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Biomedical subjects

S J Ackerman

Publications and source records attributed to S J Ackerman.

At least 73 records · Page 4Linked to original sources
PubMed1994

Toxicity of an antitumor ribonuclease to Purkinje neurons.

Purkinje cell toxicity is one of the characteristic features of the Gordon phenomenon, a syndrome manifested by ataxia, muscular rigidity, paralysis, and tremor that may lead to death (Gordon, 1933). Two members of the RNase superfamily found in humans, EDN (eosinophil-derived neurotoxin) and ECP (eosinophil cationic protein), cause the Gordon phenomenon when injected intraventricularly into guinea pigs or rabbits. We have found that another member of the RNase superfamily, an antitumor protein called onconase, isolated from Rana pipiens oocytes and early embryos, will also cause the Gordon phenomenon when injected into the cerebrospinal fluid of guinea pigs at a dose similar to that of EDN (LD50, 3-4 micrograms). Neurologic abnormalities of onconase-treated animals were indistinguishable from those of EDN-treated animals, and histology showed dramatic Purkinje cell loss in the brains of onconase-treated animals. The neurotoxic activity of onconase correlates with ribonuclease activity. Onconase modified by iodoacetic acid to eliminate 70% and 98% of the ribonuclease activity of the native enzyme displays a similar decrease in ability to cause the Gordon phenomenon. In contrast, the homologous bovine pancreatic RNase A injected intraventricularly at a dose 5000 times greater than the LD50 dose of EDN or onconase is not toxic and does not cause the Gordon phenomenon. A comparison of the RNase activities of EDN, onconase, and bovine pancreatic RNase A using three pancreatic RNA substrates demonstrates that onconase is orders of magnitude less active enzymatically than EDN and RNase A. Thus, another member of the RNase superfamily in addition to EDN and ECP can cause the Gordon phenomenon.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Human eosinophil Charcot-Leyden crystal protein: cloning and characterization of a lysophospholipase gene promoter.

The Charcot-Leyden crystal (CLC) protein is a lysophospholipase expressed exclusively by eosinophils and basophils. During eosinophilic differentiation of eosinophil-committed cell lines, CLC steady state mRNA levels increase significantly. This increased expression is transcriptionally regulated during butyrate induction of an eosinophilic subline (C15) of the promyelocytic leukemia cell line HL-60, as shown by nuclear run-on assays. The transcriptional start site of the CLC gene was identified 43 bp upstream of the 5' end of the longest available cDNA sequence. The gene encoding CLC protein was cloned from a chromosome 19-specific library and a fragment overlapping the transcriptional start site was isolated and sequenced. Plasmid constructs (in the pXP2 luciferase expression vector) containing 411 and 292 bp of genomic sequence upstream of the CLC transcriptional start site directed reporter gene expression in transient transfections of HL-60-C15 cells, as well as other myeloid (U937) and nonmyeloid (HeLa and RPMI 8402) cell lines. However, the differential expression of the two CLC promoter constructs in these cell lines suggests that the -292 to -411 bp region of the promoter may confer some specificity for expression in the eosinophil lineage. The CLC promoter sequence contains two consensus GATA binding sites, a purine-rich sequence that presents potential binding sites for PU.1, a member of the ets family of genes, as well as sequences described in other myeloid-specific promoters. This is the first demonstration of a functional eosinophil promoter that could serve as a model for identifying DNA elements and trans-activating factors that regulate gene expression during the commitment and differentiation of the eosinophil lineage.

Amino Acid Sequence↗
PubMed1993

Expression of mRNA for the GATA-binding proteins in human eosinophils and basophils: potential role in gene transcription.

The expression of the hematopoietic transcription factors GATA-1, GATA-2, and GATA-3 was studied in eosinophils and basophils. Eosinophils express mRNA for GATA-1, GATA-2, and GATA-3. Basophils express GATA-2 and GATA-3. Treatment of HL-60 eosinophilic sublines with either interleukin-5 or butyric acid increased the expression of GATA-1 mRNA concomitant with the expression of eosinophil-specific genes, whereas levels of GATA-2 mRNA remained relatively constant. The presence of mRNA for these proteins in eosinophils and basophils suggests that gene transcription in these lineages may be regulated by GATA-binding proteins.

Base Sequence↗
PubMed1993

Characterization of a distinct binding site for the prokaryotic chaperone, GroEL, on a human granulocyte ribonuclease.

Although ribonucleases fold into correct tertiary conformation in vitro guided solely by information contained in the primary amino acid sequence (Sela, M., White, F. H., and Anfinsen, C. B. (1957) Science 124, 691-693), it is not clear whether folding of these proteins proceeds unassisted in a complex intracellular environment. We describe here the specific and high affinity binding of groEL, the prokaryotic homolog of the heat shock protein 60 family of molecular chaperones, to recombinant eosinophil cationic protein and eosinophil-derived neurotoxin, two members of the human ribonuclease gene family. We have determined that groEL binds to a unique peptide sequence near the amino terminus of nascent eosinophil cationic protein that includes the first of eight cysteine residues. This binding site functions independently and can confer groEL binding activity on an unrelated carrier protein. GroEL dissociates from the binding site upon addition of ATP and Mg2+; no other cations or cofactors are necessary. These findings suggest the possibility that interaction with a groEL-like molecular chaperone may be a requirement for correct folding and/or translocation of eukaryotic ribonucleases in vivo.

Amino Acid Sequence↗
PubMed1993

Molecular cloning and characterization of human eosinophil Charcot-Leyden crystal protein (lysophospholipase). Similarities to IgE binding proteins and the S-type animal lectin superfamily.

We have isolated and sequenced a 598-bp full length cDNA clone for the human Charcot-Leyden crystal (CLC) protein (eosinophil lysophospholipase), the unique and prominent constituent of human eosinophils and basophils that forms the hexagonal bipyramidal crystals classically observed in tissues and secretions from sites of eosinophil-associated inflammation. A 426-bp open reading frame encoded a 142-amino acid polypeptide with a predicted molecular mass of 16.5 kDa and isoelectric point of 7.28. The deduced amino acid sequence of CLC protein showed 20 to 30% similarity over regions of approximately 100 amino acids with the carboxyl-terminal domains of four IgE-binding proteins, including the 31-kDa human and rat IgE-binding proteins, the 35-kDa mouse carbohydrate binding protein (CBP35), Mac-2, the murine macrophage cell surface protein that is identical to CBP35, and the human homologue of Mac-2. These proteins are members of a superfamily of beta-galactoside binding S-type animal lectins, which includes a group of highly conserved 14-kDa lectins isolated from human lung, heart, placenta, bovine heart, chicken skin, mouse fibroblasts, and the electric organ of the electric eel; CLC protein also showed sequence similarities to these 14-kDa animal lectins, including conservation of 7 of 16 invariant amino acid residues thought to comprise the carbohydrate-binding domain of these proteins, with conservative amino acid changes at others; thus, CLC protein could potentially possess carbohydrate or IgE-binding activities. Northern analyses revealed an approximately 900-bp mRNA species that was present in peripheral blood eosinophils from patients with eosinophilia, basophils from patients with chronic myelogenous leukemia, and in HL-60 cells induced towards eosinophilic differentiation with B cell growth factor-II (IL-5) or granulocytic differentiation with DMSO, but was absent in neutrophils, monocytes, T cells, B cells, or HL-60 cells induced towards monocytic differentiation with vitamin D3. Southern analyses revealed a gene of approximately 5 to 6 kb in length. The cDNA clone and complete amino acid sequence data for CLC protein will facilitate structure-function analyses of its unusual hydrophobic properties, unique propensity for crystallization, lysophospholipase, and potential lectin-like activities.

Amino Acid Sequence↗
PubMed1993

Receiver operating characteristic analysis of fracture and pneumonia detection: comparison of laser-digitized workstation images and conventional analog radiographs.

Forty pneumonia and 40 fracture cases were matched by patient age and gender with normal cases. Pediatric cases constituted 25% of this sample. All 160 examinations were laser-digitized at a spot size of 210 microns, a contrast resolution of 12 bits, and a spatial resolution of 2.35 line pairs per millimeter. The digitized images were transmitted 5 miles over a dedicated telephone line at a T1 rate from Francis Scott Key Hospital to Johns Hopkins Hospital (both in Baltimore, Md). At Johns Hopkins Hospital, eight radiologists interpreted the original analog radiograph and corresponding soft-copy display (1,280 x 1,024 pixels). Findings, confidence ratings, and image quality were reported for each reading. A receiver operating characteristic (ROC) analysis was conducted to compare observer performance under the analog and digital reading conditions. The overall sensitivity of the analog method was 89% versus 78% for the digital method (P < .001), while the specificity values were 96% and 92%, respectively (P > .20). ROC analysis similarly indicated a statistically significant difference in favor of analog radiographs (P = .028).

Child↗
PubMed1993

Subtle orthopedic fractures: teleradiology workstation versus film interpretation.

The purpose of this study was to evaluate whether radiologists perform equally well with plain radiographs or digitized images displayed on a video monitor in interpretation of difficult orthopedic trauma cases. Interpretations with film and those made from a teleradiology system with spatial resolution of 2.35 line pairs per millimeter were compared in 120 difficult cases, 60 with the selected abnormality (ie, fracture or dislocation) and 60 that were control cases. Seven senior radiology residents and one radiology fellow each interpreted 60 randomly ordered cases with the teleradiology system (1,280 x 1,024-pixel monitors) and 60 cases with the original radiographs. The overall accuracy of the readers was 80.6% for film interpretations and 59.6% for teleradiology screen readings (P < .001). Sensitivity was 78.5% for film and 48.8% for on-screen images (P < .001), and specificity was 83.2% for film and 72.3% for on-screen images (P < .025). Receiver operating characteristic analysis showed rejection of the null hypothesis in favor of film interpretation (P < .0049). It was concluded that the teleradiology system was not acceptable for primary diagnostic interpretation of difficult fracture cases.

Computer Terminals↗
PubMed1993

Cationic proteins induce airway hyperresponsiveness dependent on charge interactions.

It has previously been demonstrated that human eosinophil-derived granule major basic protein (MBP) can increase airway responsiveness after intratracheal administration in the rat. This effect was mimicked by synthetic polycations, suggesting that charge interactions may be important in the development of airway hyperresponsiveness (AHR). To support this hypothesis, we investigated whether two other cationic proteins, platelet factor 4 (PF4) and cathepsin G, were capable of inducing AHR. Furthermore, to determine whether these effects were dependent on their positive charge, the charge of these proteins was neutralized with low molecular weight heparin. In addition, we have examined whether the effect of a synthetic polycation, poly-L-lysine could be inhibited by low molecular weight heparin, albumin, or dextran sulphate. MBP, PF4, or cathepsin G induced a 2- to 3-fold increase in airway responsiveness 1 h after instillation, as assessed by the dose of inhaled methacholine required to increase total lung resistance by 100%. Admixing these native cationic proteins with low molecular weight heparin inhibited the development of AHR. Similarly, poly-L-lysine increased airway responsiveness that was inhibited not only by low molecular weight heparin but also by two other anionically charged molecules, albumin and dextran sulphate. These findings suggest that charge interactions in the airways are important not only in alterations in airway responsiveness induced by eosinophil-derived MBP, but also because they underlie altered airway responsiveness after treatment with other cationic proteins. The precise mechanisms involved in this phenomenon remain to be determined.

Albumins↗
PubMed1993

The effect of human eosinophil granule major basic protein on airway responsiveness in the rat in vivo. A comparison with polycations.

Major basic protein (MBP) is a highly cationic protein found in the granules of eosinophils. It has been postulated that MBP may participate in the pathogenesis of airway hyperresponsiveness exhibited by asthmatic patients. Accordingly, we used a rat model to investigate the effect of human MBP instillation on airway responsiveness and the possible role of cationic charge in the determination of this effect. Dose-response characteristics to inhaled methacholine (MDRC) were determined at baseline, and the animals were allowed to recover. Then animals in the experimental group received 100 micrograms of purified human MBP via direct instillation into the trachea. One hour after instillation, the MDRC were again assessed. Control animals received (in lieu of MBP) buffer from the void volume pool of the same chromatography column used to purify the MBP. One hour after instillation of MBP there was a significant increase in airway responsiveness to inhaled methacholine, whereas control animals exhibited no increase in airway responsiveness. Some animals from the MBP group were restudied 48 h after MBP instillation, by which time airway responsiveness had returned to baseline level. The effect of the polycations poly-L-arginine and poly-L-lysine on airway responsiveness was also examined. As with MBP, airway responsiveness to inhaled methacholine increased 1 h after the instillation of either polycation. In addition, acetylation of the charged groups on poly-L-lysine resulted in a loss of this effect. Histologic examination of the airways failed to reveal airway epithelial shedding 1 h after MBP or polycation instillation.(ABSTRACT TRUNCATED AT 250 WORDS)

Airway Resistance↗
PubMed1992

Charcot-Leyden crystal protein in the degranulation and recovery of activated basophils.

The Charcot-Leyden crystal (CLC) protein, a prominent cell constituent unique to eosinophils and basophils, possesses lysophospholipase activity. This activity and the extracellular deposition and formation of CLC in tissues and body fluids in association with eosinophils suggest an extracellular function for this protein in inflammation. During degranulation, basophils release granule-derived mediators of inflammation. We postulated that CLC protein, localized in part to the basophil granule, might be released along with other mediators during this process. The extracellular release of CLC protein was studied during the degranulation of basophils stimulated by anti-immunoglobulin E (anti-IgE), N-formyl-methionyl-leucyl-phenylalanine (fMLP), phorbol myristate acetate, eosinophil major basic protein (MBP), and calcium ionophore A23187. Histamine release was used as a marker of basophil degranulation; its release was measured utilizing the fluorometric technique. CLC protein was not released into the supernatant during this process as determined by radioimmunoassay. CLC protein in the extracellular space, either as intact crystals or aggregates, was undetectable by indirect immunofluorescent staining of basophils activated with either anti-IgE or fMLP. However, upon activation, the immunofluorescent cytoplasmic and nuclear staining pattern for CLC protein was significantly altered. Decreased cytoplasmic staining and persistent or increased nuclear staining for CLC protein were observed after activation, with recovery of the preactivation, unstimulated cellular staining pattern at 30 and 45 min after stimulation with fMLP and anti-IgE, respectively. These findings suggest that CLC protein functions intracellularly in basophils during the process of activation, degranulation, and recovery. The potential nuclear function(s) of this lysophospholipase in the basophil requires further investigation.

Antigens, Differentiation, B-Lymphocyte↗
PubMed1992

The gene for human eosinophil Charcot-Leyden crystal protein directs expression of lysophospholipase activity and spontaneous crystallization in transiently transfected COS cells.

Expression of the gene encoding human eosinophil lysophospholipase, the Charcot-Leyden crystal (CLC) protein, was studied in transiently transfected COS cells. Recombinant CLC (rCLC) protein expression was demonstrated both by Western blot and radioimmunoassay inhibition analyses of transfected COS cell extracts and by immunofluorescent staining and ultrastructural immunogold analyses of intact cells. The rCLC protein was immunochemically indistinguishable from native eosinophil-derived CLC protein, and each transfected COS cell expressed approximately 11 pg of rCLC protein as determined by radioimmunoassay and assessment of transfection efficiency. Immunofluorescent microscopy and ultrastructural immunogold analyses localized rCLC protein to the nucleus, cytoplasm, and plasma membrane of COS cells. Lysates from transfected COS cells producing CLC protein expressed significant lysophospholipase activity. Furthermore, rCLC protein expressed in COS cells spontaneously formed the distinctive intracytoplasmic and intranuclear hexagonal bipyramidal crystals characteristic of the native eosinophil and basophil-derived protein. Expression of the CLC gene confirms the authenticity of the CLC cDNA, the expression of lysophospholipase activity by this unique eosinophil and basophil constituent, and will facilitate the routine purification of the active enzyme for in vitro and animal model studies of its role (or roles) in eosinophil and basophil associated allergic inflammation and eosinophil-parasite interactions.

Animals↗
PubMed1992

The effects of eosinophil-granule major basic protein on lung-macrophage superoxide anion generation.

The effects of major basic protein (MBP) on superoxide anion release in vitro from normal guinea pig (GP) alveolar macrophages (AM) was determined. Native MBP at 55 micrograms/ml had an immediate effect (reduction) on phorbol myristate acetate (PMA)-induced (p less than 0.01) and spontaneous (p = 0.055) superoxide (O2-) release by GP AMs. A similar effect was not observed from AMs incubated for 24 hours with MBP before assessment of O2- release. However, after AMs were incubated with MBP for 48 hours, again there was a significant reduction observed in both PMA-induced (p = 0.01) and spontaneous (p = 0.002) O2- release compared to that of control cultures. The immediate effect of MBP on AM O2- release was not due to cytotoxicity of MBP for AM. In contrast, the effect observed after 48 hours of culture was due, in part, to a direct toxic effect of the MBP on the AM because the viability of AM cultured for 48 hours with MBP (55 micrograms/ml) was 63.6% +/- 13% compared to the viability of control cultures of AM that was 83.9% +/- 7%; p = 0.03. The effect of MBP on AM O2- release at 48 hours was progressive over concentrations ranging from 2 to 55 micrograms/ml. These data suggest that native MBP can affect adversely PMA-induced and spontaneous release of O2- by GP AMs and that this effect depends only in part on the cytotoxic properties of MBP.

Adolescent↗
PubMed1992

Mature eosinophils stimulated to develop in human-cord blood mononuclear cell cultures supplemented with recombinant human interleukin-5. II. Vesicular transport of specific granule matrix peroxidase, a mechanism for effecting piecemeal degranulation.

The mechanism of piecemeal degranulation by human eosinophils was investigated. Mature eosinophils that developed in rhIL-5-containing conditioned media from cultured human cord blood mononuclear cells were prepared for ultrastructural studies using a combined technique to image eosinophil peroxidase by cytochemistry in the same sections on which postembedding immunogold was used to demonstrate Charcot-Leyden crystal protein. Vesicular transport of eosinophil peroxidase from the specific granule matrix compartment to the cell surface was associated with piecemeal degranulation. This process involved budding of eosinophil peroxidase-loaded vesicles and tubules from specific granules. Some eosinophil peroxidase that was released from eosinophils remained bound to the cell surface; some was free among the cultured cells. Macrophages and basophils bound the released eosinophil peroxidase to their plasma membranes, internalized it in endocytotic vesicles, and stored it in their respective phagolysosomes and secretory granules. Charcot-Leyden crystal protein was diffusely present in the nucleus and cytoplasm of IL-5-stimulated mature eosinophils. Extensive amounts were generally present in granule-poor and subplasma membrane areas of the cytoplasm in contrast to eosinophil peroxidase, which was secreted and bound to the external surface of eosinophil plasma membranes. These studies establish vesicular transport as a mechanism for emptying the specific eosinophil granule matrix compartment during IL-5-associated piecemeal degranulation.

Blood Cells↗
PubMed1991

Cytoplasmic lipid bodies of neutrophils: formation induced by cis-unsaturated fatty acids and mediated by protein kinase C.

Lipid bodies, nonmembrane-bound cytoplasmic inclusions, serve as repositories of esterified arachidonate and are increased in cells associated with inflammatory reactions. We have evaluated stimuli and mechanisms responsible for lipid body formation within human polymorphonuclear leukocytes (PMNs). Arachidonic acid and oleic acid stimulated dose-dependent formation of lipid bodies over 0.5-1 h. Other C20 and C18 fatty acids were less active and demonstrated rank orders as follows: cis-unsaturated fatty acids were much more active than trans-fatty acids, and activity diminished with decreasing numbers of double bonds. Lipid bodies elicited in vitro with cis-fatty acids were ultrastructurally identical to lipid bodies present in PMNs in vivo. Lipid body induction was not because of fatty acid-elicited oxidants or fatty acid-induced ATP depletion. Cis-fatty acid-induced activation of protein kinase C (PKC) was involved in lipid body formation as evidenced by the capacity of other PKC activators, 1-oleoyl-2-acetyl-glycerol and two active phorbol esters, phorbol myristate acetate, and phorbol 12,13 dibutyrate, but not an inactive phorbol, to induce lipid body formation. The PKC inhibitor, 1-O-hexadecyl-2-O-methyl-glycerol, inhibited PMN lipid body formation induced by oleic and arachidonic acids and by 1-oleoyl-2-acetyl-glycerol and phorbol myristate acetate. Other PKC inhibitors (staurosporine, H-7) also inhibited lipid body formation. Formation of lipid bodies in PMNs is a specific cellular response, stimulated by cis-fatty acids and diglycerides and apparently mediated by PKC, which results in the mobilization and deposition of lipids within discrete, ultrastructurally defined cytoplasmic domains.

Adenosine Triphosphate↗
PubMed1991

Mature eosinophils stimulated to develop in human cord blood mononuclear cell cultures supplemented with recombinant human interleukin-5. Part I. Piecemeal degranulation of specific granules and distribution of Charcot-Leyden crystal protein.

Human cord blood mononuclear cells were cultured for 35 days in media containing recombinant human interleukin 5 (rhIL-5) supplemented with a fraction of the culture supernatant of phytohemagglutinin (PHA)-stimulated human T lymphocytes from which interleukin 2 (IL-2) was eliminated. Cultured cells were studied by electron microscopy and an immunogold procedure to detect subcellular site(s) of Charcot-Leyden crystal (CLC) protein. The majority of cells (greater than 70%) developing in this system were mature eosinophils, with descending frequency of other cells, including macrophages, mature basophils, eosinophilic myelocytes, and mature neutrophils. Mature eosinophils were characterized by increased numbers of primary granules, small granules, tubulovesicular structures, and decreased secondary granules. These eosinophils showed extensive piecemeal degranulation (PMD) characterized by partially empty and empty secondary granule chambers in the cytoplasm. Small, smooth vesicles were evident within empty granule chambers as well as adjacent to them. Eosinophils formed close associations with phagocytic macrophages that contained both standard-shaped and irregularly shaped CLC within phagolysosomes. Subcellular sites of CLC protein were demonstrated by immunogold in eosinophils and macrophages arising in these cultures. Charcot-Leyden crystal protein was present in the nuclear matrix and extraorganellar cytoplasm of eosinophils. Primary granules and some cytoplasmic vesicles were labeled for CLC protein, but full and empty secondary granules and the extensive network of tubulovesicles were not. Charcot-Leyden crystals were absent from eosinophils, nor were they present in the extracellular space. Charcot-Leyden crystals were absent from eosinophils, nor were they present in the extracellular space. Charcot-Leyden crystals within phagosomes of macrophages were labeled by the immunogold procedure for CLC protein. These results demonstrate that rhIL-5-supplemented, PHA-stimulated, T-cell-conditioned media induced the development of mature human eosinophils from cord blood cells. These eosinophils underwent PMD of secondary granule contents. Immunogold analysis showed eosinophil CLC protein in the cytoplasm, nucleus, and primary granules of eosinophils. Macrophages also were present in these cultures. They contained CLC protein-containing crystals in their phagosomes, suggesting active sequestration of eosinophil CLC protein by macrophages in vitro.

Cell Degranulation↗