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G F Morris

Publications and source records attributed to G F Morris.

At least 37 records · Page 2Linked to original sources
PubMed1997

Distinct transcriptional pathways of TAR-dependent and TAR-independent human immunodeficiency virus type-1 transactivation by Tat.

Tat stimulates HIV-1 gene expression during transcription initiation and elongation. Tat functions primarily through specific interactions with TAR RNA and several putative cellular cofactors to increase the processivity of RNA polymerase II complexes during HIV-1 transcription elongation. Although HIV-1 transactivation by Tat in most cell types requires intact TAR sequences, previous reports demonstrate that Tat transactivates HIV-1 long terminal repeat (LTR)-directed gene expression in several central nervous system-derived astrocytic/glial cell lines in the absence of TAR. Within this study, transient expression assays performed in the astrocytic/glial cell line, U87-MG, confirm that kappa B elements within the HIV-1 LTR mediate TAR-independent transactivation by Tat and demonstrate additionally that distinct amino acid residues within the cysteine-rich activation domain of Tat are required for TAR-independent versus TAR-dependent transactivation. Established U87-MG cell lines expressing a transdominant negative mutant of I kappa B alpha, I kappa B alpha delta N, fail to support TAR-independent transactivation by Tat, suggesting that binding of NF-kappa B to kappa B enhancer elements within the HIV-1 LTR is necessary for Tat-mediated transactivation in the absence of TAR. Ribonucleic acid protection analyses of promoter-proximal and -distal transcripts derived from TAR-deleted and TAR-containing HIV-1 LTR reporter constructs in U87-MG cells indicate that the predominant effect of Tat during TAR-independent transactivation occurs at the lavel of transcription initiation, whereas a prominent elongation effect of Tat is observed in the presence of TAR. These data suggest an alternative regulatory pathway for Tat transactivation in specific cells derived from the central nervous system that is independent of TAR and that requires direct or indirect interaction of Tat with NF-kappa B-binding sites in the HIV-1 LTR.

Amino Acid Sequence↗
PubMed1997

Repression of tumor necrosis factor-beta expression by the human immunodeficiency virus type-1 tat protein in central nervous system-derived glial cells.

HIV-1 Tat is a potent transactivator that stimulates expression from the HIV-1 LTR, from certain cellular gene promoters and from several heterologous viral promoters. Previous reports show that HIV-1 Tat transactivates tumor necrosis factor-beta (TNF-beta) promoter-directed gene expression in lymphocytic and monocytic cell lines and further demonstrate that a 'TAR-like structure' downstream of the TNF-beta promoter is essential for Tat activity. The ability of Tat to activate TNF-beta may have profound effects as TNF has been shown to be a potent activator of HIV-1 gene expression and an important immunomodulatory and growth regulatory factor. The studies presented herein demonstrate a novel finding where HIV-1 Tat specifically represses (> 10-fold) TNF-beta promoter-directed gene expression in central nervous system-derived glial cells. Amino acid residues 2 to 36 of HIV-1 Tat are required for TNF-beta repression. Tat repression of TNF-beta, a factor which upregulates HIV-1 gene expression, suggests a novel mechanism whereby HIV-1 is able to establish latent infection of glial cells that present no detectable virions and/or viral antigens.

Astrocytes↗
PubMed1997

Human T-cell leukemia virus type 1 Tax transactivates the human proliferating cell nuclear antigen promoter.

The human T-cell leukemia virus type 1 (HTLV-1) transforming protein, Tax, is a potent transactivator of both viral and cellular gene expression. The ability of Tax to transform cells is believed to depend on its transactivation of cellular-growth-regulatory genes. Expression of proliferating cell nuclear antigen (PCNA) is intimately linked to cell growth and DNA replication and repair. By testing a series of PCNA promoter deletion constructs, we have demonstrated that the PCNA promoter can be transactivated by Tax. The smallest construct that was activated did not include the ATF/CRE binding site at nucleotide -50, and mutations in the ATF/CRE element in the context of a larger promoter were still activated by Tax. In addition, a Tax mutant that is defective for activation of the CRE pathway retained the ability to activate the -397 promoter construct. When a series of linker scanner mutations that span the region from nucleotide -45 to -7 were assayed, mutations in and around a repeat sequence were found to abolish Tax transactivation. Multimerized copies of either half of the repeat were Tax responsive. A single protein complex was shown to bind specifically to the Tax-responsive region, and the binding of this complex was enhanced in the presence of Tax. These results demonstrate that the PCNA promoter contains a Tax-responsive element located between nucleotides -45 and -7 whose sequence is different from those of other, previously identified Tax-responsive elements. The ability of Tax to activate the PCNA promoter may play an important role in cellular transformation by HTLV-1.

Base Sequence↗
PubMed1997

Inhaled asbestos fibers induce p53 expression in the rat lung.

Humans and rodents exposed to an aerosol of asbestos fibers develop lung injury that can lead to a fibroproliferative response culminating in excessive scarring and impaired lung function. To define the early events that precede asbestos-induced fibrotic lung disease, rats were exposed to an aerosol of chrysotile asbestos fibers for 5 h. At various times after exposure, the lungs of the asbestos-exposed animals were evaluated immunohistochemically for expression of the p53 tumor suppressor protein, a growth regulatory protein. p53 became detectable by immunostaining at the predicted sites of fiber deposition (the bronchiolar-alveolar duct bifurcations) by 24 h after exposure. The number of cells positive for p53 immunostaining increased to a maximal level at 8 days after exposure, decreased by 14 days and returned to a low basal level at the 30-day time point. Control groups of rats that were unexposed or exposed to an aerosol of iron beads were negative for p53 immunostaining throughout the 30-day assessment period. Simultaneous detection of the proliferating cell nuclear antigen (PCNA) at the sites of fiber deposition in the asbestos-exposed animals agrees with our previous finding that p53 binds and regulates the PCNA promoter.

Administration, Inhalation↗
PubMed1997

Rapid activation of PDGF-A and -B expression at sites of lung injury in asbestos-exposed rats.

The development of interstitial pulmonary fibrosis is associated with a variety of inflammatory mediators, including peptide growth factors and cytokines. In the work presented here, we have asked whether or not platelet-derived growth factor (PDGF)-A and -B genes and proteins are expressed in anatomic and temporal patterns consistent with this factor playing a role in the disease process. Using an established rat model of asbestos-induced fibroproliferative lung disease, we demonstrate elevated levels of PDGF-A and -B mRNAs in total lung RNA immediately after a single 5-h exposure to approximately 1,000 fibers/ml of chrysotile asbestos. In situ hybridization revealed the PDGF-A and -B in RNAs primarily in macrophages and bronchiolar-alveolar epithelial cells at sites of initial fiber deposition and lung injury. There was clear evidence of PDGF-A and -B mRNAs in interstitial cells as well. The pattern of in situ hybridization was entirely consistent with the appearance (established by immunohistochemistry) of PDGF-A and -B proteins by 24 h post-exposure in the same cell types. Both mRNAs and proteins remained detectable at the fiber deposition sites for almost 2 wk post-exposures. These findings are consistent with our previous studies showing increased mesenchymal cell proliferation and fibroproliferative lesions that progress at the sites where PDGF-A and -B are expressed. Although it is clear that multiple growth factors are produced simultaneously at sites of initial injury, we suggest that the PDGF isoforms could be playing a central role in the disease process based upon their potent mitogenic effects upon mesenchymal cells.

Animals↗
PubMed1996

Wild-type and transactivation-defective mutants of human immunodeficiency virus type 1 Tat protein bind human TATA-binding protein in vitro.

SUMMARY: Tat regulates human immunodeficiency virus type 1 (HIV-1) gene expression by increasing both the rate of transcription initiation and the efficiency of transcription elongation. The ability of Tat to facilitate HIV-1 transcription preinitiation complex formation suggests that components of the basal transcriptional machinery may be targeted by Tat. Previous studies have demonstrated that Tat interacts directly with the human TATA-binding protein (TBP) and specific TBP-associated factors (TAFS) that comprise the TFIID complex. Here, in vitro glutathione S-transferase protein binding assays containing fully functional or transactivation-defective mutant Tat proteins have been used to investigate the functional significance of the direct interaction between Tat and TBP relative to Tat transactivation. Results demonstrate that full-length Tat, as well as the activation domain of Tat alone, binds human TBP in vitro. Site-directed mutations within the activation domain of Tat (C22G and P18IS) that abrogate transactivation by Tat in vivo fail to inhibit Tat-TBP binding. Full-length Tat, the activation domain of Tat alone, and a transactivation-defective mutant of Tat that lacks N-terminal amino acid residues 2-36 bind with equal efficiencies to TBP provided that the H1 alpha helical domain that maps to amino acids 167-220 within the highly conserved carboxyl terminus of TBP is maintained. These data indicate that an activity mapped within the activation domain of Tat, which is distinct from Tat-TBP binding. is required for transactivation by Tat.

Binding Sites↗
PubMed1996

TATA-dependent repression of human immunodeficiency virus type-1 transcription by the adenovirus E1A 243R oncoprotein.

Human adenovirus E1A oncoprotein activates or represses transcription from a variety of viral and cellular promoters by several complex mechanisms. The E1A products, 289R and 243R, have differential effects on transcription directed by the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR). Previous reports indicate that repression of HIV-1 LTR-directed gene expression by E1A 243R is mediated through the kappa B enhancer elements located between nucleotides -105 and -82 relative to the transcription initiation start site (+1). Results from this study suggest a novel mechanism for transcriptional repression of the HIV-1 LTR by E1A 243R that is enhancer-independent and that is mediated through basal HIV-1 promoter elements. Transient expression assays, in which 5'-truncated or site-directed mutant HIV-1 LTR-CAT reporters were tested for their response to repression mediated by wild-type or mutant 243R, demonstrate that LTR sequences upstream of -31 relative to the transcription initiation start site (+1) and inclusive of the enhancer elements are dispensable for 243R-mediated repression. The ability of 243R to repress HIV-1 basal promoter activity requires both an intact N-terminus of E1A 243R and the TATA element within the HIV-1 promoter. These results support a novel mechanism for E1A 243R-induced transcriptional repression that is enhancer-independent and that targets directly the general transcription machinery.

Adenovirus E1A Proteins↗
PubMed1996

Transcriptional activation of the human proliferating-cell nuclear antigen promoter by p53.

Proliferating-cell nuclear antigen (PCNA) is a DNA damage-inducible protein that performs an essential function in DNA replication and repair as an auxiliary factor for DNA polymerases delta and epsilon. Examination of the human PCNA promoter DNA sequence revealed a site with homology to the consensus DNA sequence bound by p53. PCNA promoter fragments with this site intact bound p53 in vitro and were transcriptionally activated by wild-type p53 in transient expression assays in SAOS-2 cells. The resident p53-binding site could be functionally substituted by a previously described p53-binding site from the ribosomal gene cluster. A plasmid expressing a mutated version of p53 derived from a patient with Li-Fraumeni syndrome failed to activate the PCNA promoter in the cotransfection assay. In different cell types, activation of the PCNA promoter by the p53-binding sequence correlated with the status of p53. Activation of the PCNA promoter by wild-type p53 depends upon the level of p53 expression. This concentration dependence and cell type specificity reconciles the observations presented here with prior results indicating that wild-type p53 represses the PCNA promoter. These findings provide a mechanism whereby p53 modulates activation of PCNA expression as a cellular response to DNA damage.

Base Sequence↗
PubMed1996

p53 and PCNA coexpression of 81 pleural and peritoneal effusion specimens: an immunohistochemical study.

Several studies have indicated that wild type p53 plays an important role in controlling cell growth and acts as a cyclin modifier. Abnormalities in p53 induce the overexpression of proliferating cell nuclear antigen. The aim of this study is to correlate immunocytochemically the expression of mutant p53 and the proliferative index (PI) as indicated by image analysis of PCNA immunoreactivity in 81 cases of pleural and peritoneal effusions. There was a strong correlation (r = 73%) between p53 immunoreactivity and PCNA PIs. Forty-three (71%) cases indicated p53 immunostaining out of 61 cases with PCNA immunoreactivity, forty of which (93%) proved to have a diagnosis of malignancy using histological or clinical data. Furthermore, 7 malignant cases showed PCNA reactivity but no p53 immunostaining. An additional four malignant cases indicated no reactivity for either p53 or PCNA. Also, there was a significant difference in the PCNA PI between benign and malignant effusions (p < 0.001). These clinical observations confirm the function of wild p53 as a check point during cell cycling, and as a strong negative feedback effect on PCNA expression. Furthermore, such co-expression represents a significant indicator for malignancy.

Antibodies, Monoclonal↗
PubMed1996

Up-regulated expression of transforming growth factor-alpha in the bronchiolar-alveolar duct regions of asbestos-exposed rats.

It has become apparent that the numerous growth factors and cytokines are produced during the development of fibroproliferative lung disease. Investigators must sort out which combinations of these factors are playing mechanistic roles in the disease process. Here we demonstrate that transforming growth factor (TGF)-alpha, a potent epithelial and mesenchymal cell mitogen, is upregulated specifically at the sites of asbestos fiber deposition in the lungs of rats exposed for 5 hours. Unexposed animals and those exposed to high concentrations of iron spheres exhibited no increase in TGF-alpha expression at any time during the experiment. Inhaled asbestos fibers deposit initially at the bronchiolar-alveolar duct regions and alveolar macrophages accumulate at these sites within hours. Non-isotopic in situ hybridization and immunohistochemistry were used to show that the mRNA that codes for TGF-alpha along with the peptide were clearly up-regulated at the bronchiolar-alveolar duct regions by 24 hours after the single asbestos exposure. The numbers of labeled cells demonstrated that expression of the mRNA and protein remained significantly above background for at least 2 weeks after exposure along with increased cell proliferation assessed by staining for proliferating cell nuclear antigen. This, to our knowledge, is the first demonstration of TGF-alpha expression at sites of lung injury in developing fibroproliferative disease. This finding supports the hypothesis that the growth factor is involved in the dramatic epithelial and mesenchymal proliferation we documented previously, although additional experiments will be essential to establish the precise role of TGF-alpha.

Administration, Inhalation↗
PubMed1994

Can parturients distinguish between intravenous and epidural fentanyl?

We tested the hypothesis that the sedative, euphoric, and analgesic effects of intravenous fentanyl would distinguish intravenous from epidural administration. One hundred ASA I and II labouring parturients received 100 micrograms fentanyl either iv or via an epidural catheter in a double-blind, randomized, cross-over fashion. Nineteen anaesthetists (8 staff and 11 residents) participated and correctly guessed the route of administration of the fentanyl in 61/66 intravenous doses and in 69/75 epidural doses yielding a sensitivity of 92.4%, a specificity of 92.0%, a positive predictive value of 91.0%, and a negative predictive value of 93.2%. Of the 41 patients that were crossed over, 38 (92.7%) were able to detect a difference between the routes of administration. Most patients experienced prompt, short-lived symptoms with iv fentanyl but no important differences in fetal heart rate pattern or in maternal desaturation were seen between the groups. This study suggests that subjective symptoms will accurately distinguish intravenous from epidural fentanyl administration in labouring parturients (P < 0.001), and should serve as a safe and reliable intravenous test dose for epidural anaesthesia in the obstetric population.

Adult↗
PubMed1994

Modulation of transcriptional activation of the proliferating cell nuclear antigen promoter by the adenovirus E1A 243-residue oncoprotein depends on proximal activators.

Previous analyses defined a proliferating cell nuclear antigen (PCNA) E1A-responsive element (PERE) in the PCNA promoter that is essential for transactivation by the 243-residue product of the adenovirus type 2 E1A 12S mRNA (E1A 243R). In this report, we show that the PERE activates a heterologous basal promoter and confers susceptibility to transactivation by E1A 243R, indicating that the PERE is both necessary and sufficient for the response of the PCNA promoter to this oncoprotein. Insertion of linker sequences between the PERE and the site of transcription initiation in the PCNA promoter severely impairs the promoter's response to E1A 243R transactivation. GAL4 sites can replace the function of the PERE in the E1A 243R response of the PCNA basal promoter if transcriptional activators of suitable strength are supplied as GAL4 fusion proteins. Weak transcriptional activators render the PCNA basal promoter subject to transactivation by E1A 243R but do not endow the adenovirus E1B basal promoter with a similar response. Strong transcriptional activators do not support transactivation by E1A 243R, however; instead, E1A reduces the ability of the strong activators to activate both the PCNA and E1B basal promoters. Although other mechanistic differences might determine the response, the data imply a relationship between the activation strength of promoter-proximal effectors and the response of the PCNA basal promoter to E1A 243R. These experiments indicate that the PERE can function autonomously in mediating transactivation by E1A 243R and that the PCNA basal promoter is configured in a manner that permits modulation by E1A 243R of transcriptional activation by promoter-proximal effectors.

Adenovirus E1A Proteins↗
PubMed1993

The adenovirus E1A 12S product displays functional redundancy in activating the human proliferating cell nuclear antigen promoter.

The adenovirus E1A 243R oncoprotein stimulates expression from the promoter of the human proliferating cell nuclear antigen (PCNA). To gain insight into the mechanism of activation, we analyzed deletion and point mutations of the 243R protein for their abilities to activate PCNA promoter-directed reporter gene expression upon cotransfection into HeLa cells. Large deletions that in combination span the entire protein severely impaired the ability of E1A 243R to induce PCNA expression. Smaller deletions and specific point mutations that target specific E1A-binding proteins were less deleterious to PCNA induction. The data suggest that E1A activates transcription of the PCNA gene by multiple mechanisms and that, of the known 243R-associated proteins, p300 and p107-cyclin A can mediate the response while p105-RB does not appear to participate. Presumably, the functional redundancy ensures that 243R can activate expression of this essential DNA replication protein regardless of cell type and physiological conditions.

Adenovirus E1A Proteins↗
PubMed1993

A complex promoter element mediates transactivation of the human proliferating cell nuclear antigen promoter by the 243-residue adenovirus E1A oncoprotein.

The adenovirus E1A oncoproteins interfere with the normal regulation of cellular proliferation through interactions with cell cycle regulatory proteins. In view of the essential role of proliferating-cell nuclear antigen (PCNA) in DNA replication, we performed a mutational analysis of the minimal human PCNA promoter (nucleotides -87 to +62) to define sequence elements which mediate transactivation by the 243-residue E1A protein (E1A 243R). Linker-scanning and site-directed mutants were examined for basal and E1A-induced expression of chloramphenicol acetyltransferase (CAT) from PCNA promoter-CAT reporter constructs transiently expressed in HeLa cells. The results define the cis-acting element required for induction of PCNA by E1A 243R as a region between -59 and -45 relative to the transcription initiation site. This PCNA E1A-responsive element (PERE), which is protected from DNase I digestion by nuclear extracts from 293 cells, includes the sequence AGCGTGG immediately upstream of the ATF binding site previously shown to be important for activation of PCNA by E1A 243R (G. F. Morris and M. B. Mathews, J. Virol. 65:6397-6406, 1991). Mutation of either the upstream component or the ATF site within the PERE diminishes basal promoter activity and abrogates transactivation by E1A 243R. This novel cis-acting element is also essential for both basal and E1A-induced expression in the context of the full-length PCNA promoter.

Adenovirus E1A Proteins↗
PubMed1991

The adenovirus E1A transforming protein activates the proliferating cell nuclear antigen promoter via an activating transcription factor site.

The transforming region of adenovirus (E1) stimulates expression of a reporter construct linked to the promoter for the human proliferating cell nuclear antigen (PCNA) gene in a cotransfection assay (G. F. Morris and M. B. Mathews, J. Biol. Chem. 264:13856-13864, 1989). The major products of the E1 region were assessed individually for their contribution to transactivation of the PCNA promoter. The E1A 13S and 12S products and the E1B 19-kDa product elevated expression from the PCNA promoter, whereas the E1B 55-kDa product did not. Induction of the PCNA promoter by E1A differed from transcriptional activation of the adenovirus E3 promoter in that the PCNA promoter is activated by the E1A 12S product whereas the E3 promoter is repressed; furthermore, the PCNA promoter is activated upon E1A overexpression, whereas the E3 promoter responds less well to high amounts of E1A. A site for the activating transcription factor ATF located approximately 50 nucleotides upstream from the transcription initiation site in the PCNA promoter mediates a positive response to the E1A 12S and 13S products.

Adenovirus Early Proteins↗