Biomedical subjects

S J Ackerman

Publications and source records attributed to S J Ackerman.

At least 19 recordsLinked to original sources

PubMed1997

Anion, cation, and zwitterion selectivity of phospholemman channel molecules.

Phospholemman (PLM), a 72-amino acid membrane protein with a single transmembrane domain, forms taurine-selective ion channels in lipid bilayers. Because taurine forms zwitterions, a taurine-selective channel might have binding sites for both anions and cations. Here we show that PLM channels indeed allow fluxes of both cations and anions, making instantaneous and voltage-dependent transitions among conformations with drastically different ion selectivity characteristics. This surprising and novel ion channel behavior offers a molecular explanation for selective taurine flux across cell membranes and may explain why molecules in the phospholemman family can induce cation- or anion-selective conductances when expressed in Xenopus oocytes.

Animals↗

PubMed1996

Atrial rhythm after atrioventricular junctional ablation.

Atrioventricular (AV) junctional ablation followed by pacemaker implantation is an established treatment for patients with refractory paroxysmal atrial arrhythmias. The stability of the underlying atrial rhythm after AV junctional ablation is unknown. This study evaluates the atrial rhythm after AV junctional ablation in 49 patients with medically refractory atrial arrhythmias. The group included 25 men and 24 women, of whom 36 had known structural heart disease. Paroxysmal atrial fibrillation was the primary rhythm disturbance in 41 patients, whereas 8 manifested either atrial tachycardias or atrial flutter. All patients had failed therapy with > or = 1 antiarrhythmic drug. Chronic pacing modes were DDIR or DDDR, with mode switching in 15 patients and VVIR in 34 patients. After AV junctional ablation, chronic antiarrhythmic drug therapy was prescribed in only 4 patients (8%). Routine electrocardiograms (ECGs; 6.5 +/- 6.1/patient) during long-term follow-up (18.6 +/- 15.6 months) showed that 7 patients (14%) had an atrial arrhythmia detected on all ECGs, 30 patients (61%) had sinus or atrial-paced rhythms on all recordings, and 12 patients (25%) had both atrial arrhythmias and sinus rhythm documented. Sinus or an atrial-paced rhythm was present on the last available ECG in 33 of 49 patients (67%). Pacing mode was not a predictor of continued sinus rhythm. In conclusion, most patients with a history of paroxysmal atrial tachyarrhythmias will not convert to chronic atrial arrhythmias after AV junctional ablation, even in the absence of antiarrhythmic drug therapy. Use of dual-chamber pacing modes will allow maintenance of at least intermittent atrial function in these patients.

Aged↗

PubMed1996

Eosinophil-fibroblast interactions. Granule major basic protein interacts with IL-1 and transforming growth factor-beta in the stimulation of lung fibroblast IL-6-type cytokine production.

To test the hypothesis that eosinophil major basic protein (MBP) is an important regulator of fibroblast effector function, we characterized the effects of MBP on human lung fibroblast production of the IL-6-type cytokines, IL-6, IL-11, and leukemia inhibitory factor. Unstimulated fibroblasts did not produce substantial quantities of these cytokines, while IL-1 and TGF-beta(1) stimulated these cytokines in a potent fashion. MBP at doses < or = 44 micrograms/ml did not stimulate IL-6-type cytokine production. It did, however, interact in a synergistic, dose- and time-dependent fashion with rIL-1-alpha and TGF-beta(1) to further increase IL-6-type cytokine elaboration. These MBP-induced increases in cytokine production were associated with proportionate alterations in mRNA accumulation. In contrast, eosinophil-derived neurotoxin did not regulate fibroblast cytokine production, and MBP did not augment fibroblast granulocyte-macrophage-CSF, or type I collagen production, or fibroblast proliferation in this culture system. The effects of MBP could not be attributed to cell cytotoxicity or contaminants in the MBP preparations. They were, however, at least partially charge mediated, since heparin abolished the effects of MBP on IL-1-stimulated cells, and the surrogate cationic molecule poly-L-arginine mimicked the stimulatory effects of MBP on fibroblast IL-6-type cytokine elaboration. These studies demonstrate that MBP interacts in a synergistic fashion with rIL-1-alpha or TGF-beta(1) to further augment fibroblast IL-6-type cytokine production. They also demonstrate that this stimulation is pretranslationally mediated and due, in part, to the cationic nature of the MBP molecule. MBP regulation of fibroblast cytokine production may play an important role in the pathogenesis of eosinophilic disorders of the airway or other organs.

Blood Proteins↗

PubMed1996

Interleukin-5 receptor alpha subunit gene regulation in human eosinophil development: identification of a unique cis-element that acts lie an enhancer in regulating activity of the IL-5R alpha promoter.

Further functional and biochemical characterization of the nuclear factor(s) which interacts with the EOS1 enhancer-like element in the IL-5R alpha promoter is currently in progress. Since different transcription factors recognize and interact with DNA in distinct fashions and with distinct structural motifs, we have modeled potential binding of the EOS1 factor to its cis-element based upon its methylation interference pattern (Fig. 2), using a cylindrical DNA helical projection (Fig. 6). Over a length of two helical turns, all nuclear protein contacts indicated by methylation interference map to one side of the DNA helix, suggesting that EOS1 binds in the major groove, across the minor groove, and on only one side of the helix. Further review of the model also reveals a potential diad symmetry for the binding site, suggestive of binding by a homodimer and consistent with the formation of the two DNA-protein complexes in our electrophoretic mobility shift experiments that could represent interactions with monomer versus dimer. Comparison of the EOS1 binding motif to similar models for the binding of other transcription factor families for which structural crystallographic and/or binding data is available suggests a similarity of the EOS1 complex to that of the bacterial helix-turn-helix phage lambda and 434 repressor-operator complexes, and the Cys4 zinc finger glucocorticoid response element (GRE) DNA-binding motifs, all of which show similar diad symmetry and binding in the major groove on one side of the DNA. The possibility that EOS1 functions as a GRE is being investigated, especially since there is a consensus AP-1 site at bp -440 to -432 of the IL-5R alpha promoter, immediately adjacent to the EOS1 binding site (see Fig. 5 in reference [36]) and AP-1/GRE interactions have been identified for composite response elements in the regulation of a number of different genes. The identification or cloning of EOS1, a potentially novel and eosinophil lineage-active transcription factor, should enhance our understanding of the processes involved in eosinophil development in particular and myeloid lineage commitment and differentiation in general.

Animals↗

PubMed1996

Vesicular transport of Charcot-Leyden crystal protein in tumor-promoting phorbol diester-stimulated human basophils.

Secretion from human basophils (HB) that are stimulated with a phorbol ester takes place with slower kinetics than typically regulated secretion, which is stimulated by the IgE-mediated mechanism associated with classical granule exocytosis. Phorbol ester stimulation of HB induces emptying of granule contents (with retention of granule containers) and increases the number of cytoplasmic vesicles, an anatomic process similar to one termed piecemeal degranulation (PMD), which is a secretory process originally described in HB that migrate from the blood in vivo into contact allergy skin lesions. Charcot-Leyden crystal (CLC) protein is a basophil granule-associated protein that is readily imaged by using a postembedding immunogold procedure. This method was used to localize this granule protein in phorbol ester-stimulated, isolated human peripheral blood basophil cytoplasmic vesicles in samples collected within a time frame for which histamine was secreted. The results of this study showed that the proportion of cytoplasmic vesicles that were gold-labeled in stimulated HB, which indicated the presence of CLC protein, increased significantly over unstimulated cells at 2, 5, and 10 minutes after stimulation. Additionally, CLC protein-labeled vesicles in the cells that were stimulated for 10 minutes significantly exceeded the number in stimulated cells at 0, 30, and 45 minutes after exposure to phorbol ester. Thus, transport vesicles carrying a granule-associated protein (CLC protein) were increased in phorbol ester-stimulated HB in the time frame for histamine secretion and the anatomic development of PMD. These findings support vesicular transport as a major mechanism for effecting PMD, which is morphologically the most frequent activation anatomy displayed by HB in human disease in vivo.

Basophils↗

PubMed1995

Crystal structure of human Charcot-Leyden crystal protein, an eosinophil lysophospholipase, identifies it as a new member of the carbohydrate-binding family of galectins.

BACKGROUND: The Charcot-Leyden crystal (CLC) protein is a major autocrystallizing constituent of human eosinophils and basophils, comprising approximately 10% of the total cellular protein in these granulocytes. Identification of the distinctive hexagonal bipyramidal crystals of CLC protein in body fluids and secretions has long been considered a hallmark of eosinophil-associated allergic inflammation. Although CLC protein possesses lysophospholipase activity, its role(s) in eosinophil or basophil function or associated inflammatory responses has remained speculative. RESULTS: The crystal structure of the CLC protein has been determined at 1.8 A resolution using X-ray crystallography. The overall structural fold of CLC protein is highly similar to that of galectins -1 and -2, members of an animal lectin family formerly classified as S-type or S-Lac (soluble lactose-binding) lectins. This is the first structure of an eosinophil protein to be determined and the highest resolution structure so far determined for any member of the galectin family. CONCLUSIONS: The CLC protein structure possesses a carbohydrate-recognition domain comprising most, but not all, of the carbohydrate-binding residues that are conserved among the galectins. The protein exhibits specific (albeit weak) carbohydrate-binding activity for simple saccharides including N-acetyl-D-glucosamine and lactose. Despite CLC protein having no significant sequence or structural similarities to other lysophospholipase catalytic triad has also been identified within the CLC structure, making it a unique dual-function polypeptide. These structural findings suggest a potential intracellular and/or extracellular role(s) for the galectin-associated activities of CLC protein in eosinophil and basophil function in allergic diseases and inflammation.

Acute Disease↗

PubMed1995

Changing the differentiation program of hematopoietic cells: retinoic acid-induced shift of eosinophil-committed cells to neutrophils.

The mechanisms by which hematopoietic progenitor cells become lineage-committed remain poorly understood. A cloned subline of the AML14 cell line (AML14.3D10) that spontaneously differentiates to eosinophilic myelocytes in the absence of cytokine stimulation was obtained by limiting dilution. This subline exhibits augmented expression of interleukin-5 (IL-5) receptor alpha subunit mRNA and synthesizes all major eosinophil granule proteins. Exposure of this cell line to all-trans retinoic acid (ATRA) causes loss of eosinophilic granules and fast green staining within 48 hours, without cell death. In addition, mRNA for the IL-5 receptor alpha subunit becomes undetectable by 48 hours and the cells lose responsiveness to IL-5. Major basic protein, measured as a marker of eosinophilic granule content, decreases from more than 16 pg/cell to undetectable levels by 5 days after ATRA. Concomitant with the loss of major basic protein and fast green staining, surface expression of CD16 becomes detectable and is maximum by 10 days after ATRA. mRNA for the granulocyte colony-stimulating factor (G-CSF) receptor becomes detectable by day 5, and the cells become responsive to G-CSF. At this time, the cells appear morphologically as mature neutrophils and can reduce nitroblue tetrazolium. With continued culture, the neutrophilic cells die and the culture becomes repopulated with eosinophilic myelocytes. These findings show that it is possible to change the differentiation program of hematopoietic cells even after they show evidence of advanced lineage commitment. The AML14.3D10 subclone of AML14 will be a valuable model for study of the transcriptional regulation of the eosinophil and neutrophil differentiation programs and lineage-specific gene expression.

Base Sequence↗

PubMed1995

Unitary anion currents through phospholemman channel molecules.

Phospholemman (PLM) is a 72-amino-acid peptide with a single transmembrane domain, the expression of which induces chloride currents in Xenopus oocytes. It has remained unknown whether PLM is an ion channel or acts as a channel regulator. Here we show, by measuring unitary anion currents across planar phospholipid bilayers to which immunoaffinity-purified recombinant PLM was added, that it does indeed form ion channels. Excised patches of oocytes expressing PLM had similar currents. Of the ions tested, the sulphonic amino acid taurine was the most permeant, and expression of PLM increased fluxes of radiolabelled taurine in oocytes. Phospholemman is the smallest protein in cell membranes known to form an ion channel and the taurine selectivity suggests that it is involved in cell volume regulation.

Animals↗

PubMed1995

Identification and characterization of a functional promoter region in the human eosinophil IL-5 receptor alpha subunit gene.

The molecular basis for the commitment of multipotential myeloid progenitors to the eosinophil lineage, and the transcriptional mechanisms by which eosinophil-specific genes are subsequently expressed and regulated during eosinophil development are currently unknown. Interleukin-5 (IL-5) is a T cell and mast cell-derived cytokine with actions restricted to the eosinophil and closely related basophil lineages in humans. The high affinity receptor for IL-5 (IL-5R) is composed of an alpha subunit (IL-5R alpha) expressed by the eosinophil lineage, that associates with a beta c subunit shared with the receptors for IL-3 and granulocyte-macrophage colony stimulating factor (GM-CSF). As a prerequisite to studies of the transcriptional regulation of the IL-5R alpha subunit gene, we used three different methods, including primer extension, RNase protection, and 5'-RACE to precisely map the transcriptional start site to a position 15 base pairs (bp) upstream of the 5' end of the published sequence of IL-5R alpha exon 1. To initially identify the IL-5R alpha promoter, 3.5 kilobases (kb) and 561 bp of the 5' sequence flanking the transcriptional start site were subcloned into the promoterless pXP2-luciferase vector. Transient transfection of these constructs into an eosinophil-committed HL-60 subline, clone HL-60-C15, induced the expression of approximately 240-fold greater luciferase activity than the promoterless vector, identifying a strong functionally active promoter region within the 561 bp of sequence proximal to the transcriptional start site and with activity equivalent to pXP2 constructs containing the entire 3.5 kb of upstream sequence. To more precisely localize the cis-acting regulatory elements in this region important for promoter activity, a series of 5' deletion mutants of the 561-bp region were generated in the pXP2-luciferase vector. Deletion of the region between bp -432 and -398 reduced promoter activity by more than 80% in the HL-60-C15 cell line. Further analyses of the activity of the IL-5R alpha promoter constructs in various other eosinophil, myeloid, and non-myeloid cell lines indicated that the promoter was relatively myeloid and eosinophil lineage-specific in its expression. Consensus sequences for known transcription factor binding sites were not present in the 34-bp region of the promoter required for maximal activity, suggesting unique myeloid- and possibly eosinophil-specific regulatory elements.(ABSTRACT TRUNCATED AT 400 WORDS)

Base Sequence↗

PubMed1995

Human eosinophil-granule major basic protein and synthetic polycations induce airway hyperresponsiveness in vivo dependent on bradykinin generation.

In the current series of experiments we investigated the role of bradykinin in airway hyperresponsiveness induced by human eosinophil-granule major basic protein (MBP). Bronchoalveolar lavage was performed after intratracheal instillation of MBP or poly-L-lysine in anesthetized, intubated rats, and levels of immunoreactive kinins and kallikrein-like activity were determined. Both MBP and poly-L-lysine induced a three- and eightfold increase in levels of kallikrein-like activity and i-kinins, respectively. To determine whether kinin production is required for the development of airway hyperresponsiveness induced by cationic proteins, dose-response curves to methacholine were constructed before and 1 h after intratracheal instillation of either MBP or poly-L-lysine (100 micrograms). MBP and poly-L-lysine induced an increase in airway responsiveness, which was inhibited by pretreatment with a selective BK-2 receptor antagonist, NPC 17713 (250 micrograms/ml). Our results demonstrate that MBP and poly-L-lysine activate kallikrein and stimulate the generation of i-kinins in vivo, an effect that may be related to the cationic charge of these proteins. Furthermore, the ability of these proteins to increase airway responsiveness appears to be dependent on the generation of i-kinins.

Amino Acid Sequence↗

PubMed1995

Methacholine challenge does not affect bronchoalveolar fluid cell number and many indices of cell function in asthma.

Methacholine (MCh) challenge testing is often incorporated into clinical studies prior to performing bronchoscopy as a measure of bronchial hyperresponsiveness (BHR). However, the effect of methacholine on many aspects of bronchoalveolar lavage (BAL) fluid cell count and function have not been fully evaluated. Ten patients with asthma, maintained on inhaled beta 2-agonists, were studied. Each subject underwent two bronchoscopies in a random order, one preceded by methacholine challenge within 30 min of the BAL. The investigators were blinded to the regimen. Several markers of BAL fluid cell number and function were studied: cell count and differential histamine, eosinophil products, including eosinophil cationic protein and Charcot-Leyden crystal protein, macrophage production of thromboxane B2 and leukotriene B4, neutrophil lysozyme and lactoferrin, and lymphocyte typing and activation markers measured via flow cytometry. No significant differences were noted in any of these markers of cell number or function which could be ascribed to methacholine challenge. Thus, methacholine challenge does not appear to affect these markers of cell number and function. These findings indicate that a methacholine challenge can be used as a measure of bronchial hyperresponsiveness within 30 min prior to bronchoscopy without altering bronchoalveolar lavage fluid characteristics.

Adult↗

PubMed1995

Major basic protein regulation of lung fibroblast cytokine production. Role of cytokine synergy and charge.

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Blood Proteins↗

PubMed1995

Validation of transcranial Doppler ultrasound with a stereotactic neurosurgical technique.

Vessel identification during a transcranial Doppler (TCD) ultrasound examination is usually based on well-established inferential criteria without confirmation by imaging. Part of a routine study involves taking measurements from the M1 segment of the middle cerebral artery (MCA) and the A1 segment of the anterior cerebral artery (ACA) at the points of maximum mean linear blood flow velocity (LBFV). The authors tested the hypothesis that insonation is from the midpoints of the M1 and A1 segments during clinical TCD examinations. Conventional hand-held TCD examinations were performed on five volunteers. The points of maximum mean LBFV of the M1 and A1 segments of the MCA and ACA were located. Measurements were also taken from the midpoints of the M1 and A1 segments using a magnetic resonance (MR) imaging-guided stereotactic TCD technique. Values for depths of insonation and maximum mean LBFV obtained with the two techniques were compared. There was no significant difference between the two techniques for the measured values of depth of insonation of either the individual vessels (p > 0.11) or the aggregate (p = 0.46). There was a significant difference between the aggregate maximum mean LBFV measurements (p = 0.0022). The hand-held technique systematically produced higher maximum mean LBFV than the MR-guided stereotactic technique. The authors conclude that when using traditional criteria for TCD examination of the ACA and MCA, the points of insonation approximate the middle of the A1 and M1 segments.

Adult↗

PubMed1994

Functional characterization of the promoter for the gene encoding human eosinophil peroxidase.

The molecular basis for commitment of progenitors to the eosinophil lineage and mechanisms by which eosinophil-specific genes are expressed and regulated during differentiation is unknown. Expression of eosinophil peroxidase (EPO) is restricted to the eosinophil lineage. To understand the mechanisms involved in transcriptional regulation of EPO gene expression, we clone the region of the EPO gene upstream of the transcriptional start site and analyzed the cis-acting elements required for EPO promoter activity in an eosinophil-inducible leukemic cell line, HL-60-C15. The 5'-flanking region of the EPO gene containing 1.5 kilobases of sequence upstream of the transcriptional start site was subcloned into the promoterless pXP2-luciferase vector. The EPO-pXP2 construct and 5' deletion mutants were electroporated into HL-60-C15 cells and luciferase reporter activity assessed. The -1.5-kilobase EPO-pXP2 promoter construct reproducibly expressed > 120-fold more luciferase activity than did promoterless pXP2, and a 12-fold (90%) decrease in promoter activity was obtained when sequences between -122 and -45 base pairs (bp) were deleted. The specificity of the EPO promoter for the eosinophil lineage was analyzed by transfecting the EPO-pXP2 constructs and deletion mutants into HL-60-C15 cells and the parental HL-60 line; EPO promoter activity was 8-10-fold less in the HL-60 parental line, suggesting lineage specific elements in the -122 to -45 bp region. To further characterize regulatory sequences important for promoter activity, we performed linker-scanning analysis on the -122 to -45 bp region and identified a number of positively and negatively acting elements in the promoter. DNase I footprinting was performed with HL-60-C15, HL-60, and HeLa nuclear extracts to identify nuclear proteins that may bind to the functional elements; these experiments identified three protected regions of the EPO promoter which correspond to the functional segments defined by linker-scanning analysis and which contain consensus, potential binding sites for Egr-1, H4TF-1, PuF, CTCF, UBP-1, and GaEII transcription factors. Further study of EPO promoter regulation should elucidate unique transcriptional features of eosinophil gene regulation in granulocyte development.

Base Sequence↗

PubMed1994

Cytokine induction of granule protein synthesis in an eosinophil-inducible human myeloid cell line, AML14.

Study of eosinophil growth and differentiation has been hampered by the difficulty of obtaining adequate numbers of highly purified eosinophil progenitors or mature eosinophils for analysis. The AML14 myeloid leukemia cell line has the unusual ability to exhibit eosinophilic differentiation in response to stimulation by combinations of the eosinophil-active cytokines interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor, and IL-5. We now demonstrate that AML14 cells can be stimulated by a combination of these cytokines to produce mRNA encoding all the eosinophil granule proteins, including major basic protein (MBP), eosinophil peroxidase (EPO), eosinophil cationic protein (ECP), eosinophil-derived neurotoxin (EDN), and the Charcot-Leyden crystal (CLC) protein (eosinophil lysophospholipase). The production of the mature proteins was demonstrated by Western blotting, and ultrastructural analysis demonstrated the presence of immature secondary granules in cells that had been induced to differentiate to eosinophils. These findings demonstrate the utility of the AML14 cell line as a model for the study of cytokine induction of eosinophil growth and differentiation.

Base Sequence↗

PubMed1994

Hyperpolarization-activated chloride currents in Xenopus oocytes.

During hyperpolarizing pulses, defolliculated Xenopus oocytes have time- and voltage-dependent inward chloride currents. The currents vary greatly in amplitude from batch to batch; activate slowly and, in general, do not decay; have a selectivity sequence of I- > NO3- > Br- > Cl- > propionate > acetate; are insensitive to Ca2+ and pH; are blocked by Ba2+ and some chloride channel blockers; and have a gating valence of approximately 1.3 charges. In contrast to hyperpolarization-activated chloride currents induced after expression of phospholemman (Palmer, C. J., B. T. Scott, and L. R. Jones. 1991. Journal of Biological Chemistry. 266:11126; Moorman, J. R., C. J. Palmer, J. E. John, J. E. Durieux, and L. R. Jones. 1992. 267:14551), these endogenous currents are smaller; have a different pharmacologic profile; have a lower threshold for activation and lower voltage-sensitivity of activation; have different activation kinetics; and are insensitive to pH. Nonetheless, the endogenous and expressed current share striking similarities. Recordings of macroscopic oocyte currents may be inadequate to determine whether phospholemman is itself an ion channel and not a channel-modulating molecule.

Animals↗

PubMed1994

Transcriptional regulation of the human eosinophil peroxidase genes: characterization of a peroxidase promoter.

Eosinophil Peroxidase↗

PubMed1994

Role of cationic proteins in the airway. Hyperresponsiveness due to airway inflammation.

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 have employed a rat system to investigate the effect of human MBP instillation on airway responsiveness and the possible role of cationic charge in the determination of this effect. Major basic protein caused a significant increase in airway responsiveness to inhaled methacholine. Two polycations, poly-L-arginine and poly-L-lysine, also increased airway responsiveness to inhaled methacholine. Moreover, two other very different cationic proteins, platelet factor 4 (PF4) and cathepsin G were also capable of inducing airway hyperresponsiveness. These effects were dependent on their positive charge, since the charge--and, hence the effect--of these proteins was neutralized with low molecular weight heparin. In addition, other polyanions, such as low molecular weight heparin, albumin, or dextran sulfate, were also effective. We investigated whether two synthetic cationic proteins, poly-L-arginine and poly-L-lysine, could modify epithelial-dependent responses using a perfused guinea pig tracheal tube preparation. With an intact epithelium, methacholine was some 150 times less potent when applied intraluminally than when applied extraluminally. Perfusion of the luminal surface with cationic proteins increased the potency of intraluminally applied methacholine without modifying the responses to extraluminally applied methacholine. Cationic proteins also attenuated the relaxant effects of intraluminally applied KCl. These effects occurred in the absence of any overt epithelial cell damage. Our data demonstrates that cationic proteins can modify epithelial-dependent responses in the airways.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗