-SP-A to isolated type II cells. The functional activity of this 32-kDa protein was studied by examining surfactant secretion in isolated type II cells. Surfactant phospholipid secretion was measured in cells that were exposed to various surfactant phospholipid secretagogues (ATP, dibutyryl cAMP, terbutaline, or ionomycin), +/-SP-A (100 ng/ml), +/-A2C or A2R. Both antibodies block the negative feedback loop by which SP-A inhibits surfactant secretion. This activity of A2C and A2R is dose-dependent and is independent of the secretagogue used. Thus, the 32-kDa type II cell membrane protein bound by A2C and A2R is the functional receptor on alveolar type II cell membranes and regulates type II cell surfactant secretion.

Animals↗
PubMed1996

Antibody to surfactant protein A increases sensitivity of pulmonary surfactant to inactivation by fibrinogen in vivo.

It has been suggested that surfactant protein-A (SP-A) protects surfactant activity from inhibitors such as fibrinogen. Substantial evidence indicates that inhibition of surfactant activity is often important in the pathogenesis of acute respiratory failure. Studies on surfactant function in the pulsating bubble surfactometer imply that SP-A helps to maintain low surface tension in the presence of inhibitors such as fibrinogen. We tested whether SP-A acts in this way in vivo. Rabbit pups, 29 d gestational age, were treated with a monoclonal antibody to rabbit SP-A (R5) followed by fibrinogen, or with control preparations (normal IgG and saline, respectively). Lung compliance was measured during ventilation throughout these experiments. Air-space volume and pulmonary edema were quantitated morphometrically. Animals receiving anti-SP-A antibody + fibrinogen showed substantial and significant impairment in lung compliance compared with control littermates receiving normal IgG and/or saline. Lungs from these animals showed decreased pulmonary air-space volume and increased alveolar edema. We conclude that SP-A protects pulmonary surfactant from inhibition by fibrinogen in vivo. This protective activity may be important in the pathogenesis of both adult and neonatal respiratory distress syndromes, and it may also be useful in devising therapies for these diseases.

Animals↗
PubMed1996

SV40 mediates stable gene transfer in vivo.

Gene transfer in vivo requires an efficient, nonreplicating, transfer agent. We report here the efficacy of recombinant, replication-deficient SV40 in transferring firefly luciferase (luc) production to murine hematopoietic cells and selected internal organs in vivo. Replication-deficient SV40 was made by replacing the large T antigen gene (Tag) with a polylinker, into which luc cDNA (luc) was cloned. Luc expression was controlled by SV40 early promoter. Tag-, luc+ SV40 DNA was transfected into Tag-expressing cells to yield a replication-deficient SV40-derivative virus containing luc (SVluc). The ability of SVluc to transfer luc production in vivo was tested in two ways: SVluc was inoculated into BALB/C mice intravenously; also bone marrow cells treated with SVluc were infused into syngeneic hosts. Luc production was followed for 105 days by immunochemical analysis of peripheral blood and selected internal organs using anti-luciferase antibody, and by assay of luc enzyme activity in peripheral blood. Luc was found in 20-25% of peripheral blood nucleated cells from day 20 until > or = 105 days. Luc-producing cells were also identified in liver, spleen, brain, kidney, skin and colon from day 20, also until > or = 105 days. Analysis of whole blood showed fluctuating levels of functionally active luc enzyme beginning on day 21, and remaining substantially and significantly greater than control values to day 105. Thus, SV40 may transfer sustained expression of foreign genes to bone marrow and other organs, for at least 3 months.

Animals↗
PubMed1995

The upstream region of the SP-B gene: intrinsic promoter activity and glucocorticoid responsiveness related to a new DNA-binding protein.

We identified and cloned the rabbit SP-B gene, encoding the pulmonary surfactant-associated protein, and sequenced its upstream region from -2635 to +428, including a much larger fragment of the upstream region than has previously been reported for an SP-B for any species. Rabbit SP-B showed substantial homology to its human counterpart in the coding and noncoding regions immediately upstream from the TATAA box. Using a luciferase (Luc) reporter gene (luc) construct we measured promoter activity with a 212-bp fragment (SPB212) from nucleotides (nt) -41 to -252, inclusive. SPB212 functioned as an active promoter in this assay. Further, we identified, cloned and sequenced the cDNA encoding a unique DNA-binding protein, N, that bound SPB212 at approx. -195. When the N cDNA was cloned into the expression vector pKC4 and cotransfected with the luc reporter construct, N significantly enhanced Luc production, but only in the presence of dexamethasone. Therefore, we identified and sequenced a functional promoter region upstream from rabbit SP-B, and isolated and characterized a DNA-binding protein that confers enhanced glucocorticoid responsiveness on this promoter.

Amino Acid Sequence↗
PubMed1995

DNA binding proteins that amplify surfactant protein B gene expression: isolation and characterization.

We identified and characterized two proteins that bind the promoter of surfactant protein B (SP-B) and affect its expression. Proteins A2 and B were identified and their cDNAs cloned and sequenced. Both were novel. They bound a 212-bp functional promoter region at an NF1 site, located between -184 and -198. Effects of these DNAbp on SP-B promoter activity were studied by contransfecting a reporter construct of this 212-bp sequence + luciferase, together with expression constructs for A2 and B into H441 cells. Alone, A2 and B expression elicited modest but statistically significant increases in SP-B promoter activity. When dexamethasone was added, B further increased SP-B promoter activity. For SP-B, basal expression and glucocorticoid responsiveness may involve a number of hitherto unknown gene activators.

Adult↗
PubMed1995

Levels of SP-A-anti-SP-A immune complexes in neonatal respiratory distress syndrome correlate with subsequent development of bronchopulmonary dysplasia.

As part of a double-blind, randomized, placebo-controlled study of human surfactant therapy for neonatal respiratory distress syndrome (NRDS), we measured circulating immune complexes between surfactant protein-A and anti-surfactant protein-A antibodies (SAS). Plasma from almost all infants contained detectable immune complexes. Immune complex levels in surfactant-treated infants were comparable with those of placebo-treated controls. Despite the relatively small sample size, maximum SAS immune complex values between 2 and 4 weeks after birth correlated significantly with subsequent development of BPD. Levels of these immune complexes correlated with eventual BPD independently of, and more strongly than, gestational age and birth weight. Thus, plasma SAS immune complex measurements may be useful in analyzing the course and outcome of NRDS, in particular the likelihood of subsequent development of BPD. This assay may also help to identify infants at risk for BPD and to target preventative therapy to them.

Antigen-Antibody Complex↗
PubMed1995

Glucocorticoid responsiveness conferred by a cloned DNA binding protein.

Glucocorticoids stimulate surfactant protein-B (SP-B) (expression in type II alveolar cells) by unknown mechanisms. We identified, cloned, and characterized a protein that binds the SP-B promoter. This protein, D, increases the activity of the SP-B promoter in response to glucocorticoid stimulation. Protein D was identified by its ability to bind the SP-B promoter region, which it binds at an NF1 site from -184 to -198 bp. Its binding was abolished by digestion of promoter DNA with BalI, which cuts at -194. Protein D was cloned and sequenced. It is a new DNA binding protein of 33 kDa whose carboxyl end contains a modified basic leucine zipper-like DNA binding motif (bzip). The effects of D on SP-B promoter activity were studied in H441 cells, using a reporter construct containing 212 bp from the SP-B promoter with a luciferase reporter gene (p2121uc), which was cotransfected with a protein D expression construct in which D expression was controlled by the SV40 early promoter. These two plasmids were cotransfected into H441 cells. Without added glucocorticoids, D did not alter SP-B promoter activity. When dexamethasone was added, D strongly enhanced SP-B promoter activity. Identification of this protein suggests that, at least for SP-B, glucocorticoid responsiveness may involve one or more hitherto unknown gene activators.

Amino Acid Sequence↗
PubMed1993

Surfactant protein A-binding proteins. Characterization and structures.

An alveolar cell membrane protein acts as a surfactant protein A (SP-A) receptor; it binds SP-A and regulates surfactant secretion. We identified such alveolar cell membrane SP-A-binding proteins using anti-idiotype antibodies directed against the surfactant protein binding region of anti-surfactant antibodies. These monoclonal anti-idiotype antibodies, A2C and A2R, also recognize an alveolar cell membrane protein of approximately 30 kDa. A pulmonary protein of approximately 30 kDa binds SP-A. Unique cDNAs encoding this protein were identified in human (4.1-kilobase) and porcine (1.8-kilobase) lung expression libraries. Coding regions of these cDNAs cross-hybridize with each other under stringent conditions. Both cDNAs encode similar approximately 32-kDa proteins that bind SP-A. The human and porcine SP-A recognition (SPAR) proteins resemble each other, as well as other cell membrane receptors. Their projected structures are consistent with cell membrane receptors. Recombinant human and porcine SPAR proteins bind SP-A as well as the two anti-idiotype antibodies just as do native lung proteins of approximately 30 kDa. SPAR transcripts are expressed primarily in lung. The cellular distribution of these transcripts, as determined by in situ hybridization, is similar to that of SPAR protein, as determined by immunohistochemistry; both are found in cells consistent with type II pneumocytes. SPAR-producing cells resemble the alveolar cells expressing SP-B and SP-C transcripts in appearance, location, and distribution. Therefore, cDNAs for pulmonary SP-A-binding proteins from two disparate species have been isolated and sequenced, and the recombinant proteins they encode bind the same ligand. Further structural, functional, and genetic studies of these proteins may help explain how pulmonary surfactant secretion is regulated.

Amino Acid Sequence↗
PubMed1993

Effect of virus infection on levels of transcripts for cellular genes.

Malignant rabbit fibroma virus (MV) induces tumors composed of proliferating cells, principally fibroblasts, and vasculature. These tumors are associated with large amounts of collagen and other connective tissue proteins. We studied the effect of MV infection on levels of mRNA for alpha 1 chains of collagens type I, III and V in RK-13 fibroblasts and alpha 2 chain of collagen type I. MV infection induces expression of specific collagen genes at particular time points after infection in vitro. Expression of these collagen genes is clearly different in MV-infected cells compared to uninfected cells. Transcript levels for a cellular transcription factor that regulates expression of alpha 1 chain type I collagen, cbf-a, were increased in MV-infected cells prior to the increase in type I collagen mRNA. The virus infection also specifically induced increased levels of mRNA for the cellular transcription factors c-fos and SP1. MV infection is therefore associated with increased levels of specific cellular mRNAs, and is correspondingly associated with increased mRNA for transcription factors that may regulate transcription of these genes. The ability of malignant fibroma virus to influence expression of cellular genes may be exerted through the cells' own transcription regulatory apparatus.

Animals↗
PubMed1993

Epidermal growth factor-like growth factors. I. Breast malignancies and other epithelial proliferations in transgenic mice.

BACKGROUND: Growth factors recognized by the epidermal growth factor receptor are important in tumor production in some organs. The family of epidermal growth factor-like growth factors includes a group of poxviral growth factors: Shope growth factor (SGF), myxoma growth factor MGF), and vaccinia growth factor. These viral growth factors are glycoproteins, whereas all other members of the epidermal growth factor family are proteins. EXPERIMENTAL DESIGN: To understand the potential significance of poxviral growth factors, we made transgenic mice using three different constructs: SGF and MGF cloned downstream from the metallothionein (MT) promoter (MTSGF), and SGF downstream from Rous sarcoma virus long terminal repeat. RESULTS: Founder transgenic mice for each construct were identified, and lines established. Expression of transgenes in MT-SGF mice and MT-MGF mice was induced by feeding animals Zn at 2 months of age. Two months later, both MT-SGF and MT-MGF mice showed proliferation and arborization of breast ducts and ductules, with slight intraductal proliferation in virgin mice. They also showed gastric epithelial hyperplasia, particularly in MT-MGF mice. Stromal and epithelial hyperplasia were found in several organs. The transgenes were expressed in epithelia and stroma of breast, lungs, liver and stomach. Rous sarcoma virus long terminal repeat-SGF transgenic mice developed atypical preneoplastic mammary ductal proliferations in both virgin and nonvirgin females by 6 months of age. In 1/3 of 8-month-old females, invasive secretory adenocarcinoma developed. These mice also developed severe epithelial atypia in the stomach, and papillary gastric tumors. CONCLUSIONS: Poxviral growth factors may thus be helpful in the study of mammary and gastric oncogenesis and provide insight into growth factor-induced tumor development.

Animals↗
PubMed1993

A 34-kd protein with strong homology to ras-like proteins inhibits epidermal growth factor activity.

Epidermal growth factor (EGF) and its analog, transforming growth factor-alpha, are felt to be important in oncogenesis. When malignant rabbit fibroma virus infects RK-13 rabbit kidney cells, a 34-kd protein that inhibits the effects of EGF on certain target cell lines is produced. We have purified this protein using high-pressure liquid chromatography and gel electrophoresis. This purified protein abolishes EGF-induced cellular proliferation. It also causes the EGF receptor-bearing A431 carcinoma cell line to stop proliferating in vitro. This purified 34-kd EGF inhibitor (EGFI) redirects cellular protein phosphorylation in the presence or absence of EGF. Whereas EGF increases phosphorylation of cellular proteins in normal rat kidney cells, clone 49F, and A431 EGFI generally decreases it. Both EGF and EGFI cause increased protein production in A431 and normal rat kidney cells. The major species of protein synthesized by cells seem invariant to EGFI, with or without EGF. The partial protein sequence of two fragments of EGFI shows striking similarity to two ras like proteins. Possible means by which such a ras-like protein might inhibit EGF-induced cellular proliferation are discussed. Therefore, a purified 34-kd ras-like protein inhibits EGF-induced cellular proliferation and changes the targets for cellular protein phosphorylation. Studies are in progress to characterize this protein further, both structurally and functionally.

Amino Acid Sequence↗
PubMed1992

Sequence and analysis of the BamHI "D" fragment of Shope fibroma virus: comparison with similar regions of related poxviruses.

Differences observed in the virulence of two related leporipoxviruses are closely tied to a particular region of their genomes. For the virulent poxvirus of this pair, malignant rabbit fibroma virus (MV), this region is the BamHI "C" fragment, which is 10.7 kb. For the avirulent poxvirus, Shope fibroma virus, SFV, this region is the corresponding BamHI "D" fragment, which is 13.1 kb. As part of our attempt to understand the virulence of these two viruses, we sequenced these two DNA fragments. The sequence for the BamHI "C" fragment of MV is reported elsewhere (Strayer et al., 1991). We report here the sequence for SFV's BamHI "D" fragment and resultant open reading frames, and compare both DNA and open reading frame structures to those of MV and other known poxviruses. The BamHI "D" fragment of SFV contains 12 open reading frames of 100 amino acids or more, arranged similarly to orf's in MV and vaccinia. Striking similarities between SFV and MV are seen in certain parts of this restriction fragment, including substantial stretches of DNA in which the two viruses are identical. Clear homologies exist between these leporipox virus genomes and those of other related poxviruses. To understand the pathogenesis of virus infection, one must appreciate the structure of those viral genes that play important roles in infection.

Base Sequence↗
PubMed1992

Applications of anti-curosurf antibodies to understanding the biology of alveolar surfactant.

The structure of surfactant-binding proteins from alveolar cell membranes was investigated using monoclonal anti-idiotypic antibodies directed against surfactant-binding regions of anti-surfactant monoclonal antibodies. Two different series of monoclonal anti-surfactant antibodies were used. One series included antibodies against rabbit surfactant, and the other antibodies against porcine surfactant. In addition, two monoclonal anti-idiotype antibodies were made. These anti-idiotype antibodies, called A2R and A2C, bind both anti-Curosurf and anti-rabbit surfactant antibodies comparably. They recognize a 30-kDa cell membrane protein on alveolar and bronchial epithelial cells. In addition, A2C and A2R block the binding of radiolabeled surfactant to these cells. Using a combination of A2C and A2R cDNA expression libraries from both human and porcine lungs were screened. One independent clone was identified in each library that produced protein that bound these monoclonal antibodies. The protein encoded by the cDNA in each clone bound radiolabeled recombinant surfactant protein-A. It is concluded that human and porcine alveolar cell SP-A binding proteins have been identified and its cDNA cloned. The potential implications of this finding for the understanding and treatment of surfactant deficiency states are considerable.

Animals↗
PubMed1991

Sequence and analysis of a portion of the genomes of Shope fibroma virus and malignant rabbit fibroma virus that is important for viral replication in lymphocytes.

The 10.7-kb BamHI "C" restriction fragment of malignant rabbit fibroma virus (MV) contains genes that are important for its immunosuppressive activity. When this fragment is transferred to a related avirulent leporipoxvirus, Shope fibroma virus (SFV), recombinant viruses show clinical features characteristic of MV: they replicate in lymphocytes and alter immune function in vitro, induce disseminated tumors in recipient rabbits, and are immunosuppressive in vivo. The 10.7-kb BamHI "C" restriction fragment of MV was sequenced in its entirety. Its DNA sequence and the 14 ORF's derived from analyzing this sequence are discussed. Analysis of known open reading frames to which the ORF's from MV's Bam "C" fragment show homology permits us to identify some MV ORF's showing high degrees of similarity to known and postulated proteins produced by vaccinia virus. Functions for some of these vaccinia proteins are known, while functions for others are hypothetical or unknown. Further analysis of genetic determinants of MV's virulence has indicated that two overlapping restriction subfragments of the BamHI "C" fragment can transfer MV's virulent behavior to SFV. The 0.7-kb region in which these two subfragments overlap includes the C-terminus of MV orf C-7 and the N terminus of MV orf C-8. These correspond to the C- and N-termini, respectively, of SFV orf's D-9 and D-10 and to vaccinia orf's D-6 (early transcription factor) and D-7 (subunit of RNA polymerase). We sequenced the region of SFV's BamHI "D" fragment in this area and illustrate here the comparative sequences of this portion of SFV's genome and orf's. On the basis of comparisons between MV, SFV, and vaccinia in this area we discuss the potential significance of these observations.

Amino Acid Sequence↗