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Biomedical subjects

G R Boss

Publications and source records attributed to G R Boss.

At least 55 records · Page 3Linked to original sources
PubMed1989

cGMP-induced differentiation of the promyelocytic cell line HL-60.

cGMP is a second messenger that mediates numerous metabolic events; in the present work a role in myeloid cell differentiation was demonstrated. Nitroprusside and NaNO2, which activate cytosolic guanylate cyclase and increase the intracellular cGMP concentration, induced granulocytic differentiation of the human promyelocytic cell line HL-60; differentiation was measured by acquisition of the OKM1 antigen, morphological changes, and nitroblue tetrazolium reduction. When theophylline, a phosphodiesterase inhibitor, which by itself induced modest differentiation, was added to nitroprusside or NaNO2, differentiation increased in an additive fashion. The degree of differentiation correlated with the increase in the intracellular cGMP concentration. 8-Bromoguanosine 3',5'-cyclic monophosphate, a membrane-permeable cGMP analogue, also induced differentiation of HL-60 cells but was much more effective in the presence of theophylline, with the two agents interacting synergistically. The effect of theophylline in these studies could not be attributed to increasing the intracellular cAMP concentration. Dimethyl sulfoxide, and established inducer of differentiation of HL-60 cells, markedly enhanced the differentiation induced by nitroprusside and NaNO2.

Cell Differentiation↗
PubMed1987

Purine deoxynucleosides and adenosine dialdehyde decrease 5-amino-4-imidazolecarboxamide (Z-base)-dependent purine nucleotide synthesis in cultured T and B lymphoblasts.

Deoxyadenosine (dAdo) and deoxyguanosine (dGuo) decrease methionine synthesis from homocysteine in cultured lymphoblasts; because of the possible trapping of 5-methyltetrahydrofolate this could lead to decreased purine nucleotide synthesis. Since purine deoxynucleosides could also inhibit purine synthesis de novo at an early step not involving folate metabolism, we measured in azaserine-treated cells 5-amino-4-imidazolecarboxamide (Z-base)-dependent purine nucleotide synthesis using [14C]formate. In the T lymphoblasts, Z-base-dependent purine nucleotide synthesis was decreased 26% by 0.3 microM-dAdo, 21% by 1 microM-dGuo and 28% by 1 microM-adenosine dialdehyde, a potent S-adenosylhomocysteine hydrolase inhibitor; homocysteine fully reversed the inhibitions. The B lymphoblasts were considerably less sensitive to the deoxynucleoside-induced decrease in Z-base-dependent purine nucleotide synthesis, with 100 microM-dAdo required for significant inhibition and no inhibition by dGuo at this concentration; homocysteine partly reversed the inhibition by dAdo. The observed decrease in Z-base-dependent purine nucleotide synthesis could not be attributed either to dUMP depletion changing the folate pools or to decreased ATP availability because dUrd was without effect and during the experimental period the intracellular ATP concentration did not change significantly. Cells with 5,10-methylenetetrahydrofolate reductase deficiency were relatively resistant to inhibition of Z-base-dependent purine nucleotide synthesis by dAdo and adenosine dialdehyde. Our results suggest that deoxynucleosides decrease purine nucleotide synthesis by trapping 5-methyltetrahydrofolate.

5,10-Methylenetetrahydrofolate Reductase (FADH2)↗
PubMed1987

The polyol pathway and glucose 6-phosphate in human endothelial cells cultured in high glucose concentrations.

In an attempt to identify the mechanisms underlying the ill effects of high glucose previously described in cultured human endothelial cells, we have investigated in these cells the activity of the polyol pathway and accumulation of glucose 6-phosphate, a powerful agent of non-enzymatic glycosylation. Sorbitol accumulation varied among different batches of cells (primary cultures). In presence of 5 mmol/l glucose the cellular sorbitol content ranged from 0.04 to 0.12 nmol/10(6) cells. When cells were exposed to 20 mmol/l glucose the sorbitol content increased by 2- to 3-fold to concentrations of 0.08-0.38 nmol/10(6) cells (p less than 0.01). Addition to the culture medium of 100 mumol/l Sorbinil, an inhibitor of aldose reductase, resulted in a substantial inhibition of sorbitol accumulation throughout the 14 days in culture, but the degree of inhibition varied inversely with the duration of cell exposure to high glucose (70% inhibition in cells exposed to high glucose and Sorbinil for 1-3 days versus 14% inhibition in cells exposed for 14 days, p less than 0.01). Sorbinil treatment failed to improve even slightly the abnormalities in cellular replication induced by high glucose. The cellular content of glucose 6-phosphate was augmented 3-fold by exposure to 20 mmol/l glucose (p less than 0.001). In conjunction with other studies these results indicate that in this model the polyol pathway is not an osmotically or metabolically important mechanism of glucotoxicity, and that the inhibitory activity of Sorbinil on the polyol pathway of human tissues may be a function of their length of exposure to hyperglycaemia. The consequences of intracellular accumulation of glucose 6-phosphate await investigations.

Aldehyde Reductase↗
PubMed1987

Induction of HL-60 differentiation by starvation for a single essential amino acid but not by protein synthesis inhibitors.

Starvation for a single essential amino acid induced differentiation of the human promyelocytic leukemia line HL-60 into morphologically and functionally mature granulocytes. Differentiation occurred when protein synthesis was inhibited up to 90% but was not simply secondary to growth arrest or protein synthesis inhibition, because neither glucose starvation nor treatment with protein synthesis inhibitors induced differentiation. Induction of differentiation by an aminoacyl tRNA synthetase inhibitor and the effect of cycloheximide and puromycin on amino acid-starved cells suggested an important regulatory role of tRNA molecules during differentiation.

Amino Acids, Essential↗
PubMed1987

Adenosine dialdehyde and nitrous oxide induce HL-60 differentiation.

Adenosine dialdehyde and nitrous oxide, specific S-adeno-sylhomocysteine hydrolase and methionine synthetase inhibitors, respectively, induced differentiation of the human promyelocytic cell line HL-60. Their effect did not appear to be mediated through changes in transmethylation or decreased S-adenosylmethionine synthesis because (1) there was little correlation between the concentrations of adenosine dialdehyde that induced differentiation and those that changed the ratio of the intracellular concentrations of S-adenosylmethionine to S-adenosylhomocysteine, and (2) inhibition of methionine adenosyltransferase by cycloleucine did not induce differentiation. The differentiation induced by adenosine dialdehyde was prevented by homocysteine and that by nitrous oxide was inversely related to the medium methionine concentration. This suggested that differentiation was secondary to decreased methionine synthesis.

Adenosine↗
PubMed1986

Decreased methylation rates of DNA in SV40-transformed human fibroblasts.

The rates of methylation of total cellular DNA and newly synthesized DNA were measured in four unrelated SV40-transformed human fibroblast lines and in four control parent fibroblast lines. Rates of methylation of total cellular DNA were decreased by a factor of 1.8-2.3 in the transformed cells relative to control cells. Methylation was largely (75%-87%) restricted to newly synthesized DNA in control and transformed fibroblasts, and methylation rates of newly synthesized DNA were diminished in transformed cells by 12- to 19-fold relative to control cells. Growth rates were similar in the normal and transformed cells. The cellular uptake of methionine and conversion to S-adenosylmethionine were similar in the normal and transformed cells, suggesting no major differences between the normal and transformed cells in the cellular transport of methionine, methionine S-adenosyltransferase activity, or the intracellular concentrations of methionine and S-adenosylmethionine. The diminished rates of DNA methylation that we have observed suggest a possible mechanism for altered gene expression and growth control in transformed cells.

Cell Line↗
PubMed1985

Phosphoribosylpyrophosphate synthesis from glucose decreases during amino acid starvation of human lymphoblasts.

When cultured human lymphoblasts are starved 3 h for an essential amino acid, rates of purine nucleotide synthesis decrease markedly because of a decrease in the intracellular phosphoribosylpyrophosphate concentration (Boss, G.R., and Erbe, R.W. (1982) J. Biol. Chem. 257, 4242-4247; Boss, G. R. (1984) J. Biol. Chem. 259, 2936-2941). In amino acid-starved cells, glucose transport was not changed, whereas total glucose consumption and lactate production decreased by approximately 25 and 10%, respectively. Carbon flow through the oxidative pentose phosphate pathway, measured by 14CO2 release from [1-14C]glucose, decreased by 18% during amino acid starvation. However, kinetic studies of ribulose-5-phosphate 3-epimerase and phosphoriboisomerase suggested that the ribulose 5-phosphate produced by this pathway is converted mostly to xylulose 5-phosphate instead of to ribose 5-phosphate so that this pathway produces little phosphoribosylpyrophosphate. The activity of the nonoxidative pentose phosphate pathway, measured by high performance liquid chromatography following the incorporation of [1-14C]glucose into phosphoribosylpyrophosphate, ATP, and GTP, decreased by approximately 55% during amino acid starvation. None of the enzymes of either pathway changed in specific activity during amino acid starvation. We conclude that the nonoxidative pentose phosphate pathway is the major source of phosphoribosylpyrophosphate for purine nucleotide synthesis and that this pathway is regulated by a metabolite which changes in concentration during amino acid starvation.

Aldose-Ketose Isomerases↗
PubMed1985

Cobalamin inactivation decreases purine and methionine synthesis in cultured lymphoblasts.

The megaloblastic anemia of cobalamin deficiency appears secondary to decreased methionine synthetase activity. Decreased activity of this enzyme should cause 5-methyltetrahydrofolate to accumulate intracellularly, and consequently, decrease purine and DNA synthesis; this is the basis of the "methylfolate trap" hypothesis of cobalamin deficiency. However, only some of the clinical and biochemical manifestations of cobalamin deficiency can be explained by the methylfolate trap. We investigated cobalamin deficiency by treating cultured human lymphoblasts with N2O since N2O inhibits methionine synthetase activity by inactivating cobalamin. We found that 4 h of N2O exposure reduced rates of methionine synthesis by 89%. Rates of purine synthesis were not significantly reduced by N2O when folate and methionine were present at 100 microM in the medium; however, at the physiologic methionine concentration of 10 microM, N2O decreased rates of purine synthesis by 33 and 57% in the presence of 100 microM folate and in the absence of folate, respectively. The dependency of rates of purine synthesis on methionine availability would be expected in cells with restricted methionine synthetic capacity because methionine is the immediate precursor of S-adenosylmethionine, a potent inhibitor of 5-methyltetrahydrofolate synthesis; methionine serves as a source of formate for purine synthesis; and rates of purine synthesis are dependent on the intracellular availability of essential amino acids. We conclude that cobalamin inactivation decreases purine synthesis by both methylfolate trapping and reduction of intracellular methionine synthesis.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed1984

Decreased phosphoribosylpyrophosphate as the basis for decreased purine synthesis during amino acid starvation of human lymphoblasts.

Rates of de novo and salvage purine synthesis decrease by approximately 80 and 60%, respectively, when normal human lymphoblasts are starved 3 h for an essential amino acid (Boss, G. R., and Erbe, R. W. (1982) J. Biol. Chem. 257, 4242-4247). Amino acid starvation decreased the intracellular phosphoribosylpyrophosphate (PP-Rib-P) and ribose 5-phosphate concentrations by approximately 40%, but neither the specific activities of PP-Rib-P synthetase and glutamine amidophosphoribosyltransferase nor the intracellular concentrations of purine nucleotides and inorganic phosphate changed significantly. In mutant cells with either an increased capacity to generate PP-Rib-P (superactive PP-Rib-P synthetase), or an increased PP-Rib-P concentration (inosinate-guanylate:pyrophosphate phosphoribosyltransferase deficiency), the intracellular PP-Rib-P concentration decreased by less than 15% during amino acid starvation and de novo purine synthesis decreased significantly less than in normal cells. When normal cells were treated with drugs that simultaneously decreased feed-back inhibition by purine nucleotides and increased the intracellular concentration of ribose 5-phosphate and PP-Rib-P rates of de novo purine synthesis were stimulated 3-fold in nonstarved cells and more than 8-fold in starved cells. This greater stimulation in the starved cells appeared to be from the increased PP-Rib-P production; moreover, in starved cells in which the increase of the PP-Rib-P concentration by the drugs was impaired because of purine nucleoside phosphorylase deficiency, rates of de novo purine synthesis increased only 3.5-fold. The data suggest that amino acid starvation decreases purine synthesis by decreasing the generation of PP-Rib-P from glucose.

Amidophosphoribosyltransferase↗
PubMed1984

The influence of ribose 5-phosphate availability on purine synthesis of cultured human lymphoblasts and mitogen-stimulated lymphocytes.

The intracellular ribose 5-phosphate concentration was found to be an important determinant of rates of de novo purine synthesis. When ribose 5-phosphate production was reduced in cultured human lymphoblasts by glucose starvation, the intracellular phosphoribosylpyrophosphate concentration and rates of de novo purine synthesis decreased. Inosinate-guanylate:pyrophosphate phosphoribosyltransferase (HPR transferase)-deficient cells were relatively more resistant to glucose starvation. To minimize the effect of purine nucleotide feedback inhibition on the de novo pathway, cells were treated with inhibitors of IMP dehydrogenase and adenylosuccinate synthetase. In normal lymphoblasts, purine synthesis was stimulated only at glucose concentrations greater than 100 microM while in HPR transferase-deficient lymphoblasts, stimulation occurred even in the absence of glucose. The differences between the normal and HPR transferase-deficient cells were lost when ribose reutilization from endogenous nucleotide breakdown was impaired in the HPR transferase-deficient cells by incubation with 2'-deoxyinosine. Endogenous ribose reutilization for purine synthesis is, therefore, important when either glucose availability is limited or synthesis is stimulated. In the absence of glucose, exogenous purine nucleotides restored the intracellular concentrations of ribose 5-phosphate, phosphoribosylpyrophosphate, and purine nucleotides to almost 100% and rates of purine synthesis to 50-75% of those at 10 mM glucose. When ribose 5-phosphate production was increased in peripheral blood lymphocytes by phytohemagglutinin activation, the intracellular phosphoribosylpyrophosphate concentration and rates of de novo purine synthesis increased.

Cell Line↗
PubMed1984

Decreased methionine synthesis in purine nucleoside-treated T and B lymphoblasts and reversal by homocysteine.

Purine nucleosides, which accumulate in adenosine deaminase and purine nucleoside phosphorylase deficiency, are toxic to lymphoid cells. Since adenine nucleosides inhibit S-adenosylhomocysteine hydrolase, they could potentially decrease intracellular methionine synthesis. To test this hypothesis, we measured methionine synthesis by the use of [14C]formate as a radioactive precursor in cultured human T and B lymphoblasts treated with varying concentrations of purine nucleosides; 2'-deoxycoformycin and 8-aminoguanosine were added to inhibit adenosine deaminase and purine nucleoside phosphorylase, respectively. In the T lymphoblasts methionine synthesis was inhibited approximately 50% by 10 microM of 2'-deoxyadenosine, adenine arabinoside, or 2'-deoxyguanosine. By contrast, in the B lymphoblasts methionine synthesis was considerably less affected by these nucleosides, with 50% inhibition occurring at 100 microM of 2'-deoxyadenosine and adenine arabinoside; 100 microM of 2'-deoxyguanosine yielded less than 10% inhibition. Adenosine and guanosine were considerably less potent inhibitors of methionine synthesis in both the T and B lymphoblasts. An adenosine deaminase-deficient and a purine nucleoside phosphorylase-deficient cell line, both of B cell origin, exhibited sensitivities to the nucleosides similar to those of the normal B cell lines. In both the T and B cell lines homocysteine reversed the methionine synthesis inhibition induced by the adenine nucleosides and guanosine and largely reversed that induced by 2'-deoxyguanosine. Methionine synthesis from homocysteine generates free tetrahydrofolate from 5-methyltetrahydrofolate, the main intracellular storage form of folate. We conclude that purine nucleoside toxicity may be partly mediated through (a) decreased intracellular methionine synthesis, and (b) altered folate metabolism.

Adenosine↗
PubMed1983

Assay of glutamine phosphoribosylpyrophosphate amidotransferase using [1-14C]phosphoribosylpyrophosphate.

Glutamine phosphoribosylpyrophosphate amidotransferase (EC 2.4.2.14) catalyzes the transfer of the amide group of glutamine to 5-phospho-alpha-D-ribose-1-pyrophosphate. It is the first enzyme committed to the synthesis of purines by the de novo pathway. Previous assays of enzyme activity have either measured the phosphoribosylpyrophosphate-dependent disappearance of radioactive glutamine or have linked this reaction to subsequent steps in the purine pathway. A new assay for activity of the enzyme by directly measuring the synthesis of the product of the reaction. 5-beta-phosphoribosyl-1-amine, using [1-14C]phosphoribosylpyrophosphate as substrate is described. Substrate and product are separated by thin-layer chromatography and identified by autoradiography. Glutamine or ammonia may be used as substrates; the apparent Km values of the human lymphoblast enzyme are 0.46 mM for glutamine and 0.71 mM for ammonia. GMP is a considerably more potent inhibitor of the human lymphoblast enzyme than is AMP; 6-diazo-5-oxo-L-norleucine inhibits only glutamine-dependent activity and has no effect on ammonia-dependent activity.

Amidophosphoribosyltransferase↗
PubMed1982

Decreased purine synthesis during amino acid starvation of human lymphoblasts.

Normal human lymphoblasts starved for each of several essential, but not essential, amino acids had decreased DNA and RNA synthesis but no change in free intracellular purine nucleotides. The rates of purine nucleotide synthesis via the de novo and salvage pathways were measured by incorporating [14C]formate and [14C]hypoxanthine labels, respectively, into lymphoblasts starved for an amino acid or treated with a protein synthesis inhibitor. After 3 h of starvation, purine synthesis via the de novo pathway decreased 90% and via the salvage pathway decreased 60%. Cycloheximide and puromycin each reduced de novo synthesis by 96% and salvage synthesis by 72%. The decrease in purine synthesis de novo after removal of the amino acid was of first order kinetics and was fully and rapidly reversed by reconstitution with the amino acid. The synthesis of alpha-N-formylglycinamide ribonucleotide declined 97% after amino acid starvation; the synthesis of purines from 5-aminoimidazole-4-carboxamide riboside decreased 41%. The synthesis of guanylates decreased more than the synthesis of adenylates during amino acid starvation.

Amino Acids↗