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G R Boss

Publications and source records attributed to G R Boss.

At least 37 records · Page 2Linked to original sources
PubMed1998

Energy deprivation and a deficiency in downstream metabolic target genes during the onset of embryonic heart failure in RXRalpha-/- embryos.

RXRalpha null mutant mice display ocular and cardiac malformations, liver developmental delay, and die from cardiac failure around embryonic day (E) 14.5 pc. To dissect the molecular basis of the RXRalpha-associated cardiomyopathy, we performed subtractive hybridization and systematically characterized putative downstream target genes that were selectively lacking in the mutant embryos, both at early (E10.5) and late (E13.5) stages of mouse embryonic development. Approximately 50% of the subtracted clones (61/115) encoded proteins involved in intermediary metabolism and electron transport, suggesting an energy deficiency in the RXRalpha-/- embryos. In particular, clone G1, which encodes subunit 14.5b of the NADH-ubiquinone dehydrogenase complex, displayed a dose-dependent expression in the wild-type, heterozygous and RXRalpha mutant mice. This gene was also downregulated in a retinoid-deficient rat embryo model. ATP content and medium Acyl-CoA dehydrogenase mRNA were lower in RXRalpha mutant hearts compared to wild-type mice. Ultrastructural studies showed that the density of mitochondria per myocyte was higher in the RXRalpha mutant compared to wild-type littermates. We propose a model whereby defects in intermediary metabolism may be a causative factor of the RXRalpha-/- phenotype and resembles an embryonic form of dilated cardiomyopathy.

Acyl-CoA Dehydrogenase↗
PubMed1997

A low molecular weight substance purified from human placenta inhibits cAMP-dependent protein kinase and activates protein kinase C.

We have purified from human placenta a low molecular mass substance that inhibits cAMP-dependent protein kinase and activates protein kinase C. This protein kinase regulator was purified in three steps: (1) homogenizing placentas in chloroform/methanol and extracting the regulator into water; (2) eluting a strong anion exchange high performance liquid chromatography (HPLC) column with a quaternary gradient; and (3) eluting a reversed-phase HPLC column with a binary gradient. The regulator was found to be highly purified by HPLC, thin-layer chromatography (TLC) and laser desorption ionization mass spectrometry with a molecular mass of 703 Daltons by the latter procedure. The physical and biochemical properties of this protein kinase regulator suggest that it is a phospholipid but it did not co-elute by HPLC or by TLC with any of the known phospholipid activators of protein kinase C.

Biological Factors↗
PubMed1997

A decrease in the intracellular guanosine 5'-triphosphate concentration is necessary for granulocytic differentiation of HL-60 cells, but growth cessation and differentiation are not associated with a change in the activation state of Ras, the transforming principle of HL-60 cells.

We found that when human promyelocytic leukemic cells (HL-60 cells) were induced to differentiate along the granulocytic lineage by two diverse mechanisms (starvation for an essential amino acid or treatment with DMSO), there was a marked decrease in the intracellular guanosine 5'-triphosphate (GTP) concentration with no change in the guanosine 5'-diphosphate (GDP) concentration. Differentiation was prevented by guanine or guanosine in a dose-dependent manner. We showed that: (a) guanine had to be converted to a nucleotide because it did not prevent differentiation of hypoxanthine-guanine phosphoribosyltransferase-deficient HL-60 cells; (b) the effect of guanine correlated with a return of the cytosolic GTP:GDP ratio to normal; and (c) other purine bases were not effective. We hypothesized that the decreased GTP:GDP ratio in differentiating HL-60 cells might decrease the relative amount of GTP bound to Ras, a key regulatory GTP-binding protein important to cell growth and differentiation. Consistent with data showing that HL-60 cells harbor an activating N-Ras mutation, we found that the percentage of Ras molecules in the GTP-bound state was high in proliferating HL-60 cells (27 +/- 3%) compared with other cultured mammalian cells (< 1%); however, we found no change in the activation state of Ras when cells ceased to proliferate and differentiated in response to DMSO, amino acid deprivation, or inhibitors of guanylate synthesis. We conclude that: (a) a decrease in the intracellular GTP concentration is necessary for HL-60 cells to undergo granulocytic differentiation; and (b) although a high degree of Ras activation contributes to the malignant phenotype of the cell, there is no change in the activation state of Ras during granulocytic differentiation.

Amino Acids↗
PubMed1996

Regional deficits of myocardial blood flow and function in left ventricular pacing-induced heart failure.

BACKGROUND: Pacing-induced congestive hear, failure has become a preferred model for the study of the pathogenesis of dilated cardiomyopathy. However, little is known regarding regional myocardial blood flow and function during the development of heart failure in this model. METHODS AND RESULTS: To determine whether regional differences in myocardial blood flow are associated with regional dysfunction in ventricular pacing-induced heart failure, regional myocardial blood flow (radioactive microspheres) and regional wall thickening (transthoracic echocardiography) were measured in pigs studied at weekly intervals during the progression of heart failure induced by rapid pacing from the lateral wall of the left ventricle (220 +/- 9 bpm for 26 +/- 4 days). Echocardiography and hemodynamic measurements with the pacemaker off showed progressive, severe global left ventricular dysfunction. During pacing over the 3- to 4-week period, a progressive decrease in systolic wall thickening in the lateral wall occurred compared with the interventricular septum (IVS; P = .001); at 21 to 28 days, the difference was 50% (lateral wall, 14 +/- 6%; IVS, 28 +/- 6%; P = .0001). A difference in subendocardial blood flow per beat between the left ventricular lateral wall (the site of stimulation) and the IVS was found immediately on the initiation of pacing (IVS, 0.009 +/- 0.002 mL.min-1.g-1.beat-1; lateral wall, 0.005 +/- 0.001 mL.min-1.g-1.beat-1; P = .001), a difference that was sustained during pacing throughout the study. Subendocardial blood flow per beat was normal in both regions with the pacemaker off throughout the study. CONCLUSIONS: These data indicate that regional myocardial ischemia is associated with the development of contractile dysfunction of the paced wall during prolonged rapid left ventricular pacing and that regional stunning contributes to persistent global left ventricular dysfunction when pacing is discontinued.

Adenosine Diphosphate↗
PubMed1996

Enhanced tumorigenic behavior of glioblastoma cells expressing a truncated epidermal growth factor receptor is mediated through the Ras-Shc-Grb2 pathway.

A mutant epidermal growth factor receptor (DeltaEGFR) containing a deletion of 267 amino acids from the extracellular domain is common in human glioblastomas. We have previously shown that the mutant receptor fails to bind EGF, is constitutively phosphorylated, and confers upon U87MG glioblastoma cells expressing it (U87MG. DeltaEGFR), an increased ability to form tumors in mice. Here we demonstrate that the constitutively phosphorylated DeltaEGFR enhances growth of glioblastoma cells through increased activity of Ras: 1) there was an increase in the proportion of Ras present in the GTP-bound form, and 2) introduction of neutralizing anti-Ras 259 antibodies into U87MG and U87MG.DeltaEGFR cells by microinjection inhibited DNA synthesis to the same low level in both cell populations. We also show that the truncated EGF receptor constitutively associates with the adapter proteins Shc and Grb2 which are involved in the recruitment of Ras to activated receptors. Several derivatives of DeltaEGFR containing single, or multiple mutations at critical autophosphorylation sites were constructed and used to demonstrate that the major Shc binding site is Tyr-1148, and that Grb2 association occurs primarily through Tyr-1068. We conclude that the increased tumorigenic potential of glioblastoma cells expressing the truncated EGF receptor is due at least in part to Ras activation presumably involving the Shc and Grb2 adapter proteins.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Regulation of gene expression by cGMP-dependent protein kinase. Transactivation of the c-fos promoter.

The cAMP/cAMP-dependent protein kinase (A-kinase) and Ca2+/calmodulin-dependent protein kinase (Cam-kinase) signal transduction pathways are well known to regulate gene transcription, but this has not been demonstrated directly for the cGMP/cGMP-dependent protein kinase (G-kinase) signal transduction pathway. Here we report that transfection of G-kinase into G-kinase-deficient cells causes activation of the human c-fos promoter in a strictly cGMP-dependent manner. The effect of G-kinase appeared to be mediated by several sequence elements, most notably the serum response element (SRE), the AP-1 binding site (FAP), and the cAMP response element (CRE). The magnitude of G-kinase transactivation of the fos promoter was similar to that of A-kinase, but there were significant differences between G-kinase and A-kinase activation of single enhancer elements and of a chimeric Gal4-CREB transcription factor. Our results indicate that G-kinase transduces signals to the nucleus independently of A-kinase or Ca2+, although it may target some of the same transcription factors as A-kinase and Cam-kinase.

Animals↗
PubMed1995

Determination of absolute amounts of GDP and GTP bound to Ras in mammalian cells: comparison of parental and Ras-overproducing NIH 3T3 fibroblasts.

We devised enzyme-based methods to measure fmol amounts of GDP and GTP and applied these methods to measure absolute amounts of Ras-bound GDP and GTP in NIH 3T3 fibroblasts. We found that parental NIH 3T3 cells contained 509 and 1.3 fmol of Ras-bound GDP and GTP, respectively, per mg of cellular protein and that stable transfectants of NIH 3T3 cells overexpressing wild-type Ha-Ras contained 7008 and 21.3 fmol of Ras-bound GDP and GTP, respectively, per mg of cellular protein; thus, in both cell types < 0.3% of Ras was in the active GTP-bound state. In contrast, NIH 3T3 cells overexpressing an activated form of Ha-Ras contained 5013 and 2049 fmol of Ras-bound GDP and GTP, respectively, per mg of protein, yielding 29% of Ras in the GTP-bound state. Since intracellular Ras is probably all in a guanine-nucleotide bound state, this method allows one to calculate the number of Ras molecules in each cell: parental NIH 3T3 cells and the Ha-Ras overproducing cells contain approximately 20,000 and approximately 275,000 Ras molecules per cell, respectively. We also incubated parental NIH 3T3 cells with 32PO4 and determined the radioactivity in Ras-bound GDP and GTP and the specific activity of cytosolic GDP and GTP; these experiments indicated that Ras-bound GTP may not be in equilibrium with the total cytosolic GTP pool.

3T3 Cells↗
PubMed1995

Nitric oxide and cGMP analogs activate transcription from AP-1-responsive promoters in mammalian cells.

Nitric oxide (NO) increases cytosolic guanylate cyclase activity and thereby activates the cGMP signal transduction pathway. The cAMP and Ca2+/phospholipid signal transduction pathways activate transcription factors that bind to the cAMP response element (CRE) and phorbol ester response element (TRE), respectively. Little is known about transcriptional regulation of gene expression by NO/cGMP. In transient and stable transfection experiments and in microinjection studies we found that three different NO-releasing agents and two membrane-permeable cGMP analogs activated TRE-regulated but not CRE-regulated reporter genes in rodent fibroblast and epithelial cell lines. Activation of TRE-regulated genes by NO-releasing agents and cGMP analogs appeared to be mediated by the AP-1 (Jun/Fos) transcription factor complex because we observed increased DNA binding of AP-1 and increased junB and c-fos mRNA in cells treated with these agents. The mechanism of gene activation by NO/cGMP was distinct from that used by phorbol esters and cAMP because it was not associated with c-jun mRNA induction and was not observed with CRE-containing promoters.

Animals↗
PubMed1995

Nitric oxide-releasing agents and cGMP analogues inhibit murine erythroleukemia cell differentiation and suppress erythroid-specific gene expression: correlation with decreased DNA binding of NF-E2 and altered c-myb mRNA expression.

Differentiation of murine erythroleukemia (MEL) cells induced by hexamethylene bisacetamide (HMBA) and DMSO was inhibited by several structurally unrelated nitric oxide (NO)-releasing agents and two membrane-permeable cGMP analogues. Since the effect of the NO-releasing agents was augmented by a cGMP phosphodiesterase inhibitor, at least some of their effect appeared to be mediated by activation of cytosolic guanylate cyclase. The drugs did not globally block differentiation since hemin-induced differentiation was undisturbed. In HMBA-treated cells, the NO-releasing agents and cGMP analogues reduced beta-globin and delta-aminolevulinate synthetase mRNA expression and inhibited the late down-regulation of c-myb mRNA that is required for HMBA-induced differentiation of MEL cells; the regulation of c-myc mRNA was not changed by the drugs. Nuclear run-off analyses showed that the drugs inhibited the HMBA-induced changes in beta-globin and c-myb transcription rates, and transient transfection of a reporter gene construct demonstrated that the drugs inhibited HMBA-inducible enhancer function of the alpha-globin control region, which contains binding sites for the erythroid transcription factors NF-E2 and GATA-1. The NO-releasing agents and cGMP analogues largely prevented HMBA-induced increases in DNA binding of NF-E2, whereas DNA binding of GATA-1 and SP-1 was not affected. The inhibition of erythroid gene expression by NO and cGMP analogues may be physiologically important under conditions of high NO production by endothelial cells and macrophages, i.e. during acute or chronic inflammation.

5-Aminolevulinate Synthetase↗
PubMed1994

Isolation and characterization of HL-60 cells resistant to nitroprusside-induced differentiation.

Sodium nitroprusside and sodium nitrite, which generate nitric oxide and increase the intracellular cGMP concentration, and 8-bromo-cGMP, a membrane-permeable cGMP analog, induce myelomonocytic differentiation of HL-60 cells (Boss, G. R. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 7174-7178). We have selected HL-60 cells resistant to nitroprusside-induced differentiation as assessed by acquisition of the OKM-1 antigen, reduction of nitro blue tetrazolium, and morphologic maturation. The variant cells were also resistant to differentiation induced by sodium nitrite and two cGMP analogs but still differentiated in response to other inducing agents such as dimethyl sulfoxide and cAMP analogs and showed the same changes in c-myc and c-fos expression in response to the latter drugs as occurred in parental cells. We studied the early steps of the NO/cGMP signal transduction pathway in the variant cells and found that basal and nitroprusside-stimulated guanylate cyclase activity was similar in parental and variant cell extracts and that nitroprusside increased the intracellular cGMP concentration to the same extent in parental and variant cells. As part of these studies we found that HL-60 cells expressed only alpha 2 and beta 2 guanylate cyclase mRNA; the abundance of these two mRNA species was similar in parental and variant cells. Neither nitroprusside nor 8-bromo-cGMP changed the intracellular calcium concentration in parental or variant cells. The data suggest that the defect in the variant cells is after guanylate cyclase activation in the NO/cGMP transduction pathway and that the cGMP and cAMP transduction pathways operate independently in inducing differentiation of HL-60 cells.

Cell Differentiation↗
PubMed1994

Identification of a ras-related protein in murine erythroleukemia cells that is a cAMP-dependent protein kinase substrate and is phosphorylated during chemically induced differentiation.

Murine erythroleukemia (MEL) cells deficient in cAMP-dependent protein kinase (A-kinase) activity are impaired in chemically induced differentiation (Pilz, R. B., Eigenthaler, M., and Boss, G. R. (1992) J. Biol. Chem. 267, 16161-16167). We identified by two-dimensional polyacrylamide gel electrophoresis two low molecular weight proteins (referred to as pp 21-1 and 21-2) that were phosphorylated when parental MEL cells, but not A-kinase-deficient MEL cells, were treated with the membrane-permeable cAMP analog 8-bromo-cAMP. We showed that pp 21-1 and 21-2: (a) were direct A-kinase substrates; (b) bound GTP; and (c) belonged to the ras superfamily of proteins. The only ras-related proteins that are clearly A-kinase substrates both in vitro and in vivo are Rap 1A and 1B while H- and K-Ras can be A-kinase substrates in vitro; we showed by immunological methods, phosphopeptide mapping, and migration on two-dimensional gels that pp 21-1 and 21-2 were not identical to one of these four proteins. We found a 3-fold increase of 32PO4 incorporation into pp 21-2 in hexamethylene bisacetamide-treated parental MEL cells which was not secondary to an increase in pp 21-2 protein but appeared secondary to increased phosphorylation of pp 21-2 by A-kinase. Thus, pp 21-1 and 21-2 are either new ras-related proteins or are previously identified ras-related proteins not known to be A-kinase substrates, and increased phosphorylation of pp 21-2 occurs during differentiation of MEL cells.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1993

Microinjection of an nm23 specific antibody inhibits cell division in rat embryo fibroblasts.

Expression of the nm23/NDP-K gene correlates with reduced metastasis in some tumors and with increased proliferation in both nontransformed and transformed cells in culture. Decreased nm23/NDP-K expression results in mitotic arrest in neuroblasts of developing Drosophila. In order to better understand the biological role(s) of nm23 in non-transformed cells, an nm23-specific antibody was introduced into rat embryo fibroblasts and effects on DNA synthesis and cell cycle progression were analyzed. Microinjection of the nm23 antibody inhibited cell division with no apparent effect on DNA synthesis. Control experiments revealed that the survival of cells injected with the nm23 antibody was similar to that of control antibody injected cells in the absence of cell division. These results suggest that in mammalian fibroblasts, as in Drosophila, nm23 expression may be necessary for progression through the cell cycle.

Animals↗
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↗