Search PubMed⌕ Search

Biomedical subjects

C Upton

Publications and source records attributed to C Upton.

At least 37 records · Page 2Linked to original sources
PubMed1999

Cytotoxic response of ovarian cancer cell lines to IFN-gamma is associated with sustained induction of IRF-1 and p21 mRNA.

Interferon-gamma (IFN-gamma) has some anti-tumour activity in human ovarian cancer. This cytokine inhibited proliferation in three of four ovarian cancer cell lines in vitro. We then compared the action of IFN-gamma in two cell lines, one sensitive and one resistant to its growth inhibitory effects. IFN-gamma signalling appeared normal in both cell lines, with stat1 DNA binding activity detectable at 30 min. Continuous exposure to IFN-gamma for 2-3 days was necessary for an irreversible effect on cell growth and apoptosis in cells sensitive to growth inhibition. During this time there was an increase in mRNA for the CKI p21, but no alterations in mRNA levels for other members of the CKI family. Maintenance of p21 mRNA required continuous mRNA synthesis. mRNA for the transcription factor IRF-1 was also induced in growth inhibited cells with similar kinetics to those observed for p21. Maximal induction of both p21 and IRF-1 mRNA was observed after 2-3 days IFN-gamma exposure as the cells became committed to cell death. There was also a rapid increase in p21 and IRF-1 mRNA in cells resistant to the growth inhibitory effects of IFN-gamma, but this increase was not maintained. Thus, continuous interaction with the IFN-gamma receptor, together with a sustained induction of p21 and IRF-1, is associated with growth inhibitory and apoptotic effects of IFN-gamma in ovarian cancer cells.

Adenocarcinoma↗
PubMed1999

Novel nitrated derivatives of 5,8-diazabenzo[c]phenanthrene and 9,14-diazadibenz[a,e]acephenanthrylene: new classes of potent mutagenic compounds.

We report the synthesis of 4-nitro-5,8-diazabenzo[c]phenanthrene (4-NDBP) and 11-nitro-9,14-diazadibenz[a,e]acephenanthrylene (11-NDDA) and the remarkable mutagenic activity of the latter. These two compounds and their non-nitrated parents, 5, 8-diazabenzo[c]phenanthrene (DBP) and 9,14-diazadibenz[a, e]acephenanthrylene (DDA), were screened in Ames plate incorporation assays against Escherichia coli WP2uvrA and Salmonella typhimurium TA98 both in the presence and absence of S9 liver fraction from Aroclor 1254-induced rats. None of the four compounds were cytotoxic up to the limits of their solubility and none showed mutagenic activity in E.coli WP2uvrA, which suggested that any such activity they may have had was not mediated via a base substitution mechanism. DBP and DDA also displayed a lack of activity in TA98 up to their precipitating doses (560 and 33.5 microg/plate, respectively). The two nitrated compounds, however, were genotoxic. 4-NDBP was active at a dose of 500 ng/plate, in the absence of S9, producing 80.0 +/- 28.0 prototrophic organisms (equivalent to 44 revertants/nmol) and at 0.5 ng/plate, in the presence of S9, giving 147 +/- 6.6 revertants (equivalent to 81 000/nmol) and allowed the description of this tetracycle as a potent mutagen. Much more striking was the activity of 11-NDDA: in the absence of S9 a dose of 8.0 ng produced 2000 revertants/nmol and, remarkably, in the presence of S9 80 pg produced the equivalent of 643 000 revertants/nmol. This makes the hexacyclic 11-NDDA the most potent mutagen to date, in the Ames procedures described here.

Coumarins↗
PubMed1998

Shope fibroma virus RING finger protein N1R binds DNA and inhibits apoptosis.

Shope fibroma virus (SFV) N1R gene encodes a RING finger protein that localizes to virus factories within the cytoplasm of infected cells. Altered proteins, with deletions and site-specific mutations, were transiently expressed in vaccinia virus-infected cells to discern regions of the protein that are required for localization. We have determined that at least part of the RING finger region is necessary for localization but that the RING motif alone is not sufficient. A chimeric protein, however, in which the RING finger region of the herpes simplex virus-1 ICP0 protein replaces the SFV N1R RING motif does localize to virus factories. A region of five highly conserved amino acids at the amino terminus of SFV N1R is also critical for localization. We report that the SFV N1R protein binds double- and single-stranded DNA, suggesting a mechanism for localization, and that overexpression of this protein in vaccinia virus-infected cells reduces apoptosis-associated fragmentation of nuclear DNA.

Amino Acid Sequence↗
PubMed1998

Stereochemical studies of the isomerization of novel 2-alkyl-9-phenyl-2,3,4,4a- and 2,3,4,9-tetrahydro-1H-indeno[2,1-c]pyridines.

The published synthetic route to the antihistaminic tetrahydroindeno[2,1-c]pyridines (phenindamines) relies on catalytic reduction of the precursor dihydroindenopyridines. This reduction gives mixtures of 9,9a- and 4a,9a-enes and the clinically active 4a,9a isomer has to be isolated by recrystallization of an appropriate salt. The structure of the product recovered depends on the anion used to isolate the proton salt and appears to be arbitrary. To rationalize this outcome a series of novel N-2 alkylated tetrahydroindeno[2,1-c]pyridines and their diene precursors has been synthesized from accessible piperidines. The structures and geometry of the piperidines and the dihydro- and tetrahydroindenopyridines have been determined by 1H and 13C NMR. An unusual feature of the proton spectra of the piperidines is the resonance of the axial protons at lower field than their equatorial counterparts. By controlling the reaction conditions for the reduction of the dihydroindenopyridines to their tetrahydro derivatives the kinetic or thermodynamic product can be selected as required. A predictable outcome for the reductions investigated was achieved and is generally applicable.

Histamine H1 Antagonists↗
PubMed1998

Novel 5,8-diazabenzo[c]phenanthrenes: synthesis and mutagenicity.

The polycyclic aromatic hydrocarbons have been recognized as carcinogens and mutagens since the early part of this century. More recently their aza and polyaza derivatives have been shown to have the same biological activity. A major source of these compounds is the combustion of fresh or metamorphosed plant materials; this contributes to the environmental burden of, and exposure to, these carcinogens. We report the synthesis and characterization of a series of novel 5,8-diazabenzo[c]phenanthrenes which are isosteric with the known epidermal carcinogen benzo[c]phenanthrene but have not yet been reported as components of soot or diesel particulate matter. The synthesis of the compounds exploits a versatile, double Friedlander reaction between the appropriately substituted 2,2'-diaminobenzophenone and beta-diketones, with yields of purified product ranging from 30-90%. The nucleophilic substitution of these diazabenzophenanthrenes with ethanolamine is also described. This strategy will enable further elaboration of these heterocyclic nuclei at a later date. Mutagenicity testing of these agents was performed using spot tests and in Ames plate-incorporation assays using Escherichia coli WP2 and WP2uvrA as test organisms. The plate-incorporation assays were performed in the presence or absence of metabolic enzymes contained in the S9 liver fraction from Aroclor 1254-induced rats, to investigate whether bioactivation of the diazabenzophenanthrenes contributed to their toxicity. No differences between these two protocols were observed, with neither test showing reversion to prototrophic behaviour. Furthermore, the compounds were not toxic to the test organism. These initial results suggest that these compounds are not mutagenic in the Ames tests employed.

Animals↗
PubMed1998

Subversion of cytokine networks by viruses.

Viruses and the immune system have been competitors throughout their co-evolution. It is therefore not surprising that the viruses in circulation today possess a variety of strategies to counteract those aspects of the immune system that are involved in virus clearance. Examination of these virus encoded functions provides an important view of immune function and an appreciation of the complexity of the virus-host interaction. It is clear that viruses, seeking to subvert the immune system, have become adept in blocking the communication channels of the immune system. There are numerous examples of viral proteins that target the cytokine networks, disrupting the processes by which the delicately balanced immune system is regulated. This review focuses on the gene products of poxviruses, adenoviruses and herpesviruses that function primarily as immune-modulators.

Adenoviridae↗
PubMed1997

Generation of an anti-tumour immune response in a non-immunogenic tumour: HSVtk killing in vivo stimulates a mononuclear cell infiltrate and a Th1-like profile of intratumoural cytokine expression.

Direct delivery of the herpes simplex virus thymidine kinase (HSVtk) gene, in combination with the prodrug ganciclovir (GC), has been used for the treatment of localised, inoperable tumours. Several groups have shown that when rodent tumours are ablated in vivo with suicide genes, anti-tumour immunity can also be generated. Hence, this approach may also be useful in treating disseminated disease. Here we have studied the mechanisms associated with this anti-tumour immunity. In B16 HSVtk+ tumours being killed in vivo with GC treatment, we observed the induction of a pronounced intratumoural infiltrate of macrophages, CD4+ and CD8+ T cells. In addition, using reverse transcriptase polymerase chain reaction, expression of interleukin (IL)-2, IL-12, interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha) and granulocyte/macrophage colony-stimulating factor (GM-CSF) but not IL-4, IL-6 or IL-10, was observed, a profile of cytokine expression which resembles that of a Th1 immune response. To complement these findings, we also investigated the mechanisms by which expression of HSVtk leads to cell death. Our data show that B16/HSVtk+ cells die predominantly by necrosis, rather than apoptosis, on exposure to GC, a process which may be associated with the generation of anti-tumour inflammatory responses. From these data we propose a model for the induction of anti-tumour immunity using suicide genes and discuss the development of improved vectors for gene therapy to augment these effects in vivo.

Animals↗
PubMed1997

A human homolog of the vaccinia virus HindIII K4L gene is a member of the phospholipase D superfamily.

We have identified a human gene encoding a protein with 48% amino acid identity to the vaccinia virus (VV) K4L gene product. Both contain motifs characteristic of the phospholipase D (PLD) protein superfamily. These proteins are also related to vaccinia virus p37, encoded by the F13L gene, which is required for envelopment and spread of the virus. The similarity to phospholipase D provides insight into the mechanisms and evolution of these processes.

Amino Acid Sequence↗
PubMed1997

A novel poxvirus gene and its human homolog are similar to an E. coli lysophospholipase.

A novel poxvirus gene has been characterized within the genome of ectromelia virus. It has significant similarity to a family of lysophospholipases suggesting that it may function in the degradation of lysophospholipids. Since these molecules are active in the stimulation of inflammation, we hypothesize that this gene may play a role in virus virulence. This gene is expressed early in the ectromelia virus replication cycle, before DNA replication. We have also characterized a human cDNA that encodes a protein which is 49.5% identical to the ectromelia virus protein. By its presence in multiple cDNA libraries, this human gene is known to be expressed in a variety of body tissues and is likely to function in the normal regulation of lysophospholipid levels. This family of proteins have conserved blocks of amino acids that are indicative of a serine-aspartic acid-histidine catalytic triad, similar to those used by true lipases and a number of esterases.

Amino Acid Sequence↗
PubMed1997

Interferon gamma induces cell cycle arrest and apoptosis in a model of ovarian cancer: enhancement of effect by batimastat.

Locoregional human IFN-gamma may have activity against refractory ovarian cancer. We investigated this further in an ovarian cancer xenograft model. Administered at clinically relevant doses, intraperitoneal IFN-gamma prolonged the survival of mice bearing multiple established peritoneal tumours, with optimal treatment giving a 3-6-fold increase in median survival time. Daily dosing, which was superior to intermittent treatment, decreased DNA synthesis and induced apoptosis in tumour cells with maximal effects after 7-21 days treatment. This was preceded by an increase in p53 protein at 48 h. The effect of IFN-gamma was not enhanced by sequential treatment with carboplatin. However, the matrix metalloprotease inhibitor, batimastat, further increased mouse survival when given after IFN-gamma. Thus IFN-gamma is cytotoxic to ovarian epithelial cells in vivo and intensive locoregional dosing over short periods is effective. Sequential administration of novel agents that perturb the host/tumour relationship may be of benefit.

Animals↗
PubMed1996

Blocking effects of promethazine, triprolidine and their analogues on the excitation caused by the peptide, achatin-I.

An Achatina endogenous tetrapeptide, achatin-I (Gly-D-Phe-Ala-Asp), applied by brief pressure, produced an inward current (Iin) on an Achatina giant neurone type, PON (periodically oscillating neurone). Promethazine, triprolidine and their analogues tested, applied by perfusion, showed a tendency to inhibit the Iin, suggesting that the effective structures vary to a wide extent. With respect to promethazine and its analogues, the presence of 2-bromo, 5-oxo, 3-dimethylsulfamido and 2-methoxy weakened the effects. 10-(2-methylamino-2-methylethyl) instead of 10-(2-dimethylamino-2-methylethyl) of promethazine and the azepine ring instead of phenothiazine ring potentiated the effects. From the dose (pressure duration)-response study of achatin-I, the two promethazine analogues, RP 6497 and RP 6549 (the structures are shown in Fig. 1), inhibited the Iin in partly competitive and partly noncompetitive manners. Regarding triprolidine and its analogues, the compounds in Z-configuration seemed to be more effective than those in E-configuration. The presence of 4-methyl in 1-phenyl, and 1-(4-pyridyl) instead of 1-(2-pyridyl) potentiated the effects. 3-Dimethylamino instead of 3-pyrrolidino weakened the effects. The two triprolidine analogues, Trip Der 3 and Trip Der 6 (the structures in Fig. 2), inhibited the Iin in an uncompetitive manner.

Animals↗
PubMed1996

Approaches to detection of distantly related proteins by database searches.

The searching of protein databases as a method of identifying newly sequenced genes is commonplace in molecular biology laboratories. However, it is a procedure that is not usually formally taught to students, and method cookbooks discuss it only briefly. This article uses a single family of highly diverged uracil-DNA glycosylases, which fall into two distinct groups, to highlight some of the difficulties associated with identification of such proteins by database searching.

Amino Acid Sequence↗
PubMed1995

A new family of lipolytic plant enzymes with members in rice, arabidopsis and maize.

We have noted a striking similarity between the sequences of proteins in a novel family of lipases we recently reported [Upton, C. and Buckley, J. T. (1995) Trends Biol. Sci. 20, 178-9] and more than 120 sequences from the database of Expressed Sequence Tags (dbEST) which correspond to at least 30 unique genes from arabidopsis, rice and maize. A cDNA (Arab-1) corresponding to one of these sequences was isolated, sequenced and translated. There was significant similarity to sequences in the new lipase family over the entire open reading frame of Arab-1 and when expressed in E. coli, the gene product was lipolytic. Arab-1 and genes for some of the other plant proteins appear to be differentially expressed. They may play a role in the regulation of lipid metabolism during plant development.

Amino Acid Sequence↗
PubMed1995

Species specificity of ectromelia virus and vaccinia virus interferon-gamma binding proteins.

Interferon-gamma functions within the immune system as a potent anti-viral and immunoregulatory cytokine. In order to successfully replicate within a host cell, poxviruses have evolved a number of strategies to counteract the pleiotropic effects of interferon-gamma. In particular, the leporipoxvirus myxoma virus was shown to express an extracellular soluble interferon-gamma receptor homolog, denoted M-T7, which is capable of inhibiting the anti-viral activities of rabbit interferon-gamma (C. Upton, K. Mossman, and G. McFadden, 1992, Science 258, 1369-1372). Here, we demonstrate that expression of soluble interferon-gamma receptor homologs appears to be characteristic of all poxviruses tested, including Shope fibroma virus, vaccinia virus (strains WR and IHDW), ectromelia virus, cowpox virus, and rabbitpox virus. We have cloned, sequenced, and characterized the interferon-gamma binding protein in supernatants from ectromelia virus-infected cells, and demonstrate the capability of this soluble protein to bind human, murine, and rabbit interferon-gamma with similar affinity. We also investigate the properties of the vaccinia virus interferon-gamma binding protein and demonstrate that this protein binds human and rabbit interferon-gamma with similar affinity and binds murine interferon-gamma with a significantly lower relative affinity. The implications of these studies with respect to viral pathogenesis and the evolutionary relationship between a virus and its host are discussed.

Amino Acid Sequence↗
PubMed1995

The myxoma virus-soluble interferon-gamma receptor homolog, M-T7, inhibits interferon-gamma in a species-specific manner.

The myxoma virus M-T7 protein contains significant sequence similarity to the ligand binding domain of the mammalian interferon-gamma receptors, and functions as a soluble homolog which can bind and inhibit the biological activities of rabbit interferon-gamma (Upton, C., Mossman, K., and McFadden, G. (1992) Science 258:1369-1372). M-T7, the most abundantly secreted protein from myxoma virus-infected cells, was shown to be expressed in significant biological amounts as a typical poxvirus early gene product, efficiently secreted at early times of infection to levels that exceed 5 x 10(7) molecules/cell, and function as a stable inhibitory protein in infected cell supernatants until late times of infection. M-T7 was specific in binding and inhibiting rabbit interferon-gamma, and did not bind either human or murine interferon-gamma. Scatchard analysis of rabbit interferon-gamma binding curves yielded a single high affinity binding site on M-T7, with a Kd of 1.2 x 10(-9) M, which is comparable to the affinity between soluble forms of cellular interferon-gamma receptors and their cognate ligands. In comparison, rabbit interferon-gamma was shown to bind its cellular receptor with a Kd of 5.9 x 10(-10) M, again comparable to the affinity of membrane bound forms of other mammalian interferon-gamma receptors for interferon-gamma. Thus, the myxoma virus M-T7 protein is a functional soluble interferon-gamma receptor homolog which binds and inhibits interferon-gamma with high affinity in a species-specific manner.

Amino Acid Sequence↗