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Biomedical subjects

B M Babior

Publications and source records attributed to B M Babior.

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

Activation of the leukocyte NADPH oxidase in a cell-free system: phosphorylation vs. amphiphiles.

We examined the ability of C-terminal deletion mutants of p47PHOX, a cytosolic subunit of the leukocyte NADPH oxidase, to support the activity of the oxidase in two different cell-free systems, one using protein kinase C and the other an anionic amphiphile (SDS or arachidonic acid) as the oxidase-activating agent. Two deletion mutants were studied: p47PHOXdelta330 and p47PHOXdelta348, each named according to the first residue of the deleted polypeptide. Wild-type (WT) p47PHOX and both mutants were phosphorylated by protein kinase C, but the WT protein was the most heavily phosphorylated, containing 6.0 +/- 0.5 mol phosphate/mol protein. Of the two deletion mutants, only p47PHOXdelta348 could support oxidase activity, and then only in the amphiphile-activated system; neither of the mutants supported oxidase activity in the system activated by protein kinase C. Translocation correlated with activity: WT p47PHOX translocated to the membrane in response to both protein kinase C and amphiphile, but p47PHOXdelta348 translocated only in the amphiphile-activated system. Comparison of these findings with the results of earlier studies suggests that the phosphorylation of p47PHOX is an important component of oxidase activation. The findings provide no information, however, about whether amphiphiles participate in the activation process in intact cells. Consequently, a mechanism of in vivo oxidase activation involving both phosphorylation and the generation of an amphiphile remains a distinct possibility.

Cell-Free System↗
PubMed1997

Granulocyte colony-stimulating factor upregulates the vacuolar proton ATPase in human neutrophils.

We have previously shown that granulocyte colony-stimulating factor (G-CSF ) delays spontaneous neutrophil apoptosis through activation of the vacuolar proton ATPase (v-ATPase). We have now examined the regulation of the v-ATPase in neutrophils exposed to G-CSF in vitro. When neutrophils were cultivated in the absence of G-CSF, the 57-kD cytosolic B subunit of the v-ATPase disappeared within 1 to 2 hours, its loss preceding the nuclear changes of apoptosis and coinciding with the onset of acidification. By contrast, in neutrophils cultured for 2 hours in the presence of G-CSF, the amount of the 57-kD subunit was similar to that in freshly isolated neutrophils. However, inhibition of protein synthesis with cycloheximide and actinomycin D led to loss of the 57-kD subunit even in the presence of G-CSF. These results indicated that ongoing protein synthesis was required to maintain the v-ATPase, and further suggested that G-CSF acted, at least in part, by maintaining synthesis of the 57-kD cytosolic subunit. G-CSF also promoted the translocation of the 57-and 33-kD cytosolic v-ATPase subunits to the membrane. Our findings suggested two coordinate mechanisms by which the activity of the v-ATPase could be increased by G-CSF: the synthesis of cytosolic v-ATPase subunits and their translocation to the membrane.

Adult↗
PubMed1997

Bcl-2 and the outer mitochondrial membrane in the inactivation of cytochrome c during Fas-mediated apoptosis.

Fas-driven apoptosis in Jurkat cells results in the inactivation of cytochrome c with cessation of oxygen consumption. Overexpression of Bcl-2 was found to protect against acidification and apoptosis mediated by Fas ligation in these cells. Bcl-2 is present in the outer mitochondrial membrane, but the molecular mechanism by which it protects cells is unknown. Because Bcl-2 projects into the mitochondrial intermembrane space and cytochrome c is located in the intermembrane space, we considered the possibility that Bcl-2 might protect cytochrome c from inactivation during Fas-mediated apoptosis. The present study shows that 1) in Jurkat cells, cytochrome c inactivation during Fas-driven apoptosis requires the permeabilization of the outer mitochondrial membrane; and 2) the post-mitochondrial fraction from CEM cells that overexpress Bcl-2 both prevents and reverses cytochrome c inactivation.

Apoptosis↗
PubMed1997

Activation of the leukocyte NADPH oxidase subunit p47phox by protein kinase C. A phosphorylation-dependent change in the conformation of the C-terminal end of p47phox.

The leukocyte NADPH oxidase of neutrophils is a membrane-bound enzyme that catalyzes the production of O2- from oxygen using NADPH as electron donor. Dormant in resting neutrophils, the enzyme acquires catalytic activity when the cells are exposed to appropriate stimuli. During activation, the cytosolic oxidase components p47phox and p67phox migrate to the plasma membrane, where they associate with cytochrome b558, a membrane-bound flavohemoprotein, to assemble the active oxidase. An essential element of the activation process is the phosphorylation of p47phox, an event that accompanies oxidase activation in whole cells and can activate the oxidase in a cell-free system. We show here that the phosphorylation of p47phox leads to a substantial decrease in the reactivity of cysteine C378 toward N-ethylmaleimide, indicating the occurrence of a conformational change involving the C-terminal region of p47phox. A similar conformational change occurs when p47phox is exposed to arachidonate, one of a number of anionic detergents that activate the oxidase in the cell-free system. We propose that this change in conformation results in the appearance of a binding site through which p47phox interacts with cytochrome b558 during the activation process.

Amino Acid Sequence↗
PubMed1997

Kinase-dependent activation of the leukocyte NADPH oxidase in a cell-free system. Phosphorylation of membranes and p47(PHOX) during oxidase activation.

The leukocyte NADPH oxidase catalyzes the 1-electron reduction of oxygen to O2- at the expense of NADPH: 2 O2 + NADPH --> 2 O2- + NADP+ + H+. The oxidase is dormant in resting cells but acquires activity when the cells are stimulated with a suitable agent. Activation in whole cells is accompanied by extensive phosphorylation of p47(PHOX), an oxidase subunit located in the cytosol of resting cells that during oxidase activation migrates to the plasma membrane to complex with cytochrome b558, an oxidase-specific flavohemoprotein. Oxidase activation can be mimicked in a cell-free system using an anionic amphiphile as activating agent. We now report a cell-free system in which the oxidase can be activated in two stages using phosphorylated p47(PHOX). The first stage, which effects a change in the membrane, requires ATP and GTP and is blocked by the protein kinase inhibitor GF-109203X, suggesting a protein kinase requirement. The second stage requires phosphorylated p47(PHOX) and GTP, but no ATP, and is unaffected by GF-109203X; assembly of the oxidase may take place during this stage. Activation is accomplished by p47(PHOX) phosphorylated by protein kinase C but not protein kinase A or mitogen-activated protein kinase. We believe that activation by phosphorylated p47(PHOX) is more physiological than activation by amphiphiles, because the mutant p47(PHOX) S379A, which is inactive in whole cells, is also inactive in this system but works in systems activated by amphiphiles.

Adenosine Triphosphate↗
PubMed1997

Superoxide: a two-edged sword.

Superoxide (O2-) is the compound obtained when oxygen is reduced by one electron. For a molecule with an unpaired electron, O2- is surprisingly inert, its chief reaction being a dismutation in which it reacts with itself to form H2O2 and oxygen. The involvement of O2- in biological systems was first revealed by the discovery in 1969 of superoxide dismutase, an enzyme that catalyzes the dismutation of O2-. Since then it has been found that biological systems produce a bewildering variety of reactive oxidants, all but a few arising ultimately from O2-. These oxidants include O2- itself, H2O2 and alkyl peroxides, hydroxyl radical and other reactive oxidizing radicals, oxidized halogens and halamines, singlet oxygen, and peroxynitrite. These various oxidants are able to damage molecules in their environment, and are therefore very dangerous. They are thought to participate in the pathogenesis of a number of common diseases, including among others malignancy, by their ability to mutate the genome, and atherosclerosis, by their capacity for oxidizing lipoproteins. Their properties are put to good use, however, in host defense, where they serve as microbicidal and parasiticidal agents, and in biological signalling, where their liberation in small quantities results in redox-mediated changes in the functions of enzymes and other proteins.

Animals↗
PubMed1996

Activation of p38 in stimulated human neutrophils: phosphorylation of the oxidase component p47phox by p38 and ERK but not by JNK.

Incubation of human neutrophils with FMLP, a chemotactic peptide, or PMA, a stimulator of protein kinase C, resulted in the activation of p38, a proline-directed kinase. Previous studies had shown that extracellular signal-regulated kinase (ERK), another proline-directed kinase, was activated with similar kinetics in neutrophils stimulated with FMLP and PMA (1, 2). Because one possible target for these proline-directed kinases is p47phox, a component of the respiratory burst oxidase, we examined the phosphorylation of this protein by p38 and ERK, as well as JNK, another proline-directed kinase present in neutrophils. We found that both p38 and ERK phosphorylated p47phox at the same site and at similar rates, but that p47phox was not a substrate for JNK. These data show that p38, like ERK, can be activated in neutrophils exposed to an appropriate stimulus, and that some but not all proline-directed kinases are able to participate in the phosphorylation of a protein essential for normal neutrophil function.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1996

Loss of function of cytochrome c in Jurkat cells undergoing fas-mediated apoptosis.

Mitochondrial function was examined in Jurkat cells undergoing Fas-mediated apoptosis. With succinate or ascorbate/tetramethylphenylenediamine as substrate, oxygen uptake by digitonin-permeabilized apoptotic mitochondria was greatly decreased as compared with control. Assessment of the function of the cytochrome c-cytochrome oxidase segment of the electron transport chain of apoptotic mitochondria showed that the activity of cytochrome oxidase appeared to be normal, but that of cytochrome c was greatly diminished. A death protease was found to participate in the events leading to the loss of cytochrome c activity, but the cytochrome did not seem to be extensively degraded during the course of apoptosis. Our results suggest that a rapid loss in mitochondrial function due at least in part to the inhibition or inactivation of cytochrome c is a potentially fatal component of the apoptosis program of Jurkat cells.

Apoptosis↗
PubMed1996

The cytosolic subunit p67phox contains an NADPH-binding site that participates in catalysis by the leukocyte NADPH oxidase.

The NADPH-dependent respiratory burst oxidase of human neutrophils catalyzes the reduction of oxygen to superoxide using NADPH as the electron donor and is essential for normal host defenses. To gain insight into the function of the various oxidase subunits that are required for the full expression of catalytic activity, we studied the interactions between the 2',3'-dialdehyde derivative of NADPH (NADPH dialdehyde) and neutrophil cytosol. NADPH dialdehyde treatment of cytosol resulted in the loss of the ability of the cytosol to participate in cell-free oxidase activation; this inactivation was blocked by NADPH but not by NAD, NADP, or GTP. Partial purification of neutrophil cytosol yielded a single peak which could restore the activity lost in cytosol treated with NADPH dialdehyde. This peak contained p67phox but not p47phox or Rac2. Purified recombinant p67phox was similarly able to restore the activity lost in NADPH dialdehyde-treated cytosol and bound [32P]NADPH dialdehyde in a specific fashion. The activity of recombinant p67phox in cell-free oxidase assays was lost on treatment with NADPH dialdehyde. Together, these data suggest p67phox contains the catalytic NADPH-binding site of the leukocyte NADPH oxidase.

Aldehydes↗
PubMed1996

Events in apoptosis. Acidification is downstream of protease activation and BCL-2 protection.

Cytoplasmic acidification is now recognized as a feature of apoptosis in a variety of systems. However, its relation to other events in the process of apoptosis is not yet characterized. In this work, we examined the effect of BCL-2 overexpression on acidification mediated by cycloheximide treatment or Fas ligation in Jurkat T-lymphoblasts. We find that BCL-2 overexpression attenuates cytoplasmic acidification and apoptosis detected by annexin V labeling. Acidification and phosphatidylserine externalization were found to occur concurrently. We also examined the requirement for protease activation for cytoplasmic acidification to occur and found that inhibition of interleukin-1beta converting enzyme/CED-3 family proteases (using carbobenzoxy-Val-Ala-Asp-fluoromethylketone, an inhibitor of these proteases) prevents acidification and apoptosis mediated by Fas ligation. These studies suggest that BCL-2 acts at a point upstream of acidification and that protease activation is also upstream of acidification.

Amino Acid Chloromethyl Ketones↗
PubMed1996

Enhancement of protein kinase C-dependent O2 production in Epstein-Barr virus-transformed B lymphocytes by p120Ras-GAP antisense oligonucleotide.

The mammalian Ras GTPase-activating protein (p120Ras-GAP) interacts with activated members of the Ras superfamily of GTP-binding proteins to accelerate their deactivation by sharply increasing their rates of GTP hydrolysis. Among the Ras-family proteins interacting with p120Ras-GAP is Rap1A/Krev1, whose activity is not affected by p120Ras-GAP but which competes with Ras for p120Ras-GAP. A second protein that interacts with p120Ras-GAP is P190Rac-GAP, which activates the GTPase of guanine nucleotide-binding proteins of the Rho family (including Rac1 and Rac2). Both these p120Ras-GAP-binding proteins are of interest in connection with the regulation of the respiratory burst oxidase, Rap1A/Krev1 because it copurifies with cytochrome b558 and p190Ras-GAP because it inhibits the Rac2-dependent activation of the respiratory burst oxidase in a cell-free system. Using an 18-mer antisense oligonucleotide, we were able to decrease the expression of p120Ras-GAP in Epstein-Barr virus-transformed B lymphocytes. Under conditions where p120Ras-GAP expression was significantly depressed by antisense oligonucleotides, we observed a 40% increase in protein kinase C-dependent but not receptor-dependent O2 production. In contrast, sense and scrambled oligonucleotides had no effect on either p120Ras-GAP expression or O2 production. Our results suggest a role for p120Ras-GAP as a negative regulator in the protein kinase C-mediated activation of the respiratory burst oxidase.

B-Lymphocytes↗
PubMed1996

Phosphorylation of the respiratory burst oxidase subunit p47phox as determined by two-dimensional phosphopeptide mapping. Phosphorylation by protein kinase C, protein kinase A, and a mitogen-activated protein kinase.

The respiratory burst oxidase is responsible for superoxide (O2) production by phagocytes and B lymphocytes. This multicomponent enzyme is dormant in resting cells but is activated on exposure of the cells to an appropriate stimulus. Upon activation, several serine residues on the cytosolic oxidase subunit p47phox become phosphorylated. Using two-dimensional tryptic phosphopeptide mapping, we studied the phosphorylation of p47phox in 32Pi-loaded Epstein-Barr virus-transformed B lymphoblasts expressing wild type p47phox or any of several P47phox Ser -> Ala mutants. We were able to identify the labeled peptides from wild type p47phox as those contain- ing Ser303/304 Ser315, Ser320, Ser328 and/or Ser359/370, and Ser345/348 ; no 32P-labeled Ser310-containing peptide was found. When purified p47phox, was phosphorylated in vitro by various protein kinases, varying phosphopeptide patterns were observed. Protein kinase C phosphorylated all the peptides except the one containing Ser345/348; protein kinase A phosphorylated the peptide containing Ser320 and one or both of the peptides containing Ser328 and Ser359/370; while mitogen-activated protein kinase phophorylated only the peptide containing Ser345/348. These findings suggest that these three kinases play distinct roles in the activation of the respiratory burst oxidase, each of them catalyzing the phosphorylation of a different group of serines in p47phox.

Amino Acid Sequence↗
PubMed1996

Apoptosis induced in Jurkat cells by several agents is preceded by intracellular acidification.

We have previously shown that in neutrophils deprived of granulocyte colony-stimulating factor, apoptosis is preceded by acidification and that the protection against apoptosis conferred on neutrophils by granulocyte colony-stimulating factor is dependent upon delay of this acidification. To test the hypothesis that acidification could be a general feature of apoptosis, we examined intracellular pH changes in another cell line. Jurkat cells, a T-lymphoblastoid line, were induced to undergo apoptosis with anti-Fas IgM, cycloheximide, or exposure to short-wavelength UV light. We found that acidification occurred in response to treatment with these agents and that acidification preceded DNA fragmentation. Jurkat cells were also found to possess an acid endonuclease that is active below pH 6.8, compatible with a possible role for this enzyme in chromatin digestion during apoptosis. Incubation of the cells with the bases imidazole or chloroquine during treatment with anti-Fas antibody or cycloheximide or after UV exposure decreased apoptosis as assessed by nuclear morphology and DNA content. The alkalinizing effect of imidazole and chloroquine was shown by the demonstration that the percentage of cells with an intracellular pH below 6.8 after treatment with anti-Fas antibody, cycloheximide, or UV was diminished in the presence of base as compared with similarly treated cells incubated in the absence of base. We conclude that acidification is an early event in programmed cell death and may be essential for genome destruction.

Acids↗
PubMed1996

Partial reconstitution of the respiratory burst oxidase in lymphoblastoid B cell lines lacking p67-phox after transfection with an expression vector containing wild-type and mutant p67 -phox cDNAs: Deletions of the carboxy and amino terminal residues of p67-phox are not required for activity.

The respiratory burst oxidase of phagocytes and B lymphocytes in a multicomponent enzyme that catalyzes the reduction of oxygen by NADPH. It is responsible for O-(2) production in response to stimulation with phorbol 12-myristate 13-acetate (PMA). The study of patients with chronic granulomatous disease (CGD), an inherited disorder characterized by deficient of absent respiratory burst activity, has contributed greatly to our understanding of the NADPH-oxidase. The absence of any one of four components results in the clinical expression of CGD: the two membrane-bound components of the cytochrome b-558, gp91-phox and p22-phox, or the cytosolic factors, p47-phox and p67-phox. We used a system to investigate the activity of mutant p67-phox proteins expressed in a reconstitution assay. This system is characterized by the partial reconstitution of O-(2) production in an Epstein-Barr virus (EBV)-transformed lymphoblastoid B cell line from a patient with p67-phox-deficient CGD by transfection with an expression plasmid containing the 67-phox cDNA in the sense orientation. No O-(2) production was detectable in p67-phox-deficient lymphoblastoid B cell lines transfected with an antisense plasmid or in untransfected p67-phox lymphoblastoid cells stimulated by PMA. We tested two mutants, pEBOp67delta1-22 and pEBOp67delta512-526, and found that both recombinant proteins are active in our system. Thus, we conclude that the first 22 amino acid residues and the last 14 amino acid residues are not critical for initiation of O-(2) production

B-Lymphocytes↗
PubMed1995

The acid deoxyribonuclease of neutrophils: a possible participant in apoptosis-associated genome destruction.

Human neutrophils are terminally differentiated cells that spontaneously undergo apoptosis in tissue culture. Apoptosis in these cells can be delayed by culture in the presence of granulocyte colony-stimulating factor or other inflammatory mediators. Neutrophils were found to contain an acid endonuclease that appeared to be responsible for the internucleosomal DNA cleavage that accompanies apoptosis. As measured by a plasmid nicking assay, this endonuclease had a molecular weight (M(r)) of 35,000, a pH optimum of 5.5, and a threshold for activity of pH 6.6 to 6.8. It was weakly inhibited by divalent cations (Ca2+, Mg2+, and Zn2+) and more strongly inhibited by aurintricarboxylic acid and N-bromosuccinimide. DNA from neutrophils treated with nigericin in buffers of defined pH displayed nucleosomal ladders whose prominence varied with pH in a manner that paralleled the pH dependence of the plasmid cleavage assays, consistent with internucleosomal DNA cleavage by the acid endonuclease. We have previously shown that neutrophils undergo acidification to a pH value as low as 6.0 during apoptosis; we suggest that this endonuclease may be responsible for the DNA cleavage seen in apoptotic neutrophils.

Apoptosis↗
PubMed1995

Cell acidification in apoptosis: granulocyte colony-stimulating factor delays programmed cell death in neutrophils by up-regulating the vacuolar H(+)-ATPase.

Neutrophils in tissue culture spontaneously undergo programmed cell death (apoptosis), a process characterized by well-defined morphological alterations affecting the cell nucleus. We found that these morphological changes were preceded by intracellular acidification and that acidification and the apoptotic changes in nuclear morphology were both delayed by granulocyte colony-stimulating factor (G-CSF). Among the agents that defend neutrophils against intracellular acidification is a vacuolar H(+)-ATPase that pumps protons out of the cytosol. When this proton pump was inhibited by bafilomycin A1, G-CSF no longer protected the neutrophils against apoptosis. We conclude that G-CSF delays apoptosis in neutrophils by up-regulating the cells' vacuolar H(+)-ATPase and that intracellular acidification is an early event in the apoptosis program.

Anti-Bacterial Agents↗