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D J Harrison

Publications and source records attributed to D J Harrison.

At least 109 records · Page 6Linked to original sources
PubMed1996

Transcriptional regulation of gamma-glutamylcysteine synthetase-heavy subunit by oxidants in human alveolar epithelial cells.

We studied the regulation of glutathione (GSH) synthesis and characterised the 5'-promoter region of the gamma-glutamylcysteine synthetase-heavy subunit (gammaGCS-HS) gene in human alveolar type II cells (A549) following exposure to menadione (MQ) and hydrogen peroxide (H2O2). Both MQ (100 microM) and H2O2 (100 microM) exposure increased intracellular GSH levels associated with increased gammaGCS activity. This was concomitant with enhanced expression of gammaGCS-HS mRNA. Transfection of deletion constructs of the gammaGCS-HS promoter (-1050 to +82 bp) in a chloramphenicol acetyl transferase (CAT) reporter system revealed that an human antioxidant response element (hARE), present within the proximal region of the promoter (-1050 to -818 bp), is not required for oxidant-mediated gene induction. We conclude that oxidant stress-induced gammaGCS-HS mRNA expression is associated with AP-1 or AP-1 like responsive elements (-817 to +45 bp).

Cells, Cultured↗
PubMed1996

Induction of gamma-glutamylcysteine synthetase by cigarette smoke is associated with AP-1 in human alveolar epithelial cells.

Increased levels of glutathione (GSH) occur in the epithelial lining fluid (ELF) of chronic cigarette smokers. Therefore we investigated the effect of cigarette smoke condensate solution (CSC) on GSH synthesis and the regulation of gamma-glutamylcysteine synthetase (gammaGCS) in human type II alveolar epithelial cells (A549). CSC exposure increased GSH levels, gammaGCS activity and gammaGCS heavy subunit (HS) mRNA, as well as increasing DNA binding of the activator protein-1 (AP-1) and the human antioxidant response element (hARE). Transfection of deletion constructs of the gammaGCS-HS promoter in a chloramphenicol acetyl transferase (CAT) reporter system revealed that an hARE, present within promoter, is not required for the CSC mediated induction. We conclude that CSC induction of gammaGCS-HS expression is associated with AP-1/AP-1-like responsive elements.

Antioxidants↗
PubMed1996

Ganciclovir-induced ablation non-proliferating thyrocytes expressing herpesvirus thymidine kinase occurs by p53-independent apoptosis.

In adult mice of the transgenic strain TG66.19, in which expression of herpes simplex type 1 virus thymidine kinase (HSVI-TK) is driven in thyrocytes from the thyroglobulin promoter, the drug Ganciclovir causes the death (ablation) of thyrocytes. Ablation occurred in the absence of thyrocyte proliferation or nuclear DNA synthesis, but was accompanied by transient expression of proliferating cell nuclear antigen and the dying thyrocytes exhibited the ultrastructural features of apoptosis. Control experiments show that the apoptosis is a result of the production of Ganciclovir phosphates in thyrocytes that express HSV1-TK. However, cell death was not dependent upon the presence of a functional copy of the oncosuppressor gene p53. We conclude that the apoptosis is probably not mediated by induction of DNA damage and occurs via a pathway that is independent of p53. The fact that Ganciclovir phosphate can kill cells by a p53-independent apoptotic pathway is encouraging in relation to tumour ablation by methods based on transfection with HSV1-tk genes and administration of Ganciclovir.

Animals↗
PubMed1996

Design and synthesis of a highly immunogenic, discontinuous epitope of HIV-1 gp120 which binds to CD4+ve transfected cells.

Here we report the design and synthesis of a novel 32-mer peptide, Lys364-378Val445-459.oxidized (named GC-1), which represents a discontinuous epitope from the C3 and C4 domains of gp120 from the HIV-1 IIIB isolate. This peptide induces high titre IgG antibody responses in mice, indicating that it has both B and T cell epitopes. Epitope mapping using reduced GC-1 and appropriate linear peptides demonstrated that a large proportion of the antibodies raised in mice were directed against discontinuous epitope(s). Furthermore, antibodies to GC-1 peptide cross-reacted with purified HIV-1 strain IIIB gp120, indicating the GC-1 mimicked at least one epitope of the native protein. The peptide, which incorporates three gp120 residues Asp 368, Glu 370 and Asp 457, previously shown to be critical for CD4 ligation, bound to the surface of a CD4 transfected human epithelial cell line HeLa, but not to the parent cell line and inhibited binding of recombinant HIV-1 gp120 to recombinant soluble CD4. We have synthesized the first of a series of discontinuous peptides which will be useful for the probing of interactions of HIV-1 gp120 with the CD4 molecule.

Amino Acid Sequence↗
PubMed1996

Transjugular intrahepatic portosystemic stent-shunt (TIPSS) occlusion and the role of biliary venous fistulae.

BACKGROUND/AIM: The aim of this study was to assess the pathology and pathogenic mechanisms involved in the occlusion of transjugular intrahepatic portosystemic stent-shunts. METHODS: Thirty-four patients with transjugular intrahepatic portosystemic stent-shunt who had at least two portographic assessments of shunt function were the subjects of this study. The contents of any shunt demonstrating > 70% stenosis were biopsied before balloon dilatation. Further assessment was made of 10 livers obtained at either post mortem (8) or at liver transplantation (2). Cholangiography was performed in these explanted livers, which were then perfused and fixed with formaldehyde. The shunts were dissected out, sectioned, opened and the contents and the surrounding liver examined macroscopically, histopathologically and immunohistochemically. RESULTS: Fourteen patients with TIPSS developed shunt stenosis. In eight patients the stenosis was greater than 70% and significant re-stenosis occurred in all at repeat portography. Three of these patients who were managed by insertion of new shunts showed no further shunt-related problems, whereas the five who were treated solely by dilatation developed further re-stenosis. Organising thrombus was found in all eight patients and bile was incorporated in the thrombus in four. Biliary epithelium was found in two. Four of the ten explanted livers showed evidence of shunt stenosis, of which three were severe and one was mild (< 70%). The occluding material in patients with severe stenosis was composed of organising thrombus containing bile and a granulomatous inflammatory response. This was associated with a transected bile duct, and the degree of stenosis was related closely to the size of the bile duct transected. The shunts free of bile showed no stenosis. CONCLUSIONS: The results of this study suggest that transection of a major bile duct and bile leak play an important role in the stenosis and occlusion of the intraparenchymal portion of transjugular intrahepatic portosystemic stent-shunt. This has important implications for patient management and stent design.

Biliary Fistula↗
PubMed1996

Lymphocytes from orally tolerized mice display enhanced susceptibility to death by apoptosis when cultured in the absence of antigen in vitro.

The mechanism responsible for the induction of immunological tolerance by oral administration of soluble antigen remains unclear. Here we show that, when cultured in vitro in the absence of antigen, lymphocytes from mice tolerized with a single feed of 25 mg of ovalbumin display an enhanced mortality in comparison with cells from immunized control animals. This increased cell death affects both CD4+ and CD8+ T-lymphocyte subsets, and morphological and flow cytometric analyses suggest that it occurs via apoptosis. All of the changes associated with the propensity of tolerant cells to die by apoptosis in vitro are reduced by the inclusion of the tolerizing antigen in the cultures. These results suggest that tolerance to dietary proteins is accompanied by functional changes in T lymphocytes that render them susceptible to apoptosis. This mechanism may underlie the profound and permanent tolerance to food antigens found under physiological conditions and may provide a useful basis for immunotherapy.

Administration, Oral↗
PubMed1995

Interaction between murine germline mutations in p53 and APC predisposes to pancreatic neoplasia but not to increased intestinal malignancy.

Murine strains which bear constitutive inactivating mutations of either the APC or the p53 tumor suppressor genes are characterised by spontaneous tumors. APC mutated (Min) mice develop large and small bowel adenomas, a small proportion of which, in time, become malignant. p53 deficient mice develop predominantly lymphoma and sarcoma. By interbreeding these strains we have shown that there is co-operativity between these mutations, leading to a shift in phenotype. Most notably, this was characterised by a range of abnormalities of the exocrine pancreas in 83% of animals heterozygous for the APC mutation and constitutively null for functional p53. Dysplasia and preneoplastic foci were seen in 61% of these animals and pancreatic acinar cell adenocarcinoma in 22%. Analysis of these tumors showed them to have lost the remaining wild-type copy of APC. Similar loss of APC was not associated with the development of other extra-intestinal tumors. Surprisingly, given the proposed role for loss of function mutations of the p53 gene in the development of human colorectal cancer, we have found no evidence for either an increase in the rate of adenoma formation in APC +/-, p53 -/- animals, or an increased rate of progression to malignancy compared with APC +/- p53 +/+ mice. These findings highlight striking tissue-specific differences in the tumor suppressor effects of p53.

Adenocarcinoma↗
PubMed1995

High-frequency developmental abnormalities in p53-deficient mice.

BACKGROUND: Several strains of mice carrying null mutations of the tumour suppressor gene p53 have been developed. It has been reported that homozygous mice from all of these strains develop normally to birth, but then succumb rapidly to neoplasia. RESULTS: Here, we report that a significant proportion of female p53-/- mice die during embryogenesis or in the period between birth and weaning, being subject to a spectrum of abnormalities. In a significant proportion (23%) of p53-/- female embryos, the normal process of neural tube closure failed, leading to exencephaly and subsequent anencephaly. Although this phenomenon was predominantly associated with females, we observed one affected male embryo. In addition to a spectrum of neural tube defects, many of these embryos exhibited a range of craniofacial malformations, including ocular abnormalities and defects in upper incisor tooth formation. We observed a significant reduction in the number of p53-/- female progeny of p53+/- x p53+/- matings, and also in an in utero analysis of the p53+/- female progeny of p53-/- x p53+/+ matings. When male mice were exposed to irradiation prior to mating, a significant increase in the rate of abnormality was seen in the progeny, which was specifically associated with p53 deficiency. CONCLUSIONS: We have identified a high rate of developmental abnormalities associated with p53 deficiency. This manifests itself as a spectrum of lesions, predominantly female-associated defects in neural tube closure. These defects may arise either because p53 plays a physiological role at the time of neural tube closure, or because of an abnormally high frequency of mutation within the haploid gametes of p53-null parents.

Animals↗
PubMed1995

The identification of two myoinhibitory peptides, with sequence similarities to the galanins, isolated from the ventral nerve cord of Manduca sexta.

Two new myoinhibitory peptides, Mas-MIP I and Mas-MIP II, were identified from the ventral nerve cord of the adult tobacco hornworm, Manduca sexta. Sequences obtained by a combination of automated Edman degradation and electrospray mass spectrometry were, respectively, AWQDLNSAW and GWQDLNSAW. The native peptides were found to co-elute with synthetic C-terminal amides on a reverse phase HPLC system. When applied to isolated ilea (anterior hindgut) of adult M. sexta, both peptides were found to significantly reduce the rate of peristalsis, or abolish peristalsis entirely, at concentrations of 1 x 10(-9) M. Both peptides share sequence similarities with Lom-MIP, a previously identified myoinhibitory peptide from Locusta migratoria, and with the N-terminal portion of vertebrate peptides in the galanin family.

Abdomen↗
PubMed1995

Effects of heterozygosity for the Rb-1t19neo allele in the mouse.

We describe the pathology of a cohort of 80 mice heterozygous for an inactive allele of the Rb-1 tumour suppressor gene. The majority of these mice developed locally invasive tumours, arising from the pituitary gland. The time of onset of overt signs of disease in mice known to have inherited their mutant allele paternally shows a small but statistically significant shift to the lower end of the spectrum, suggesting that tumorigenesis is influenced by gametic imprinting. In situ hybridisation analysis demonstrates the presence in the tumours of pro-opiomelanocortin mRNA, which is normally found both in corticotroph and melanotroph cells. Mice within this cohort also develop systemic defects. Most notably, there is increased siderosis in the spleen indicating the possibility of an abnormality in red blood cell turnover. This is consistent with the abnormalities of erythropoiesis described previously in homozygous Rb-1-deficient mice. In addition, a proportion of mice developed liver steatosis, probably representing the end organ effects of hormonal imbalance as a direct consequence of tumour presence. A significant proportion showed C cell hyperplasia in the thyroid. The spectrum of pathology in mice differs from that in the human but does provide a useful model of site-specific tumour predisposition.

Alleles↗
PubMed1995

Molecular mechanisms of drug resistance in tumours.

Despite advances in the design and use of chemotherapeutic drugs, the majority of human cancers are resistant to therapy at presentation or become resistant after an initial partial response. This suggests that resistance may be inherent in a tumour cell or may evolve under the selection pressure of drug administration. A number of possible molecular explanations for drug resistance exist. There may be exclusion of drug from the cell, failure to activate the prodrug to its active form, increased detoxification, alteration in the drug target, enhanced repair capability of the cell after injury, or failure to engage an appropriate response, leading to apoptosis in the damaged cell. Many of these factors may co-exist in vivo in human tumours; some are a feature of cell lineage whilst others appear de novo during disease progression. Modulation of these mechanisms has been of some value in laboratory studies but widespread clinical application and benefit remain some way off.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Cell death in health and disease: the biology and regulation of apoptosis.

Apoptosis is a morphologically stereotyped form of cell death, prevalent in multicellular organisms, by which single cells are deleted from the midst of living tissues. Recognition of the cellular corpses and their removal by phagocytosis occurs without disturbance to tissue architecture or function and without initiating inflammation. Apoptosis is regulable and is of fundamental importance to tissue development and homeostasis. Cellular susceptibility to apoptosis is determined by a variety of signals, of both extracellular and internal origin, including proliferative status. Dysregulated apoptosis is important in the pathogenesis of several important human diseases including neoplasia, and recognition of the defects involved is prompting development of new therapeutic strategies.

Acquired Immunodeficiency Syndrome↗
PubMed1995

Magnetic resonance imaging before chemonucleolysis for lumbar disc prolapse.

Chemonucleolysis is an established non-operative treatment of a prolapsed symptomatic lumbar disc. It was introduced as a form of treatment in the early 1960s by Smith [18]. One of the main causes of failure is the difficulty in pre-operative assessment of a contained disc prolapse. Reducing failure rates is very important for the morale of surgeon and patient alike. We investigated 58 patients with magnetic resonance imaging (MRI) to select those with a contained protrusion prior to chemonucleolysis. Per-operative discography confirmed contained protrusion in 96.5% (56/58) of cases, increasing the specificity of selection. At 6 months post chemonucleolysis 86% of our patients were asymptomatic. We would commend MRI as the investigation method of choice prior to chemonucleolysis for a prolapsed symptomatic lumbar disc, thus avoiding separate diagnostic discography, thereby reducing inconvenience to the patient and wastage of prepared chymopapain.

Adult↗
PubMed1995

Accurate determination of skin flux from flow-through diffusion cell data.

PURPOSE: The goal of this investigation was to demonstrate whether the intrinsic flux of a drug diffusing across a membrane mounted in a flow-through diffusion cell may be accurately and easily determined by accounting for the accumulation in the receiver chamber. METHODS: Mathematical modeling, applied to transdermal diffusion, was used to calculate receiver concentration data for single layer and bilayer membranes. The data were interpreted using two apparent flux values, Japp1 and Japp2. Japp1 has been used extensively in the literature, but did not account for accumulation in the receiver. Japp2 did take the accumulation into consideration. RESULTS: The results confirm that, generally, Japp1 values were not accurate estimates of the intrinsic flux. Japp2 values were significantly more accurate, especially prior to the maximum in receiver concentration. CONCLUSIONS: Japp2 was an accurate measurement of intrinsic flux over the entire experimental time period, except at time zero. It was more accurate because it accounted for solute accumulation in the receiver compartment. The accuracy of the Japp2 approximation was practically independent of receiver volume, flow rate and donor volume. For very slowly permeating drugs, or a very small receiver volume combined with a high flow rate, the Japp1 estimate accurately reflected the intrinsic flux. Early time data were required to properly account for accumulation in the receiver cell. If such data were not available, the inverse Laplace method of determining intrinsic flux was preferable to the Japp2 calculation.

Diffusion↗