Biomedical subjects

D J Harrison

Publications and source records attributed to D J Harrison.

At least 73 records · Page 4Linked to original sources

PubMed1999

Method of preparing emergency medicine residents for giving legal depositions.

This study was performed to determine if a simulated legal deposition increases emergency medicine (EM) residents' knowledge, self-confidence, and understanding of a legal deposition. This prospective study included a convenience sample of EM 1-3 residents. A knowledge and a self-assessment pretest were given, followed by a didactic session moderated by local attorneys, followed by knowledge and a self-assessment posttest. The total time involved was 2 hours. The mean score on the knowledge pretest was 4.5 and 5.25 on the posttest. Using a paired t-test, the authors found this difference to be statistically significant. (P < 0.01) Using Hotelling's T2 test, the authors compared presimulation and postsimulation self-assessment questions. The results revealed that there was a difference between these scores (P < 0.001). Participants in the deposition significantly improved their self-assessment ranking and knowledge inventory test scores by participating in a simulated legal deposition.

Adult↗

PubMed1999

Cellular response to cancer chemopreventive agents: contribution of the antioxidant responsive element to the adaptive response to oxidative and chemical stress.

Cancer chemopreventive agents can act by inhibiting either the acquisition of mutations or the neoplastic processes that occur subsequent to mutagenesis. Compounds that reduce the rate at which mutations arise, referred to as blocking agents, exert their effects largely through their ability to induce the expression of antioxidant and detoxification proteins. This is achieved by the transcriptional activation of a small number of genes that are co-regulated through the presence of an antioxidant responsive element (ARE) in their promoters. Blocking agents can cause gene induction by producing oxidative and/or chemical stress within the cell and, as the inducible proteins act to ameliorate the metabolic insult, the process represents a form of adaptive response. The transcription factors which mediate this response through the ARE are members of the basic leucine zipper superfamily. The mechanism whereby cells sense and respond to the chemical signal(s) generated by chemopreventive blocking agents is discussed.

Adaptation, Physiological↗

PubMed1999

Apoptosis and carcinogenesis.

Apoptosis has long been known to be effected through a common sequence of structural changes, despite the wide variety of initiating stimuli. These common structural events appear to depend upon activation of a set of enzymes (caspases) which direct a strongly conserved, terminal effector pathway. The regulation of this pathway, and in particular its coupling to DNA damage, appears to be critical in maintaining at low levels the number of mutated cells within tissues. The frequency with which tumours (experimental and human) bear deficiency in p53 or MSH-2 repair function may indicate the importance of these proteins in coupling DNA damage to apoptosis.

Animals↗

PubMed1998

Life and death in the genesis of the tumour cell.

Most tumours arise because of an aberrant response of cells following exposure to chemicals deliberately ingested, for example cigarette smoke, or present as an environmental pollutant, for example dietary aflatoxin. Recent evidence has highlighted the importance of tumour suppressor genes and oncogenes in determining the response of a cell to potentially mutagenic or growth disrupting events. Many toxicants in vivo can cause apoptosis in a dose dependent manner. At low dose apoptosis is engaged, but with high exposure cells may undergo necrosis as cellular metabolism is catastrophically overwhelmed preventing the ordered set of events that constitute apoptosis from occurring. Mutations in genes that control deletion of potentially damaged cells result in overriding of death signals and may result in survival of a cell that otherwise should have been deleted. This gave rise to the concept of the 'undead' cell--the aberrant cell that has escaped normal growth controls taking the first step towards cancer. However, not all cell lineages respond to injury in the same ways, and even the same gene may have quite varied effects depending on the cellular and tissue environment.

Apoptosis↗

PubMed1998

Hepatitis B x protein inhibits p53-dependent DNA repair in primary mouse hepatocytes.

The mechanisms by which the hepatitis B x protein (HBx) contributes to hepatocarcinogenesis remain unclear. However, interaction with the tumor suppressor gene p53 and inhibition of p53-dependent cellular functions, including nucleotide excision repair, could be central to this process. We studied the levels of global repair (removal of cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts) and transcription-coupled repair (removal of CPDs in both strands of the dihydrofolate reductase gene) in primary wild-type and p53-null mouse hepatocytes. We show that global repair of CPDs appears to be more efficient in mouse hepatocytes than in other commonly studied rodent cells and approaches the levels of human cells and that p53 is required for global genomic DNA repair of CPDs but not for transcription-coupled repair. We then investigated the effect of HBx expression on hepatocyte nucleotide excision repair. We demonstrate that HBx expression affects DNA repair in a p53-dependent manner. Transient HBx expression reduces global DNA repair in wild-type cells to the level of p53-null hepatocytes and has no effect on the repair of a transfected damaged plasmid. Therefore, in viral hepatitis, the hepatitis B virus could inhibit the p53-dependent component of global repair leading, over time, to accumulation of genetic defects and fostering carcinogenesis.

Animals↗

PubMed1998

Molecular cloning, expression and catalytic activity of a human AKR7 member of the aldo-keto reductase superfamily: evidence that the major 2-carboxybenzaldehyde reductase from human liver is a homologue of rat aflatoxin B1-aldehyde reductase.

The masking of charged amino or carboxy groups by N-phthalidylation and O-phthalidylation has been used to improve the absorption of many drugs, including ampicillin and 5-fluorouracil. Following absorption of such prodrugs, the phthalidyl group is hydrolysed to release 2-carboxybenzaldehyde (2-CBA) and the pharmaceutically active compound; in humans, 2-CBA is further metabolized to 2-hydroxymethylbenzoic acid by reduction of the aldehyde group. In the present work, the enzyme responsible for the reduction of 2-CBA in humans is identified as a homologue of rat aflatoxin B1-aldehyde reductase (rAFAR). This novel human aldo-keto reductase (AKR) has been cloned from a liver cDNA library, and together with the rat protein, establishes the AKR7 family of the AKR superfamily. Unlike its rat homologue, human AFAR (hAFAR) appears to be constitutively expressed in human liver, and is widely expressed in extrahepatic tissues. The deduced human and rat protein sequences share 78% identity and 87% similarity. Although the two AKR7 proteins are predicted to possess distinct secondary structural features which distinguish them from the prototypic AKR1 family of AKRs, the catalytic- and NADPH-binding residues appear to be conserved in both families. Certain of the predicted structural features of the AKR7 family members are shared with the AKR6 beta-subunits of voltage-gated K+-channels. In addition to reducing the dialdehydic form of aflatoxin B1-8,9-dihydrodiol, hAFAR shows high affinity for the gamma-aminobutyric acid metabolite succinic semialdehyde (SSA) which is structurally related to 2-CBA, suggesting that hAFAR could function as both a SSA reductase and a 2-CBA reductase in vivo. This hypothesis is supported in part by the finding that the major peak of 2-CBA reductase activity in human liver co-purifies with hAFAR protein.

Aflatoxin B1↗

PubMed1998

p53-independent DNA repair and cell cycle arrest in embryonic stem cells.

The role of p53 in DNA repair and cell cycle checkpoint after ultraviolet irradiation was investigated in an embryonic stem cell line homozygous for a targeted deletion of p53. Results indicate that loss of p53 does not alter the capacity of ES cells to respond to DNA damage. Wild-type and p53-deficient cells showed similar cessation of DNA synthesis after UV damage and similar ultimate capacity to repair a transiently transfected reporter plasmid. Interestingly, in the absence of DNA damaging treatment, the transit of p53-deficient cells through S phase was slower than wild-type cells. We suggest that this may result from the absence of a p53-dependent response to endogenous DNA damage: without p53 sensing endogenous damage leading to immediate repair, such damage may persist and thus delay DNA synthesis.

Animals↗

PubMed1998

Chemiluminescence detection in integrated post-separation reactors for microchip-based capillary electrophoresis and affinity electrophoresis.

Chemiluminescence (CL) detection based on the horseradish peroxidase (HRP) catalyzed reaction of luminol with peroxide was investigated as a post-separation detection scheme for microchip-based capillary electrophoresis. An integrated injector, separator and post-separation reactor was fabricated on planar glass wafers. The fluorescein conjugate of HRP (HRP-F1) was used as a sample for optimization of the CL detector response. In devices etched 10 microm deep, with an aluminum mirror integrated onto the backside of the detection zone to enhance collection efficiency, the detection limit, estimated at 3 standard deviations (SD) above background noise, for 1 nL injected sample plugs was 35 nM in HRP-F1. In devices etched 40 microm deep, 8 nL plugs gave a detection limit of 7 nM. Separation and CL detection of the products of an immunological reaction of a F(ab')2 fragment of the HRP conjugate of goat anti-mouse immunoglobulin G (IgG) with mouse IgG was performed on-chip. A linear calibration curve was obtained for the decrease in peak height of the HRP conjugate (53 microg/mL) with increasing mouse IgG (0-60 microg/mL). When microperoxidase was used as an internal standard, the R2 value of a linear least-squares fit was 0.9867, and the relative errors in the slope and intercept were +/- 5.8 and +/- 1.3 %, respectively.

Animals↗

PubMed1998

Monoclonal antibody binding affinity determined by microchip-based capillary electrophoresis.

The affinity constant of a monoclonal antibody to fluorescently labeled bovine serum albumin (BSA) was measured in diluted mouse ascites fluid using a microfluidic chip to perform affinity capillary electrophoresis. Borofloat glass-based devices could be used repeatedly with samples for many months. On-chip separations were performed in less than 60 s, and 30-60 s was required for manual sample exchange. The change in peak height for BSA with increasing BSA/anti-BSA concentration ratio was used to determine concentration changes in bound and free BSA. A Scatchard plot analysis gave an affinity constant (more exactly the intrinsic association constant) of 3.5+/-0.6 x 10(7) M(-1) for a 1:1 stoichiometric ratio. Two affinity complexes were separated. One complex was identified by the Scatchard method as having a 1:1 stoichiometric ratio. The other complex is proposed to have a stoichiometry with an excess of anti-BSA to BSA, most likely (anti-BSA)2-BSA, on the basis of a faster migration time than the 1:1 complex, a decrease in the amount of this complex with increasing [BSA], and predictions of theoretical models for multi-valent antigens. Potential applications of microchip-based devices in affinity measurements are discussed.

Animals↗

PubMed1998

N-acetyl transferase 1: two polymorphisms in coding sequence identified in colorectal cancer patients.

Increased cancer risk has been associated with functional polymorphisms that occur within the genes coding for the N-acetyltransferase enzymes NAT1 and NAT2. We detected two NAT1 polymorphisms in colorectal cancer patients by heteroduplex analysis. DNA sequencing revealed the wild-type sequence (NAT1*4) and two single base substitutions at adjacent positions 999 bp (C to T, NAT1*14) and 1000 bp (G to A, NAT1*15) of the gene, changing Arg187 to a stop codon and Arg187 to Gln respectively. NAT1 alleles NAT1*4 (0.98) and NAT1*15 (0.02) were present at a similar frequency in patients with colorectal cancer (n=260) and in a Scottish control group (n=323). The third allele, NAT1*14, was present only in the colorectal cancer group at a frequency of 0.006. NAT1 genotype NAT1*4/ NAT1*15 was significantly less frequent in individuals that had a slow NAT2 genotype. This was observed in both cancer and control groups and suggests that this association was unrelated to cancer risk. We conclude that polymorphisms within the coding region of the NAT1 gene are infrequent and do not appear to have an independent association with colorectal cancer risk. However, the relationship between NAT1 and NAT2 polymorphisms appears non-random, suggesting a linkage between these enzymes.

Acetyltransferases↗

PubMed1998

Intrahepatic proliferation of 'naive' and 'memory' T cells during liver allograft rejection: primary immune response within the allograft.

Liver allograft rejection is mediated by a primary response of T lymphocytes, followed by infiltration of the graft with a mixed inflammatory reaction. Using single and double label immunocytochemistry, we examined the proliferation index and the phenotype of leukocytes on liver biopsies from 10 patients with acute rejection before and after treatment with i.v. steroids, 10 patients with chronic rejection, 10 patients without rejection posttransplant, and 15 nongrafted, nonimmunosuppressed patients. Proliferation of mononuclear leukocytes (assessed by expression of Ki-67, a nuclear antigen associated with the cell cycle) inside the allograft was a prominent feature of acute and chronic rejection and was down-regulated by steroid treatment. Leukocytes in cell cycle were located predominantly in the portal tracts at the site of the inflammatory infiltrate. The majority of 'naive' (CD45RA+) and 'memory' (CD45RO+) CD4+ T lymphocytes were also periportally distributed. In contrast, CD8+ T lymphocytes, CD57+ natural killer cells, and CD68+ macrophages were located intraparenchymally throughout the liver lobules, whereas CD20+ B lymphocytes were only present in some of the portal tracts. Predominantly CD4+ and occasionally CD8+ lymphocytes were proliferating (assessed by double staining). The proliferating CD4+ cells were of both naive (CD4+, CD45RA+) and memory (CD4+, CD45RO+) phenotypes. To our knowledge, this is the first description of proliferating naive T lymphocytes in situ in liver allografts. These findings suggest that there may be a primary immune response generated within the allograft as well as in draining lymphatic tissue. This implicates not only intrahepatic proliferation of T lymphocytes as a prominent feature of rejection, but also suggests that the liver has a special immunological status comparable to that of lymphatic tissue.

Adult↗

PubMed1998

Synthetic peptides representing discontinuous CD4 binding epitopes of HIV-1 gp120 that induce T cell apoptosis and block cell death induced by gp120.

A vaccine against HIV-1 virus would block initial infection and must target conserved residues. Since initial infection depends on binding of the viral envelope protein gp120 to CD4 on the cell surface, the CD4 binding site of gp120 is a target for vaccine design. To identify the optimal biologically active site, we synthesized a series of 32-mer peptides, based on conserved residues in the C3 and C4 regions of gp120. These included three of five sequence discontinuous residues known to be involved in CD4 binding, one or two of which were substituted with alanine. We also synthesized a 44-mer peptide with an additional branch to incorporate an extra C4 region sequence including a fourth CD4 binding residue. All these peptides used an oxidized Cys-X-Cys bridge to link the discontinuous sequence elements in a manner suggested by the known conserved disulfide bridges in gp120. Polyclonal sera raised to these peptides indicate that they all contain both B and T lymphocyte epitopes. Binding of the peptides to CD4-transfected HeLa cells reveals a hierarchy dependent on the number of relevant CD4 binding residues present. Furthermore, antibody cross-linking of peptides bound to the surface of human T cells results in apoptosis that is similar to the known properties of gp120. The peptide incorporating three CD4 binding residues competitively inhibited gp 120-induced T lymphocyte apoptosis. Thus, we have synthesized novel, branched peptides incorporating conserved discontinuous sequences from two different conserved domains of HIV-1 gp120 that contain T and B lymphocyte epitopes and mimic biological functions of the native protein. These synthetic peptides are candidates for future vaccine development.

Amino Acid Sequence↗

PubMed1998

Altered DNA repair and dysregulation of p53 in IRF-1 null hepatocytes.

The tumor suppressor proteins IRF-1 and p53 are involved in response pathways after DNA damage. In different cell types, IRF-1 and p53 can cooperate to produce cell cycle arrest (embryo fibroblasts) or can independently trigger apoptosis (lymphoid cells). p53 may also regulate DNA repair, but there is no information on IRF-1 and repair. The cell lineage dependency of these effects precludes extrapolation of findings to other tissues of relevance to human cancer. Here, we report the consequences of IRF-1 deficiency for apoptosis, cell cycle arrest, and DNA repair in primary hepatocytes after DNA damage and extend previous work on the role of p53 in hepatocytes. IRF-1-deficient hepatocytes showed reduced DNA repair activity compared with wild-type, as assessed by unscheduled DNA synthesis after UV irradiation (10J/m2) and by host reactivation of a UV-damaged reporter construct. p53-deficient hepatocytes also showed reduced unscheduled DNA synthesis after UV, but there was no impairment of specific repair in host reactivation assays. IRF-1 deficiency did not affect the p53-dependent G1/S arrest after UV irradiation. Hepatocyte apoptosis after UV treatment, previously reported to be independent of p53, was also independent of IRF-1. However, IRF-1 deficiency produced dysregulation of p53, manifested as increased transactivation of a p53-reporter plasmid in undamaged hepatocytes, and accelerated p53 stabilization after DNA damage. Hence, in hepatocytes, IRF-1 is not required for growth arrest or apoptosis after DNA damage, but the results suggest for the first time a role in DNA repair regulation.

Animals↗

PubMed1998

Bile duct cells in primary biliary cirrhosis are 'primed' for apoptosis.

OBJECTIVE: Primary biliary cirrhosis (PBC) is characterized by progressive, immune-mediated destruction of bile ducts (<75 microm diameter) and secondary changes related to cholestasis which may involve apoptosis. In this study we sought to examine the protein expression of genes involved in apoptosis in biliary epithelium of PBC cases. DESIGN: In order to investigate the susceptibility of biliary epithelial cells to apoptosis and their ability to proliferate, we examined the expression of a number of apoptosis related proteins in early and late stage PBC and histologically normal liver control tissue using immunohistochemistry. METHODS: Liver biopsies from 15 early (stages I and II) and 14 late (stages III and IV) cases of PBC and 15 normal cases were examined immunohistochemically for expression of p53, CD95/Fas, bax, bcl-x, bcl-2 and the proliferation marker Ki-67. RESULTS: CD95/Fas, bax and bcl-x were identified in biliary epithelium in 8/15, 11/15 and 8/15 normal biopsies. Weak expression of bcl-2 was found, but p53 was not identified. In cases of PBC surviving bile ducts showed strong bax and bcl-x expression. Inflammatory infiltrates were strongly bcl-2 positive. In cases showing a marked ductular reaction there was increased reactivity for bax and bcl-x in ductules. No change in CD95/Fas or p53 expression was seen. An increase in Ki-67 positive biliary epithelial cells was seen in PBC cases, indicating cell cycle activity. CONCLUSIONS: Bile duct epithelium constitutively expresses several genes involved in the execution of apoptosis but these cells also retain the ability to proliferate.

Apoptosis↗

PubMed1998

Direct detection of eae-positive bacteria in human and veterinary colorectal specimens by PCR.

A PCR test based on the amplification of an eae-specific sequence was designed and evaluated for its ability to directly detect homologous sequences in enteropathogenic Escherichia coli and Citrobacter spp. (amplification of eae open reading frame, 178 bp) in sections of the intestines of humans and animals with colonic lesions. Positive PCR results were observed with eae-positive reference strains of E. coli and Citrobacter rodentium (Citrobacter freundii biotype 4280). Known eae-negative reference strains of E. coli and other laboratory strains of enteric bacteria were negative by the amplification test. The sensitivity of the PCR for detection of eae-positive E. coli and C. rodentium was between 1 and 2 CFU. To detect these sequences directly from sections of fixed colon from human and veterinary sources, PCR conditions were modified by the addition of 0.1 mM 8-methoxypsoralen to eliminate extraneous bacterial DNA from the PCR amplification cocktail without added template. Sections of colon from three pigs experimentally affected with colon lesions due to enteropathogenic (attaching and effacing) E. coli were PCR positive for bacterial eae genome. Sections from control animals were negative. Sections of colon from one of 18 biopsies from confirmed AIDS patients and from 22 of 35 colorectal cancer patients were PCR positive for bacterial eae genome. The PCR test was a simple and quick method of detecting bacterial eae genome in human and veterinary clinical specimens. This method may remove the need for initial culture and detection of the gene by DNA probing from potential associated lesions. The clear relationship of bacteria containing the eae gene with colonic lesions in the pigs and mice indicates that a similar relationship is possible for human patients having similar lesions.

Acquired Immunodeficiency Syndrome↗

PubMed1998

Clinical significance of intrahepatic hepatitis C virus levels in patients with chronic HCV infection.

BACKGROUND: The clinical significance of a single assessment of circulating hepatitis C virus (HCV) RNA and its relation to the level of intrahepatic HCV RNA remains unclear. AIMS: To investigate the relation between intrahepatic HCV levels and clinicopathological characteristics of chronic HCV infection. PATIENTS: Ninety eight consecutive patients with chronic HCV infection were studied; none had received alpha interferon therapy. Of these, 12 patients were repeatedly negative for HCV RNA in serum by reverse transcriptase polymerase chain reaction (RT-PCR). METHODS: After diagnostic laparoscopy and liver biopsy, semiquantitative analysis of intrahepatic HCV RNA levels was carried out by limiting dilution of HCV cDNA. HCV genotypes were assessed in 96 patients by restriction fragment length polymorphism analysis of HCV cDNA. RESULTS: Ten out of 12 patients who were RT-PCR negative for HCV RNA in serum were RT-PCR positive in liver; however, this group had a significantly lower intrahepatic HCV level and serum aminotransferase level than the remaining 86 patients. Histological severity (cirrhosis: n = 10); histological activity index; HCV genotype (genotype 1: n = 41; genotype 2: n = 12; genotype 3: n = 36; genotype 4: n = 7); mode of infection (intravenous drug abuse: n = 58; post-transfusion: n = 10; haemophiliac: n = 4; sporadic: n = 26) and alcohol abuse did not affect the intrahepatic virus level. There was no correlation between patient age, duration of infection, and intrahepatic HCV level. CONCLUSIONS: Intrahepatic virus levels were not determined by host factors (age of patient, mode or duration of infection) or by virus factors (HCV genotype). Repeatedly negative RT-PCR for HCV RNA in serum does not indicate absence of HCV from the liver.

Adult↗

PubMed1998

Microchip systems for immunoassay: an integrated immunoreactor with electrophoretic separation for serum theophylline determination.

A glass microchip is described in which reagents and serum samples for competitive immunoassay of serum theophylline can be mixed, reacted, separated, and analyzed. The device functions as an automated microfluidic immunoassay system, creating a lab-on-a-chip. Electroosmotic pumping was used to control first the mixing of 50-fold-diluted serum sample with labeled theophylline tracer in a 1:1 ratio, followed by 1:1 mixing and reaction with anti-theophylline antibody. The 51-nL on-chip mixer gave the same concentration as dilution performed off-chip, within 3%. A 100-pL plug of the reacted solution was then injected into an electrophoresis separation channel integrated within the same chip. Measurements of free and bound tracer by fluorescence detection gave linear calibration curves of signal vs log[theophylline] between 0 and 40 mg/L, with a slope of 0.52 +/- 0.03 and an intercept of -0.04 +/- 0.04 after a 90-s reaction time. A detection limit of 0.26 mg/L in serum (expressed before the dilution step, actual concentration of 1.3 micrograms/L at the detector) was obtained. Recovery values were 107% +/- 8% for 15 mg/L serum samples.

Automation↗

PubMed1997

Microchip-based capillary electrophoresis of human serum proteins.

The separation and relative quantitation of human serum proteins is important to the clinical diagnosis of various states of disease. Microchip-based capillary electrophoresis (CE) of human serum proteins offers several advantages over sodium dodecyl sulfate-poly(acrylamide) gel electrophoresis and conventional CE methods, including decreased sample consumption and analysis time and the possibility of on-chip sample manipulation (dilution, labelling, etc.). The microchip used in these studies was designed to allow for on-chip, post-separation labelling of the proteins and subsequent laser-induced fluorescence detection. 2-Toluidinonaphthalene-6-sulfonate (TNS) is a virtually non-fluorescent reagent which, upon non-covalent association with the protein and excitation at 325 nm, produces a fluorescent product with an emission maximum near 450 nm. After optimization of buffer conditions (100 mM borate with 2 mM lactate, pH 10.5), individual serum proteins (IgG to mimic the gamma zone, transferrin the beta zone, alpha-1-antitrypsin the alpha 1 zone and albumin its own zone) were successfully resolved on-chip, as was a "synthetic" serum solution composed of a mixture of all four of the previously mentioned proteins. Analysis of all five protein zones in a true human serum sample, however, has not yet been achieved on-chip due to the poor sensitivity of the TNS label for several of the serum proteins.

Blood Protein Electrophoresis↗