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Biomedical subjects

R M Bell

Publications and source records attributed to R M Bell.

At least 145 records · Page 8Linked to original sources
PubMed1987

Distinct patterns of expression of different protein kinase C mRNAs in rat tissues.

The identification of multiple protein kinase C (PKC) cDNA sequences from rat, bovine, and human tissues has led to the discovery of a family of PKC genes. Using probes complementary to three cDNA sequences from rat brain, designated PKC-I, -II, and -III, the distribution of PKC transcripts in rat tissues was studied by in situ hybridization histochemistry. In brain, PKC-II and PKC-III transcripts colocalized except in the brain stem and spinal cord, where some cells appeared to contain only PKC-III message. PKC-I mRNA had a different, but partially overlapping, pattern of expression. In spleen, PKC-II and PKC-III, but not PKC-I, transcripts were concentrated in the marginal zone of white pulp, the major B-lymphocyte compartment of rat spleen. The distinct distributions of rat PKC mRNAs suggest that different members of the PKC family have specialized functions.

Animals↗
PubMed1987

Mutants of Saccharomyces cerevisiae defective in sn-1,2-diacylglycerol cholinephosphotransferase. Isolation, characterization, and cloning of the CPT1 gene.

A colony autoradiographic assay for the sn-1,2-diacylglycerol cholinephosphotransferase activity in Saccharomyces cerevisiae was developed. Twenty-two mutants defective in cholinephosphotransferase activity were isolated. Genetic analysis revealed that all of these mutations were recessive, and three complementation groups were identified. The cholinephosphotransferase activities in membranes prepared from cpt1 mutants were reduced 2-10-fold compared to wild-type activity. The cholinephosphotransferase activities of two cpt1 isolates differed from wild-type activity with respect to their apparent KM for CDP-choline. The residual cholinephosphotransferase activities of cpt1 isolates were more sensitive to inhibition by CMP than the wild-type activity. The CPT1 gene was cloned by genetic complementation of cpt1 using a yeast genomic library. In strains transformed with the CPT1-bearing plasmid, a 5-fold overproduction of cholinephosphotransferase activity with wild-type kinetic properties was observed. The CPT1 gene was localized to a 1.2-2.4-kilobase region of DNA by transposon Tn5 mutagenesis and deletion mapping. An insertional mutant of the CPT1 gene was constructed and introduced into the chromosome by integrative transformation. The resulting cpt insertional mutant fell into the cpt1 complementation group. The cholinephosphotransferase activity in membranes prepared from the cpt1 insertional mutant was reduced 5-fold and exhibited CMP sensitivity. The sn-1,2-diacylglycerol ethanolaminephosphotransferase activities in membranes from all of the cpt1 isolates including the insertional mutant were normal. The data indicate that the cloned CPT1 gene represents the yeast cholinephosphotransferase structural gene, that the yeast choline- and ethanolaminephosphotransferase activities are encoded by different genes, and that the CPT1 gene is nonessential for growth.

Cell Membrane↗
PubMed1987

Diacylglycerol mass measurements in stimulated HL-60 phagocytes.

The mass of sn-1,2-diacylglycerol in crude lipid extracts from differentiated HL-60 phagocytes was measured by quantitative conversion of the diacylglycerol to [32P]-labeled phosphatidic acid catalyzed by E. coli diacylglycerol kinase. The chemotactic peptide N-formyl-Met-Leu-Phe caused a time- and concentration-dependent increase in diacylglycerol that was maximal at 4 min. Diacylglycerol returned toward basal levels by 15 min. The basal level of diacylglycerol was 290 +/- 25 pmol/10(7) cells (n = 36). Maximally effective concentrations of N-formyl-Met-Leu-Phe and N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys increased diacylglycerol to 176% +/- 16 of basal (n = 8) and 198% +/- 15 of basal (n = 4), respectively. t-Boc-Phe-Leu-Phe-Leu-Phe, a competitive antagonist of formyl peptide receptor function, competitively inhibited the N-formyl-Met-Leu-Phe-induced diacylglycerol increase. Pretreatment of the cells with pertussis toxin abolished the stimulated rise in diacylglycerol, whereas depletion of extracellular Ca2+ markedly inhibited the increase. The Ca2+ ionophore A23187 stimulated a large (450% of basal) and persistent (greater than 30 min) increase in diacylglycerol. These data suggest that agents which raise intracellular Ca2+ levels in differentiated HL-60 cells produce a prolonged increase in cellular diacylglycerol which may activate protein kinase C.

Calcimycin↗
PubMed1987

Lysosphingolipids inhibit protein kinase C: implications for the sphingolipidoses.

Lysosphingolipids potently and reversibly inhibited protein kinase C activity and binding of phorbol dibutyrate in vitro and in human platelets. As with activation of protein kinase C by phosphatidylserine and sn-1,2-diacylglycerol, inhibition was subject to surface dilution. Accordingly, inhibition in mixed micelle assays was dependent on the molar percentage of lysosphingolipids rather than the bulk concentration. Lysosphingolipids inhibited protein kinase C activity at molar percentages similar to those required for activation by phosphatidylserine and sn-1,2-diacylglycerol. Since lysosphingolipids accumulate in Krabbe's disease, Gaucher's disease, and other sphingolipidoses, the hypothesis that lysosphingolipid inhibition of protein kinase C represents the missing functional link between the accumulation of sphingolipids and the pathogenesis of these disorders appears to unify existing data. The accumulation of lysosphingolipids would cause progressive dysfunction of signal transduction mechanisms vital for neural transmission, differentiation, development, and proliferation and would eventually lead to cell death.

Blood Platelets↗
PubMed1987

Membrane phospholipid bilayer assembly: phospholipid biosynthetic enzymes and phospholipid transporters.

Assembly of the phospholipid bilayer of cellular membranes is a fundamental aspect of cell growth and proliferation. Phospholipids are concomitantly synthesized and inserted at the cytoplasmic surface of the endoplasmic reticulum. Following this asymmetric assembly, transmembrane movement to the lumenal leaflet of the endoplasmic reticulum must occur in order to ensure coordinated growth of the bilayer. For phosphatidylcholine, the predominant phospholipid of eukaryotic membranes, this latter process appears to be facilitated by a specific transport protein.

Animals↗
PubMed1986

sn-1,2-Diacylglycerol kinase of Escherichia coli. Structural and kinetic analysis of the lipid cofactor dependence.

The lipid cofactor requirement of Escherichia coli sn-1,2-diacylglycerol kinase was studied using a beta-octylglucoside mixed micellar assay (Walsh, J. P., and Bell, R. M. (1986) J. Biol. Chem. 261, 6239-6247). The enzyme was shown to have an absolute requirement for a lipid activator. sn-1,2-Dioleoylglycerol was both an activator and a substrate for the enzyme, 1,3-dioleoylglycerol was an activator but not a substrate, and sn-1,2-dioctanoylglycerol was a substrate but not an activator. Activation was observed with a large number of phospholipids, sulfolipids, neutral lipids, and detergents. Lipids with longer alkyl/acyl chains stimulated activity to a greater extent and at lower concentrations than their shorter chain homologs. Anionic lipids were the best activators, and neutral lipids were somewhat less effective. Cationic lipids were poor activators. Lipid activation was cooperative in all cases, with Hill coefficients ranging from 2.9 to 4.7. Lipid activators stabilized the enzyme against inactivation induced by diacylglycerols. The effectiveness of several lipids in stabilizing the enzyme correlated with their effectiveness as kinetic activators, suggesting a common mechanism. Kinetic analyses also suggested that a lipid cofactor-induced conformational change occurs as a part of the activation process. beta-Octylglucoside was shown not to function as a lipid cofactor for diacylglycerol kinase. The requirement for detergent in the assay was related, instead, to the need to disperse and deliver water-insoluble substrates and cofactors to the enzyme. beta-Octylglucoside also provided an inert matrix to which lipid substrates and cofactors could be added, enabling study of their concentration dependencies.

Adenosine Triphosphate↗
PubMed1986

The lipid binding, regulatory domain of protein kinase C. A 32-kDa fragment contains the calcium- and phosphatidylserine-dependent phorbol diester binding activity.

Trypsinization of rat brain protein kinase C (80 kDa) into 50- and 32-kDa fragments occurred without inhibition of [3H]phorbol dibutyrate ([3H]PDBu) binding activity. The 50-kDa fragment, the catalytic domain (Inoue, M., Kishimoto, A., Takai, Y., and Nishizuka, Y. (1977) J. Biol. Chem. 252, 7610-7616), was further degraded by trypsin, whereas the 32-kDa fragment was resistant. Protein kinase activity and the [3H]PDBu binding activity were completely separated upon gel filtration of a solution containing Triton X-100/phosphatidylserine mixed micelles and trypsinized protein kinase C. Pooled fractions of the [3H]PDBu binding activity contained a 32-kDa fragment exclusively. The binding of [3H]PDBu to this fragment was dependent on calcium and phosphatidylserine and was of high affinity (Kd = 2.8 nM) and of essentially identical specificity to that of native protein kinase C. It is concluded that the 32-kDa fragment represents a lipid binding, regulatory domain of protein kinase C.

Animals↗
PubMed1986

Attenuation of sn-1,2-diacylglycerol second messengers. Metabolism of exogenous diacylglycerols by human platelets.

The metabolism of exogenous [3H]diacylglycerols by intact human platelets was studied in order to examine: the metabolic fate of these second messengers in an intact cell, the effect of diacylglycerol kinase and diacylglycerol lipase inhibitors on this metabolism, the effect of agonist stimulation on metabolism, and the dependence of metabolism on diacylglycerol chain length. When 2.5 microM [3H]dioctanoylglycerol (diC8) was added to 10(9) platelets it was rapidly metabolized; 80% was converted to various products in 2.5 min. Initially, 40% was recovered as 3H-labeled phospholipid (predominantly phosphatidic acid) reflecting the action of diacylglycerol kinase, 20% was recovered as [3H]glycerol due to the action of diacylglycerol and monoacylglycerol lipases, and small amounts were recovered as triacylglycerol and monoacylglycerol. Thrombin stimulation of platelets did not affect the rate or pathway of metabolism. Pretreatment of platelets with the diacylglycerol kinase inhibitors, diC8ethyleneglycol or 1-monooleoylglycerol, inhibited 3H-labeled phospholipid production 47% and 75%, respectively, and resulted in a longer lived diC8 signal. The diacylglycerol lipase inhibitor, RHC 80267, inhibited the production of water-soluble metabolites 75%. Despite inhibition of the lipase, the overall metabolism of exogenous [3H]diC8 occurred at a similar rate as in control platelets due to an increased flux towards phospholipid. The ability of exogenous diacylglycerols to be metabolized by diacylglycerol kinase correlated well with their ability to activate protein kinase C in platelets. [3H]Dibutyroylglycerol, didodecanoylglycerol, and ditetradecanoylglycerol, were not metabolized by this route. These diacylglycerols were still metabolized via the lipase pathway. The results indicate that platelets possess potent attenuation systems to defend against the accumulation of diacylglycerol second messengers, and that the primary metabolic fate of cell-permeable, exogenous diacylglycerols is conversion to phosphatidic acid.

Blood Platelets↗
PubMed1986

Inhibition of the oxidative burst in human neutrophils by sphingoid long-chain bases. Role of protein kinase C in activation of the burst.

The neutrophil oxidative burst is characterized by increased cellular O2 consumption due to the activation of a membrane-associated superoxide-generating NADPH-oxidase. The response is triggered by a variety of stimuli, including opsonized zymosan, formylmethionylleucinephenylalanine (FMLP), arachidonate, short-chain diacylglycerols, and phorbol myristate acetate (PMA). We herein demonstrate that incubation of cells with sphinganine or sphingosine blocks or reverses activation by these agonists. The inhibition is reversible, does not affect cell viability, and does not affect another complex cell function, phagocytosis. Inhibitory concentrations of sphinganine did not significantly affect cytoplasmic calcium levels or FMLP-generated calcium transients. Structural requirements for inhibition of the oxidative burst include a long aliphatic chain and an amino-containing head-group, and there is modest specificity for the native (erythro) isomer of sphinganine. Inhibition involves stimulus-induced activation mechanisms rather than a direct effect on the NADPH oxidase, since sphinganine did not inhibit NADPH-dependent superoxide generation in isolated membranes containing the active enzyme. Activation by FMLP, diacylglycerol, PMA, opsonized zymosan, and arachidonate was blocked by the same concentrations of sphinganine, indicating that these agonists share a common inhibited step. Three lines of evidence indicate that this step involves protein kinase C. First, in a micelle system and in platelets, long-chain bases are inhibitors of this enzyme (Hannun, Y., Loomis, C., Merrill, A., and Bell, R. M. (1986) J. Biol. Chem. 261, 12604-12609). Second, sphinganine blocks PMA-stimulated incorporation of 32PO4 into neutrophil proteins. Third, sphinganine inhibits the binding of [3H]phorbol dibutyrate to its cellular receptor, known to be protein kinase C. We suggest that long-chain bases function as physiologic modulators of cellular regulatory pathways involving protein kinase C.

Arachidonic Acid↗
PubMed1986

Sphingosine inhibition of protein kinase C activity and of phorbol dibutyrate binding in vitro and in human platelets.

Sphingosine inhibited protein kinase C activity and phorbol dibutyrate binding. When the mechanism of inhibition of activity and phorbol dibutyrate binding was investigated in vitro using Triton X-100 mixed micellar methods, sphingosine inhibition was subject to surface dilution; 50% inhibition occurred when sphingosine was equimolar with sn-1,2-dioleoylglycerol (diC18:1) or 40% of the phosphatidylserine (PS) present. Sphingosine inhibition was modulated by Ca2+ and by the mole percent of diC18:1 and PS present. Sphingosine was a competitive inhibitor with respect to diC18:1, phorbol dibutyrate, and Ca2+. Increasing levels of PS markedly reduced inhibition by sphingosine. Since protein kinase C activity shows a cooperative dependence on PS, the kinetic analysis of competitive inhibition was only suggestive. Sphingosine inhibited phorbol dibutyrate binding to protein kinase C but did not cause protein kinase C to dissociate from the mixed micelle surface. Sphingosine addition to human platelets blocked thrombin and sn-1,2-dioctanoylglycerol-dependent phosphorylation of the 40-kDa (47 kDa) dalton protein. Moreover, sphingosine was subject to surface dilution in platelets. The mechanism of sphingosine inhibition is discussed in relation to a previously proposed model of protein kinase C activation. The possible physiological role of sphingosine as a negative effector of protein kinase C is suggested and a plausible cycle for its generation is presented. The potential physiological significance of sphingosine inhibition of protein kinase C is further established in accompanying papers on HL-60 cells (Merrill, A. H., Jr., Sereni, A. M., Stevens, V. L., Hannun, Y. A., Bell, R. M., Kinkade, J. M., Jr. (1986) J. Biol. Chem. 261, 12010-12615) and human neutrophils (Wilson, E., Olcott, M. C., Bell, R. M., Merrill, A. H., Jr., and Lambeth, J. D. (1986) J. Biol. Chem. 261, 12616-12623). These results also suggest that sphingosine will be a useful inhibitor for investigating the function of protein kinase C in vitro and in living cells.

Amines↗
PubMed1986

Inhibition of phorbol ester-dependent differentiation of human promyelocytic leukemic (HL-60) cells by sphinganine and other long-chain bases.

The effects of long-chain (sphingoid) bases on the phorbol ester-dependent differentiation of HL-60 cells were investigated since these molecules are potent inhibitors of protein kinase C (Hannun, Y. A., Loomis, C. R., Merrill, A. H., Jr., and Bell, R. M. (1986) J. Biol. Chem. 261, 12604-12609). After 24 h, low concentrations of sphinganine (1-5 microM blocked both cell adherence and the inhibition of growth in response to phorbol 12-myristate 13-acetate, as measured by cell number and acid phosphatase activity. Sphinganine and sphingosine decreased adherence by 50% at 1-3 microM; other long-chain bases were effective in parallel to their inhibition of protein kinase C. Sphinganine decreased the binding of [3H]phorbol dibutyrate by the phorbol receptor of HL-60 cells, protein kinase C, and inhibited the response of HL-60 cells to dioctanoylglycerol, a cell permeable activator of this enzyme. Long-chain base uptake by HL-60 cells was demonstrated with [3-3H]sphinganine and within 1-3 days much had been converted to ceramides. By day 3, most of the cells had recovered the ability to adhere and exhibited macrophage characteristics, whereas cells in suspension did not differentiate. The level of free sphinganine in HL-60 cells was determined to be 12.3 +/- 1.2 pmol/10(6) cells. These results establish that sphingoid bases inhibit protein kinase C in HL-60 cells and may function physiologically as negative effectors of this enzyme.

Acid Phosphatase↗
PubMed1986

Regulation of diacylglycerol kinase biosynthesis in Escherichia coli. A trans-acting dgkR mutation increases transcription of the structural gene.

The mechanism of a trans-acting mutation, dgkR1, which causes a 7-fold elevation of diacylglycerol kinase activity in membranes (Raetz, C. R. H., Kantor, G. D., Nishijima, M., and Jones, M. L. (1981) J. Biol. Chem. 256, 2109-2112) was investigated by direct measurement of diacylglycerol kinase polypeptide by high performance liquid chromatography and by construction of fusions of the dgkA promoter to beta-galactosidase and galactokinase. The dgkR1 mutation was demonstrated to act by increasing the transcription of the structural gene for diacylglycerol kinase, dgkA. Additionally, sn-glycerol-3-phosphate acyltransferase activities were shown to be decreased 30-50% in membranes from dgkR1 mutant strains. Increased diacylglycerol levels occurred when cells were grown on low osmolarity media. This did not affect dgkA expression. In a dgkR+ background, enhanced expression of sn-1,2-diacylglycerol kinase activity in cells containing a high copy number plasmid bearing dgkA decreased sn-1,2-diacylglycerol levels. However, overproduction of diacylglycerol kinase in a dgkR1 genetic background did not affect diacylglycerol levels, suggesting that the dgkR1 mutation affects diacylglycerol metabolism by mechanisms additional to enhancement of dgkA transcription.

Chromatography, High Pressure Liquid↗