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D Glick

Publications and source records attributed to D Glick.

At least 19 recordsLinked to original sources
PubMed1997

In vitro phosphorylation of acetylcholinesterase at non-consensus protein kinase A sites enhances the rate of acetylcholine hydrolysis.

Here, we report that the catalytic subunit of cAMP-dependent protein kinase (PKA) but not casein kinase II or protein kinase C phosphorylates recombinant human acetylcholinesterase (AChE) in vitro. This enhances acetylthiocholine hydrolysis up to 10-fold as compared to untreated AChE, while leaving unaffected the enzyme's affinity for this substrate and for various active and peripheral site inhibitors. Alkaline phosphatase treatment enhanced the electrophoretic migration, under denaturing conditions, of part of the AChE proteins isolated from various mammalian sources and raised the isoelectric point of some of the treated AChE molecules, indicating that part of the AChE molecules are also phosphorylated in vivo. Enhancement of acetylthiocholine hydrolysis also occurred with Torpedo AChE, which has no consensus motif for PKA phosphorylation. Further, mutating the single PKA site in human AChE (threonine-249) did not prevent this enhancement, suggesting that in both cases it was due to phosphorylation at non-consensus sites. In vivo suppression of the acetylcholine hydrolyzing activity of AChE and consequent impairment in cholinergic neurotransmission occur under exposure to both natural and pharmacological compounds, including organophosphate and carbamate insecticides and chemical warfare agents. Phosphorylation of AChE may possibly offer a rapid feedback mechanism that can compensate for impairments in cholinergic neurotransmission, modulating the hydrolytic activity of this enzyme and enabling acetylcholine hydrolysis to proceed under such challenges.

Acetylcholine↗
PubMed1996

IgG immunoglobulins induce activation of the sphingomyelin cycle in HL-60 cells.

In HL-60 cells signal transduction via sphingomyelin hydrolysis (sphingomyelin cycle) is induced by binding of tumor necrosis factor alpha (TNFalpha) to cell surface TNFalpha receptor. We found that IgG immunoglobulins activate sphingomyelin hydrolysis in plasma membrane of HL-60 cells, with kinetics similar to that of activation by TNFalpha. Activation was induced by different IgG isotypes (most of which are irrelevant to known inducers of the sphingomyelin cycle) and also by Fcgamma fragments of IgG. The facts that inhibiting the binding of the antibodies to the cell surface by protein A prevents activation of sphingomyelin hydrolysis and that soluble TNF receptor of 55-kDa subtype (TBP55) inhibits activation, suggest that the mechanism of IgG-induced sphingomyelin hydrolysis involves binding of IgGs through their Fcgamma domain to Fcgamma surface receptors which mediate autocrine secretion of TNFalpha. The latter is responsible for inducing sphingomyelin hydrolysis. This study suggests that TBP55 may be an effective inhibitor of the sphingomyelin cycle.

HL-60 Cells↗
PubMed1996

Overlapping drug interaction sites of human butyrylcholinesterase dissected by site-directed mutagenesis.

Butyrylcholinesterase [BuChE (acylcholine acyl hydrolase); EC 3.1.1.8] limits the access of drugs, including tacrine, to other proteins. The "atypical" BuChE variant, in which Asp70 at the rim of the active site gorge is substituted by glycine, displayed a more drastically weakened interaction with tacrine than with cocaine, dibucaine, succinylcholine, BW284c51 [1,5-bis(4-allyldimethylammoniumphenyl)pentan-3-one dibromide], or alpha-solanine. To delineate the protein domains that are responsible for this phenomenon, we mutated residues within the rim of the active site gorge, the region parallel to the peripheral site in the homologous enzyme acetylcholinesterase [AChE (acetylcholine acetyl hydrolase); EC 3.1.1.7], the oxyanion hole, and the choline-binding site. When expressed in microinjected Xenopus laevis oocytes, all mutant DNAs yielded comparable amounts of immunoreactive protein products. Most mutants retained catalytic activity close to that of wild-type BuChE and were capable of binding ligands. However, certain modifications in and around the oxyanion hole caused a dramatic loss in activity. The affinities for tacrine were reduced more dramatically than for all other ligands, including cocaine, in both oxyanion hole and choline-binding site mutants. Modified ligand affinities further demonstrated a peripheral site in residues homologous with those of AChE. BuChE mutations that prevented tacrine interactions also hampered its ability to bind other drugs and inhibitors, which suggests a partial overlap of the binding sites. This predicts that in addition to their genetic predisposition to adverse responses to tacrine, homozygous carriers of "atypical" BuChE will be overly sensitive to additional anticholinesterases and especially so when exposed to several anticholinesterases in combination.

Animals↗
PubMed1995

Engineering of human cholinesterases explains and predicts diverse consequences of administration of various drugs and poisons.

The acetylcholine hydrolyzing enzyme, acetylcholinesterase, primarily functions in nerve conduction, yet it appears in several guises, due to tissue-specific expression, alternative mRNA splicing and variable aggregation modes. The closely related enzyme, butyrylcholinesterase, most likely serves as a scavenger of toxins to protect acetylcholine binding proteins. One or both of the cholinesterases probably also plays a non-catalytic role(s) as a surface element on cells to direct intercellular interactions. The two enzymes are subject to inhibition by a wide variety of synthetic (e.g., organophosphorus and carbamate insecticides) and natural (e.g., glycoalkaloids) anticholinesterases that can compromise these functions. Butyrylcholinesterase may function, as well, to degrade several drugs of interest, notably aspirin, cocaine and cocaine-like local anesthetics. The widespread occurrence of butyrylcholinesterase mutants with modified activity further complicates this picture, in ways that are only now being dissected through the use of site-directed mutagenesis and heterologous expression of recombinant cholinesterases.

Carbamates↗
PubMed1995

Successive organophosphate inhibition and oxime reactivation reveals distinct responses of recombinant human cholinesterase variants.

To explore the molecular basis of the biochemical differences among acetylcholinesterase (AChE), butyrylcholinesterase (BuChE) and their alternative splicing and allelic variants, we investigated the acylation phase of cholinesterase catalysis, using phosphorylation as an analogous reaction. Rate constants for organophosphate (DFP) inactivation, as well as for oxime (PAM)-promoted reactivation, were calculated for antibody-immobilized human cholinesterases produced in Xenopus oocytes from natural and site-directed variants of the corresponding DNA constructs. BuChE displayed inactivation and reactivation rates 200- and 25-fold higher than either product of 3'-variable AChE DNAs, consistent with a putative in vivo function for BuChE as a detoxifier that protects AChE from inactivation. Chimeric substitution of active site gorge-lining residues in BuChE with the more anionic and aromatic residues of AChE, reduced inactivation 60-fold but reactivation only 4-fold, and the rate-limiting step of its catalysis appeared to be deacylation. In contrast, a positive charge at the acyl-binding site of BuChE decreased inactivation 8-fold and reactivation 30-fold. Finally, substitution of Asp70 by glycine, as in the natural 'atypical' BuChE variant, did not change the inactivation rate yet reduced reactivation 4-fold. Thus, a combination of electrostatic active site charges with aromatic residue differences at the gorge lining can explain the biochemical distinction between AChE and BuChE. Also, gorge-lining residues, including Asp70, appear to affect the deacylation step of catalysis by BuChE. Individuals carrying the 'atypical' BuChE allele may hence be unresponsive to oxime reactivation therapy following organophosphate poisoning.

Acetylcholinesterase↗
PubMed1995

Genetic predisposition to adverse consequences of anti-cholinesterases in 'atypical' BCHE carriers.

Normal butyrylcholinesterase (BuChE), but not several of its common genetic variants, serves as a scavenger for certain anti-cholinesterases (anti-ChEs). Consideration of this phenomenon becomes urgent in view of the large-scale prophylactic use of the anti-ChE, pyridostigmine, during the 1991 Persian Gulf War, in anticipation of nerve gas attack and of the anti-ChE, tacrine, for improving residual cholinergic neurotransmission in Alzheimer's disease patients. Adverse symptoms were reported for subjects in both groups, but have not been attributed to specific causes. Here, we report on an Israeli soldier, homozygous for 'atypical' BuChE, who suffered severe symptoms following pyridostigmine prophylaxis during the Persian Gulf War. His serum BuChE and recombinant 'atypical' BuChE were far less sensitive than normal BuChE to inhibition by pyridostigmine and several other carbamate anti-ChEs. Moreover, atypical BuChE demonstrated 1/200th the affinity for tacrine of normal BuChE or the related enzyme acetylcholinesterase (AChE). Genetic differences among BuChE variants may thus explain at least some of the adverse responses to anti-ChE therapies.

Acetylcholinesterase↗
PubMed1994

Population diversity and distinct haplotype frequencies associated with ACHE and BCHE genes of Israeli Jews from trans-Caucasian Georgia and from Europe.

Variant alleles of the butyrylcholinesterase gene, BCHE, have often been used to trace the genetic histories of populations. The D70G substitution in BCHE causes prolonged postanesthesia apnea ("atypical" phenotype); H322N substitution in the closely related acetylcholinesterase gene, ACHE, is the basis of the mutually incompatible Yt blood groups. In both genes, additional point mutations were reported to be linked to these phenotypically evident ones. To examine whether the intragenic linkage reported for the ACHE and BCHE mutations in Americans is universal, we studied frequencies of these mutations in trans-Caucasian Georgian Jews, a population that has remained relatively isolated for 1500 years. To this end we employed PCR amplification followed by DNA sequencing and enzymatic restriction and compared the frequencies we found to corresponding reported phenotype data. Georgian Jews' N322 ACHE was a rather low 7.0% and was totally linked to a P446 mutation, in agreement with a recent report. In BCHE, however, G70 was a relatively high 5.8%, and the V497 and T539 mutations were not found, either in Georgian or in Ashkenazi Jews, in contrast to reported findings in Americans. Our findings reveal distinct displays of ACHE and BCHE haplotypes in Georgian Jews and suggest different founder effects, genetic drifts, and/or selection pressures in the evolution of each of these genes.

Acetylcholinesterase↗
PubMed1994

Mutations and impaired expression in the ACHE and BCHE genes: neurological implications.

The acetylcholine hydrolysing cholinesterases control the termination of cholinergic signalling in multiple tissues and are targets for a variety of drugs, natural and man-made poisons and common insecticides. Molecular cloning and gene mapping studies revealed the primary structure of human acetyl- and butyrylcholinesterase and localized the corresponding ACHE and BCHE genes to the chromosomal positions 3q26-ter and 7q22, respectively. Several different point mutations in the coding region of BCHE were found to be particularly abundant in the Israeli population. Analytical expression studies in microinjected Xenopus oocytes have demonstrated that the biochemical properties of cholinesterases may be modified by rationalized site-directed mutagenesis and in chimeric ACHE/BCHE constructs. These properties are differently altered in the various allelic BCHE variants, conferring resistance to several anti-cholinesterases, which may explain the evolutionary emergence of these multiple alleles. At the clinical level, abnormal expression of both ACHE and BCHE and the in vivo amplification of the ACHE and BCHE genes has been variously associated with abnormal megakaryocytopoiesis, leukemias and brain and ovarian tumors. Moreover, antisense oligonucleotides blocking the expression of these genes were shown to interfere with hemocytopoiesis in culture, implicating these genes in cholinergic influence on cell growth and proliferation.

Acetylcholinesterase↗
PubMed1993

Structural organization of the maxicircle variable region of Trypanosoma brucei: identification of potential replication origins and topoisomerase II binding sites.

The maxicircle of the parasitic protozoan Trypanosoma brucei, one component of the mitochondrial genome, has size differences among isolates that localize to the variable region (VR) between the ND5 and 12S rRNA genes. We present here the nucleotide sequence of this entire region, thus completing the sequence of the maxicircle genome. We also find heterogeneously sized transcripts from throughout most of the VR. The VR has three distinct sections, each with characteristic repeated sequences. The repeated sequences in two sections are short and highly reiterated; the intraspecies size variation occurs within this region. The third section contains non-repetitive sequences and a large duplication immediately upstream of the 12S rRNA gene. Two repeat units within section I contain a sequence that has homology to the DNA replication origin of minicircles. This region also contains sequences with homology to topoisomerase II binding and cleavage sites. These findings suggest a role for the VR in DNA replication of the maxicircle.

Animals↗
PubMed1988

Intraoperative decisions based on angioscopy in peripheral vascular surgery.

We describe the technique of intraoperative angioscopy for delineation of peripheral vascular anatomy. Angioscopes with outer diameters of 0.85-2.9 mm have been used during 86 peripheral vascular procedures. Angioscopic inspections were performed during 68 femoral popliteal bypasses, four aortofemoral bypass grafts, one abdominal aortic aneurysm, two extra anatomic axillary femoral bypass grafts, and 11 other vascular procedures. We obtained useful images in 73 of 86 procedures (85%), thereby yielding 118 angioscopic inspections (53 arteries, 37 anastomoses, and 28 vein grafts). Changes in intraoperative management based on angioscopic findings included revision of five of 37 (14%) anastomoses, deletion of four of 31 (13%) completion angiograms, revision of eight of 17 (47%) in situ venous valves, and repetition of thrombectomy in six of seven (86%) cases. In 22 of 73 (30%) peripheral angioscopies, potential causes of graft occlusion were recognized. Complications from intraoperative angioscopy have included one anastomotic flap from intimal disruption that required anastomotic revision. Three small flaps, possibly resulting from angioscopic trauma, were recognized but appeared to have no clinical significance. In conclusion, intraoperative angioscopy provides visual assessment of luminal patency and anastomotic anatomy. This assessment alters intraoperative procedures in some cases and cannot be obtained by angiography.

Adult↗
PubMed1982

Role of lipids in the Neurospora crassa membrane: IV. Biochemical and electrophysiological changes caused by growth on phytanic acid.

Neurospora crassa strain cel, which is deficient in fatty acid synthesis, was grown with phytanic acid supplementation. The temperature dependence of membrane potential is increased by growth on phytanic acid. A temperature change of 40 degrees C produces a change of 184 mV in phytanic acid-grown cells as compared to a 50 mV change for cel grown on palmitic acid or wild-type. Membrane resistance (measured as DC input resistance) of phytanic acid-grown cells did not differ from cel grown on palmitic acid or wild-type. Lipid analysis of cel grown on phytanic acid revealed approximately 7 mole percent phytanic acid incorporation into phospholipids, no change in phospholipid base composition, a reduction of ergosterol content from 80 to 30 percent, and the induction of beta sitosterol, a sterol not usually present in Neurospora. beta sitosterol accounted for approximately 60 percent of the sterol present. Incorporation of 7 mole percent phytanic acid into phospholipids lowers the phase transition temperature by approximately 5 degrees C, and decreases the heat content of the phase transition (delta H) slightly. Results are discussed in relation to Refsum's disease, a human neurological disorder associated with high plasma levels of phytanic acid. It is proposed that high intracellular phytanic acid concentration induces novel sterol synthesis and that the incorporation of the novel sterol into the membrane is responsible for the increased temperature sensitivity of membrane potential. The excitable membrane deficits observed in patients with Refsum's disease may be explained by such a mechanism.

Cell Membrane↗