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E J Ackermann

Publications and source records attributed to E J Ackermann.

12 recordsLinked to original sources
PubMed2001

Suppression of vascular endothelial growth factor-mediated endothelial cell protection by survivin targeting.

The protective genes that mediate endothelial cell (EC) survival during angiogenesis have not been completely characterized. Here, we show that an antisense oligonucleotide to the apoptosis inhibitor survivin suppressed de novo expression of survivin in ECs by vascular endothelial cell growth factor (VEGF). In contrast, the survivin antisense oligonucleotide did not affect anti-apoptotic bcl-2 levels in endothelium. When assessed in cell death assays, antisense targeting of survivin abolished the anti-apoptotic function of VEGF against tumor necrosis factor-alpha- or ceramide-induced cell death, enhanced caspase-3 activity, promoted the generation of a approximately 17-kd active caspase-3 subunit, and increased cleavage of the caspase substrate, polyADP ribose polymerase. In contrast, the survivin antisense oligonucleotide had no effect on EC viability in the absence of VEGF. Antisense oligonucleotides to platelet-endothelial cell adhesion molecule-1 (PECAM-1, CD31), lymphocyte function-associated molecule-3 (LFA-3, CD58), or intercellular adhesion molecule-1 (ICAM-1, CD54) did not reduce the anti-apoptotic function of VEGF in endothelium. When tested on other angiogenic activities mediated by VEGF, survivin antisense treatment induced rapid regression of three-dimensional vascular capillary networks, but did not affect EC migration/chemotaxis. These data suggest that the anti-apoptotic properties of VEGF during angiogenesis are primarily mediated by the induced expression of survivin in ECS: Manipulation of this pathway may increase EC viability in compensatory angiogenesis or facilitate EC apoptosis and promote vascular regression during tumor angiogenesis.

Apoptosis↗
PubMed1999

The role of antiapoptotic Bcl-2 family members in endothelial apoptosis elucidated with antisense oligonucleotides.

In this study, we utilized potent antisense oligonucleotides to examine the role of two Bcl-2 family members found in human umbilical vein endothelial cells (HUVEC). The first, A1, is thought to be a TNF-alpha-inducible cytoprotective gene, and the second, Bcl-XL, is constitutively expressed. Inhibition of the constitutive levels of Bcl-XL caused 10-25% of the cell population to undergo apoptosis and increased the susceptibility of cells to treatment with low concentrations of staurosporin or ceramide. The caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-CH2 prevented DNA fragmentation and DeltaYm loss caused by Bcl-XL inhibition or Bcl-XL inhibition combined with staurosporin. However, disruption of DeltaYm caused by Bcl-XL inhibition combined with ceramide treatment was not inhibited by benzyloxycarbonyl-Val-Ala-Asp(OMe)-CH2, although DNA fragmentation was completely prevented. Taken together, these results demonstrate a direct protective role for Bcl-XL under normal resting conditions and under low level apoptotic challenges to HUVEC. Furthermore, Bcl-XL protects cells from caspase-dependent and -independent mechanisms of DeltaYm disruption. In contrast to Bcl-XL, A1 inhibition did not show a marked effect on the susceptibility of HUVEC to undergo apoptosis in response to TNF-alpha, ceramide, or staurosporin. These results demonstrate that although A1 may be a cytoprotective gene induced by TNF-alpha, it is not primarily responsible for HUVEC resistance to this cytokine.

Apoptosis↗
PubMed1999

Pleiotropic cell-division defects and apoptosis induced by interference with survivin function.

Here we investigate the role of the control of apoptosis in normal cell division. We show that interference with the expression or function of the apoptosis inhibitor survivin causes caspase-dependent cell death in the G2/M phase of the cell cycle, and a cell-division defect characterized by centrosome dysregulation, multipolar mitotic spindles and multinucleated, polyploid cells. Use of a dominant-negative survivin mutant or antisense survivin complementary DNA disrupts a supramolecular assembly of survivin, caspase-3 and the cyclin-dependent-kinase inhibitor p21Waf1/Cip1 within centrosomes, and results in caspase-dependent cleavage of p21. Polyploidy induced by survivin antagonists is accentuated in p21-deficient cells, and corrected by exogenous expression of p21. These findings show that control of apoptosis and preservation of p21 integrity within centrosomes by survivin are required for normal mitotic progression.

Apoptosis↗
PubMed1998

Identification of pairwise interactions in the alpha-neurotoxin-nicotinic acetylcholine receptor complex through double mutant cycles.

alpha-Neurotoxins are potent inhibitors of the nicotinic acetylcholine receptor (nAChR), binding with high affinity to the two agonist sites located on the extracellular domain. Previous site-directed mutagenesis had identified three residues on the alpha-neurotoxin from Naja mossambica mossambica (Lys27, Arg33, and Lys47) and four residues on the mouse muscle nAChR alpha-subunit (Val188, Tyr190, Pro197, and Asp200) as contributing to binding. In this study, thermodynamic mutant cycle analysis was applied to these sets of residues to identify specific pairwise interactions. Amino acid variants of alpha-neurotoxin from N. mossambica mossambica at position 33 and of the nAChR at position 188 showed strong energetic couplings of 2-3 kcal/mol at both binding sites. Consistently smaller yet significant linkages of 1.6-2.1 kcal/mol were also observed between variants at position 27 on the toxin and position 188 on the receptor. Additionally, toxin residue 27 coupled to the receptor residues 190, 197, and 200 at the alphadelta binding site with observed coupling energies of 1.5-1.9 kcal/mol. No linkages were found between toxin residue Lys47 and the receptor residues studied here. These results provide direct evidence that the two conserved cationic residues Arg33 and Lys27, located on loop II of the toxin structure, are binding in close proximity to the alpha-subunit region between residues 188-200. The toxin residue Arg33 is closer to Val188, where it is likely stabilized by adjacent negative or aromatic residues on the receptor structure. Lys27 is positioned closer to Tyr190, Pro197, and Asp200, where it is likely stabilized through electrostatic interaction with Asp200 and/or cation/pi interactions with Tyr190.

Amino Acid Sequence↗
PubMed1998

Toxins selective for subunit interfaces as probes of nicotinic acetylcholine receptor structure.

The pentameric structure of the nicotinic acetylcholine receptor with two of the five subunit interfaces serving as ligand binding sites offers an opportunity to distinguish features on the surfaces of the subunits and their ligand specificity characteristics. This problem has been approached through the study of assembly of subunits and binding characteristics of selective peptide toxins. The receptor, with its circular order of homologous subunits (alpha gamma alpha delta beta), assembles in only one arrangement, and through mutagenesis, the residues governing assembly can be ascertained. Selectivity of certain toxins is sufficient to readily distinguish between sites at the alpha gamma and alpha delta interfaces. By interchanging residues on the gamma and delta subunits, and ascertaining how they interact with the alpha-subunit, determinants forming the binding sites can be delineated. The alpha-conotoxins, which contain two disulfide loops and 12-14 amino acids, show a 10,000-fold preference for the alpha delta over the alpha gamma subunit interface with alpha epsilon falling between the two. The waglerins, as 22-24 amino acid peptides with a single core disulfide loop, show a 2000-fold preference for alpha epsilon over the alpha gamma and alpha delta interfaces. Finally, the 6700 Da short alpha-neurotoxin from N. mossambica mossambica shows a 10,000-fold preference for the alpha gamma and alpha delta interfaces over alpha epsilon. Selective mutagenesis enables one to also distinguish alpha-neurotoxin binding at the alpha gamma and alpha delta subunits. This information, when coupled with homology modeling of domains and site-directed residue modification, reveals important elements of receptor structure and conformation.

Amino Acid Sequence↗
PubMed1997

Nonidentity of the alpha-neurotoxin binding sites on the nicotinic acetylcholine receptor revealed by modification in alpha-neurotoxin and receptor structures.

alpha-Neurotoxins constitute a large family of polypeptides that bind with high affinity to the nicotinic acetylcholine receptor (nAChR). Using a recombinant DNA-derived alpha-neurotoxin (Naja mossambica mossambica, NmmI) and mouse muscle nAChR expressed transiently on the surface of HEK 293 cells, we have delineated residues involved in the binding interaction on both the alpha-neurotoxin and the receptor interface. Several of the studied NmmI mutations, including two residues conserved throughout the alpha-neurotoxin family (K27 and R33), resulted in substantial decreases in the binding affinity. We have also examined 23 mutations located on the receptor alpha subunit and have identified 4 positions that appear to be important to NmmI recognition. These determinants represent a conserved aromatic residue (Y190), two positions where neuronal and muscle receptors differ (V188 and P197), and a negatively charged residue (D200). Unlike many of the nAChR agonists and antagonists which bind to the alphadelta and alphagamma binding sites on the receptor with different affinities, the wild-type NmmI-wild-type nAChR interaction showed a single affinity. However, by mutating critical toxin or receptor residues, we were able to produce site-selectivity between the alphagamma and alphadelta interfaces. These results suggest a nonequivalence in the binding interaction at the two sites, sensitive to discrete structural changes at key contact points on either the toxin or the receptor protein, and underscore the importance of delta and gamma receptor subunits in governing binding affinity.

Amino Acid Sequence↗
PubMed1997

Expression and activity of mutants of fasciculin, a peptidic acetylcholinesterase inhibitor from mamba venom.

Fasciculin, a selective peptidic inhibitor of acetylcholinesterase, is a member of the three-fingered peptide toxin superfamily isolated from snake venoms. The availability of a crystal structure of a fasciculin 2 (Fas2)-acetylcholinesterase complex affords an opportunity to examine in detail the interaction of this toxin with its target site. To this end, we constructed a synthetic fasciculin gene with an appropriate leader peptide for expression and secretion from mammalian cells. Recombinant wild-type Fas2, expressed and amplified in Chinese hamster ovary cells, was purified to homogeneity and found to be identical in composition and biological activities to the venom-derived toxin. Sixteen mutations at positions where the crystal structure of the complex indicates a significant interfacial contact point or determinant of conformation were generated. Two mutants of loop I, T8A/T9A and R11Q, ten mutants of the longest loop II, R24T, K25L, R27W, R28D, H29D, DeltaPro30, P31R, K32G, M33A, and V34A/L35A, and two mutants of loop III, D45K and K51S, were expressed transiently in human embryonic kidney cells. Inhibitory potencies of the Fas2 mutants toward mouse AChE were established, based on titration of the mutants with a polyclonal anti-Fas2 serum. The Arg27, Pro30, and Pro31 mutants each lost two or more orders of magnitude in Fas2 activity, suggesting that this subset of three residues, at the tip of loop II, dominates the loop conformation and interaction of Fas2 with the enzyme. The Arg24, Lys32, and Met33 mutants lost about one order of magnitude, suggesting that these residues make moderate contributions to the strength of the complex, whereas the Lys25, Arg28, Val34-Leu35, Asp45, and Lys51 mutants appeared as active as Fas2. The Thr8-Thr9, Arg11, and His29 mutants showed greater ratios of inhibitory activity to immunochemical titer than Fas2. This may reflect immunodominant determinants in these regions or intramolecular rearrangements in conformation that enhance the interaction. Of the many Fas2 residues that lie at the interface with acetylcholinesterase, only a few appear to provide substantial energetic contributions to the high affinity of the complex.

Amino Acid Sequence↗
PubMed1995

Inhibition of calcium-independent phospholipase A2 prevents arachidonic acid incorporation and phospholipid remodeling in P388D1 macrophages.

Cellular levels of free arachidonic acid (AA) are controlled by a deacylation/reacylation cycle whereby the fatty acid is liberated by phospholipases and reincorporated by acyltransferases. We have found that the esterification of AA into membrane phospholipids is a Ca(2+)-independent process and that it is blocked up to 60-70% by a bromoenollactone (BEL) that is a selective inhibitor of a newly discovered Ca(2+)-independent phospholipase A2 (PLA2) in macrophages. The observed inhibition correlates with a decreased steady-state level of lysophospholipids as well as with the inhibition of the Ca(2+)-independent PLA2 activity in these cells. This inhibition is specific for the Ca(2+)-independent PLA2 in that neither group IV PLA2, group II PLA2, arachidonoyl-CoA synthetase, lysophospholipid:arachidonoyl-CoA acyltransferase, nor CoA-independent transacylase is affected by treatment with BEL. Moreover, two BEL analogs that are not inhibitors of the Ca(2+)-independent PLA2--namely a bromomethyl ketone and methyl-BEL--do not inhibit AA incorporation into phospholipids. Esterification of palmitic acid is only slightly affected by BEL, indicating that de novo synthetic pathways are not inhibited by BEL. Collectively, the data suggest that the Ca(2+)-independent PLA2 in P388D1 macrophages plays a major role in regulating the incorporation of AA into membrane phospholipids by providing the lysophospholipid acceptor employed in the acylation reaction.

Animals↗
PubMed1995

Inhibition of macrophage Ca(2+)-independent phospholipase A2 by bromoenol lactone and trifluoromethyl ketones.

A novel Ca(2+)-independent phospholipase A2 (PLA2) has recently been purified from the murine macrophage-like cell line P388D1 (Ackermann, E. J., Kempner, E. S., and Dennis, E. A. (1994) J. Biol. Chem. 269, 9227-9233). This enzyme is now shown to be inhibited by palmitoyl trifluoromethyl ketone (PACOCF3), arachidonyl trifluoromethyl ketone (AACOCF3), and a bromoenol lactone (BEL). Both PACOCF3 and AACOCF3 were found to inhibit the macrophage PLA2 in a concentration-dependent manner. PACOCF3 was found to be approximately 4-fold more potent than AACOCF3, with IC50 values of 3.8 microM (0.0075 mol fraction) and 15 microM (0.028 mol fraction), respectively. Reaction progress curves in the presence of either inhibitor were found to be linear, and the PACOCF3.PLA2 complex rapidly dissociated upon dilution. BEL was also found to inhibit the macrophage PLA2 in a concentration-dependent manner, with half-maximal inhibition observed at 60 nM after a 5-min preincubation at 40 degrees C. Inhibition was not reversed after extensive dilution of the enzyme into assay buffer. Treatment of the PLA2 with BEL resulted in a linear, time-dependent inactivation of activity, and the rate of this inactivation was diminished in the presence of PACOCF3. In addition, PLA2 treated with [3H]BEL resulted in the covalent labeling of a major band at M(r) 80,000. Inactivation of the PLA2 by 5,5'-dithiobis(2-nitrobenzoic acid) prior to treatment with [3H]BEL resulted in the near complete lack of labeling consistent with covalent irreversible suicide inhibition of the enzyme. The labeling of a M(r) 80,000 band rather than a M(r) 40,000 band upon treatment with [3H]BEL distinguishes the macrophage Ca(2+)-independent PLA2 from a previously identified myocardial Ca(2+)-independent PLA2 and provides strong evidence that the M(r) 80,000 protein is the catalytic subunit.

Animals↗
PubMed1994

Ca(2+)-independent cytosolic phospholipase A2 from macrophage-like P388D1 cells. Isolation and characterization.

A novel form of an ATP-regulated, oligomeric, Ca(2+)-independent phospholipase A2 (iPLA2) has been purified from the cytosol of the murine macrophage-like cell line P388D1. The purification procedure included ammonium sulfate precipitation and sequential column chromatography on octyl-Sepharose, ATP-agarose, Mono Q fast protein liquid chromatography (FPLC), and hydroxyapatite FPLC. The resulting enzyme preparation was purified over 400,000-fold with a final specific activity of approximately 5 mumol/min/mg using a mixed micelle assay system of Triton X-100 and dipalmitoyl phosphatidylcholine (PC). The purified enzyme was Ca(2+)-independent and did not show a preference for either sn-2 arachidonic acid or sn-1 alkyl-ether containing phospholipids when utilizing mixed micelles as substrate. It was found to hydrolyze dipalmitoyl-PC approximately 4-fold faster than 1-palmitoyl-2-arachidonyl-PC and approximately 15-fold faster than 1-O-hexadecyl-2-arachidonyl-PC. Triton X-100 increased the P388D1 iPLA2 activity with optimal activity found at a Triton/phospholipid molar ratio of 4:1. The purified enzyme was activated 2-6-fold by ATP as well as other di- and triphosphate nucleosides. This activation was sensitive to the concentration of Triton X-100 present in the assay. SDS-polyacrylamide gel electrophoresis carried out on the purified enzyme yielded a single major band at a molecular weight of about 80,000. However, radiation inactivation experiments, carried out on the cell homogenate, demonstrated a target size of 337 +/- 25 kDa, indicating that the catalytically active iPLA2 exists as a large oligomeric complex, either through self-aggregation or association of the enzyme with other proteins.

Adenosine Triphosphate↗