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S Seidman

Publications and source records attributed to S Seidman.

At least 37 records · Page 2Linked to original sources
PubMed1992

Cloning and antisense oligodeoxynucleotide inhibition of a human homolog of cdc2 required in hematopoiesis.

Mechanisms triggering the commitment of pluripotent bone marrow stem cells to differentiated lineages such as mononuclear macrophages or multinucleated megakaryocytes are still unknown, although several lines of evidence suggested correlation between cholinergic signaling and hematopoietic differentiation. We now present cloning of a cDNA coding for CHED (cholinesterase-related cell division controller), a human homolog of the Schizosaccharomyces pombe cell division cycle 2 (cdc2)-like kinases, universal controllers of the mitotic cell cycle. Library screening, RNA blot hybridization, and direct PCR amplification of cDNA reverse-transcribed from cellular mRNA revealed that CHED mRNA is expressed in multiple tissues, including bone marrow. The CHED protein includes the consensus ATP binding and phosphorylation domains characteristic of kinases, displays 34-42% identically aligned amino acid residues with other cdc2-related kinases, and is considerably longer at its amino and carboxyl termini. An antisense oligodeoxynucleotide designed to interrupt CHED's expression (AS-CHED) significantly reduced the ratio between CHED mRNA and actin mRNA within 1 hr of its addition to cultures, a reduction that persisted for 4 days. AS-CHED treatment selectively inhibited megakaryocyte development in murine bone marrow cultures but did not prevent other hematopoietic pathways, as evidenced by increasing numbers of mononuclear cells. An oligodeoxynucleotide blocking production of the acetylcholine-hydrolyzing enzyme, butyrylcholinesterase, displayed a similar inhibition of megakaryocytopoiesis. In contrast, an oligodeoxynucleotide blocking production of the human 2Hs cdc2 homolog interfered with production of the human 2Hs cdc2 homolog interfered with cellular proliferation without altering the cell-type composition of these cultures. Therefore, these findings strengthen the link between cholinergic signaling and cell division control in hematopoiesis and implicate both CHED and cholinesterases in this differentiation process.

Amino Acid Sequence↗
PubMed1992

Intramolecular relationships in cholinesterases revealed by oocyte expression of site-directed and natural variants of human BCHE.

Structure-function relationships of cholinesterases (CHEs) were studied by expressing site-directed and naturally occurring mutants of human butyrylcholinesterase (BCHE) in microinjected Xenopus oocytes. Site-directed mutagenesis of the conserved electronegative Glu441,Ile442,Glu443 domain to Gly441,Ile442,Gln443 drastically reduced the rate of butyrylthiocholine (BTCh) hydrolysis and caused pronounced resistance to dibucaine binding. These findings implicate the charged Glu441,Ile442,Glu443 domain as necessary for a functional CHE catalytic triad as well as for binding quinoline derivatives. Asp70 to Gly substitution characteristic of 'atypical' BCHE, failed to alter its Km towards BTCh or dibucaine binding but reduced hydrolytic activity to 25% of control. Normal hydrolytic activity was restored to Gly70 BCHE by additional His114 or Tyr561 mutations, both of which co-appear with Gly70 in natural BCHE variants, which implies a likely selection advantage for these double BCHE mutants over the single Gly70 BCHE variant. Gly70 BCHE variants also displayed lower binding as compared with Asp70 BCHE to cholinergic drugs, certain choline esters and solanidine. These effects were ameliorated in part by additional mutations or in binding solanidine complexed with sugar residues. These observations indicate that structural interactions exist between N' and C' terminal domains in CHEs which contribute to substrate and inhibitor binding and suggest a crucial involvement of both electrostatic and hydrophobic domains in the build-up of the CHE active center.

Amino Acid Sequence↗
PubMed1991

Recombinant human acetylcholinesterase is secreted from transiently transfected 293 cells as a soluble globular enzyme.

1. Coding sequences for the human acetylcholinesterase (HuAChE; EC 3.1.1.7) hydrophilic subunit were subcloned in an expression plasmid vector under the control of cytomegalovirus IE gene enhancer-promoter. The human embryonic kidney cell line 293, transiently transfected with this vector, expressed catalytically active acetylcholinesterase. 2. The recombinant gene product exhibits biochemical traits similar to native "true" acetylcholinesterase as manifested by characteristic substrate inhibition, a Km of 117 microM toward acetylthiocholine, and a high sensitivity to the specific acetylcholinesterase inhibitor BW284C51. 3. The transiently transfected 293 cells (100 mm dish) produce in 24 hr active enzyme capable of hydrolyzing 1500 nmol acetylthiocholine per min. Eighty percent of the enzymatic activity appears in the cell growth medium as soluble acetylcholinesterase; most of the cell associated activity is confined to the cytosolic fraction requiring neither detergent nor high salt for its solubilization. 4. The active secreted recombinant enzyme appears in the monomeric, dimeric, and tetrameric globular hydrophilic molecular forms. 5. In conclusion, the catalytic subunit expressed from the hydrophilic AChE cDNA species has the inherent potential to be secreted in the soluble globular form and to generate polymorphism through self-association.

Acetylcholinesterase↗
PubMed1990

Anionic site interactions in human butyrylcholinesterase disrupted by two single point mutations.

Structure-function relationships of recombinant human butyrylcholinesterase (CHE) variants were investigated by Xenopus oocyte microinjection. A Ser-425 to Pro-425 mutation failed to modify ligand binding properties. In contrast, Asp-70 to Gly-70 substitution significantly reduced CHE binding capacity for succinylcholine and specific inhibitors, demonstrating Asp-70 as a key anionic site component for certain ligands. Furthermore, the presence of both mutations rendered CHE totally resistant to succinylcholine and dibucaine inhibition, while all mutant proteins bound butyrylthiocholine, benzoylcholine, and propionylcholine normally. These findings imply structural interactions between the conserved Asp-70 and Ser-425 regions in cholinesterases and suggest the contribution of additional electronegative amino acids to anionic site binding.

Anions↗
PubMed1990

Molecular cloning and construction of the coding region for human acetylcholinesterase reveals a G + C-rich attenuating structure.

To study the primary structure of human acetylcholinesterase (AcChoEase; EC 3.1.1.7) and its gene expression and amplification, cDNA libraries from human tissues expressing oocyte-translatable AcChoEase mRNA were constructed and screened with labeled oligodeoxynucleotide probes. Several cDNA clones were isolated that encoded a polypeptide with greater than or equal to 50% identically aligned amino acids to Torpedo AcChoEase and human butyrylcholinesterase (BtChoEase; EC 3.1.1.8). However, these cDNA clones were all truncated within a 300-nucleotide-long G + C-rich region with a predicted pattern of secondary structure having a high Gibbs free energy (-117 kcal/mol) downstream from the expected 5' end of the coding region. Screening of a genomic DNA library revealed the missing 5' domain. When ligated to the cDNA and constructed into a transcription vector, this sequence encoded a synthetic mRNA translated in microinjected oocytes into catalytically active AcChoEase with marked preference for acetylthiocholine over butyrylthiocholine as a substrate, susceptibility to inhibition by the AcChoEase inhibitor BW284C51, and resistance to the BtChoEase inhibitor tetraisopropylpyrophosphoramide. Blot hybridization of genomic DNA from different individuals carrying amplified AcChoEase genes revealed variable intensities and restriction patterns with probes from the regions upstream and downstream from the predicted G + C-rich structure. Thus, the human AcChoEase gene includes a putative G + C-rich attenuator domain and is subject to structural alterations in cases of AcChoEase gene amplification.

Acetylcholinesterase↗
PubMed1990

Children with congenital anomalies: the preschool period.

To address a gap regarding the development of preschool children with physical congenital anomalies and the adaptational responses of their caretakers, 37 children with anomalies and 44 control children were assessed. Children were administered the Stanford-Binet and the Preschool Language Scale, and the children and their mothers were observed in a series of semi-structured situations. Performance on linguistic and intellectual tests was related to socioeconomic status, not to medical risk. Children with anomalies were more socially reticent and compliant than peers, and their mothers were more controlling and nonverbally active during a teaching task. Results are discussed in terms of Bell's lower-limit adaptational pattern.

Child, Preschool↗
PubMed1990

Manipulations of cholinesterase gene expression modulate murine megakaryocytopoiesis in vitro.

Megakaryocytopoiesis was selectively inhibited in cultured murine bone marrow cells by a 15-mer oligodeoxynucleotide complementary to the initiator AUG region in butyrylcholinesterase mRNA. Furthermore, conditioned medium from Xenopus oocytes producing recombinant butyrylcholinesterase stimulated megakaryocytopoiesis. These observations implicate butyrylcholinesterase in megakaryocytopoiesis and suggest application of oligodeoxynucleotides for modulating bone marrow development.

Amino Acid Sequence↗
PubMed1990

Acetylcholinesterase and butyrylcholinesterase genes coamplify in primary ovarian carcinomas.

The genes for acetylcholinesterase (ACHE) and butyrylcholinesterase (CHE) are expressed in multiple tumor tissues, including ovarian carcinomas. Both CHE and ACHE genes coamplify in leukemias. To examine the relationship of gene amplification to the expression of these genes in tumors, ACHE and CHE genes and their expression were studied in primary ovarian carcinomas. DNA blot hybridization demonstrated a significant amplification and mutagenesis of both genes in 6 of 11 malignant tumors studied. This was greater or of the same order of magnitude as the amplification of the oncogenes c-rafi, v-sis, and c-fes in these tumors. No amplification was found in normal ovarian tissues or benign ovarian cysts. Xenopus oocyte microinjections, blot and in situ hybridizations, and immuno- and cytochemical staining revealed translatable CHEmRNA and its active protein product in discrete tumor foci. The frequent coamplification in ovarian carcinomas of ACHE and CHE genes implicates cholinesterases in neoplastic growth and/or proliferation.

Acetylcholinesterase↗
PubMed1989

Expression and tissue-specific assembly of human butyrylcholine esterase in microinjected Xenopus laevis oocytes.

Cholinesterases represent a ubiquitous, polymorphic family of acetylcholine hydrolyzing enzymes. The multileveled tissue-specific heterogeneity which characterizes these enzymes makes the cholinesterases an appropriate model for studying the mechanisms involved in regulating divergent pathways in protein biogenesis. For this purpose, a cDNA coding for human butyrylcholine esterase (BuChE) was subcloned into the SP 6 transcription vector. Synthetic mRNA transcribed from this construct was microninjected into Xenopus laevis oocytes alone, and in conjunction with poly(A)+ RNAs extracted from human brain or muscle. Injected BuChE-mRNA induced the biosynthesis of a protein exhibiting the catalytic activity, substrate specificity, and sensitivity to selective inhibitors characteristic of native human serum BuChE, and clearly distinct from the related enzyme acetylcholinesterase (AChE). The nascent BuChE was reproducibly distributed into low salt-soluble and detergent-extractable pools. Sucrose gradient analysis demonstrated that the nascent human enzyme was capable of limited subunit assembly, appearing as functional dimeric molecules in both of these fractions. Co-injection with brain or muscle-derived mRNAs facilitated higher order oligomeric assembly. Co-injected brain mRNA induced the appearance of tetramers while co-injected muscle mRNA induced the appearance of an array of heavy molecular forms, including a heavy 16 S form. These results indicate that the molecular determinants which distinguish BuChE from AChE are inherent to its primary amino acid sequence and that additional, tissue-specific protein(s) are involved in the modulation of subunit assembly within particular biological milieues.

Animals↗
PubMed1989

Tissue-specific processing and polarized compartmentalization of clone-produced cholinesterase in microinjected Xenopus oocytes.

1. To approach the involvement of tissue-specific elements in the compartmentalization of ubiquitous polymorphic proteins, immunohistochemical methods were used to analyze the localization of butyrylcholinesterase (BuChE) in Xenopus oocytes microinjected with synthetic BuChEmRNA alone and in combination with tissue-extracted mRNAs. 2. When injected alone BuChEmRNA efficiently directed the synthesis of small membrane-associated accumulations localized principally on the external surface of the oocyte's animal pole. Tunicamycin blocked the appearance of such accumulations, suggesting that glycosylation is involved in the transport of nascent BuChE molecules to the oocyte's surface. Coinjection with brain or muscle mRNA, but not liver mRNA, facilitated the formation of pronounced, tissue-characteristic BuChE aggregates. 3. These findings implicate tissue-specific mRNAs in the assembly of the clone-produced protein and in its nonuniform distribution in the oocyte membrane or extracellular material.

Animals↗
PubMed1988

Cross-homologies and structural differences between human cholinesterases revealed by antibodies against cDNA-produced human butyrylcholinesterase peptides.

To study the polymorphism of human cholinesterases (ChEs) at the levels of primary sequence and three-dimensional structure, a fragment of human butyrylcholinesterase (BuChE) cDNA was subcloned into the pEX bacterial expression vector and its polypeptide product analyzed. Immunoblot analysis revealed that the clone-produced BuChE peptides interact specifically with antibodies against human and Torpedo acetylcholinesterase (AChE). Rabbit polyclonal antibodies prepared against the purified clone-produced BuChE polypeptides interacted in immunoblots with denatured serum BuChE as well as with purified and denatured erythrocyte AChE. In contrast, native BuChE tetramers from human serum, but not AChE dimers from erythrocytes, interacted with these antibodies in solution to produce antibody-enzyme complexes that could be precipitated by second antibodies and that sedimented faster than the native enzyme in sucrose gradient centrifugation. Furthermore, both AChE and BuChE dimers from muscle extracts, but not BuChE tetramers from muscle, interacted with these antibodies. To reveal further whether the anti-cloned BuChE antibodies would interact in situ with ChEs in the neuromuscular junction, bundles of muscle fibers were microscopically dissected from the region in fetal human diaphragm that is innervated by the phrenic nerve. Muscle fibers incubated with the antibodies and with 125I-Protein A were subjected to emulsion autoradiography, followed by cytochemical ChE staining. The anti-cloned BuChE antibodies, as well as anti-Torpedo AChE antibodies, created patches of silver grains in the muscle endplate region stained for ChE, under conditions where control sera did not. These findings demonstrate that the various forms of human AChE and BuChE in blood and in neuromuscular junctions share sequence homologies, but also display structural differences between distinct molecular forms within particular tissues, as well as between similarly sedimenting molecular forms from different tissues.

Acetylcholinesterase↗
PubMed1986

Productive language of premature and physically handicapped two-year-olds.

Structural and functional features of the productive language of healthy and at-risk 2-yr-olds were compared. Subjects were normal, full-term toddlers (n = 15), toddlers with either cosmetic or orthopedic anomalies (n = 15), and premature toddlers (n = 15). Transcripts of the language of each child were compiled from 30-min videotapes of mother and child engaged in a variety of semistructured activities. Although premature and physically handicapped toddlers did not differ significantly from each other, both groups of medical risk toddlers were delayed in a variety of structural indices of their productive language. In comparison to normal children, they had a shorter MLU and upper bound, and a smaller vocabulary, including fewer verbs. On functional features of language usage, however, there were few differences between normal and high-risk toddlers. Results are discussed in the context of developmental and learning models that posit a relationship between maternal and child behavior.

Child Language↗
PubMed1986

Congenital intracardiac rhabdomyoma: prenatal detection by echocardiography, perinatal management, and surgical treatment.

A case of a newborn infant with an intracardiac tumor, detected in utero by fetal echocardiography at 30 weeks gestation, is presented. The prenatal evaluation by serial fetal echocardiograms and nonstress tests is described. In the perinatal period, ventricular tachycardia occurred and was successfully managed with antiarrhythmic medications. At 20 months of age, a right ventricular rhabdomyoma was removed at open heart surgery. The patient represents the earliest in utero detection of a fetal intracardiac tumor with successful postnatal outcome.

Adult↗
PubMed1980

Location of binding sites for RNA polymerase II from wheat germ and from human placenta on adenovirus 2 DNA.

Electron microscopic visualization of binary complexes between eukaryotic RNA polymerases and Adenovirus 2 (Ad 2) DNA was used to locate specific binding sites for the enzymes. RNA polymerase II from human placenta binds to 10--16 distinct sites depending on the ratio of enzyme to DNA and the divalent cation present in the binding mixture. Wheat germ RNA polymerase binds to 12--14 strong binding sites and 2--3 weaker sites, all but one of which correspond to binding sites for the placental enzyme. At least six of the strong binding sites for both enzymes correspond to promoters known to be active in vivo. As a test of the two-state model for transcription initiation, we examined binding of wheat germ RNA polymerase II to Ad 2 DNA at 0 degrees and 37 degrees. The extent of binding was the same at the two temperatures and the distributions of binary complexes were virtually identical. This observation, in conjunction with results presented previously, is strong support for the existence of I and RS complexes in eukaryotic systems.

Adenoviridae↗