Search PubMed⌕ Search

Biomedical subjects

G S Morris

Publications and source records attributed to G S Morris.

52 records · Page 3Linked to original sources
PubMed1986

Prenatal exclusion of purine nucleoside phosphorylase deficiency.

We report on the prenatal exclusion of purine nucleoside phosphorylase (PNP) deficiency in a fetus whose parents were known to be heterozygotes for the enzyme defect. Prenatal investigation was performed in the 16th week of gestation on amniotic fluid and cultured amnion cells using sensitive techniques. The results suggested that the fetus was either normal or a heterozygote. PNP activity in cord red cells confirmed the heterozygous status of the baby.

Adult↗
PubMed1985

Use of a fundamental elution protocol for the development of reversed-phase high-performance liquid chromatography enabling rapid simultaneous determination of purines, pyrimidines and allied compounds commonly found in human biological fluids.

Many high-performance liquid chromatographic methods for the analysis of nucleosides and bases have been developed for a specific requirement. Most are not directly applicable to the separation of all the purines and pyrimidines found in biological fluids. A method has been developed to separate a wide range of compounds found in human urine and plasma in a single run without unduly long run times. Special attention has been paid to sample preparation. Isocratic systems derived from this method for the analysis of specific compounds and enzyme assay mixtures over even shorter periods are described.

Body Fluids↗
PubMed1985

Human B lymphocytes and thymocytes but not peripheral blood mononuclear cells accumulate high dATP levels in conditions simulating ADA deficiency.

Inherited adenosine deaminase (ADA) deficiency is associated with a lymphospecific cytotoxicity affecting both dividing and non-dividing cells. The metabolic basis for this was investigated using different cell types and the potentially toxic metabolite 2'-deoxyadenosine (dAR) in short-term experiments under physiological conditions simulating ADA deficiency (1 mM Pi 8.7 microM dAR). In the uncultured cells, [8-14C] dAR alone was metabolized almost completely only by thymocytes and tonsil-derived B-lymphocytes. The greater percentage of counts (greater than 75%) were in the medium (deoxyinosine, hypoxanthine). Cellular counts were predominantly in adenine nucleotides, and to a lesser extent guanine nucleotides. Interestingly, both thymocytes and tonsil-derived B-lymphocytes, and a partially ADA deficient B lymphoblast line, accumulated detectable amounts of dATP even in the absence of ADA inhibition. Peripheral blood lymphocytes (PBMs) did not, and showed little dAR metabolism. In experiments simulating ADA deficiency varying amounts of 2'-deoxycoformycin (2'dCF) were needed to completely inhibit ADA (20-60 microM), with thymocytes requiring the highest amount. ADA inhibited thymocytes and tonsillar B-lymphocytes accumulated very high dATP levels, which were sustained to an equal extent by both over a 60-min period; PBMs accumulated the lowest values. Results in cultured cells reflected findings in previous studies. Some counts were also found in ATP by a route excluding ADA or PNP. These results again question the hypothesis that B-cells are more resistant than T-cells to the toxic effects of dAR because of an inability to accumulate and sustain elevated dATP levels and underline the lack of comparability between enzyme activity in intact as distinct from lysed cells. They cast doubt on the validity of cultured cells as a model for ADA deficiency and suggest the observed toxicity in some instances might result from altered ATP or GTP pools through inadequate ADA inhibition. They indicate that combined immunodeficiency in ADA deficiency could relate to an equal sensitivity of B-cells and T-cell precursors to the toxic effects of dATP accumulation.

Adenosine Deaminase↗
PubMed1985

Superoxide radicals, immunodeficiency and xanthine oxidase activity: man is not a mouse!

The importance of intact adenosine deaminase (ADA) activity in the generation of superoxide anion by xanthine oxidase has been disputed in studies using human neutrophils or mouse macrophages. The latter demonstrated a positive correlation between ADA activity and superoxide production during phagocytosis. The immunodeficiency in inherited ADA deficiency was related to a defect in this process. Since there is considerable interspecies variation in the tissue distribution of xanthine oxidase, the metabolism of [8-14C]deoxyadenosine (dAR), the toxic metabolite which accumulates in inherited ADA deficiency, was investigated in human peritoneal macrophages. Evaluation of the distribution of radiolabel in both cell and medium demonstrated that human macrophages with intact ADA metabolize dAR under physiological conditions to deoxyinosine and hypoxanthine exclusively. The hypoxanthine is further metabolized within the cell to ATP and GTP, via IMP. No xanthine or uric acid could be detected, confirming that in human macrophages xanthine oxidase activity is insignificant, as it is in most other human cells and tissues, except liver and intestinal mucosa. Thus production of superoxide radicals in such cells via this route would be impossible, and consequently unaffected either by ADA deficiency or the xanthine oxidase inhibitor allopurinol.

Deoxyadenosines↗
PubMed1985

EHNA is a poor inhibitor of deoxyadenosine catabolism in cultured human lymphocytes.

Erythro-9-(2-hydroxy-3-nonyl)-adenine (EHNA) has been used by many workers as enzyme inhibitor in vitro to simulate the in vivo situation in inherited adenosine deaminase (ADA) deficiency. In this study the metabolism of 8-14C deoxyadenosine (dAR) has been followed in cultured lymphocytes from patients deficient in enzymes associated with the catabolism and salvage of dAR, in the absence and presence of 10 microM EHNA. The results show that EHNA, at these concentrations, does not prevent the catabolism of dAR and thus does not provide a valid model for investigating the toxicity to the immune system in inherited ADA deficiency.

Adenine↗
PubMed1984

Metabolism of deoxynucleosides by lymphocytes in long-term culture deficient in different purine enzymes.

The metabolism of 8-14C-labelled 2'-deoxyadenosine (dAR) and 2'-deoxyguanosine (dGR) has been investigated using lymphocytes in long-term culture transformed by Epstein-Barr (EB) virus (B-cells) from eight patients with different inherited purine enzyme defects. The use of such lines enabled accurate mapping of the route of metabolism by acting as a 'trap' for the radiolabel at specific points. With either substrate (25 microM) most of the label was recovered in the medium. Using dAR, less than 30% of the radiolabel was incorporated into cellular nucleotides. For dGR, values were less than 18%. Studies with dAR alone confirmed the principal route of metabolism was to hypoxanthine, with further metabolism (by lines with intact salvage pathways) to ATP and GTP in the ratio of approximately 4:1. Lack of accumulation of deoxyinosine in the purine nucleoside phosphorylase (PNP) deficient line, or hypoxanthine in the hypoxanthine guanine phosphoribosyltransferase (HGPRT) deficient line, using dAR together with the adenosine deaminase (ADA) inhibitor 2'-deoxycoformycin (dCF) at 10 microM, confirmed the effectiveness of ADA inhibition. Nevertheless, some ATP was still formed by all lines in the presence of dCF by a route as yet unknown. Only the ADA deficient lines formed dATP with dAR alone. However, some dATP was formed by all lines in the presence of dCF. A partially HGPRT deficient line formed extremely high dATP levels, well in excess of those formed by the T-cell line CEM. Studies with dGR revealed some interesting differences, a large proportion of the substrate being metabolized predominantly to xanthine by most enzyme deficient lines. In the PNP deficient line most of the substrate remained unmetabolized, but some dGTP was formed. No other enzyme deficient line formed any dGTP--with or without the PNP inhibitor 8-aminoguanosine (8-NH2GR)--with one exception. Again this was the partially HGPRT deficient line, which with the inhibitor again formed more dGTP than the T-cell line. Within the cells most of the substrate was metabolized to GTP, except in the PNP, and totally HGPRT deficient lines. Levels of GTP formed were not altered by the inhibitor, reflecting the lack of effective PNP inhibition by 8-NH2GR. Some counts were also found in ATP and IMP, confirming the existence of this route in mammalian cells of lymphoid origin. The results also support previous studies by us using cell lines with intact purine pathways, which demonstrated that, contrary to current beliefs, some B-cell lines are capable of accumulating high levels of deoxynucleotides.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Deaminase Inhibitors↗
PubMed1984

dATP accumulation and ATP depletion in platelets in adenosine deaminase deficiency: significance for the immune response?

High levels of dATP and dADP, accompanied by ATP depletion, were found in the platelets of two ADA-deficient children with severe combined immunodeficiency (SCID). In vitro studies demonstrated that even normal platelets had the ability to make dATP from deoxyadenosine (dAR) under physiological conditions. This capability was greatly enhanced by conditions simulating ADA deficiency. These results question whether the platelet has a specific role in the normal immune response.

Adenosine Deaminase↗
PubMed1984

B cells as well as T cells form deoxynucleotides from either deoxyadenosine or deoxyguanosine.

Enzyme inhibitors used to simulate the inherited immunodeficiency diseases, adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) deficiency, have been assessed in cultured human lymphocytes. Only 2'-deoxycoformycin (dCF) completely inhibited ADA in T and B cells at concentrations in excess of 5 microM. Erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) and 8-amino guanosine (8-NH2GR) did not inhibit ADA or PNP completely at any concentration. Detailed metabolic experiments comparing viability and deoxynucleotide accumulation showed that B cell lines of malignant origin also accumulated high levels of dATP from 2'-deoxyadenosine (dAR), and dGTP from 2'-deoxyguanosine (dGR) as effectively as T cells--even without inhibitors, however, dAR reduced cell viability only when ADA was inhibited by dCF, whilst dGR was equally toxic with or without inhibitor, even to a line which accumulated no dGTP. These experiments indicate that cultured lymphocytes, using either EHNA or 8-NH2GR as enzyme inhibitor, are not valid models of the toxicity to the immune system in inherited ADA or PNP deficiency. They demonstrate that the ability to accumulate high levels of dATP or dGTP is not exclusive to T cells and that the in vitro toxicity of dAR or dGR could relate to the use of excess substrate and/or accumulation in different nucleotide, not deoxynucleotide pools.

Adenine↗
PubMed1983

Ultrastructural observations of the fetal rat heart during prolonged gestation.

The ultrastructure of myocardial cells was examined from fetal rat hearts during experimentally induced prolonged gestation (GD 23, 24 and 25), and compared to those from term fetuses (GD 22) and newborn animals (N1, N2, N3). Gestation was prolonged by daily subcutaneous injections of 5 mg progesterone to gravid females on days 20 through 24. The 4 chambers of the heart were assessed individually for structural differences by morphometric analysis. Comparison between the 2 atria at each stage revealed no differences as was the case between the 2 ventricles. At term and throughout the newborn period, atrial and ventricular cells were characterised by numerous mitochondria, moderate amounts of glycogen, few lipid droplets and membrane-bounded granules, which, moreover, were peculiar to atrial tissue. During prolonged gestation (GD 24 and 25), there was a statistically significant increase in the size of mitochondria in both atria and ventricles and a reduction of membrane-bounded atrial granules. Whole heart glycogen content was also assessed, biochemically, and found reduced on GD 24 and 25. In addition to the morphological and biochemical findings, fetal rat heart weights were obtained from each designated day of prolonged gestation and compared to newborn rat hearts of comparable age. A statistically significant increase in heart weight was noted on GD 24. Meconium staining, which is indicative of fetal distress was also observed on GD 24 and 25. The morphological and biochemical changes observed suggest a response by the fetal heart to stressful conditions, namely hypoxia and hypoglycemia, which are known to exist during prolonged gestation.

Animals↗
PubMed1981

Relationship between lung intra and extracellular DSPC in fetal and neonatal rats.

Using lamellar bodies (LB) as an index of intracellular disaturated phosphatidyl choline (DSPCin) and biochemical measurements of the total lung DSPC, we studied the relationship between the intra and extracellular DSPC (DSPCex) in the lungs of fetal and neonatal rats. A linear correlation was found between the number of LB and the amount of DSPC in fetal lungs. A similar correlation was found in fetal lungs after lung lavage. The difference between the two corresponded to the DSPCex, which was greater than anticipated. Prior to birth there was a massive release of DSPC, into the extracellular space, which was lost from the lungs during labor and upon initiation of respiration at birth. The DSPCin, however, continued to increase during labor and the first 5 min after birth. Thereafter release of DSPCin and accumulation in extracellular space occurred. After age 12 hours, there was a gradual reduction in DSPCex till 24 hours. Thereafter, a linear correlation between number of LB and total DSPC resumed. The results suggest that lung lavage promotes release of DSPCin and that excess DSPCex, from birth to 24 hours, is removed by two different mechanisms: initially through the airways and subsequently by alveolar macrophages.

Animals↗
PubMed1980

Development of fetal rat lung during prolonged gestation.

Pregnancies were prolonged by daily subcutaneous injection of progesterone to the maternal rats from day 20 through 24 of gestation (Term is 22 days). The fetuses were harvested by cesarean section on gestation days (GD) 21 through 25. In spite of reduction in placental weight the fetal body weight increased during prolonged gestation. Both lung wet and dry weights decreased resulting in smaller lung per body weight. Compared to term fetuses, post term fetuses (GD 25) exhibited a 10% reduction in lung cell numbers, and a 90% reduction in lung glycogen. Lung disaturated phosphatidyl choline expressed per lung increased up to GD 23 and subsequently decreased by GD 25. Surface tension of lung extracts and pulmonary lavage fluids increased with prolongation of gestation. Histological studies revealed a decrease in the number of lamellar bodies in the Type II cells and a gradual reduction in the size of terminal sacs leading to areas of complete atelectasis at GD 25. It is suggested that lung hypocellularity, depletion of lung glycogen, reduction in intracellular reserve of phospholipids, loss of pulmonary fluid leading to atelectasis, and loss of surface activity of pulmonary fluid may be predisposing factors for the development of respiratory distress in postmaturity.

Animals↗