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B S Mitchell

Publications and source records attributed to B S Mitchell.

At least 109 records · Page 6Linked to original sources
PubMed1987

Transcriptional regulation of the c-myc protooncogene by 1,25-dihydroxyvitamin D3 in HL-60 promyelocytic leukemia cells.

Exposure of HL-60 promyelocytic leukemia cells to calcitriol results in a decrease in steady-state levels of c-myc mRNA and induces cellular differentiation. We have asked whether calcitriol has a direct effect on the transcription of the c-myc gene. 1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) decreased RNA elongation in a nuclear run-off transcription assay by 4 h after treatment. In the continuous presence of 1,25-(OH)2D3, HL-60 cell transcription of c-myc was decreased by 38% at 4 h and was abolished by 48 h. In contrast, the transcription of beta-actin was not affected by 1,25-(OH)2D3 treatment. The rate of transcription of c-myc and beta-actin was proportional to the number of nuclei and to time. Furthermore, specific hybridization of c-myc and beta-actin RNA was a linear function of RNA input. After a 48-h treatment, the c-myc/beta-actin ratio was decreased by 80-100% at [32P]RNA inputs ranging from 2 to 20 X 10(6) cpm/ml. These data temporally correlate inhibition of c-myc transcription with decreases in the steady-state levels of c-myc mRNA as assessed by Northern blot analysis. We conclude that the effect of 1,25-(OH)2D3 on c-myc expression occurs at the transcriptional level.

Actins↗
PubMed1987

Elevated adenosine deaminase activity and hereditary hemolytic anemia. Evidence for abnormal translational control of protein synthesis.

We have investigated the molecular basis of the marked elevation in erythrocyte adenosine deaminase (ADA) activity in a kindred with hereditary hemolytic anemia. Red cell ADA-specific activity was verified to be 70- to 100-fold normal levels. Western blots demonstrated a corresponding increase in erythrocyte ADA-specific immunoreactive protein. Analysis of genomic DNA revealed no evidence for amplification or major structural changes in the ADA gene. ADA-specific messenger RNA (mRNA) from proband reticulocytes was comparable in size and amount to mRNA from control reticulocytes. Translation of proband poly A+ reticulocyte mRNA in a rabbit reticulocyte lysate system and immunoprecipitation of 35S-labeled protein products with anti-ADA antibody yielded a band of approximately 42,000 apparent mol wt that was absent in translation products from control reticulocyte mRNAs. These data suggest that the increased ADA activity in red cells in this disorder results from the increased translation of an aberrant ADA mRNA.

Adenosine Deaminase↗
PubMed1987

Uptake of immunoglobulin and albumin by granulated metrial gland cells in vitro.

Single cell suspensions of metrial gland tissue from rats at Day 14 of pregnancy were prepared for maintenance in vitro. During the first 2 days of culture IgG was detected in glycoprotein granule-containing granulated metrial gland (GMG) cells. Albumin was also detected in GMG cells at the same stages. The IgG and albumin were not detected during the next 4 days in culture. When metrial gland cells, maintained in vitro for 5 days, were incubated with rat serum for a further 24 h, IgG and albumin were detected in GMG cells. When similar cultures were incubated for 24 h with purified rat IgG or purified rat albumin, GMG cells were positive for IgG and albumin respectively. Albumin was not detected in GMG cells in wax sections of metrial gland tissue, although IgG has previously been demonstrated. The uptake of serum proteins by GMG cells in vitro has been clearly shown but the difference in IgG and albumin content of these cells in paraffin-wax sections indicates that the means by which IgG accumulates intracellularly may be different in vitro and in vivo.

Albumins↗
PubMed1987

Genetic expression of adenosine deaminase in human lymphoid malignancies.

Adenosine deaminase (ADA) is an enzyme in the purine catabolic pathway that has been used as an enzymatic marker of T cell lymphoblastic malignancies due to its high specific activity in thymocytes and immature T cells. We have investigated whether the level of ADA activity in lymphoid leukemic cells correlates with the amount of ADA-specific RNA and/or immunoreactive protein in these cells as an initial step toward characterizing the nature of the genetic regulation of ADA expression during differentiation. We have found a good correlation between the steady state levels of ADA-specific RNA and ADA-immunoreactive protein in T lymphoblastic leukemic cell lines, mature T cell lines, a B lymphoblast cell line, and leukemic cells directly isolated from four patients with acute lymphoblastic leukemia and three patients with chronic lymphocytic leukemia. Southern blot analysis of DNA from these cells shows no evidence for differences in ADA gene copy number or gene rearrangement to account for the variability in ADA expression. We conclude that levels of ADA in lymphoid leukemic cells are directly related to the amount of ADA-specific mRNA present. These findings imply that ADA expression in leukemic cells reflects either the transcriptional activity of the ADA gene or the stability of ADA mRNA in these cells.

Adenosine Deaminase↗
PubMed1986

Leucocyte surface antigens in metrial gland tissue of ovariectomized pregnant rats, deciduomata-bearing rats and in uterine tissue of non-pregnant rats.

The effects of withdrawal of ovarian hormones and absence of paternal antigens on numbers and distribution of cells expressing Ia, Thy 1.1 or leucocyte-common (LC) antigens in the rat metrial gland have been assessed. The distribution of these markers in non-pregnant uterus at various stages of the oestrous cycle has also been investigated. In primigravid rats at Day 15 of gestation, following ovariectomy on Day 12, marker distribution was not different from that in uninterrupted pregnancy. However, there was a significant increase in the percentage of cells expressing LC antigen, which was not accompanied by an increase in the percentage of the glycoprotein-containing, LC antigen-positive granulated metrial gland (GMG) cells. It was suggested that the accumulation of LC antigen-positive, non-GMG cells might represent the precursors of GMG cells. In deciduomata-bearing rats, there was a significantly lower percentage of LC antigen-bearing cells at Day 13 of pseudopregnancy than in pregnant rats at Day 13, but no difference in the percentages of GMG cells was noted. Cells expressing Ia, LC or Thy 1.1 antigens were detected in the endometrial stroma, surrounding endometrial glands and the luminal epithelium of the non-pregnant uterus.

Animals↗
PubMed1985

Differential metabolism of deoxyribonucleosides by leukaemic T cells of immature and mature phenotype.

Experimental evidence has indicated that T lymphoblasts are more sensitive to deoxynucleoside toxicity than are B lymphoblasts. These data have led to the use of purine enzyme inhibitors as selective chemotherapeutic drugs in the treatment of T cell malignancies ranging from T cell acute lymphoblastic leukaemia to cutaneous T cell lymphomas. We have compared the toxicities of 2'-deoxyadenosine, 2'-deoxyguanosine, and thymidine for T cell lines derived from patients with T cell acute lymphoblastic leukaemia with those for mature T cell lines derived from patients with cutaneous T cell leukaemia/lymphoma. We have found that both deoxynucleosides are far less toxic to the mature T cell lies than to T lymphoblasts and that the mature cells accumulate much lower amounts of dATP and dGTP when exposed to deoxyadenosine and deoxyguanosine, respectively. Similar studies performed on peripheral blood cells from patients with T cell leukaemias of mature phenotype and on peripheral blood T cells demonstrate similar low amounts of deoxynucleotide accumulation. Measurements of the activities of several purine metabolizing enzymes that participate in deoxynucleoside phosphorylation or degradation do not reveal differences which would explain the toxicity of deoxynucleosides for immature, as compared to mature, T cells. We conclude that deoxynucleoside metabolism in leukaemic T cells varies with their degree of differentiation. These observations may be relevant to the design of chemotherapeutic regimes for T cell malignancies.

Adult↗
PubMed1985

Z-nucleotide accumulation in erythrocytes from Lesch-Nyhan patients.

5-Amino-4-imidazolecarboxamide riboside 5'-monophosphate (ZMP) is an intermediate in the purine de novo synthetic pathway that may be further metabolized to inosine 5'-monophosphate, degraded to the corresponding nucleoside (5-amino-4-imidazole-carboxamide riboside; Z-riboside), or phosphorylated to the corresponding 5'-triphosphate (ZTP). Accumulation of ZTP in microorganisms has been associated with depletion of folate intermediates that are necessary for the conversion of ZMP to inosine 5'-monophosphate and has been postulated to play a regulatory role in cellular metabolism. We have shown the presence of Z-nucleotides in erythrocytes derived from five individuals with the Lesch-Nyhan syndrome. Erythrocyte folate levels were within the normal range, although guanosine triphosphate levels were significantly reduced below those in normal controls (P less than 0.01). A small amount of Z-nucleotide accumulation was also found in one individual with partial deficiency of the enzyme hypoxanthine guanine phosphoribosyltransferase and in two individuals with other disorders of purine overproduction. In contrast, no Z-nucleotides were detected in 13 normal controls or in three individuals with hyperuricemia on allopurinol therapy. We conclude that Z-nucleotide formation may result from markedly increased rates of de novo purine biosynthesis. It is possible that metabolites of these purine intermediates may play a role in the pathogenesis of the Lesch-Nyhan syndrome.

Aminoimidazole Carboxamide↗
PubMed1985

Differential metabolism of guanine nucleosides by human lymphoid cell lines.

Deficiency of the enzyme purine nucleoside phosphorylase is associated with a specific depletion of T cells which is presumably mediated by its substrate, 2'-deoxyguanosine. Inhibitors of this enzyme are therefore being developed as potential immunosuppressive agents. We have compared the effects of 8-aminoguanosine, a competitive inhibitor of purine nucleoside phosphorylase, on the metabolism of 2'-deoxyguanosine by human T lymphoblasts, B lymphoblasts, and mature T-cell lines. 8-Aminoguanosine markedly potentiates the accumulation of dGTP in T lymphoblasts, but results in increased GTP levels in B lymphoblasts and mature T cells. GTP accumulation is associated with ATP depletion of a magnitude similar to that seen with an inhibitor of de novo purine biosynthesis, but does not result in inhibition of either DNA or RNA synthesis. In contrast, direct inhibition of de novo purine biosynthesis sharply decreased the incorporation of [3H]uridine into both DNA and RNA. We conclude that the mechanism of cell damage resulting from prolonged accumulation of GTP appears to involve more than inhibition of de novo purine biosynthesis and consequent ATP depletion. Perturbations in guanine nucleotide pools resulting from partial inhibition of purine nucleoside phosphorylase activity in vivo could result in cellular toxicity not limited to the target T cell population.

Adenine Nucleotides↗
PubMed1985

Effects of guanine ribonucleotide accumulation on the metabolism and cell cycle of human lymphoid cells.

A deficiency of purine nucleoside phosphorylase activity is associated with marked depletion of T-lymphocytes which is felt to be mediated by accumulation and further metabolism of the purine nucleoside phosphorylase substrate, 2'-deoxyguanosine. Human T-lymphoblasts incubated in the presence of 2'-deoxyguanosine and the purine nucleoside phosphorylase inhibitor 8-aminoguanosine accumulate deoxyguanosine 5'-triphosphate whereas B-lymphoblasts and mature T4+-cell lines accumulate GTP under identical conditions. We have compared the effects of guanine ribo- and deoxyribonucleotide accumulation on the metabolism and cell cycle of the respective cell lines. Deoxyguanosine 5'-triphosphate elevations in T-lymphoblasts are associated with inhibition of [3H]uridine incorporation into DNA and a complete block at the G1-S interface of the cell cycle. In contrast 3- to 5-fold increases in guanosine 5'-triphosphate pools in B-lymphoblasts and mature T-cell lines do not inhibit [3H]uridine incorporation into DNA or RNA but do cause a pronounced slowing in the progression of cells through S phase. B-lymphoblasts deficient in the salvage enzyme hypoxanthine guanine phosphoribosyltransferase do not accumulate guanosine 5'-triphosphate from 2'-deoxyguanosine and progress normally through the cell cycle, demonstrating a requirement for guanine salvage to inhibit cell growth. Guanine ribonucleotide accumulation was also associated with inhibition of de novo purine biosynthesis and a moderate decline in adenine nucleotide pools but not with inhibition of protein synthesis or alterations in basal levels of 3':5'-cyclic adenosine monophosphate or 3':5'-cyclic guanosine monophosphate. We conclude that the accumulation of guanine ribonucleotides by actively cycling human lymphoid cells is associated with an increase in S-phase cells and inhibition of growth. This effect is distinctly different from that produced by 2'-deoxyguanosine 5'-triphosphate and should be taken into account in pharmacological studies with 2'-deoxyguanosine and its analogues.

Cell Cycle↗
PubMed1985

Metabolism and selective cytotoxicity of 9-beta-D-arabinofuranosylguanine in human lymphoblasts.

The selective toxicity of purine deoxyribonucleosides for T-lymphoblasts appears to be mediated by the selective accumulation of the corresponding deoxyribonucleoside triphosphates in these cells. This finding has led to a search for deoxyribonucleoside analogues which may have clinical utility in T-cell lymphoproliferative disorders. 9-beta-D-arabinofuranosylguanine (ara-G) is a 2'-deoxyguanosine analogue which is 70-fold more inhibitory to the growth of T- than of B-lymphoblasts. It is less potent than ara-C but far more selective in its cytotoxic effect on T-cells. ara-G is not significantly degraded by purine nucleoside phosphorylase (EC 2.4.2.1) activity in T-lymphoblasts and is metabolized to 9-beta-D-arabinofuranosylguanine 5'-triphosphate. The accumulation of this metabolite directly correlates with inhibition of DNA but not of RNA or protein synthesis. MOLT-4 T-lymphoblasts were selected for ara-G resistance, and six clones were identified which exhibited a major degree of resistance to 2'-deoxyguanosine but little or none to ara-C. Further characterization of clone 24B3 revealed a 600-fold increase in ara-G resistance, a 36-fold increase in 2'-deoxyguanosine resistance, and only a 4-fold increase in 1-beta-D-arabinofuranosylcytosine resistance. The 24B3 cell line accumulated less than 10% of 9-beta-D-arabinofuranosylguanine 5'-triphosphate and 2'-deoxyguanosine 5'-triphosphate from the corresponding nucleosides as compared to wild-type MOLT-4 cells; in contrast, 1-beta-D-arabinofuranosylcytosine 5'-triphosphate accumulation was approximately 30% of control values. Thus, ara-G differs from 1-beta-D-arabinofuranosylcytosine in its selectivity for cultured T-lymphoblasts and may be of use as a chemotherapeutic or immunosuppressive agent.

Antineoplastic Agents↗
PubMed1984

Peripheral neuropathy in osteosclerotic myeloma: clinical and electrodiagnostic improvement with chemotherapy.

A patient with a severe remote-effect polyneuropathy and other paraneoplastic features of osteosclerotic myeloma improved dramatically with melphalan and prednisone treatment. Serial electrodiagnostic studies provided an objective means of following the response to therapy and documented the improvement. We believe this represents the first reported patient with multifocal osteosclerotic myeloma and a myelomatous polyneuropathy responding to melphalan and prednisone.

Adult↗
PubMed1984

2'-deoxyguanosine toxicity for B and mature T lymphoid cell lines is mediated by guanine ribonucleotide accumulation.

Inherited deficiency of the enzyme purine nucleoside phosphorylase (PNP) results in selective and severe T lymphocyte depletion which is mediated by its substrate, 2'-deoxyguanosine. This observation provides a rationale for the use of PNP inhibitors as selective T cell immunosuppressive agents. We have studied the relative effects of the PNP inhibitor 8-aminoguanosine on the metabolism and growth of lymphoid cell lines of T and B cell origin. We have found that 2'-deoxyguanosine toxicity for T lymphoblasts is markedly potentiated by 8-aminoguanosine and is mediated by the accumulation of deoxyguanosine triphosphate. In contrast, the growth of T4+ mature T cell lines and B lymphoblast cell lines is inhibited by somewhat higher concentrations of 2'-deoxyguanosine (ID50 20 and 18 microM, respectively) in the presence of 8-aminoguanosine without an increase in deoxyguanosine triphosphate levels. Cytotoxicity correlates instead with a three- to fivefold increase in guanosine triphosphate (GTP) levels after 24 h. Accumulation of GTP and growth inhibition also result from exposure to guanosine, but not to guanine at equimolar concentrations. B lymphoblasts which are deficient in the purine salvage enzyme hypoxanthine guanine phosphoribosyltransferase are completely resistant to 2'-deoxyguanosine or guanosine concentrations up to 800 microM and do not demonstrate an increase in GTP levels. Growth inhibition and GTP accumulation are prevented by hypoxanthine or adenine, but not by 2'-deoxycytidine. 8-Aminoguanosine appears to effectively inhibit extracellular PNP activity; thus, it prolongs the extracellular half-life of 2'-deoxyguanosine and guanosine, but does not completely inhibit intracellular PNP activity in these lymphoid cells. As a result, 2'-deoxyguanosine and guanosine are phosphorolyzed and actively salvaged within the cell, accounting for the accumulation of GTP. Partial inhibition of PNP activity in vivo, therefore, may lead to nonselective cellular toxicity by a mechanism independent of dGTP accumulation.

B-Lymphocytes↗
PubMed1984

Identification of cells bearing leucocyte surface antigens in metrial gland tissue from rats of different gestational ages, strains or parities.

The effects of gestational age, strain and parity on cells bearing leucocyte markers in the rat metrial gland have been studied on smears of single cell preparations and on cryostat sections. No cells were observed bearing T lymphocyte markers, but cells expressing leucocyte common (LC), Ia and Thy-1.1 antigens were detected. In the metrial glands from random bred primigravid rats, there was a significant increase in the percentage of cells bearing LC antigen between day 10 and day 18. There was also a significant difference in the percentage of cells expressing LC antigen between inbred rats and random bred multiparous rats at day 13. The cells expressing LC antigen were distributed around blood vessels and were shown by their glycoprotein granule content to be identical with granulated metrial gland cells. The identity of the cells bearing Ia antigen was not clear but they appeared randomly distributed in the stroma of the metrial gland. Thy-1.1 antigen was observed on some cells and was associated with some blood vessels of the metrial gland. There were no significant differences in relation to gestational age, strain or parity in the percentages of cells bearing Ia or Thy-1.1 antigens. The results were discussed in relation to the ontogeny of granulated metrial gland cells and the role that the various antigen bearing cells may play in the development and function of the metrial gland.

Animals↗
PubMed1984

Purine metabolizing enzymes as predictors of lymphoblast sensitivity to deoxyadenosine.

The toxicity of low concentrations of 2'-deoxyadenosine for T-lymphoblasts and certain null lymphoblasts has been attributed to the decreased degradation of the deoxynucleotides formed from deoxyadenosine in these cells. Low activities of the ectoenzymes ecto-5'-nucleotidase and ecto-ATPase have each been associated with deoxyadenosine sensitivity and dATP accumulation in human T-lymphoblasts. We studied a B-lymphoblast cell line, NC-37, which lacks detectable ecto-5'-nucleotidase and ecto-ATPase activities, but which is otherwise easily distinguishable from T-lymphoblasts by its low adenosine deaminase activity and its pattern of reactivity with monoclonal antibodies to cell surface antigens (Bl and IgM positive). The NC-37 B cells were completely analogous to other B-lymphoblast lines with high ectonucleotidase activities in their relative resistance to deoxyadenosine toxicity and low rates of dATP accumulation. This resistance could not be accounted for by lower rates of deoxyadenosine phosphorylating activity. Cytoplasmic nucleotidase activity in crude extracts from the NC-37 line was similar to that in other B-lymphoblasts with regard to both substrate specificity and optimal pH. We conclude that low ectonucleotidase activities are not etiologically associated with the accumulation of deoxynucleotides by human lymphoblasts, although they may serve as markers of deoxyadenosine sensitivity in certain malignant lymphoid cells.

5'-Nucleotidase↗
PubMed1983

Adenosine deaminase deficiency with normal immune function. An acidic enzyme mutation.

In most instances, marked deficiency of the purine catabolic enzyme adenosine deaminase results in lymphopenia and severe combined immunodeficiency disease. Over a 2-yr period, we studied a white male child with markedly deficient erythrocyte and lymphocyte adenosine deaminase activity and normal immune function. We have documented that (a) adenosine deaminase activity and immunoreactive protein are undetectable in erythrocytes, 0.9% of normal in lymphocytes, 4% in cultured lymphoblasts, and 14% in skin fibroblasts; (b) plasma adenosine and deoxyadenosine levels are undetectable and deoxy ATP levels are only slightly elevated in lymphocytes and in erythrocytes; (c) no defect in deoxyadenosine metabolism is present in the proband's cultured lymphoblasts; (d) lymphoblast adenosine deaminase has normal enzyme kinetics, absolute specific activity, S20,w, pH optimum, and heat stability; and (e) the proband's adenosine deaminase exhibits a normal apparent subunit molecular weight but an abnormal isoelectric pH. In contrast to the three other adenosine deaminase-deficient healthy subjects who have been described, the proband is unique in demonstrating an acidic, heat-stable protein mutation of the enzyme that is associated with less than 1% lymphocyte adenosine deaminase activity. Residual adenosine deaminase activity in tissues other than lymphocytes may suffice to metabolize the otherwise lymphotoxic enzyme substrate(s) and account for the preservation of normal immune function.

Adenosine Deaminase↗