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D Marcus

Publications and source records attributed to D Marcus.

At least 19 recordsLinked to original sources
PubMed1995

Allosteric modulation of acetylcholinesterase activity by peripheral ligands involves a conformational transition of the anionic subsite.

Replacement of residues Asp74, Trp286, and Tyr72, which are constituents of the peripheral anionic site (PAS) of human acetylcholinesterase (HuAChE), affected similarly both the binding and the inhibition constants of the PAS-specific ligand propidium, demonstrating that changes in the inhibitory activity are a direct consequence of altered binding to the PAS. In contrast, the active center HuAChE mutants W86A and Y133A show respective 350- and 25-fold increased resistance to inhibition by propidium but no change in binding affinities, demonstrating that the allosteric mechanism of PAS-mediated inhibition involves a conformational change of these Trp86 and Tyr133 residues rather than physical obstruction of substrate access by the inhibitor itself. These findings support the recent proposal that the allosteric mechanism operates via transition between active and nonactive conformations of the anionic subsite Trp86 and that replacement of Tyr133 by alanine may stabilize a nonactive Trp86 conformation that occludes the active center [Ordentlich et al. (1995) J. Biol. Chem. 270, 2082]. In further support of this mechanism and the role of Tyr133, we find that (a) the dissociation constants (Kd) for the noncovalent complexes of the irreversible inhibitors diisopropyl phosphorofluoridate or paraoxon with Y133A HuAChE are increased 20-500-fold, relative to either wild-type enzyme or its Y133F or W86A mutants; and (b) access of substrates such as 3,3-dimethylbutyl thioacetate is restored by removal of Trp86 from the Y133A enzyme (i.e., the W86A/Y133A mutant). We suggest that the conformational transition of Trp86 is coupled to the motions of the cysteine loop (Cys69-Cys96) of HuAChE and is inherent to the dynamics of the native enzyme.

Acetylcholinesterase↗
PubMed1995

Involvement of oligomerization, N-glycosylation and sialylation in the clearance of cholinesterases from the circulation.

The possible role of post-translational modifications such as subunit oligomerization, protein glycosylation and oligosaccharide processing on the circulatory life-time of proteins was studied using recombinant human acetylcholinesterase (rHuAChE). Different preparations of rHuAChE containing various amounts of tetramers, dimers and monomers are cleared at similar rates from the circulation, suggesting that oligomerization does not play an important role in determining the rate of clearance. An engineered rHuAChE mutant containing only one N-glycosylation site was cleared from the circulation more rapidly than the wild-type triglycosylated enzyme. On the other hand, hyperglycosylated mutants containing either four or five occupied N-glycosylation sites, analagous to those present on the slowly cleared fetal bovine serum acetylcholinesterase (FBS-AChE), were also cleared more rapidly from the bloodstream than the wild-type species. Furthermore, the two different tetraglycosylated mutants were cleared at different rates while the pentaglycosylated mutant exhibited the most rapid clearance profile. These results imply that though the number of N-glycosylation sites plays a role in the circulatory life-time of the enzyme, the number of N-glycan units in itself does not determine the rate of clearance. When saturating amounts of asialofetuin were administered together with rHuAChE, the circulatory half-life of the enzyme was dramatically increased (from 80 min to 19 h) and was found to be similar to that displayed by plasma-derived cholinesterases while desialylation of these enzymes caused a sharp decrease in the circulatory half-life to approximately 3-5 min. Determination of the average number of sialic acid residues per enzyme subunit of the five different N-glycosylation species generated, revealed that the rate of clearance is not a function of the absolute number of appended sialic acid moieties but rather of the number of unoccupied sialic acid attachment sites per enzyme molecule. Specifically, we demonstrate an inverse-linear relationship between the number of vacant sialic acid attachment sites and the values of the enzyme residence time within the bloodstream.

Acetylcholinesterase↗
PubMed1994

Acetylcholinesterase peripheral anionic site degeneracy conferred by amino acid arrays sharing a common core.

Several of the residues constituting the peripheral anionic site (PAS) in human acetylcholinesterase (HuAChE) were identified by a combination of kinetic studies with 19 single and multiple HuAChE mutants, fluorescence binding studies with the Trp-286 mutant, and by molecular modeling. Mutants were analyzed with three structurally distinct positively charged PAS ligands, propidium, decamethonium, and di(p-allyl-N-dimethylaminophenyl)pentane-3-one (BW284C51), as well as with selective active center inhibitors, hexamethonium and edrophonium. Single mutations of residues Tyr-72, Tyr-124, Glu-285, Trp-286, and Tyr-341 resulted in up to 10-fold increase in inhibition constants for PAS ligands, whereas for multiple mutants up to 400-fold increase was observed. The 6th PAS element residue Asp-74 is unique in its ability to affect conformation of both the active site and the PAS (Shafferman, A., Velan, B., Ordentlich, A., Kronman, C., Grosfeld, H., Leitner, M., Flashner, Y., Cohen, S., Barak, D., and Ariel, N. (1992) EMBO J. 11, 3561-3568) as demonstrated by the several hundred-fold increase in Ki for D74N inhibition by the bisquaternary ligands decamethonium and BW284C51. Based on these studies, singular molecular models for the various HuAChE inhibitor complexes were defined. Yet, for the decamethonium complex two distinct conformations were generated, accommodating the quaternary ammonium group by interactions with either Trp-286 or with Tyr-341. We propose that the PAS consists of a number of binding sites, close to the entrance of the active site gorge, sharing residues Asp-74 and Trp-286 as a common core. Binding of ligands to these residues may be the key to the allosteric modulation of HuAChE catalytic activity. This functional degeneracy is a result of the ability of the Trp-286 indole moiety to interact either via stacking, aromatic-aromatic, or via pi-cation attractions and the involvement of the carboxylate of Asp-74 in charge-charge or H-bond interactions.

Acetylcholinesterase↗
PubMed1994

Hashimoto's thyroiditis: fine-needle aspirations of 50 asymptomatic cases.

The cytologic findings from 50 cases of asymptomatic Hashimoto's thyroiditis diagnosed by fine-needle aspiration are reviewed. All patients were referred for fine-needle aspiration by the same physician: 48 because of thyroid enlargement and the clinical suspicion of Hashimoto's thyroiditis, two because of a small thyroid nodule. Positive antimicrosomal (antithyroid peroxidase) and/or antithyroglobulin antibody titers (performed by the same laboratory using the same technique) were present in only 25 of 48 patients evaluated. Cytologic findings included a predominantly lymphocytic process in 92% of the cases and a predominantly oxyphilic (Askanazy/Hürthle) cell process in 8% of the cases. It is likely that all patients were in an early stage of the autoimmune process. Our data indicate that the incidence of Hashimoto's thyroiditis is considerably higher than when recognized only by serologic tests for antibodies.

Adolescent↗
PubMed1993

Engineering resistance to 'aging' of phosphylated human acetylcholinesterase. Role of hydrogen bond network in the active center.

Recombinant human acetylcholinesterase (HuAChE) and selected mutants (E202Q, Y337A, E450A) were studied with respect to catalytic activity towards charged and noncharged substrates, phosphylation with organophosphorus (OP) inhibitors and subsequent aging of the OP-conjugates. Amino acid E450, unlike residues E202 and Y337, is not within interaction distance from the active center. Yet, the bimolecular rates of catalysis and phosphylation are 30-100 fold lower for both E450A and E202Q compared to Y337A or the wild type and in both mutants the resulting OP-conjugates show striking resistance to aging. It is proposed that a hydrogen bond network, that maintains the functional architecture of the active center, involving water molecules and residues E202 and E450, is responsible for the observed behaviour.

Acetylcholinesterase↗
PubMed1993

Human butyrylcholinesterase as a general prophylactic antidote for nerve agent toxicity. In vitro and in vivo quantitative characterization.

Butyrylcholinesterase purified from human plasma (HuBChE) was evaluated both in vitro and in vivo in mice and rats as a single prophylactic antidote against the lethal effects of highly toxic organophosphates (OP). The variation among the bimolecular rate constants for the inhibition of HuBChE by tabun, VX, sarin, and soman was 10-fold (0.47 to 5.12 x 10(7) M-1 min-1; pH 8.0, 26 degrees). The half-life of HuBChE in blood after its i.v. administration in mice and rats was 21 and 46 hr, respectively. The peak blood-enzyme level was obtained in both species approximately 9-13 hr following i.m. injection of HuBChE, and the fraction of the enzyme activity absorbed into the blood was 0.9 and 0.54 for rats and mice, respectively. The stoichiometry of the in vivo sequestration of the anti-cholinesterase toxicants was consistent with the HuBChE/OP ratio of the molar concentration required to inhibit 100% enzyme activity in vitro. Linear correlation was demonstrated between the blood level of HuBChE and the extent of protection conferred against the toxicity of nerve agents. Pretreatment with HuBChE alone was sufficient not only to increase survivability following exposure to multiple median lethal doses of a wide range of potent OPs, but also to alleviate manifestation of toxic symptoms in mice and rats without the need for additional post-exposure therapy. It appeared that in order to confer protection against lethality nerve agents had to be scavenged to a level below their median lethal dose LD50 within less than one blood circulation time. Since the high rate of sequestration of nerve agents by HuBChE is expected to underlie the activity of the scavenger in other species as well, a reliable extrapolation of its efficacy from experimental animals to humans can be made.

Animals↗
PubMed1992

Production and secretion of high levels of recombinant human acetylcholinesterase in cultured cell lines: microheterogeneity of the catalytic subunit.

To allow for structural analysis of the human acetylcholinesterase (hAChE) subunit, a series of eukaryotic vectors was designed for efficient expression. Several eukaryotic multicistronic expression vectors were tested in various mammalian cell lines. All expression vectors contained the selectable neo gene under control of a weak promoter, while the hAChE cDNA was under control of the cytomegalovirus (CMV) immediate-early or Rous sarcoma virus long terminal repeat (RSV LTR) or simian virus 40 (SV40) early promoters. Optimal production and secretion of recombinant hAChE (rehAChE) was achieved in the embryonal kidney 293 cell line transfected either with the RSV-hAChE or with CMV-hAChE expression vectors. Clones expressing and secreting as much as 5-25 pg of enzyme per cell per 24 h were obtained without resorting to coamplification techniques or continuous maintenance of cells under selective pressure. The purified (specific activity of 6000 units per mg protein) homodimer and tetramer enzyme molecules displayed typical AChE biochemical properties: a Km value of 120 microM for acetylthiocholine; a kcat value of 3.9 x 10(5)/min, and selective by AChE-specific inhibitors. Catalytic subunit dimers (130 kDa) exhibit differential N-glycosylation patterns, and upon reduction resolve into 67- and 70-kDa monomeric subunits. These two forms appear as a single discrete 62-kDa band following deglycosylation by N-glycanase. The N-terminal amino acid sequence analysis of the purified mature enzyme suggests the existence of two alternative cleavage sites for the removal of the signal peptide, in which the 'mature' position 1 is either Ala31 or Gly33. Both of these positions conform with the consensus signal peptide recognition sequences and demonstrate bidirected processing of signal peptides on a native molecule.

Acetylcholinesterase↗
PubMed1992

Biosynthesis of the blood group Pk and P1 antigens by human kidney microsomes.

On human erythrocytes, the membrane components associated with Pk and P1 blood-group specificity are glycosphingolipids that carry a common terminal alpha-D-Galp-(1----4)-beta-D-Gal unit, the biosynthesis of which is poorly understood. Human kidneys typed for P1 and P2 (non-P1) blood-group specificity have been assayed for (1----4)-alpha-D-galactosyltransferase activity by use of lactosylceramide [beta-D-Galp-(1----4)-beta-D-Glcp-ceramide] and paragloboside [beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----3)-beta-D-Galp- (1----4)-beta-D-Glcp-ceramide] as acceptor substrates. The linkage and anomeric configuration of the galactosyl group transferred into the reaction products were established by methylation analysis before and after alpha- and beta-D-galactosidase treatments, as well as by immunostaining using specific monoclonal antibodies directed against the Pk and P1 antigens. The results demonstrated that the microsomal proteins from P1 kidneys catalyze the synthesis of Pk [alpha-D-Galp-(1----4)-beta-D-Galp-(1----4)-beta-D-Glcp-ceramide] and P1 [alpha-D-Galp-(1----4)-beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----3)-beta -D-Galp-(1----4)-beta-D-Glcp-ceramide] glycolipids, whereas microsomes from P2 kidney catalyze the synthesis of the Pk glycolipid, but not of the P1 glycolipid. Competition studies using a mixture of two oligosaccharides (methyl beta-lactoside and methyl beta-lacto-N- neotetraoside) or of two glycolipids (lactosylceramide and paragloboside) as acceptors indicated that these substrates do not compete for the same enzyme in the microsomal preparation from P1 kidneys. The results suggested that the Pk and P1 glycolipids are synthesized by two distinct enzymes.

Antigens, CD↗
PubMed1992

[Nutritional changes caused by the germination of legumes commonly eaten in Chile].

The changes promoted by germination on phytates, oligosaccharides, crude protein, amino acids and riboflavin contents of black and white cultivars of beans, lentils, chicken-pea and peas, were studied. Seeds germination was carried out in darkness at 25 degrees C and 85% RH during 72 hours, previously soaked overnight in a solution of sodium hypochlorite at a concentration of 50 ppm. Germination capacity was assessed by determining hypocotyl and epicotyl lengths and percent of sprouted seed. The seeds were milled and freeze-dried for the chemical analysis. Germination promoted a significant increase in crude protein content and reduction also significant in phytates levels. These changes were attributed to an increase of proteases and phytase activities. In fact, this enzyme would make a solubilization of phytates and would release soluble protein and minerals. A significant reduction of flatulence oligosaccharides took place, which was also explained by an increase of alpha-galactosidase concentration. Sprouted seeds showed a higher content of almost all amino acid than crude legumes, although this change was variable. Significant increase of riboflavin was also found. Finally, germination decreased ashes and fat contents. These findings were determined in all legumes, although both cultivars of beans showed a higher response to the biochemical changes.

Amino Acids↗
PubMed1992

Five DNA tumor viruses undetectable in human retinoblastomas.

Retinoblastoma (RB) is a childhood eye cancer that arises when a retinal cell lacks a functional RB gene. Recent data indicate the transforming proteins of adenovirus, papillomavirus, and the polyomaviruses BK and JC all can bind to the product of the RB gene. Furthermore, adenovirus 12, JC virus, and simian virus 40 are able to induce RB-like tumors in rodents. In view of these findings, 50 human RBs were tested for the presence of five human DNA tumor viruses: adenovirus 12, BK virus, JC virus, and human papillomaviruses 16 and 18. Using the polymerase chain reaction, no viral sequences were detected in 50 RB DNAs. These data provide no evidence that these viruses have an etiologic role in human RB.

Base Sequence↗
PubMed1991

The effect of elimination of intersubunit disulfide bonds on the activity, assembly, and secretion of recombinant human acetylcholinesterase. Expression of acetylcholinesterase Cys-580----Ala mutant.

Site-directed mutagenesis was used to study the cysteine residue involved in the assembly of human acetylcholinesterase (HuAChE) catalytic subunits. Substitution of the cysteine at position 580 by alanine resulted in impairment of interchain disulfide bridge formation; the mutagenized enzyme (C580A) was secreted from recombinant cells in the monomeric form and failed to assemble into dimers. The mutant monomeric HuAChE did not differ from the native oligomeric enzyme neither in rate of catalysis nor in affinity to acetylthiocholine. Mutant monomers were also shown to retain the acetylcholinesterase characteristic sensitivity to high substrate concentrations. The mutation did not seem to affect the efficiencies of either synthesis or secretion of recombinant HuAChE polypeptides, as was demonstrated in cell lines derived from human embryonic kidney (293 cells) as well as from a human neuroblastoma (SK-N-SH). Furthermore, the mutation did not lead to an increase in accumulation of intracellular HuAChE polypeptides, suggesting that export of acetylcholinesterase from cells may not be coupled to subunit assembly.

Acetylcholinesterase↗
PubMed1991

Nutritional quality of lupine (Lupinus albus cv. Multolupa) as affected by lactic acid fermentation.

The effects of selected NRRL strains of Lactobacillus acidophilus, L. buchneri, L. cellobiosus and L. fermentum upon oligosaccharide, phytate and alkaloid contents, as well as on the nutritive value of lupine, were investigated. Lupine was processed to a 12% total solids suspension, inoculated with 1% (v/v) cultures and fermented until a final desired pH of 4.5. L. acidophilus B-2092 and L. buchneri B-1837 growth was related to a significant sucrose breakdown and decreases of phytates, whereas L. acidophilus B-1910 and L. fermentum B-585 reduced the content of flatulence oligosaccharides. The activity of L. acidophilus B-1910 was particularly associated with lowering of alkaloids and increase of riboflavin. Lactic acid fermentation produced slight changes in lysine and methionine contents. No significant differences in net protein ratio values and protein digestibility were found between fermented and unfermented lupine (P less than 0.05). A 1:1 ratio mixture of B-1910 and B-2092 strains of L. acidophilus lead to a final fermented lupine with nutritional advantages to those given by the individual cultures.

Alkaloids↗
PubMed1989

Anorexia nervosa reconceptualized from a psychosocial transactional perspective.

Anorexia nervosa is conceptualized as a number of very different instrumental actions that occur within particular family contexts. Six psychosocial transactional patterns--negativistic, attention centering, distracting, childlike, attractive, and self-punishing--are identified. This psychosocial transactional perspective is proposed as an alternative to approaches that focus on the "psychopathology" of young female anorexics.

Anorexia Nervosa↗
PubMed1989

Extended major histocompatibility complex haplotypes in patients with multiple sclerosis.

We derived complete haplotypes of the major histocompatibility complex for 33 patients with MS and their families. The DR2 allele and DR2-bearing extended haplotypes, in proportion, were overrepresented on chromosomes of MS patients compared with parental chromosomes not transmitted to MS offspring. We did not confirm previous reports that particular alleles at the BF locus are overrepresented in MS or that C2 hypocomplementemia is present. These results suggest that the DR2 allele is a risk factor for MS, and not merely a genetic marker of the population of origin.

Alleles↗
PubMed1988

Cerebral germinomas and Klinefelter syndrome. A review.

Patients with Klinefelter syndrome appear to be predisposed to the development of extragonadal cerebral germinomas. A case of a pineal region germinoma in a boy 15 years of age with Klinefelter syndrome is documented in this article. In view of three other cases of cerebral germinomas associated with Klinefelter syndrome in the literature, a hypothesis for this predisposition is suggested.

Adolescent↗
PubMed1988

Adolescent suicide and suicide attempts: a population study.

To clarify the epidemiology of adolescent suicide, a retrospective study was undertaken of suicides (1978 to 1982) and hospitalized suicide attempts (1979 to 1983) by adolescents aged 10 to 19 years in an affluent suburban area. Data included date of injury, demography (for both suicides and suicide attempts), and recorded personal and social history (available for attempts only). There were 11 deaths due to suicide (definite or possible) in the five years reviewed: seven male, 10 aged 15 to 19 years. The mean annual rate for suicide deaths (definite and possible, based on ICD codes) was 10.3 per 100,000 15 to 19 year olds, with male rates exceeding female rates. Male rates were lower in the study area than in Chicago, Illinois or the United States, but female rates were higher. Suicides represented an unusually high proportion of all adolescent deaths. Atypically, there were no firearm suicide deaths. Two hundred ten suicide attempts were studied: 77% aged 15 to 19 years, 70% female, and 82% white; 83% involved ingestion of medications or poisons. The mean annual suicide attempt rate was approximately 140 per 100,000 for 15 to 19 year olds, and 45 per 100,000 for 10 to 14 year olds, with female rates exceeding male rates. There was an association between suicide attempt dates and occurrence of holidays, and there was a peak in attempts at the end of the school year. Detailed analysis of personal and social attributes associated with suicide attempts was prevented by poor recording of relevant factors in the medical record.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1988

Disintegration of microorganisms.

The most common methods for the large-scale disintegration of microorganisms are high-pressure homogenization and wet milling. The most-used homogenizer is produced by Manton Gaulin and the common ball mill is the Dyno-Mill. Other manufacturers are now producing similar equipment (e.g., Rannie homogenizers and Netzsch ball mills). However, only relatively limited information on these systems has been published so far. An additional system that might become available for large-scale disintegration is microfluidization. When coming to choose optimal equipment and conditions for cell disintegration, it might be useful to consider some relevant topics that were compiled by the Retsch company for particle disruption and are presented, with modification, in Table XIII. Obviously, the importance of the above points will vary in different processes, but all of them should be considered in any biotechnological large-scale application. It is important to realize that although the mechanical disintegration methods are of general use, it is essential to define the optimal disintegration conditions for each microorganism and/or product. As a starting point, the published data for a similar product should be consulted. This should be followed by laboratory-scale experiments carried out with equipment and conditions that can be easily scaled up.

Cell Fractionation↗