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G R Boss

Publications and source records attributed to G R Boss.

At least 19 recordsLinked to original sources
PubMed1992

Chemically induced murine erythroleukemia cell differentiation is severely impaired when cAMP-dependent protein kinase activity is repressed by transfected genes.

During chemically induced differentiation of murine erythroleukemia (MEL) cells, cAMP-dependent protein kinase activity increases, and the enzyme's isozyme pattern changes. To examine the enzyme's role during MEL cell differentiation, we stably transfected MEL cells with recombinant plasmids in which the mouse metallothionein I promoter controlled expression of either a mutant form of the type I regulatory subunit of cAMP-dependent protein kinase (RI) or the enzyme's specific peptide inhibitor (PKI); expressing either sequence rendered cells cAMP-dependent protein kinase-deficient. Chemically induced differentiation of MEL cells as assessed by beta-globin mRNA and hemoglobin accumulation was inhibited in RI mutant and PKI transfectants; adding zinc further inhibited differentiation in the transfectants but had no effect on parental MEL cells. The inhibition of differentiation correlated with the amount of RI mutant mRNA and protein in the RI mutant transfectants and with the cells' degree of cAMP-dependent protein kinase deficiency in both the RI mutant and PKI transfectants. Overexpression of wild type RI did not interfere with differentiation or enzyme activity. We conclude that cAMP-dependent protein kinase activity is important for chemically induced differentiation of MEL cells and that the down-regulation of RI protein which occurs during MEL cell differentiation is not essential for differentiation to proceed.

Acetamides↗
PubMed1992

Studies on cytosolic guanylate cyclase from human placenta.

We have purified the soluble form of guanylate cyclase from human placenta greater than 2400-fold. The enzyme shared several characteristics with the enzyme purified from other sources including molecular mass and subunit composition, activation by divalent cations, inhibition by ATP and Michaelis constants. The enzyme, however, had a lower absorption maximum in the Soret region (417 +/- 1 nm) than the enzyme from other sources and was activated only one-fifth as much by nitric oxide as the bovine lung enzyme. It appears that the heme prosthetic group in the human placental enzyme may be hexa-coordinate and in the bovine lung enzyme the heme group may be penta-coordinate.

Cations, Divalent↗
PubMed1991

Double-mixing kinetic studies of the reactions of monoliganded species of hemoglobin: alpha 2(CO)1 beta 2 and alpha 2 beta 2(CO)1.

The kinetics of CO association to and dissociation from the two isomers of monoliganded species alpha ICO beta I(alpha II beta II) and alpha I beta I (alpha II beta COII) has been studied by double-mixing stopped-flow and microperoxidase methods. The monoliganded species were generated by hybridization between excess ferric Hb and alpha CO2 beta +2 or alpha +2 beta CO2 prepared by high-pressure liquid chromatography (HPLC). The results indicated that: 1) there were no significant differences in the reactivities of alpha and beta chains in the first step of ligation; 2) in the second step of ligation there was significant cooperativity in the reaction of deoxyhemoglobin with 0.05 or 0.1 equivalent of CO. Diliganded species were therefore formed in significant amounts. The double-mixing HPLC results suggested that in the second step of ligation alpha chains reacted faster than the beta chains, and the main diliganded species formed was alpha I beta ICO (alpha IICO beta II) or its isomer alpha ICO I(alpha II beta IICO). These results seem to indicate that the reaction of the first CO is mostly random and in the second step of ligation CO binds more to the tetramers in which one beta chain is already ligated: alpha I beta I (alpha II beta II) + CO----alpha ICO beta I (alpha II beta II) and alpha I beta ICO (alpha II beta II) + CO----alpha I beta ICO (alpha IICO beta II).

Carbon Monoxide↗
PubMed1991

The evolution of lymphadenopathy and hypergammaglobulinemia are evidence for early and sustained polyclonal B lymphocyte activation during human immunodeficiency virus infection.

To examine whether polyclonal activation of B lymphocytes as measured by hypergammaglobulinemia contributes to lymphadenopathy in human immunodeficiency virus (HIV) infection, correlates of adenopathy were examined in 240 homosexual men. Lymph node size was measured in 12 sites semiannually over 4 years. Both adenopathy and hyperglobulinemia developed within 1 year after seroconversion and persisted at high levels. Adenopathy declined near diagnosis of AIDS whereas serum IgG decreased 8-16 months after diagnosis. Adenopathy attributable to HIV occurred in all palpable node groups. By logistic regression, HIV-positive men were best discriminated from HIV-negative men by size of posterior cervical nodes and the number of sites with enlarged nodes. In a repeated measures model of covariance, adenopathy in HIV-positive men was associated with more CD4+ cells (P less than .002), elevated serum globulins (P less than .01), and lower platelet counts (P less than .05). Adenopathy declined over time (P less than .001) and with diagnosis of AIDS or AIDS-related complex (P less than .03). Thus, adenopathy and hypergammaglobulinemia are correlated and follow a similar course through various stages of HIV infection, suggesting that both are caused by polyclonal B cell activation.

AIDS-Related Complex↗
PubMed1989

Inhibition of two enzymes in de novo purine nucleotide synthesis by triciribine phosphate (TCN-P).

We previously reported that triciribine (tricyclic nucleoside, TCN, NSC-154020), after phosphorylation in cultured CCRF-CEM human leukemic lymphoblasts inhibited de novo purine nucleotide synthesis, GTP more than ATP [Moore et al. Biochem. Pharmac. 38, 4037 (1989)]. To determine the enzymes inhibited, triciribine phosphate (TCN-P, NSC-280594) was tested in dialyzed extracts of the cells. A new assay for glycinamide ribotide (GAR) synthesis was based on incorporation of [14C]glycine into GAR as a ribose-containing compound retained on boronyl gel columns. Glutamine, phosphoribosyl pyrophosphate (PRPP), ATP and glycine were required for the two-step sequence of glutamine:amidophosphoribosyltransferase (EC 2.4.2.14) and phosphoribosylamine-glycine ligase (EC 6.3.4.13). When PRPP was near the normal intracellular concentration (0.1 mM), 1.2 mM TCN-P inhibited GAR synthesis by 71-95%. To permit separate assay of the ligase step, 6-diazo-5-oxo-L-norleucine was used to inhibit amidophosphoribosyltransferase and phosphoribosylamine (PRA) was supplied in situ by chemical reaction of ribose-5-phosphate and ammonia (as ammonium acetate). The ligase was not inhibited by TCN-P. Thus, TCN-P inhibits amidophosphoribosyltransferase; it acts as an analog of the purine nucleotides which regulate this first committed step of de novo purine biosynthesis by an allosteric feedback mechanism. The measured intracellular concentration (0.1 mM) of PRPP was not changed in cells treated with TCN. IMP dehydrogenase (EC 1.1.1.205), the first de novo step committed to guanosine nucleotide synthesis, was also tested. It was inhibited by TCN-P, competitively with IMP, 66% at 1.2 mM TCN-P and 8 microM IMP. The degree of inhibition of these two enzymes was sufficient to account for the effects on purine nucleotide biosynthesis observed in intact cells treated with TCN.

Acenaphthenes↗
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↗