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M Mihovilovic

Publications and source records attributed to M Mihovilovic.

At least 19 recordsLinked to original sources
PubMed2001

Thymic epithelial cell line expresses transcripts encoding alpha-3, alpha-5 and beta-4 subunits of acetylcholine receptors, responds to cholinergic agents and expresses choline acetyl transferase. An in vitro system to investigate thymic cholinergic mechanisms.

Transcriptional and immunocytological characterization of thymic epithelial (TE) cell line TE750 shows that these cells, like primary TE cell cultures, transcribe alpha-3, alpha-5 and beta-4 acetylcholine receptor (AcChR) subunit genes while expressing cortical, medullary and epithelial differentiation thymic markers. Incubation of TE750 cells with nicotine decreases cell adherence and growth as measured through direct cytological observation and nucleic acid quantification, respectively. Physostigmine, a traditional cholinesterase inhibitor that also activates nicotinic AcChRs, reproduces the effects of nicotine. Strengthening the hypothesis that cholinergic receptors mediate the effects of physostigmine, acetylcholinesterase (AcChase) activity is not detected in TE750 cells. Also, like thymocytes, TE750 cells express choline acetyltransferase (ChAT), indicating that the natural transmitter AcCh can be produced locally within the thymic parenchyma. Taken together these findings indicate that TE750 cells in culture represent a suitable in vitro system for the analysis of cholinergic mechanisms operational in the thymic epithelium.

Cell Line↗
PubMed1997

Thymocytes and cultured thymic epithelial cells express transcripts encoding alpha-3, alpha-5 and beta-4 subunits of neuronal nicotinic acetylcholine receptors: preferential transcription of the alpha-3 and beta-4 genes by immature CD4 + 8 + thymocytes.

Thymic tissues express transcripts encoding the alpha-3, alpha-5 and beta-4 subunits of nicotinic neuronal acetylcholine receptors (AcChRs) suggesting that neuronal AcChRs similar to those expressed in ganglia are expressed in the thymus. Transcription occurs in both isolated thymocytes and thymic epithelial cells. RT-PCR analyses of thymocyte subsets indicate that immature CD4 + 8 + thymocytes express higher levels of the alpha-3 and beta-4 transcripts than more mature thymocytes. Compared to freshly isolated thymocytes, peripheral blood lymphocytes do not express alpha-3 and beta-4 AcChR subunit transcripts. Cultured thymocytes rapidly down-regulate transcription of the alpha-3 and beta-4 AcChR subunit genes by a process that is not reversed by stimulation with phytohemagglutinin and IL-2. Thus our results indicate that there is transcriptional regulation of neuronal AcChR subunit genes during the process of thymocyte maturation and that factors within the thymic microenvironment influence expression of the alpha-3 and beta-4 AcChR subunit genes by developing T cells.

Amyloid beta-Protein Precursor↗
PubMed1994

Pathogenesis of hyperacute experimental autoimmune myasthenia gravis. Acetylcholine receptor/cholinergic site/receptor function/autoimmunity.

Three mAbs, mAbs 249E, 370, and 383C, directed against the alpha-bungarotoxin (alpha BgTx) binding site of the acetylcholine receptor (AChR) induce a hyperacute form of experimental autoimmune myasthenia gravis (EAMG), characterized by death within hours of mAb injection. To analyze the mechanisms of this effect, purified AChR-mAb complexes were investigated for their ability to bind the cholinergic agonist carbamoylcholine and to undergo agonist-induced activation of the cholinergic ionophore. The three mAbs inhibited carbamylcholine binding, and, conversely, their binding to AChR was inhibited by carbamylcholine. All three completely inhibited carbamylcholine-induced T1+ influxes to AChR-rich vesicles. These data indicate that the severe hyperacute EAMG induced by these mAbs results from blockage of AChR function and that the role of such potent Abs (even if present in small amounts) in the pathogenesis of human myasthenia gravis deserves further investigation.

Animals↗
PubMed1994

A new human slow skeletal troponin T (TnTs) mRNA isoform derived from alternative splicing of a single gene.

A human muscle cDNA library was screened for slow skeletal troponin T (TnTs). Sequence analysis revealed that one of the selected clones had a cDNA coding sequence different from the two previously described. RNase protection assays confirmed the expression of this new isoform in adult skeletal muscle. In addition, results of the RNase protection assays strongly suggested the expression of a fourth isoform. These isoforms for human slow TnT most likely result from combinatorial alternative splicing of a single gene.

Adult↗
PubMed1993

Expression of alpha-3, alpha-5, and beta-4 neuronal acetylcholine receptor subunit transcripts in normal and myasthenia gravis thymus. Identification of thymocytes expressing the alpha-3 transcripts.

We used amplification of reverse transcribed RNA to investigate the thymic transcription of genes encoding for subunits of neuronal acetylcholine receptors (AcChR). All thymic tissues investigated, which include normal thymi, myasthenic hypertrophic thymi, and both myasthenic and nonmyasthenic thymomas, express transcripts encoding for the alpha-3, the alpha-5, and the beta-4 subunits of AcChR. The thymic transcription of at least three different genes encoding for neuronal AcChR subunits suggests there is expression of neuronal AcChR in thymus. Amplification of reverse transcribed RNA and Northern blotting of thymic and peripheral immunocytes showed that thymocytes but not peripheral lymphocytes express alpha-3 AcChR subunit transcripts. This finding suggests that within the T cell lineage there is transcriptional regulation of the alpha-3 AcChR gene.

Base Sequence↗
PubMed1993

Splicing of an anti-sense Alu sequence generates a coding sequence variant for the alpha-3 subunit of a neuronal acetylcholine receptor.

In this report we demonstrate that an alpha-3 acetylcholine receptor subunit transcriptional variant originates through alternative splicing of a complementary sequence of the right arm of an Alu element. This element is located within the 5.1 Kb intron found between exons 5 and 6 of the alpha-3 acetylcholine receptor subunit gene. The transcriptional variant originates from the normal splicing process and carries an in-frame stop codon. If translated, it should encode for a peptide lacking the 4th transmembrane domain of the normal subunit.

Alternative Splicing↗
PubMed1993

Nicotinic neuronal acetylcholine receptor alpha-3 subunit transcription in normal and myasthenic thymus.

Thymic transcription of the alpha-3 subunit of the AChR was studied through sequencing and PCR analysis of thymic cDNA clones, Northern blotting, and ribonuclease protection assays. This analysis revealed at least three, 3' end sequence variants for the alpha-3 subunit as well as a variant that results from the alternative splicing of an antisense 122 bp Alu sequence between exons 5 and 6 of the normal transcript. The spliced Alu sequence not only shifts the exon 6 reading frame but also carries an in-frame stop codon. If translated, this variant transcript would produce a truncated peptide lacking the fourth transmembrane domain of the subunit and carrying a carboxy terminus dodecapeptide not found in any other known AChR subunit sequence. The putative variant subunit may lack biological activity and should differ antigenically from its normal counterpart. In comparing the normal, the MG hypertrophic, and the MG thymoma for transcription of the alpha-3 subunit and its 122 bp variant, it was found that there were no qualitative or quantitative changes in alpha-3 transcript expression in the MG hypertrophic thymi. Thymomas, however, showed an overall decrease in alpha-3 transcription and a comparative increase in beta-amyloid precursor transcription. The decrease in the levels of alpha-3 transcription in thymomas may be related to the proliferation of thymic epithelial cells.

Amyloid beta-Protein Precursor↗
PubMed1991

Expression of mRNAs in human thymus coding for the alpha 3 subunit of a neuronal acetylcholine receptor.

We report the isolation of clones from a human thymus cDNA library that code for the alpha 3 subunit of a neuronal acetylcholine receptor (AcChR). The clones hybridize to one major 3.0-kb mRNA thymic species and four minor ones of approximately 2.3, 4.0, 5.0, and 6.5 kb, but they do not hybridize to human muscle mRNA. These clones may be of value in defining the cholinergic thymic makeup and the putative role that a thymic AcChR may have in the triggering and/or maintenance of an anti-AcChR response in the autoimmune condition myasthenia gravis.

Amino Acid Sequence↗
PubMed1987

Monoclonal antibodies as probes of the alpha-bungarotoxin and cholinergic binding regions of the acetylcholine receptor.

We have probed the acetylcholine receptor (AcChR) molecule with six anti-AcChR monoclonal antibodies (mAbs) whose binding to the AcChR is inhibited or blocked by alpha-bungarotoxin (alpha BgTx). mAbs bound with a maximum stoichiometry of either one mAb (387D, 247G) or two mAbs (383C, 572C, 370C, 249E) per AcChR monomer, and the extent to which they inhibited alpha BgTx binding directly correlated with their stoichiometry of binding. The effect of mAbs on the alpha BgTx and cholinergic ligand binding properties of the AcChR molecule defined three major categories of mAbs: those that block alpha BgTx and carbamylcholine (agonist) binding, but do not block d-tubocurarine (antagonist) binding (383C, 572C, 370C and 249E); mAb 387D, which blocks agonist binding and partially blocks alpha BgTx and d-tubocurarine binding; and mAb 247G, which does not affect agonist binding, blocks at most 50% of the alpha BgTx binding sites, and decreases the affinity of the high affinity component of d-tubocurarine binding (Mihovilovic, M., and Richman, D. P. (1984) J. Biol. Chem. 259, 15051-15059). Except for mAb 247G, these mAbs strongly competed with each other for binding to the AcChR. In contrast, mAb 247G blocks about 50% of the binding of all the other mAbs. The results demonstrate the ability of mAbs to stabilize different conformational states of the AcChR and to probe cholinergic epitopes of functional importance. They also indicate the nonequivalence of the two alpha-toxin binding regions of the AcChR molecule and suggest that it is possible to identify epitopes within the alpha BgTx binding region that when bound produce differential effects on the binding of the agonist (carbamylcholine) and the antagonist (d-tubocurarine).

Animals↗
PubMed1987

Human x human hybridomas from patients with myasthenia gravis: possible tools for idiotypic therapy for myasthenia.

Hybridomas secreting monoclonal antibodies directed against the nicotinic acetylcholine receptor have been developed from rats with experimental autoimmune myasthenia gravis and from a patient with myasthenia gravis. Rat monoclonal antibodies were characterized by their ability to bind to electroblotted acetylcholine receptor subunits. Of 34 tested, 22 bound to the alpha subunit. Three bound to other subunits, and the remainder appeared to bind only to the native molecule. The human monoclonal antibodies were analyzed with respect to their binding to membrane-bound and solubilized acetylcholine receptor. Many bound with greater affinity to the membrane-bound form of the antigen. Two rat monoclonal antibodies capable of passively transferring experimental autoimmune myasthenia gravis, and with reactivities to the alpha subunit of the acetylcholine receptor, were employed to produce isogeneic monoclonal antiidiotypic antibodies. When they were injected prior to immunization with acetylcholine receptor, two of the antiidiotypic antibodies directed against framework determinants prevented the development of experimental autoimmune myasthenia gravis. This observation raises the possibility that the human monoclonal antibodies will be useful in the development of idiotypic treatment of the human disease.

Animals↗
PubMed1984

Modification of alpha-bungarotoxin and cholinergic ligand-binding properties of Torpedo acetylcholine receptor by a monoclonal anti-acetylcholine receptor antibody.

The interaction between acetylcholine receptor (AcChR) and monoclonal antibody (mab) 247G--whose binding is blocked by the presence of alpha-bungarotoxin (alpha BgTx)--leads, in the absence of alpha BgTx, to a maximum binding of 0.5 mabs/alpha BgTx-binding site and, in turn, produces a maximum of 50% inhibition of alpha BgTx binding. For the solubilized AcChR, this inhibition is the result of blockade by mab 247G of the kinetically resolved slow component of alpha BgTx binding. The presence of cholinergic ligands does not significantly inhibit mab binding to the AcChR. AcChR X mab 247G complexes bind d-[3H]tubocurarine and carbamyl[3H]choline with the same stoichiometry as for free AcChR. However, while the binding isotherms for the agonist remain unaltered, the dissociation constant of the antagonist for its high-affinity site increases at least 3 times and there is a decrease in the total number of high-affinity sites and a concomitant increase in the total number of low-affinity sites. These results indicate that the binding of mab 247G to the AcChR stabilizes a new conformational state of the molecule capable of binding cholinergic ligands and confirm previous reports indicating that the cholinergic binding site can be viewed as a region of overlapping cholinergic binding subsites.

Animals↗
PubMed1984

A noncholinergic site-directed monoclonal antibody can impair agonist-induced ion flux in Torpedo californica acetylcholine receptor.

We have employed several monoclonal antibodies (mAbs) directed against several regions of the acetylcholine receptor (AcChoR) to assist in the determination of the antigenic structure of this multisubunit glycoprotein and to better understand molecular events involved in the impairment of neuromuscular transmission in the autoimmune disease myasthenia gravis. Among three mAbs shown to block agonist-induced ion fluxes, mAb 371A is a putative probe of an ion channel domain(s) of the AcChoR. It appears to bind to an antigenic determinant whose structure is maintained upon treatment with sodium dodecyl sulfate, the stoichiometry of binding being of one mAb per alpha-bungarotoxin binding site. Binding of mAb 371A to the AcChoR does not affect binding of cholinergic agonists or antagonists (carbamoylcholine and d-tubocurarine) or neurotoxins (alpha-bungarotoxin) or the ability of membrane-bound AcChoR to undergo reversible sensitization-desensitization affinity transitions. However, this mAb inhibits agonist-induced thallium (T1+) influx into AcChoR-rich membrane vesicles, as measured on a millisecond time scale by means of a rapid kinetics "stopped-flow/fluorescence quenching" technique. The stoichiometry of inhibition by bound mAb 371A coincides with that for maximal binding.

Animals↗