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W R Osborne

Publications and source records attributed to W R Osborne.

At least 55 records · Page 3Linked to original sources
PubMed1988

Design of vectors for efficient expression of human purine nucleoside phosphorylase in skin fibroblasts from enzyme-deficient humans.

Purine nucleoside phosphorylase (PNP; purine-nucleoside orthophosphate ribosyltransferase, EC 2.4.2.1) deficiency is an inherited disorder associated with a severe immune defect that is fatal. Enzyme replacement therapy is an attractive approach to treatment of this disease. To this aim we constructed retroviral vectors containing a human PNP cDNA and a selectable gene encoding neomycin phosphotransferase. PNP expression was controlled by either the early promoter from simian virus 40, the immediate early promoter from human cytomegalovirus, or the retroviral promoter. Cultured skin fibroblasts from two unrelated PNP-deficient patients that were infected with these vectors expressed mean PNP activities of 0.03, 0.74, and 5.9 mumol/hr per mg of protein, respectively. The latter infectants had PNP activities eight times the level of 0.74 mumol/hr per mg of protein observed in normal skin fibroblasts, enabling rapid metabolism of exogenous deoxyguanosine, the cytotoxic metabolite that accumulates in the plasma of PNP-deficient patients. These experiments indicate that viral long terminal repeat was the strongest promoter for expression of PNP and suggest the potential of human skin fibroblasts as vehicles for therapeutic gene expression.

Cells, Cultured↗
PubMed1987

Efficient retrovirus-mediated transfer and expression of a human adenosine deaminase gene in diploid skin fibroblasts from an adenosine deaminase-deficient human.

Skin fibroblasts might be considered suitable recipients for therapeutic genes to cure several human genetic diseases; however, these cells are resistant to gene transfer by most methods. We have studied the ability of retroviral vectors to transfer genes into normal human diploid skin fibroblasts. Retroviruses carrying genes for neomycin or hygromycin B resistance conferred drug resistance to greater than 50% of the human fibroblasts after a single exposure to virus-containing medium. This represents at least a 500-fold increase in efficiency over other methods. Transfer was achieved in the absence of helper virus by using amphotropic retrovirus-packaging cells. A retrovirus vector containing a human adenosine deaminase (ADA) cDNA was constructed and used to infect ADA-fibroblasts from a patient with ADA deficiency. The infected cells produced 12-fold more ADA enzyme than fibroblasts from normal individuals and were able to rapidly metabolize exogenous deoxyadenosine and adenosine, metabolites that accumulate in plasma in ADA-deficient patients and are responsible for the severe combined immunodeficiency in these patients. These experiments indicate the potential of retrovirus-mediated gene transfer into human fibroblasts for gene therapy.

Adenosine↗
PubMed1986

Nucleoside kinases in T and B lymphoblasts distinguished by autoradiography.

Nucleoside kinases catalyze the initial step leading to the accumulation of deoxypurine nucleotides that occurs in patients with inherited deficiencies of adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) and purine-nucleoside phosphorylase (purine-nucleoside:orthophosphate ribosyltransferase, EC 2.4.2.1). This accumulation is thought to interfere with DNA synthesis in lymphocytes and, thus, to cause the immune defects associated with these enzymopathies. However, there is controversy about the identity of the nucleoside kinases that are responsible for intracellular phosphorylation of deoxyadenosine in adenosine deaminase deficiency and deoxyguanosine in purine nucleoside phosphorylase deficiency. To distinguish the nucleoside kinases present in T and B lymphoblastoid cells, we have coupled discontinuous PAGE with autoradiography. This procedure showed that deoxycytidine kinase (NTP:deoxycytidine 5'-phototransferase, EC 2.7.1.74), deoxyadenosine kinase (ATP:deoxyadenosine 5'-phosphotransferase, EC 2.7.1.76), and adenosine kinase (ATP:adenosine 5'-phosphotransferase, EC 2.7.1.20) are all present in both T and B lymphoblasts. While adenosine kinase is expressed at nearly equal levels in B and T cells, the deoxynucleoside kinases are expressed at much lower levels in B cells than in T cells. The autoradiographic data agreed with assays of the nucleoside kinase activities. Molecular weights were determined by using 5-10% polyacrylamide gels. Mr values were 29,000 for adenosine kinase, 41,000 for deoxyadenosine kinase, and 53,000 for deoxycytidine kinase and its isozyme. The reduced expression of deoxycytidine and deoxyadenosine kinases in B lymphoblasts may account for the lower accumulation of deoxypurine nucleotides in B cells as compared with T cells.

B-Lymphocytes↗
PubMed1986

A rat model of purine nucleoside phosphorylase deficiency.

Purine nucleoside phosphorylase (NP; EC 2.4.2.1) deficiency is associated with selective T-cell dysfunction and normal B-cell immunity. In order to create an in vivo model of this immune deficiency, we administered 8-aminoguanosine to rats. This water-soluble nucleoside was rapidly converted by NP to the more potent inhibitor 8-aminoguanine, which has a Ki of 0.19 microM. The accumulation of inosine in plasma showed that administration of 8-aminoguanosine was effectively inhibiting NP activity. The administration of 8-aminoguanosine with deoxyguanosine produced increased levels of dGTP only in thymus cells, and increased levels of GTP in cells from thymus, spleen and lymph node and in red cells. This correlated with assays of deoxyguanosine kinase, which showed significantly higher activity in thymus cells than in cells from spleen and lymph node. The intraperitoneal injection of 8-aminoguanosine alone or with deoxyguanosine for 8 consecutive days caused significant decreases in the number of thymus cells (P less than 0.001) and in lymph node and spleen lymphocytes (P less than 0.01). These data showed that the administration of 8-aminoguanosine to rats provided an animal model of NP deficiency that will allow studies of the specific regulation of T-cell function.

Animals↗
PubMed1986

A canine model of induced purine nucleoside phosphorylase deficiency.

Purine nucleoside phosphorylase (NP EC 2.4.2.1) deficiency in man is associated with selective T cell dysfunction and normal B cell immunity. To create an in-vivo model of this immune deficiency, we administered 8-aminoguanosine to dogs. This water soluble nucleoside was rapidly converted by NP to the more potent product inhibitor 8-aminoguanine, which had a Ki of 0.52 microM. The accumulation of inosine and exogenous deoxyguanosine in plasma provided evidence that administration of 8-aminoguanosine was effectively inhibiting NP activity. Four dogs given 8-aminoguanosine and deoxyguanosine concurrently for 5 consecutive days showed mean reductions in peripheral blood lymphocytes of 65 +/- 9% range (55-75%) over the test period. Granulocytes, red blood cells, and plateletes remained within the normal range. Administration of 8-aminoguanosine to dogs provides a model of NP deficiency that will permit studies of the specific control of lymphopoiesis and in-vivo immune function.

Animals↗
PubMed1985

Human cyclic hematopoiesis is associated with aberrant purine metabolism.

It appeared possible that abnormal purine or pyrimidine metabolism could cause cyclic hematopoiesis by analogy with the defective lymphopoiesis associated with inherited deficiencies of adenosine deaminase and purine nucleoside phosphorylase. Therefore, we examined erythrocyte purine and pyrimidine nucleotide levels, as well as plasma purine and pyrimidine nucleosides and bases in three patients and in normal controls. These studies showed that during neutropenia there was a significant elevation in the levels of guanosine triphosphate (P = 0.005) and adenosine triphosphate (P less than 0.001) in the patients' red cells not attributable to reticulocyte variation. Serial analysis of a patient's plasma showed a fivefold elevation of hypoxanthine (10.6 mumol/L) during neutropenia, with a return to normal values (1.4 mumol/L) as neutrophil numbers increased. Plasma inosine was also significantly elevated in comparison with normal control values (2.0 mumol/L vs. 0.8 mumol/L), whereas plasma and urinary uric acid were within the normal range. Serial analysis of red cells and plasma from two patients with chronic neutropenia showed no elevations of purine or pyrimidine metabolites. These results provide evidence of a link between abnormal concentrations of purine metabolites and cyclic hematopoiesis, and permit the speculation that aberrant purine metabolism is primarily related to the defective hematopoietic cell proliferation that is characteristic of this disease.

Adenosine Deaminase↗
PubMed1984

Dissociation constants for carbonic anhydrase--sulfonamide binding by high-performance liquid chromatography.

Carbonic anhydrase-azosulfonamide dissociation constants (Kd) were determined by gel filtration with high-performance liquid chromatography. By measuring the area of the elution peak at two wavelengths, Kd values were derived without having to measure a shallow trough. The procedure proved to be fast and reliable and has a general application. The dissociation constants were measured for 7-acetamido-2-(4'-sulfamylphenylazo)-1-hydroxynaphthalene-3, 6-disulfonate (Neoprontosil) complexes with carbonic anhydrase isozymes CA I, CA II, and CA III from bovine and human sources, and chicken CA III from skeletal muscle.

Animals↗
PubMed1984

Genetic variation in the carbonic anhydrase isozymes of macaque monkeys. IV. Degradation by heat and proteolysis of normal and variant carbonic anhydrase isozymes of Macaca nemestrina.

Studies were undertaken on the heat denaturation and proteolytic degradation by alpha-chymotrypsin of the normal red cell carbonic anhydrase isozyme, CA II, and two electrophoretic variants of carbonic anhydrase I, CA Ia and CA Ib, of the pigtail macaque. The heat degradation results showed a difference of about 40-fold in the rate constants between CA Ia and CA Ib, which is due to the marked thermostability of CA Ib compared to CA Ia. The enthalpies and entropies of activation were calculated from the heat denaturation constants. These values were compared, on enthalpy-entropy compensation plots, with those values previously determined for the human CA I and CA II isozymes. They were highly correlated and clearly fell into two distinct clusters, separated by about 200 kJ mol-1; one group comprising the macaque and human CA I isozymes and the other the CA II isozymes. The proteolytic degradation results showed that CA Ia is degraded about 2.5 times more rapidly than CA Ib by alpha-chymotrypsin. Thus, the characteristic 3/1 ratio of CA Ib/CA Ia in mature red cells could be accounted for by the greater susceptibility of CA Ia to degradation at some stage in red cell development.

Animals↗
PubMed1983

The metabolism of deoxyguanosine and guanosine in human B and T lymphoblasts. A role for deoxyguanosine kinase activity in the selective T-cell defect associated with purine nucleoside phosphorylase deficiency.

Purine nucleoside phosphorylase (NP; EC 2.4.2.1) deficiency is associated with defective T-cell and normal B-cell immunity. Biochemical mechanisms were investigated by measuring deoxyguanosine and guanosine metabolism in normal T and B lymphoblasts and NP-deficient B lymphoblasts. Deoxyguanosine kinase activity was specifically measured by using an anti-NP antibody to prevent alternative-product formation. Kinase activity towards deoxyguanosine was significantly higher in T-cells, whereas NP activity was similar in both B- and T-cells. Only in T-cells was dGTP produced from exogenous deoxyguanosine, and this was prevented by the simultaneous addition of deoxycytidine, which resulted in a concomitant increase in GTP synthesis. Inhibition by 8-aminoguanosine of NP activity in T lymphoblasts increased formation of dGTP and decreased that of GTP from deoxyguanosine and decreased the formation of GTP from guanosine. These data suggest a central role for deoxyguanosine kinase activity in the T-cell selectivity of the immune defect.

B-Lymphocytes↗
PubMed1983

Canine cyclic hematopoiesis is associated with abnormal purine and pyrimidine metabolism.

Canine cyclic hematopoiesis is an autosomal recessive disease characterized by regular 11-13-d cycles of the neutrophil, reticulocyte, and platelet counts caused by a defect in regulation of marrow stem cell proliferation. Treatment with lithium abrogates cycling of the cell counts in these grey collie dogs. Aware of the defective lymphopoiesis associated with adenosine deaminase and purine nucleoside phosphorylase deficiencies, we hypothesized that abnormal purine or pyrimidine metabolism might be present in these dogs. Using high pressure liquid chromatography, we measured erythrocyte purine and pyrimidine nucleotide levels and plasma purine and pyrimidine nucleosides and bases in normal and grey collie dogs before and during lithium treatment. During neutropenic periods in the grey collies, erythrocyte ATP, GTP, and UTP levels were significantly elevated. Normal dogs made neutropenic with cyclophosphamide did not show such elevations. Lithium treatment normalized the levels of erythrocyte ATP, GTP, and UTP in the grey collies and eliminated the differences between normal and grey collie nucleotide levels. Plasma thymine levels were markedly increased during neutropenia in the grey collie but were not increased in cyclophosphamide-treated normal dogs. The finding of abnormal concentrations of purine and pyrimidine metabolites in these dogs suggest that a metabolic derangement in purine or pyrimidine metabolism may be the cause of the defective stem cell proliferation in this disease.

Animals↗
PubMed1981

The proteolytic degradation of normal and variant human carbonic anhydrase isozymes by alpha-chymotrypsin.

The proteolytic degradation by alpha-chymotrypsin of normal and variant human carbonic anhydrase isozymes (CA I and CA II) was investigated by measuring the release of the bound azosulfonamide inhibitor, Neoprontosil, from the active sites of these isozymes. The enzymes studied were the normal isozymes, CA I and CA II, a secondary isozyme of CA I, designated CA I (+1), the CA I variants CA I London (102 Glu leads to Lys), CA I Michigan-2 (100 Thr leads to Lys), and the CA II variant CA II2 (251 Asn leads to Asp). The CA I and CA II isozymes and their respective variants could be classified by their apparent Km values, which were about 40 and 180 microM, respectively. A substrate specificity for alpha-chymotrypsin degradation was given by the ratio kcat/Km, and showed that the specificity decreased such that CA I (+1) > CA I congruent to CA I London congruent to CA I Michigan-2 > CA II2 congruent to CA II. A comparison of the proteolytic data with those previously obtained from thermal inactivation studies of the same isozymes showed agreement in the order of stability to both degradative methods.

Amino Acids↗
PubMed1981

Purine nucleoside phosphorylase is associated with centrioles and basal bodies.

We have localized a fraction of the enzyme, purine nucleoside phosphorylase (PNP), to the centrioles and basal bodies of mammalian, avian, and protozoan cells. Two completely independent methods were used, one based on the ultrastructural cytochemistry of the enzyme activity and one based on immunofluorescence microscopy using an antibody raised in rabbit against purified human PNP. PNP catalyzes the reversible conversion of purine nucleosides and inorganic phosphate to the corresponding purine bases and ribose-1-phosphate. Its partial localization to centrioles and basal bodies raises the possibility that purine compounds are involved in centriole replication and/or in the regulation of microtubule assembly in vivo. No centriolar PNP could be detected in primary skin fibroblast from two infants with severe immunodeficiency disease associated with the absence of soluble PNP. This raises the possibility that defects in centriole function may contribute to the impaired division and maturation of T lymphoid precursor in this inherited disorder. Initially, the immunofluorescence analyses were complicated by a residual centriole-binding antibody that persisted in immunoglobulins from immune animals after complete removal of anti-PNP by affinity chromatography. Binding was abolished by exposure of cells to sodium periodate, indicating that this (and possibly other) "spontaneous" anticentriole antibodies in rabbit serum may be directed against carbohydrates.

Antigens↗
PubMed1980

Human red cell purine nucleoside phosphorylase. Purification by biospecific affinity chromatography and physical properties.

As part of a study of the immune defect related to the absence of purine nucleoside phosphorylase, the substrate analogue 6-hydroxy-9-p-aminobenzylpurine was synthesized. This inosine analogue was a competitive inhibitor with an inhibition constant of about 200 muM. Using trichloro-s-triazine, the inhibitor was coupled to Sepharose, producing an efficient, reusable biospecific affinity gel. The gel was used to purify purine nucleoside phosphorylase from human red cells with an 85% yield and a specific activity of 95 mumol/min/mg. The molecular weight of native purine nucleoside phosphorylase was estimated as 90,400 using high pressure liquid chromatography. A subunit molecular weight of 31,600 was established using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Thus, a trimeric structure for the native enzyme is indicated, which is in accordance with the evidence derived from genetic studies.

Chromatography, Affinity↗
PubMed1980

Enzyme-linked immunosorbent assay for the indirect anti-human globulin test.

An enzyme-linked immunosorbent assay has been developed for detecting antibodies combined with red cell surface antigens. In the test, the fluorescence released from 4-methylumbelliferylphosphate is measured after exposure of antibody-coated red cells to alkaline phosphatase coupled to anti-human globulin. The assay is sensitive, reproducible, quantitative and potentially adaptable to an automatic analyzer system.

Alkaline Phosphatase↗
PubMed1980

Formycin B, purine nucleoside phosphorylase and lymphocyte function.

A genetic deficiency of purine nucleoside phosphorylase (NP) is associated with immune dysfunctin that specifically affects T cells. An absence of adenosine deaminase (ADA), the preceding enzyme in the pathway of purine recycling, is associated with failure of both T and B lymphocyte function. Formycin B, an analogue of inosine and an inhibitor of purine nucleosidase phosphorylase, was used in cell culture systems to stimulate deficiency of this enzyme. The cells used were normal circulating lymphocytes and lymphoblastoid cell lines (LCL) with T and B cell characteristics. Formycin B inhibited the growth of these cells, but its primary effect was found to be due to a mechanism other than purine nucleoside phosphorylase inhibition. The possibility that formycin B was inhibiting adenosine deaminase, for which it is a product analogue, was studied by the analysis of reaction progress curves using the integrated rate equation. The molecular structure of formycin B, whilst preventing its phosphorylytic cleavage by purine nucleoside phosphorylase, could enable this compound to function as a substrate for adenosine kinase. The resultant formation of formycin B nucleotide probably causes the observed inhibition of growth in cultured cells.

Adenosine Deaminase↗
PubMed1980

Purine nucleoside phosphorylase deficiency. Measurement of variant protein in four families with enzyme-deficient members by an enzyme-linked immunosorbent assay.

By a sensitive enzyme-linked immunosorbent assay, inactive variant nucleoside phosphorylase (NP) protein could be quantitated in red cells and cultured skin fibroblasts from the probands and parents in four families with enzyme-deficient members. Three different mutant alleles could be identified that, in the homozygous state, could lead to T-cell immunodeficiency. The mutant alleles formed proteins that differed from normal NP protein by their stability, catalytic activity, or isoelectric charge. Thus, sensitive biochemical techniques can demonstrate the presence of different mutations for purine NP. Any of the mutations can be responsible for a lack of purine NP and the development of the characteristic T-cell deficiency syndrome.

B-Lymphocytes↗