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B M Babior

Publications and source records attributed to B M Babior.

At least 91 records · Page 5Linked to original sources
PubMed1988

Neutrophils and host defense.

Neutrophils, the predominant phagocytes of circulating blood, are the first cells to arrive at sites of infection. Although neutropenia has long been recognized to predispose to infection, recently other syndromes marked by frequent infections have been shown to be caused by an underlying neutrophil dysfunction. Efforts to define the molecular pathology of such disorders have helped delineate the molecular basis of normal neutrophil function. Advances have been made in defining the roles of the neutrophil's varied receptors in recognition, movement, and adhesive phenomena. Progress in establishing the pathogenesis of chronic granulomatous disease has provided important insights into the enzymatic machinery that normal neutrophils use to produce antimicrobial oxidants. The identification and precise characterization of antimicrobial components, such as defensins, have outlined the potential roles of "natural antibiotics" in neutrophil-mediated host-defense functions. These areas of neutrophil function will be reviewed and placed in a clinical context to guide physicians in evaluating children and adults with frequent or unusual infections.

Blood Bactericidal Activity↗
PubMed1988

Relationship of protein phosphorylation to the activation of the respiratory burst in human neutrophils. Defects in the phosphorylation of a group of closely related 48-kDa proteins in two forms of chronic granulomatous disease.

When 32P-labeled human neutrophils were activated by exposure to phorbol myristate acetate, three 48-kDa proteins (designated pp48/6.8, pp48/7.3, and pp48/7.8, from their isoelectric points) were found to have become labeled. With maximal stimulation, labeling was complete by 30 s. With lesser degrees of stimulation, the extent of labeling at 2 min correlated with rates of production by the phorbol-treated cells. Increased labeling of these 48-kDa proteins was also seen in cells exposed to f-Met-Leu-Phe. In phorbol-treated neutrophils from patients with X-linked cytochrome b558-negative chronic granulomatous disease, pp48/7.8 was labeled in a normal fashion, but pp48/6.8 and pp48/7.3 failed to take up 32P. In cells from patients with autosomal recessive cytochrome b558-positive chronic granulomatous disease, however, none of the three proteins took up 32P in response to phorbol. The three proteins appear to be very closely related, as indicated by the findings that phosphoserine was the only phosphoamino acid found in any of the three, and all three yielded identical one-dimensional phosphopeptide maps after digestion with either chymotrypsin or staphylococcal proteinase V8. These results reconcile earlier observations on protein phosphorylation in chronic granulomatous disease and provide further evidence for a relationship between the phosphorylation of this group of 48-kDa proteins and the activation of the respiratory burst oxidase.

Blood Proteins↗
PubMed1988

Kinetics of activation of the respiratory burst oxidase in a fully soluble system from human neutrophils.

In a fully soluble system from resting human neutrophils, activation of the respiratory burst oxidase under defined conditions was found to follow first-order kinetics. The manner in which this first-order activation process varied with the concentrations of the individual components in the activating system suggested the following. 1) The respiratory burst oxidase occurs in two forms that can be distinguished by their Km values for NADPH. The low-affinity form contains one component (M) from the membrane and two components (S and C alpha) from the cytosol, while the high-affinity form contains an extra cytosolic component (C beta). 2) The active forms of the oxidase are generated in the following reactions: (formula; see text) where S is a stabilizing component and where M.S is an activated form of M.S that is capable of binding C alpha and C beta to produce the active oxidase species M.S.C alpha (the low-affinity form) and M.S.C alpha C beta (the high-affinity form). 3) SDS activates the oxidase by mediating the conversion of M.S to M.S.

Cytosol↗
PubMed1988

Evidence that de novo protein synthesis participates in a time-dependent augmentation of the chemotactic peptide-induced respiratory burst in neutrophils. Effects of recombinant human colony stimulating factors and dihydrocytochalasin B.

We examined the effects of the recombinant human colony stimulating factors GM-CSF and G-CSF, cycloheximide (a protein synthesis inhibitor) and dihydrocytochalasin B (a microfilament disrupting agent) upon FMLP (N-formyl-methionyl-leucylphenylalanine)-stimulated O2- production by neutrophils. We confirmed a time dependent augmentation of O2- production following preincubation of neutrophils either alone or with colony stimulating factors. Furthermore, we found that GM-CSF, but not G-CSF, increased O2- production at some concentrations of the stimulus. Preincubation of neutrophils with cycloheximide in the absence of CSF caused a marked fall in O2- -production that was first evident at 2 hours. The fall in O2- -forming capacity caused by cycloheximide was much less pronounced if dihydrocytochalasin B was also included in the preincubation buffer. These findings suggest a previously unrecognized role for de novo protein synthesis in maintaining the ability of neutrophils to manufacture O2-, and support earlier studies indicating that the cycling of FMLP receptors between the cell membrane and an intracellular compartment is important in determining the magnitude of the respiratory burst in FMLP-stimulated neutrophils.

Blood Proteins↗
PubMed1988

The respiratory burst oxidase.

The respiratory burst oxidase is an activatable, membrane-bound flavo(?hemo) protein that catalyzes the NADPH-dependent reduction of oxygen to superoxide (O2-) in stimulated phagocytes. Chronic granulomatous disease, a group of disorders in which phagocytes cannot manufacture O2-, is caused by a biochemical lesion involving this oxidase or its activating system.

Cytochrome b Group↗
PubMed1988

The mechanism of adenosylcobalamin-dependent rearrangements.

Adenosylcobalamin (AdoCbl)-dependent rearrangements are a group of reactions with no obvious precedents in organic chemistry. In every case, they are characterized by a mechanism in which a hydrogen atom on one carbon atom exchanges places with a group X on an adjacent carbon: (formula; see text) Much experimental work indicates that an AdoCbl rearrangement is initiated by homolysis of the C-Co bond of the cofactor. The migrating hydrogen is then abstracted from the substrate by the resulting 5'-deoxyadenosyl radical, or by a second radical that is generated elsewhere at the active site, and, after the migration of group X, is returned to the product in a similar reaction. In at least some of the rearrangements, group X migration may occur via a cation radical intermediate that formed by the departure of X with its electrons, a process assisted by the unpaired electron left behind on the adjacent carbon after the abstraction of the migrating hydrogen. Once C-Co bond cleavage has initiated the reaction by producing a free radical at the active site, the corrin ring plays no further role in the rearrangements.

Cobamides↗
PubMed1988

Plasmids in bacteria exposed to activated neutrophils mediate mutagenesis when transferred to new hosts.

The plasmid pUC18 contains a lacZ alpha-complementation gene that codes for a small peptide that can complement the delta M15 mutation of the Escherichia coli lacZ (beta-galactosidase) gene, converting bacteria carrying that mutated gene from the lacZ- to the lacZ+ phenotype. This plasmid was used in experiments designed to study mutagenesis by human neutrophils. E coli carrying pUC18 were incubated with neutrophils under conditions in which little ingestion of the bacteria took place; the plasmid was then isolated and transformed into an E coli strain (BOZO) that carries the lacZ delta M15 mutation. Of these transformants, 11 of 205,000 were lacZ, suggesting that in these 11, alpha-complementation had been lost through a mutation. No lac- colonies were detected among several hundred thousand BOZO transformed with plasmid isolated from incubations in which phagocytosis could take place, nor from incubations from which neutrophils were omitted. Despite the lac- phenotype of these 11 transformants, plasmids reisolated from nine of them showed normal alpha-complementing ability when transformed into fresh BOZO. These findings indicated that in these nine, the mutations were located in the chromosomes of the transformed BOZO. It thus appears that on exposure to activated neutrophils, a plasmid may acquire a lesion (? mutation) that can somehow be transferred to the genome of a recipient microorganism, resulting in repair of the damaged plasmid accompanied by mutation of the recipient's chromosome. Restriction mapping of the DNA from four of these nine chromosomal mutants suggested that the mutations did not represent major insertions or deletions in the portion of the bacterial chromosome corresponding to the pUC18 lac operon insert, nor in the remainder of the lacZ delta M15 gene. These results confirm previous work showing that exposure to activated neutrophils can induce mutations in biological systems, and provides an experimental model in which the mechanism of neutrophil-mediated mutagenesis may be examined.

Blood Bactericidal Activity↗
PubMed1988

Phosphorylation of the oxidase-related 48K phosphoprotein family in the unusual autosomal cytochrome-negative and X-linked cytochrome-positive types of chronic granulomatous disease.

Activation of 32P-loaded neutrophils with phorbol myristate acetate causes the labeling of a family of three 48K proteins that focus near neutral pH. The relationship between these phosphoproteins and the activation of the respiratory burst has been supported by the previous finding that phosphorylation was defective in the two most common types of chronic granulomatous disease (CGD): X-linked cytochrome-negative (X/-) and autosomal cytochrome-positive (A/+). In this report, these studies have now been extended to the rare A/- and X/+ forms of the disease. In all three patients with A/- CGD examined, the two most acidic 48K proteins failed to undergo enhanced phosphorylation in response to phorbol stimulation, a finding similar to that seen in X/- patients. In contrast, neutrophils from two patients with X/+ CGD appeared to phosphorylate the neutral 48K proteins in a normal fashion. It thus appears that the different phosphorylation patterns seen in chronic granulomatous disease are a reflection of the genetic heterogeneity of this disorder. These findings lend further support to the conclusion that the 48K phosphoprotein family is related to the respiratory burst, although not necessarily in a straightforward manner.

Adult↗
PubMed1987

Activation of the respiratory burst oxidase in a fully soluble system from human neutrophils.

The O2(-)-forming respiratory burst oxidase is present in a dormant state in a fully soluble system containing both cytosol and a deoxycholate extract of membranes from resting human neutrophils. Sodium dodecyl sulfate at low concentrations converts this soluble dormant oxidase into its catalytically active form. The Vmax for the activated oxidase was 2.1 mumol of O2-/min/mg of membrane protein. Michaelis constants for NADPH and NADH (38 microM and 1.7 mM, respectively) were similar to those measured previously in other systems. Oxidase activity was not detected after sodium dodecyl sulfate treatment of systems containing solubilized neutrophil membranes obtained from patients with X-linked chronic granulomatous disease. These results suggest that the deoxycholate extract contains both the resting oxidase and those membrane-associated components needed for its activation, all in functioning states.

Enzyme Activation↗
PubMed1987

Chronic granulomatous disease--pieces of a cellular and molecular puzzle.

Chronic granulomatous disease (CGD) is an inherited disorder in which phagocytes are unable to manufacture microbicidal oxidants. The disorder may be classified into subtypes depending upon the mode of transmission (X-linked or autosomal recessive) and the presence or absence of a heme protein designated 'cytochrome b558' (see below). Patients with CGD suffer recurrent deep-seated bacterial infections that respond poorly to therapy, slowly destroy affected tissues and eventually claim the patients' lives. Clinical deterioration is slowed, though not stopped, by the current practice of placing CGD patients on prophylactic antibiotics. Microbicidal oxidant production fails in CGD because of a defect in the enzyme responsible for the production of superoxide (O2-), the single precursor from which all the microbicidal oxidants ultimately arise. This enzyme, the respiratory burst oxidase, is a membrane-bound oxidase that catalyses the one-electron reduction of oxygen to O2- at the expense of NADPH. The oxidase is dormant in resting phagocytes, but comes to life when the phagocytes are exposed to bacteria or other appropriate stimuli. Components related to the respiratory burst oxidase include cytochrome b558 (one of those subunits has recently been shown to be encoded by a gene that is defective in the most common form of CGD), a flavoprotein that participates directly in O2- production, a cytosolic factor needed for the activation of the oxidase, and a group of 48K phosphoproteins. How these components relate to each other and to the O2- -forming activity of the oxidase is currently under active investigation.

Granulomatous Disease, Chronic↗
PubMed1987

Fate of the DNA in plasmid-containing Escherichia coli minicells ingested by human neutrophils.

Escherichia coli minicells containing the plasmid pSC101 (approximately 10 kb) or pBR322 (approximately 4 kb) were opsonized and incubated with human neutrophils. The neutrophils responded to the minicells as they would to native E coli: they ingested the minicells, discharged their granule contents into the minicell-containing phagosomes, and expressed a respiratory burst. After one hour of incubation, the fate of the ingested plasmid DNA was examined. No DNA degradation was detected by trichloroacetic acid precipitation or agarose gel electrophoresis. Moreover, when pBR322 recovered from ingested minicells was transformed into E coli, no mutations in either of the antibiotic resistance genes carried by the plasmid were detected out of many thousand transformants screened. These findings confirm the surprisingly limited effect of neutrophils on ingested DNA.

Blood Bactericidal Activity↗
PubMed1986

The respiratory burst oxidase of human neutrophils. Further studies of the purified enzyme.

A superoxide-forming oxidase from activated human neutrophil membranes was solubilized by two slightly different methods, then purified by "dye-affinity" chromatography. Kinetic studies of the purified preparations gave Vmax values of 5-10 mumol of O-2/min/mg of protein, and Km values for NADH and NADPH that were in reasonable agreement with values determined previously using particulate and crude solubilized preparations of the respiratory burst oxidase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed prominent bands at 67, 48, and 32 kDa, together with some minor contaminants, whereas gel electrophoresis under non-denaturing conditions gave a single major band that when eluted and re-electrophoresed in the presence of sodium dodecyl sulfate showed bands at 67, 48, 32 kDa. We believe that all three bands represent oxidase components. The flavin content of the purified enzyme was 20.4 +/- 2.0 S.E. pmol of FAD/microgram of protein, whereas heme averaged 0.1 +/- 0.02 pmol/microgram and ubiquinone could not be detected. Assuming that the enzyme is composed of one 67-kDa subunit, one 48-kDa subunit, and one 32-kDa subunit (i.e. that its molecular mass is approximately 150 kDa), it can be calculated to have a turnover number of 700-1500 min-1, in agreement with a value reported previously for oxidase in a particulate O-2-forming system (Cross, A. R., Parkinson, J. F., and Jones, O. T. G. (1985) Biochem. J. 226, 881-884), and to contain the following quantities of redox carriers (mol/mol): FAD, 3.0; heme, 0.015; ubiquinone, less than 0.06. It remains to be determined whether this preparation represents the complete respiratory burst oxidase or is only the pyridine nucleotide dehydrogenating component of a more complex enzyme.

Chromatography, High Pressure Liquid↗
PubMed1986

A possible role for protein phosphorylation in the activation of the respiratory burst in human neutrophils. Evidence from studies with cells from patients with chronic granulomatous disease.

Two-dimensional gel electrophoresis was used to study protein phosphorylation in granules, membranes, and soluble fractions from human neutrophils that had been loaded with 32Pi. In resting cells, label was incorporated primarily into proteins of the membranes and the soluble supernatant; little appeared in the granules. Activation of 32P-loaded neutrophils resulted in an increase in the 32P content of a small number of membrane and soluble proteins without a change in the labeling of the granule fraction. The identity of the proteins affected by activation depended on the activating agent used; all of the activating agents, however, caused an increase in the labeling of a group of approximately 48-kDa proteins that appeared to be distributed between the membranes and the soluble supernatant. To investigate the role of phosphorylation in the activation of the respiratory burst oxidase, the incorporation of 32P into phosphoproteins was studied in neutrophils from patients with chronic granulomatous disease. When these cells were exposed to phorbol myristate acetate, one of the agents used for the activation of normal neutrophils, the 48-kDa proteins in the membranes and supernatants failed to take up additional 32P. Phosphorylation patterns in normal neutrophils activated under nitrogen were similar to the patterns seen with cells activated in air, suggesting that the differences in phosphorylation between normal and chronic granulomatous disease neutrophils did not represent secondary effects of the oxidants produced by the normal cells, but reflected primary biochemical differences between the normal and the defective phagocytes. We postulate from these results that the uptake of phosphate by the 48-kDa protein group may be involved in the activation of the respiratory burst oxidase.

Arachidonic Acid↗
PubMed1986

Electron spin-echo studies of the composition of the paramagnetic intermediate formed during the deamination of propanolamine by ethanolamine ammonia-lyase, and AdoCbl-dependent enzyme.

During the deamination of S-2-aminopropanol by the AdoCbl-dependent ethanolamine ammonia-lyase of Clostridia sp., a catalytic intermediate accumulates whose active site contains two paramagnetic species: cob(II)alamin and a free radical derived from the substrate molecule. Spin-echo spectroscopy has revealed that the unpaired electron on the substrate-derived radical is delocalized over a nitrogen atom that from its quadrupole splittings is probably a component of a secondary amide group. Experiments with 15N- and deuterium-labeled propanolamine gave no evidence of an interaction between this unpaired electron and the nitrogen originally attached to the substrate molecule. These results strongly suggest that the substrate-derived radical in this intermediate has already lost its nitrogen, and that this radical is stabilized by delocalization of the unpaired electron onto a nitrogen most likely situated in one of the peptide bonds of the enzyme backbone.

Ammonia-Lyases↗
PubMed1986

Production of C5a antagonist by synovial and peritoneal tissue fibroblasts.

Fibroblasts grown from synovial and peritoneal tissues release into the medium an inhibitor of neutrophil chemotaxis. The inhibitor resembles the antagonist previously described in synovial and peritoneal fluids. It is a heat stable (56 degrees C) protein of MW approximately 40 kDa that counteracts the chemotactic activity of zymosan-activated serum or purified C5a but not the peptide chemoattractant F-met-leu-phe. No chemotactic inhibitor was detected in media from skin fibroblast cultures or in formal human sera. It is suggested that the inhibitor is produced locally by synovial and peritoneal fibroblasts and that it might play a role in the regulation of inflammation at sites lined with mesothelium.

Chemotactic Factors↗