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H L Malech

Publications and source records attributed to H L Malech.

At least 91 records · Page 5Linked to original sources
PubMed1990

Induction of the respiratory burst in HL-60 cells. Correlation of function and protein expression.

Differentiation of myeloid cells is associated with the gradual acquisition of functional capacity to produce a respiratory burst. In our study HL-60 cells were differentiated to the monocyte phenotype with IFN-gamma or 1,25-dihydroxyvitamin D3, or to the neutrophil phenotype with retinoic acid or DMSO to compare the time-course of expression of membrane and cytosolic oxidase components, and to correlate this with the appearance of a functional oxidase. Over a 6-day period of induction the rank order of the ability of these agents to induce expression of PMA-stimulated superoxide production was: IFN-gamma greater than 1,25(OH)2D3 greater than retinoic acid greater than DMSO. Immunoblot analysis of HL-60 membranes and cytosol was used to assess the amount of specific phagocyte oxidase factors (91 and 22 kDa subunits of membrane cytochrome b558 (gp91 and p22), and 47 and 67 kDa cytosol oxidase factors (p47 and p67)). HL-60 cell membranes or cytosol were tested in a cell-free assay of superoxide production by mixing with normal neutrophil cytosol or membranes, respectively. p47 was first detected at 16 h of differentiation, increasing similarly thereafter with all induction regimens and reaching a maximum by 3 to 4 days. The earliest detection of p67 varied from 2 to 6 days depending on the inducing agent and appeared to be the limiting cytosol component. Small amounts of both subunits of cytochrome b558 were detected in uninduced HL-60 membranes, but were sufficient to support substantial superoxide production when combined with normal neutrophil cytosol. Both cytochrome b558 subunit proteins and membrane oxidase activity increased during differentiation in parallel. We conclude that membrane and cytosol components of the NADPH oxidase complex appear at different times and increase differently during HL-60 differentiation. The production of p67 is the major factor limiting the respiratory burst during HL-60 differentiation.

Blotting, Western↗
PubMed1990

The phagocyte 47-kilodalton cytosolic oxidase protein is an early reactant in activation of the respiratory burst.

Activation of the phagocyte NADPH oxidase requires participation of membrane-bound cytochrome b558 and cytosol proteins of 47 kDa (p47) and 67 kDa (p67). We examined the sequence of participation of p47 and p67 in activation of the oxidase using an arachidonate-activated cell-free superoxidase (O2-) generating assay requiring phagocyte membrane and cytosol. Neutrophil cytosol from patients with certain forms of autosomal recessive chronic granulomatous disease (CGD) lack either p47 or p67. Initial incubation of membrane and arachidonate with CGD cytosol deficient in either p47 or p67 fails to generate superoxide in the cell-free assay until addition of complementary cytosol. CGD cytosol was incubated with arachidonate and membrane for 5-15 min and the lag time of O2- generation was measured after addition of complementary CGD cytosol. The lag time is shortened when p47, but not p67, is present in the initial incubation. We have previously shown that the peptide, RGVHFIF, corresponding to a cytoplasmic carboxyl-terminal domain of the large subunit of cytochrome b558, inhibits activation of NADPH oxidase in the cell-free assay, but does not affect the enzyme activity of fully assembled oxidase. Experiments with sequential addition of complementary CGD cytosols were performed as above, except that RGVHFIF was added after the initial incubation. The peptide failed to inhibit when added after initial incubation if p47 was present during that incubation. In contrast, the peptide markedly inhibited oxidase activity if p47 was absent during the initial incubation. These results suggest that p47, but not p67, is a participant with membrane and/or other cytosol components in early arachidonate-dependent reactions. In the absence of p67, these reactions culminate in the irreversible formation of a metastable activation intermediate that is insensitive to inhibition by RGVHFIF. After addition of p67, this activation intermediate subsequently reacts to form the active NADPH oxidase.

Adult↗
PubMed1990

Production of myeloid cell cytosols functionally and immunochemically deficient in the 47 kDa or 67 kDa NADPH oxidase cytosolic factors.

Professional phagocytes contain a unique NADPH oxidase responsible for the production of microbicidal oxidants. Activation of this oxidase requires participation of cytosolic and membrane proteins, but the interactions of these components are incompletely understood. Patients with autosomal recessive Chronic Granulomatous Diseases (CGD) are characterized by functional defects in phagocyte oxidase activity resulting from a deficiency of either a 47 kDa (p47) or a 67 kDa (p67) cytosolic oxidase component. Cytosols from such patients are valuable for biochemical studies of the oxidase, but are not generally available because CGD is a rare disorder. The present study illustrates means of producing cytosols functionally and immunochemically deficient in either p47 or p67. Cytosol from monocytes cultured for 6 days is immunochemically deficient in p47 but not p67, while cytosol from HL-60 cells induced with retinoic acid for 3 days is deficient in p67 but not p47. Each of these cytosols fail to generate superoxide when added to neutrophil membranes in a cell-free assay but complement each other when combined. Complementation studies in which these cytosols were mixed in the cell-free assay with p47- or p67- deficient CGD cytosol established the functional characteristics of the experimentally produced cytosols.

Cell-Free System↗
PubMed1990

Evidence for a functional cytoplasmic domain of phagocyte oxidase cytochrome b558.

Cytoplasmic domains of transmembrane proteins play a critical role in cellular processes involving interactions between membrane and cytosolic components. Activation of the phagocytic cell respiratory burst oxidase, the electron transport chain responsible for superoxide anion (O2-.) production, requires membrane components including cytochrome b558 and several cytosolic proteins; but the biochemical interactions of these components are poorly understood. Cytochrome b558 is an electron transport component of the oxidase. A role for cytochrome b558 in the organization or integration of other oxidase components has also been hypothesized. Antibodies binding the cytoplasmic carboxyl-terminal tail of the transmembrane 91-kDa subunit of cytochrome b558 specifically inhibited an amphiphile-activated cell-free O2-.-generating system that requires neutrophil membranes and cytosol. Synthetic peptides encompassing a 7-amino acid carboxyl-terminal sequence (RGVHFIF) within the same region of the 91-kDa subunit blocked activation of the oxidase by arachidonate, but did not affect activity of the assembled oxidase when added after arachidonate to the cell-free O2-.-generating system. The same peptides inhibited activation of the respiratory burst when allowed to diffuse into electrically permeabilized neutrophils before stimulation with formyl-methionyl-leucyl-phenylalanine or phorbol myristate acetate. These studies define a functional cytoplasmic domain of the transmembrane 91-kDa subunit of cytochrome b558 which may mediate interactions with other cellular proteins essential to activation of the phagocyte respiratory burst.

Amino Acid Sequence↗
PubMed1990

Cloning of a 67-kD neutrophil oxidase factor with similarity to a noncatalytic region of p60c-src.

Chronic granulomatous diseases (CGDs) are characterized by recurrent infections resulting from impaired superoxide production by a phagocytic cell, nicotinamide adenine dinucleotide phosphate (reduced) (NADPH) oxidase. Complementary DNAs were cloned that encode the 67-kilodalton (kD) cytosolic oxidase factor (p67), which is deficient in 5% of CGD patients. Recombinant p67 (r-p67) partially restored NADPH oxidase activity to p67-deficient neutrophil cytosol from these patients. The p67 cDNA encodes a 526-amino acid protein with acidic middle and carboxyl-terminal domains that are similar to a sequence motif found in the noncatalytic domain of src-related tyrosine kinases. This motif was recently noted in phospholipase C-gamma, nonerythroid alpha-spectrin (fodrin), p21ras-guanosine triphophatase-activating protein (GAP), myosin-1 isoforms, yeast proteins cdc-25 and fus-1, and the 47-kD phagocyte oxidase factor (p47), which suggests the possibility of common regulatory features.

Amino Acid Sequence↗
PubMed1990

The formyl peptide chemoattractant receptor is encoded by a 2 kilobase messenger RNA. Expression in Xenopus oocytes.

Activation of the formyl peptide chemoattractant receptor (FPCR) of phagocytic cells mobilizes intracellular calcium stores and affects the plasma membrane potential. Affinity crosslinking of FPCR has demonstrated a 60-80 kDa glycoprotein, with core peptide of 32 kDa. It is not known whether functional FPCR is this single peptide or requires multiple subunits. We used Xenopus oocyte expression system to determine the size of mRNA required for synthesis of functional FPCR. Injection of oocytes with poly(A)+ RNA from HL60 cells differentiated to the granulocyte phenotype resulted in acquisition of formyl peptide-specific responses (inward transmembrane current with a reversal potential consistent with a chloride conductance, and calcium efflux). FPCR activity expressed in oocytes had a ligand concentration dependence, ligand structure dependence and pertussis toxin sensitivity similar to those reported in phagocytic cells. When RNA was size fractionated, a single peak of FPCR activity at 2 kilobases was observed after injection of mRNA into oocytes. Our data strongly suggest that FPCR is composed of a single-sized polypeptide.

Amino Acid Sequence↗
PubMed1990

Invasive infection with Sarcinosporon inkin in a patient with chronic granulomatous disease.

Sarcinosporon inkin, a rare skin fungus, was found to have caused progressive pneumonia in a young male with chronic granulomatous disease. Histologic sections of right upper lobe lung tissue showed clusters of globose to oblong hyalin-walled, septate, sporangia throughout the necrotic areas within the pyogranulomas. Pure cultures of S. inkin were recovered from the surgical specimen of the lung. Current status of the taxonomy of S. inkin is reviewed and clarified. Treatment of the patient with Amphotericin B and white blood cell transfusions led to clinical and radiographic response. This is the first documented case of systemic infection caused by S. inkin.

Adolescent↗
PubMed1990

Normal and deficient neutrophils can cooperate to damage Aspergillus fumigatus hyphae.

Using a metabolic test of hyphal viability, the interaction between neutrophils and Aspergillus hyphae was investigated over a broad range of hyphae-to-neutrophil ratios. Normal neutrophils were found to damage hyphae whereas neutrophils from patients with both chronic granulomatous disease (CGD) and myeloperoxidase (MPO) deficiency did not. Further, both azide and catalase + superoxide dismutase inhibited the ability of normal neutrophils to damage hyphae, suggesting that this damage is mediated by products of the respiratory burst and by the MPO-halide system. Also, mixtures of small numbers of normal neutrophils with larger numbers of CGD neutrophils (range, 1:5 to 1:15) damaged hyphae more efficiently than either population of cells alone. Further, mixtures of CGD and MPO-deficient neutrophils, neither of which alone could efficiently damage hyphae, were able to damage the hyphae almost as well as a comparable number of normal neutrophils. These data demonstrate that intact neutrophils can cooperate to synergistically damage Aspergillus hyphae, possibly by extracellular mixing of hydrogen peroxide and MPO.

Aspergillus fumigatus↗
PubMed1990

Genes for two autosomal recessive forms of chronic granulomatous disease assigned to 1q25 (NCF2) and 7q11.23 (NCF1).

Chronic granulomatous disease (CGD) is a heterogeneous group of inherited disorders of impaired superoxide production in phagocytes. The most common X-linked recessive form involves the CYBB locus in band Xp21.1 that encodes the membrane-bound beta subunit of the cytochrome b558 complex. Two autosomal recessive forms of CGD result from defects in cytosolic components of the phagocyte NADPH oxidase system, p47phox (NCF1) and p67phox (NCF2). By using human cDNA probes we have mapped the genes for these proteins to chromosomal sites. The combined data from Southern analysis of somatic cell hybrid lines and chromosomal in situ hybridization localize NCF1 to 7q11.23 and NCF2 to band 1q25. The NCF1 localization corrects an erroneous preliminary assignment to chromosome 10. In the mouse, the locus corresponding to NCF2 (Ncf-2) was mapped with somatic cell hybrid panels and recombinant inbred strains to mouse chromosome 1 near Xmv-21 within a region of conserved homology with human chromosome 1 region q21-q32. A second site, probably a processed pseudogene, was identified on mouse chromosome 13.

Animals↗
PubMed1989

Asparagine-linked glycosylation of cytochrome b558 large subunit varies in different human phagocytic cells.

Cytochrome b558, an essential component of the respiratory burst of phagocytic cells, is the terminal electron donor to molecular oxygen that results in the formation of superoxide anion (O2-.). It is an integral membrane heterodimer that in neutrophils consists of a 22-kDa small subunit and a highly glycosylated 91-kDa large subunit. Identical core proteins often differ in glycosylation in different cell types and with some membrane glycoproteins, the glycosylation state may markedly affect function. In the present study, antisera reactive with cytochrome b558 large subunit was used for immunoblot analysis of the glycosylation pattern of this subunit from different types of phagocytic cells. Striking variability in the apparent m.w. of this broadly banding subunit was detected in five different phagocytic cell types (neutrophils 78,000 to 93,000; eosinophils 74,000 to 115,000; monocytes 82,000 to 99,000; dibutyryl cyclic AMP-induced HL-60 cells 79,000 to 103,000; dimethyl sulfoxide-induced HL-60 cells 77,000 to 110,000). However, after complete cleavage of N-linked oligosaccharides with endoglycosidase F, the core peptide of cytochrome b558 large subunit from these different cell types had the same Mr (58,000). Inhibition of N-glycosylation with tunicamycin in differentiating HL-60 cells resulted in the synthesis of immunoreactive protein of the same m.w. and banding pattern as seen after endoglycosidase F cleavage. These tunicamycin treated cells retained some capacity to generate superoxide anion when stimulated with PMA. We conclude that the identity of the N-linked oligosaccharides of the cytochrome b558 large subunit differ in various phagocytic cells. All N-linked glycans on cytochrome b558 in all cell types examined were of the complex type as defined by resistance to endoglycosidase H cleavage. N-linked glycosylation of the cytochrome b558 large subunit may not be essential for activation of the respiratory burst.

Acetylglucosaminidase↗
PubMed1989

Intravenous endotoxin recruits a distinct subset of human neutrophils, defined by monoclonal antibody 31D8, from bone marrow to the peripheral circulation.

Human neutrophils label with fluorochrome-labeled monoclonal antibody 31D8 as bright or dull. We determined the source and fate of 31D8 dull neutrophils by studying volunteers injected with endotoxin, epinephrine, or hydrocortisone, by examining bone marrow, and by examining skin blister exudate. We find that 31D8 dull neutrophils are normally not present in significant numbers in the circulation, are present in large numbers in normal marrow, and are recruited from the marrow by endotoxin, to a lesser extent by steroid, but not at all by epinephrine. 31D8 dull pattern correlates with morphologic immaturity in postendotoxin peripheral blood and bone marrow; however, blister exudate neutrophils contain only morphologically mature neutrophils, of which a significant number are 31D8 dull. We conclude that 31D8 dull neutrophils reside primarily in bone marrow and are released by agents which enhance bone marrow release of neutrophils. Their accumulation in skin blister exudate is unexplained, but suggests a special role in the inflammatory process.

Adult↗
PubMed1989

Genetic variants of chronic granulomatous disease: prevalence of deficiencies of two cytosolic components of the NADPH oxidase system.

Chronic granulomatous disease, a syndrome of recurrent infections and failure of oxidative microbicidal activity in phagocytes, results from defects in the gene for one of several components of an oxidase system that can undergo activation. To determine the relative prevalence of certain of the genetic variants of this disorder, we used immunoblotting to detect two specific neutrophil cytosolic proteins of 47 and 67 kd recently shown to be required for oxidase activation. Chronic granulomatous disease is usually an X-linked disorder associated with the absence of membrane cytochrome b558. Of our 94 patients with chronic granulomatous disease, however, 36 had a phenotype characterized by autosomal inheritance, normal membrane oxidase components (including cytochrome b558), and functionally defective cytosolic activity in a cell-free oxidase system. We studied 25 of these 36 patients and found that 22 lacked the 47-kd cytosolic protein, and the remaining 3 were missing the 67-kd component. Patients with chronic granulomatous disease whose functional defect was localized to the neutrophil membrane (classic X-linked cytochrome b-negative type and two other rare variants) had normal amounts of both cytosolic components. We estimate that approximately 33 percent of all patients with chronic granulomatous disease are missing the 47-kd cytosolic oxidase component and about 5 percent of patients are missing the 67-kd component. Chronic granulomatous disease caused by a defect in any cytosolic factors other than the 47-kd and 67-kd proteins, if it exists, is apparently rare.

Adolescent↗
PubMed1989

Recombinant 47-kilodalton cytosol factor restores NADPH oxidase in chronic granulomatous disease.

A 47-kilodalton neutrophil cytosol factor (NCF-47k), required for activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase superoxide (O2-.) production, is absent in most patients with autosomal recessive chronic granulomatous disease (AR-CGD). NCF-47k cDNAs were cloned from an expression library. The largest clone predicted a 41.9-kD protein that contained an arginine and serine-rich COOH-terminal domain with potential protein kinase C phosphorylation sites. A 33-amino acid segment of NCF-47k shared 49% identity with ras p21 guanosine triphosphatase activating protein. Recombinant NCF-47k restored O2-. -producing activity to AR-CGD neutrophil cytosol in a cell-free assay. Production of active recombinant NCF-47k will enable functional regions of this molecule to be mapped.

Amino Acid Sequence↗
PubMed1989

Glandular secretion of lactoferrin in a patient with neutrophil lactoferrin deficiency.

Patients with specific granule deficiency (SGD) develop recurrent severe bacterial skin infections. Neutrophils from patients with SGD are deficient in lactoferrin (Lf), an antimicrobial protein commonly found in many mucosal secretions. Unstimulated and stimulated nasal secretions, saliva, and tears were collected from a patient with SGD and from normal control subjects and were analyzed for Lf. The secretions from the patient contained normal values of Lf, suggesting that the glands secrete Lf from a source other than neutrophils. Immunohistochemical staining of normal nasal mucosa demonstrated that Lf is localized within serous submucosal gland cells and that neutrophils are not normally observed in the nasal mucosa. These findings suggest that glandular tissues produce and locally secrete Lf by processes that are independent of neutrophil degranulation.

Child↗
PubMed1989

The molecular biology of selected phagocyte defects.

Advances in molecular genetic understanding of disease processes has been extended to a number of phagocytic disorders. Most of these disorders were extensively characterized at the functional and protein level prior to cloning of the relevant genes. Nucleotide sequence data has been essential for establishing the mechanism and mode of inheritance of genetically transmitted phagocyte disorders. Such data provides insights into the functionally important regions of affected proteins and information regarding regulation of these genes and homologies to other known proteins. From such data it is also possible to determine the evolutionary history of these genes. Chronic granulomatous disease, a phenotypic classification of a heterogeneous group of defects in oxidative metabolism, has now been defined in terms of specific molecular defects. Cloning of the two subunits of cytochrome b558 has led the way to characterization of the X-linked form and one of the autosomal recessive forms of this disease and confirmed the importance of this protein in the phagocyte oxidative burst. The absence of lactoferrin associated with hereditary specific granule deficiency is a result of decreased transcription of the lactoferrin gene in myeloid cells. Myeloperoxidase deficiency is likely a result of a mutation of the gene coding for myeloperoxidase. More precise understanding of expression of the lactoferrin and myeloperoxidase genes may be important in elucidating some of the underlying mechanisms in the pathogenesis of myeloid malignancies. A rare disorder, leukocyte adhesion deficiency, has provided a model for establishing the relationship between the several distinct alpha subunits and the shared common beta subunit of leukocyte adhesion proteins. These proteins have been shown to be genetically related to the superfamily of extracellular matrix receptors termed 'integrins'. Because these proteins have been highly conserved at the genetic level across a variety of species, comparison of nucleotide sequence data has illuminated some of the evolutionary history of these genes as they arose from ancestral genes. Studies of these adhesion protein genes may contribute new information in the broader context of how the functions of these genes evolved.

Humans↗