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Biomedical subjects

D J Harrison

Publications and source records attributed to D J Harrison.

At least 145 records · Page 8Linked to original sources
PubMed1993

Performance of subcutaneously implanted needle-type glucose sensors employing a novel trilayer coating.

A miniature, needle-type glucose sensor based on a new trilayer membrane configuration has been prepared and evaluated both in vitro and in vivo. The perfluorinated ionomer, Nafion, was used as a protective, biocompatible, outer coating, and poly(o-phenylenediamine) as an inner coating to reduce interference by small, electroactive compounds. Glucose oxidase immobilized in a bovine serum albumin matrix was sandwiched between these coatings. The entire sensor assembly of Pt working electrode and Ag/AgCl reference electrode was 0.5 mm in diameter and could be inserted subcutaneously through an 18-gauge needle. The sensor current closely followed the plasma glucose level during a glucose tolerance test in active dogs, with a delay of 3 min, corresponding to the known lag time for subcutaneous glucose levels. The sensor remained functional after 1 week of implantation, but failed after 2 weeks due to degradation of the reference electrode. In vitro tests in pH 7.4 buffer or whole blood show the sensors have good selectivity, sensitivity of about 25 nA/mM, precision of 2-5%, and a 90% response time of 33 s. Stabilization following polarization requires 10-30 min in vitro and 30-40 min in vivo.

Animals↗
PubMed1993

Thymocyte apoptosis induced by p53-dependent and independent pathways.

Death by apoptosis is characteristic of cells undergoing deletion during embryonic development, T- and B-cell maturation and endocrine-induced atrophy. Apoptosis can be initiated by various agents and may be a result of expression of the oncosuppressor gene p53 (refs 6-8). Here we study the dependence of apoptosis on p53 expression in cells from the thymus cortex. Short-term thymocyte cultures were prepared from mice constitutively heterozygous or homozygous for a deletion in the p53 gene introduced into the germ line after gene targeting. Wild-type thymocytes readily undergo apoptosis after treatment with ionizing radiation, the glucocorticoid methylprednisolone, or etoposide (an inhibitor of topoisomerase II), or after Ca(2+)-dependent activation by phorbol ester and a calcium ionophore. In contrast, homozygous null p53 thymocytes are resistant to induction of apoptosis by radiation or etoposide, but retain normal sensitivity to glucocorticoid and calcium. The time-dependent apoptosis that occurs in untreated cultures is unaffected by p53 status. Cells heterozygous for p53 deletion are partially resistant to radiation and etoposide. Our results show that p53 exerts a significant and dose-dependent effect in the initiation of apoptosis, but only when it is induced by agents that cause DNA-strand breakage.

Animals↗
PubMed1993

Regulation of cytochrome P450 gene expression in human colon and breast tumour xenografts.

It is extremely difficult to identify the factors which regulate the expression of drug-metabolising enzymes in man. To address this problem, we have developed a model involving the use of human tumours grown as xenografts in immune deficient mice. Mice bearing human colon or breast tumours as xenografts were challenged with a range of compounds, known from animal studies to be inducers of cytochrome P450s from a variety of gene families. Almost all of the compounds tested could induce human tumour P450 expression, measured either by Western blot or immunohistochemical analysis. Indeed, the levels of P450s from several distinct gene families or subfamilies including CYP2A, CYP2B, CYP2C, CYP3A and CYP4A were induced. Of particular interest was the profound induction of human P450s by 1,4 bis 2-(3,5dichloro-pyridyloxybenzene)(TCPOBOP), a compound which exhibits a marked species specificity in its ability to induce P450 expression in experimental animals. Induction of a human CYP2B protein by this compound was confirmed by Northern blot analysis and in situ hybridisation for mRNA, indicating that induction occurred at the level of transcription. These studies have a variety of implications: they provide a method for approaching the previously intractable problem of how environmental, hormonal and metabolic factors regulate human P450 genes and other genes involved in drug metabolism; they demonstrate that human tumours express P450s constitutively and that the levels of these proteins can be modulated by exogenous agents.

Animals↗
PubMed1993

Mice with DNA repair gene (ERCC-1) deficiency have elevated levels of p53, liver nuclear abnormalities and die before weaning.

Defects in nucleotide excision repair are associated with the human condition xeroderma pigmentosum which predisposes to skin cancer. Mice with defective DNA repair were generated by targeting the excision repair cross complementing gene (ERCC-1) in the embryonic stem cell line, HM-1. Homozygous ERCC-1 mutants were runted at birth and died before weaning with liver failure. Examination of organs revealed polyploidy in perinatal liver, progressing to severe aneuploidy by 3 weeks of age. Elevated p53 levels were detected in liver, brain and kidney, supporting the hypothesised role for p53 as a monitor of DNA damage.

Animals↗
PubMed1993

Primary culture of bovine gall bladder epithelial cells.

Gall bladder epithelial cells serve numerous biological functions and abnormalities in their function are important in the pathogenesis of several gall bladder diseases. Direct studies on cell function are rare due to lack of reliable methods to culture this epithelium. This study reports a reliable and reproducible method of harvesting and culturing gall bladder epithelial cells. Normal bovine gall bladder epithelium, obtained within 20 minutes of slaughter, was rinsed with modified Hanks's balanced salt solution, the mucosa separated and incubated in trypsin--EDTA solution at 37 degrees C. The cells were isolated and resuspended in Dulbeco's modification of Eagles' medium containing 10% fetal calf serum and, after filtration and centrifugation, were plated under aseptic conditions. The growth rate was established by flow cytometry and the morphological characteristics of the growing cells by electron microscopy. Gall bladder epithelial cells grew successfully and visible clusters of cells were present by day two, confluency being reached at 8 to 10 days in collagen coated plates and 12 to 14 days in uncoated plates. Electron microscopy showed typical gall bladder epithelia with microvilli, tight junctions, and mucus droplets. This method proved reliable and reproducible for the culture of gall bladder epithelial cells and should allow direct studies of the biological properties of these cells in human tissue.

Animals↗
PubMed1992

Glutathione S-transferase expression in the human testis and testicular germ cell neoplasia.

Glutathione S-transferase (GST) isoenzyme expression is altered in a variety of neoplasms and the enzymes are implicated in metabolism of carcinogens and resistance to drugs, including cisplatin. We have studied GST Alpha, Pi, Mu and microsomal isoenzyme expression by immunohistochemistry in normal and cryptorchid testes, intratubal germ cell neoplasia (ITGCN), seminoma and non-seminomatous germ cell tumours. In 16 stage II-IV malignant teratoma intermediate (MTI) both orchidectomy and post-treatment residual surgical masses were studied. All four isoenzymes were strongly expressed in Leydig and Sertoli cells. GST Pi was absent from normal spermatogonia but strongly expressed by the neoplastic germ cells of ITGCN and seminoma. GST Pi was strongly expressed in all elements of teratoma, irrespective of differentiation. There were no qualitative differences in expression between primary and post-chemotherapy metastases. GST Alpha expression in teratoma correlated with epithelial differentiation. GSTs may be important in normal spermatogenesis and protection of germ cells from teratogens and carcinogens. They may have a role in testicular tumour drug resistance but this role is not well defined. GST Pi is a new marker for ITGCN.

Adolescent↗
PubMed1992

Modulation of glutathione S-transferases and glutathione peroxidase by the anticarcinogen butylated hydroxyanisole in murine extrahepatic organs.

Induction of glutathione S-transferases (GST) by the anticarcinogen butylated hydroxyanisole (BHA) has been examined in lung, kidney and small intestine of male and female BALB/c mice. BHA produced maximal induction of GST in the gut and although it increased GST levels in the kidney, it had little effect on pulmonary GST. Dietary BHA induced Alpha (Ya and Yk), Mu (Yb) and Pi (Yf) class GST subunits at least 10-fold in the small intestine but, by contrast, selenium-dependent glutathione peroxidase activity was reduced by approximately 4-fold in this organ following BHA treatment. In the kidney, all of the GST subunits, apart from Yk in males, showed modest levels of induction by BHA. However, a pronounced sex difference in the expression of renal alpha class subunits in both control and BHA-treated mice was observed, with female mice expressing approximately 4-fold greater levels of Ya and Yk than male mice. All renal GST were localized primarily in the proximal tubules. Dietary BHA was found to have the least inductive effect in the lung, where the GST were localized solely in the bronchi. The pulmonary Mu class GST subunits were induced approximately 2-fold by BHA; the expression of other GST was marginally increased by this inducer. Alpha class GST was also subject to sexual differentiation in the lung with female mice possessing higher levels of Yc and Yk than males. The Ya-type subunit was not detected in the lung nor was it induced by BHA.

Animals↗
PubMed1992

Evidence of hydrogen ion secretion from the human gall bladder in vitro.

Gall bladder bile is more acid that hepatic bile and this has been attributed to bicarbonate absorption by the gall bladder epithelium. The aim of this study was to investigate in vitro the acid base changes that occur across the human gall bladder mucosa. Fresh gall bladder tissue was obtained at cholecystectomy and placed in an Ussing Chamber and perfused with Ringer-Krebs glucose bicarbonate solution. The viability of the gall bladder was assessed by measuring the potential differences across the epithelium and by the morphology of the epithelial cells at the end of the experiments. Aliquots from the solutions were taken at two, 45 and 70 minutes and pCO2, hydrogen ion and bicarbonate concentrations were measured. In the mucosal side of the chamber a consistent and significant decrease was observed from two minutes to 70 minutes in bicarbonate concentration while pCO2 and hydrogen ion concentrations significantly increased. The degree of inflammation correlated well with the ability for acidification, the more inflamed the tissue the less its ability to acidify. When the gall bladder was exposed to amiloride or sodium free solution acidification was abolished in the mucosal side. When tissue metabolism was irreversibly inhibited by exposure to formaldehyde, hydrogen ion concentration and pCO2 were significantly decreased in the mucosal side of the chamber compared with the viable gall bladder. The human gall bladder is capable of secreting acid and this may be an important mechanism for preventing calcium precipitation and gall stone formation.

Bicarbonates↗
PubMed1992

Molecular genetics of the human cytochrome P450-dependent monooxygenases.

In this work, the role of genetic as well as environmental factors in determining cytochrome P450 isozyme levels in man have been studied. Simple DNA based assays for the identification of individuals nulled at the CYP2D6 locus are described and have been applied to investigate whether this gene defect is associated with altered cancer susceptibility. In contrast to literature reports, in no cancer type were poor metabolizers underrepresented, indeed in several cancers the mutant allele frequency was increased. A model using human tumours grown as xenografts is described that should help elucidate the factors which regulate P450 levels in man.

Chromosome Mapping↗
PubMed1991

Variation in the expression of Mu-class glutathione S-transferase isoenzymes from human skeletal muscle. Evidence for the existence of heterodimers.

The cytosolic glutathione S-transferases (GST) from human skeletal muscle were purified by a combination of affinity chromatography and anion-exchange chromatography followed by either chromatofocusing or hydroxyapatite chromatography. Pi-class and Mu-class GST, but not Alpha-class GST, were isolated from muscle. In addition to a Pi-class GST subunit, which exists as a homodimer, this tissue also contains a total of three distinct neutral-type Mu-class GST subunits, which hybridize to form homodimers or heterodimers. The neutral-type subunits are referred to as N1-N3 and are defined by the decreasing isoelectric points of the homodimers; GST N1N1, N2N2 and N3N3 have estimated pI values of 6.1, 5.3 and less than 5.0 respectively. SDS/PAGE showed that N1, N2 and N3 have Mr values of 26,700, 26,000 and 26,300 respectively. The N1, N2 and N3 subunits are catalytically distinct, with N1 possessing a high activity for trans-4-phenylbut-3-en-2-one and N2 having high activity with 1,2-dichloro-4-nitrobenzene. In skeletal muscle the expression of the N1 subunit, but not of N2 and N3 subunits, was found to differ from specimen to specimen. The N1 subunit was absent from about 50% of samples examined, and the purification results from two different specimens are presented to illustrate this inter-individual variation. Skeletal muscle from one individual (M1), which did not express N1, contained only GST N2N2, N2N3 and pi, whereas the second sample examined (M2) contained GST N1N2, N2N2 and N2N3 as well as GST pi. N-Terminal amino acid sequence analysis supported the electrophoretic evidence that the N2 subunit in GST N1N2, N2N2 and N2N3 represents the same polypeptide. The peptides obtained from CNBr digests of N2 were subjected separately to automated amino acid sequencing, and the results indicate that N2 is distinct but closely related to the protein encoded by the human Mu-class cDNA clone GTH4 [DeJong, Chang, Whang-Peng, Knutsen & Tu (1988) Nucleic Acids Res. 16, 8541-8554]. GST N2N2 is probably identical with GST 4 [Board, Suzuki & Shaw (1988) Biochim. Biophys. Acta 953, 214-217], as over the 24 N-terminal residues of GST 4 there is complete identity between the two enzymes. Our data suggest that the GST 1 and GST 4 loci are part of the same multi-gene family.

Aged↗
PubMed1991

Preliminary in vivo biocompatibility studies on perfluorosulphonic acid polymer membranes for biosensor applications.

The first biocompatibility studies on the DuPont perfluorosulphonic acid polymer (Nafion) are presented. Presterilized samples of commercially cast and solution cast Nafion membranes were implanted subcutaneously, intraperitoneally and intravenously in male Sprague-Dawley rats. Scanning electron microscopy and histological examination of explanted samples and surrounding tissues reveal little, if any, evidence of acute or chronic foreign body inflammatory response. The fibrous capsules surrounding the implant remain nominally thin (less than 100 microns) after more than 3 months in situ, while the surrounding tissue remains well vascularized. Nafion polymer exhibits sufficient biocompatibility to make it a viable candidate for some implantable biosensor applications. It may, however, be necessary to compensate for the effect of the progression of fibrous encapsulation on sensor performance, particularly during the acute response stage.

Abdominal Muscles↗
PubMed1991

Antibodies to neutrophil cytoplasmic antigens: serologic marker for Sweet's syndrome.

Seven patients with a clinical and histologic diagnosis of Sweet's syndrome were tested for the presence of circulating antibodies to neutrophil cytoplasmic antibodies. Six of the seven patients had detectable antibodies to neutrophil cytoplasmic antibodies at a serum dilution of at least 1:20. Antibodies to neutrophil cytoplasmic antibodies were not found in serum from patients with a range of cutaneous diseases, some known to cause clinical or histologic confusion with Sweet's syndrome. The detection of circulating antibodies to the neutrophil cytoplasm may be of possible diagnostic value in Sweet's syndrome.

Adult↗
PubMed1991

The effect of the somatostatin analogue SMS 201-995 on experimental pancreatic carcinogenesis in the Syrian golden hamster.

Somatostatin analogues have been suggested as possible therapy for human pancreatic cancer. This paper investigates the effect of the somatostatin analogue SMS 201-995 (Sandoz) in the Syrian golden hamster model of nitrosamine-induced pancreatic carcinogenesis. Step-wise increasing doses of i.v. SMS 201-995 suppressed pancreatic juice output from a median basal value of 212 mg/kg body wt/h (Q1:Q3 = 121:334) to a median basal value of 70 mg/kg body wt/h during infusion of 5 micrograms/kg body wt/h of SMS 201-995 (Q1:Q3 = 64:102, P less than 0.05). Chronic s.c. injection of 5 and 10 micrograms/kg body wt SMS 201-995 twice daily for 3 days each week, did not affect pancreatic wet wt or pancreatic total DNA content after 1 or 6 weeks of treatment when compared to controls. The most interesting and unexpected finding in our study was that SMS 201-995 seemed to promote pancreatic carcinogenesis when administered in low dosage. More SMS 201-995 treated animals receiving 5 micrograms/kg body wt developed invasive pancreatic adenocarcinoma than controls after 15 weeks of carcinogen (4/10 animals versus 0/10, P less than 0.05, Fisher's exact test) and pancreatic involvement by tumour was more extensive (17/75 pancreatic blocks affected versus 0/71, P less than 0.001). When carcinoma in situ and microcarcinomata were analysed with invasive lesions, animals injected with 5 micrograms/kg body wt SMS 201-995 were still significantly more affected than controls (33/75 blocks versus 9/71, P less than 0.001). Hamsters injected with the higher SMS 201-995 dose (10 micrograms/kg body wt) did not show any increase in malignancy over the controls. These results suggest that the effect of SMS 201-995 on pancreatic carcinogenesis in the Syrian hamster is complex and varies with dose administered. Further work is required before its use on man can be justified.

Animals↗
PubMed1991

Heterogeneity of glutathione S-transferase isoenzyme expression in renal disease.

Recent work has suggested that glutathione S-transferase (GST) enzymuria may be used to assess renal injury in transplant kidneys. There is little work investigating the possibility of using glutathione S-transferase enzymuria to assess other renal diseases and this study was undertaken to evaluate the localisation of GST isoenzymes in various glomerular and tubular pathologies so that the specificity of these enzymes as markers of tubular injury could be defined. Immunostaining was carried out to establish the location of alpha and pi class GST in renal biopsies from patients with a wide variety of renal diseases including membranous glomerulonephritis, minimal-lesion glomerulonephritis, mesangial proliferative glomerulonephritis, loin pain haematuria syndrome, and renal allograft rejection. In the cases of glomerulonephritis studied, alpha class GST was detected in proximal tubules and pi class GST in distal convoluted tubules, podocytes, and Bowman's capsule. The majority of cases of glomerulonephritis showed a heterogeneous pattern of expression of both isoenzymes: that is, there was variation in intensity of staining both in single tubules and also between tubules as opposed to the uniform staining pattern observed in normal kidneys. The location of enzymes in the cases of glomerulonephritis was the same as that in normal kidneys and we were unable to demonstrate any de novo expression of GST isoenzymes. However, seven out of ten renal allograft biopsies showed expression of pi class GST in proximal tubules which were atrophic. Provided there is no significant tubular atrophy, urinary release of these enzymes may be used to localise renal tubular injury.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1991

Glutathione S-transferases in human liver cancer.

An immunohistochemical study of glutathione S-transferase (GST) expression in hepatocellular carcinoma and cholangiocarcinoma is described. Unlike most animal models of hepatic malignancy pi class GST was not consistently overexpressed in hepatocellular carcinoma. This tumour type either predominantly expressed alpha class GST or failed to express GST. By contrast, cholangiocarcinoma always expressed pi class GST, presumably reflecting the tissue of origin, since in human biliary epithelium pi class GST is the predominant GST. The variable expression of pi class GST which was observed in hepatocellular carcinoma may reflect transformation of hepatocytes damaged by toxins, since this GST can be induced after a chemical insult such as alcohol. As well as indicating the biochemical heterogeneity of hepatocellular carcinoma with respect to GST, this study indicates the need for further study of the nature of inherent drug resistance in these tumour types.

Adenoma, Bile Duct↗
PubMed1991

Distribution of glutathione S-transferase isoenzymes in human ovary.

Glutathione S-transferases (GST) are drug-metabolizing and detoxification enzymes involved in the intracellular transport and metabolism of steroid hormones. We studied expression of pi, alpha, mu and microsomal GST by immunohistochemistry in normal human ovaries at different stages of the menstrual cycle and pregnancy and after the menopause. Antibodies were raised in rabbits to purified GST subunits and formalin-fixed, paraffin-embedded sections were studied using the peroxidase-antiperoxidase method. Staining density was graded from very strong to negative. All four isoenzymes were identified in the ovary and their distribution was heterogeneous. The staining pattern of follicles varied with the stage of the menstrual cycle for each isoenzyme. All the ovaries contained abundant GST pi in stroma. GST alpha is closely associated with the glutathione-dependent enzyme delta-5,3-ketosteroid isomerase, which catalyses the conversion of pregnenolone to progesterone and dehydroepiandrosterone to androstenedione. GST alpha was localized to the steroid-producing cells and thus may be useful in studying ovaries in conditions where there are assumed alterations in steroid production.

Corpus Luteum↗