Interactions of dithiols with p-aminophenyldichloroarsine and nicotinic acetylcholine receptors.

Interactions of dithiols with p-aminophenyldichloroarsine (APA) and with Torpedo nicotinic receptors were studied using two approaches. First, the stability of dithiol-APA complexes in solution was studied based on quenching thiol reactions with dithiobis-(nitrobenzoic acid). A peptide corresponding to a portion of the Torpedo alpha-subunit and various 1,2-dithiols such as 2,3-dimercaptopropanesulfonic acid (DMPS), 2,3-dimercaptosuccinic acid formed stable complexes with APA, while 1,4-dithiols, such as dithiothreitol (DTT) and 2,5 dimercapto-N,N,N'N'-tetradipamide (DTA) did not. The Kd of APA association with DTT in Tris buffer is 2 microM. These data suggest that APA has greater affinity for reduced nicotinic receptors than for either DTT or DTA, a prediction that was experimentally confirmed, since these reagents do not reverse the effects of APA on nicotinic receptors. Second, application of DMPS and BAL, but not 2,3-dimercaptosuccinic acid, to DTT-treated receptors both reversed the effects of APA-receptor complexes and prevented alkylation by bromoacetylcholine, suggesting that DMPS and BAL "oxidize" reduced nicotinic receptors. The presence of air is required for this "oxidizing" effect, but no clear mechanism was discovered, since prevention of formation of the reactive oxygen species superoxide, hydrogen peroxide, or hydroxyl radicals failed to block oxidation. These data suggest that oxygen reacts with dithiols to produce unknown reactive species that directly oxidize reduced nicotinic receptors, although other interpretations are still possible.

A reporter system using a flow cytometer to detect promoter/enhancer activity in lymphoid cell lines.

We have devised an experimental system using a flow cytometer to examine the promoter/enhancer activity of DNA fragments in human lymphoid cell lines. Murine CD8 alpha gene cDNA used as a reporter gene was inserted in the reporter constructs under the control of various promoter/enhancers. Furthermore, the Epstein-Barr virus (EBV) OriP, which supports a high transient expression, was also included in the reporter constructs. Cell lines expressing EBV nuclear antigen-1 (EBNA-1) were transfected with the reporter constructs by electroporation. The expression of the reporter gene was measured by a flow cytometric analysis. This experimental system is quite simple and may be especially useful for the analysis of transcriptional elements functioning in lymphocytes.

A novel culture system for induction of T cell development: modification of fetal thymus organ culture.

We have previously found that the development of T cells predominantly of the T cell receptor alpha beta T cell lineage, which is comparable to that in the organ culture (OC) of fetal thymus at the air-medium border (AMB), could be induced in submersion OC of murine fetal thymus if the cultivation was performed in an environment containing O2 at 60-80%. This culture method is named high oxygen submersion (HOS)-OC. In the present work, we established a culture system where T cell development can be induced from thymic as well as fetal liver progenitors by cocultivating them in a 96-well U-bottom plate with a deoxyguanosine (dGuo)-treated fetal thymus lobe under HOS conditions. Differentiation and growth of T cells in this culture system were comparable to those seen in the previously devised micro i.t. system, where progenitor cells were injected with a microinjector into dGuo-treated lobes and were cultured under AMB conditions. A similar level of T cell development was induced by cocultivating such progenitors with small fragments of thymus lobes under HOS conditions. Moreover, it was possible to induce T cell development by culturing fetal thymus cells with thymic stromal cells prepared by treating dGuo-treated lobes with trypsin plus EDTA in a V-bottom plate under HOS conditions.

Frequency analysis of the T cell precursors in the thymus.

A frequency determination of the T cell precursors in murine adult and fetal thymuses as well as in the bone marrow and fetal liver was made. Cells were serially diluted and injected into deoxyguanosine-treated fetal thymus lobes with a microinjector, and the lobes were cultured for 12 to 21 days. The lobes in which T cell development did not occur were discriminated from those in which T cells developed, and the precursor frequency was determined by Poisson probability distribution analysis. The precursor cell frequencies in adult bone marrow cells (2.4 x 10(-5)) and fetal liver cells (1.7 x 10(-4)) were comparable to those determined previously in in vivo intrathymus transfer experiments. The present study further shows that only a small fraction of fetal thymus cells (0.9-5.0 x 10(-2)), CD4-8- adult thymocytes (1.6 x 10(-2)), and Thy-1 low positive adult thymocytes (3.3 x 10-4)) retain the precursor activity.

Aromatic trivalent arsenicals: covalent yet reversible reagents for the agonist binding site of nicotinic receptors.

The agonist binding site of nicotinic acetylcholine receptors (AChRs) includes a disulfide bond that is easily reduced with dithiothreitol to a pair of thiols, and can be then either reoxidized with dithiobis(nitrobenzoic acid) (DTNB) or irreversibly alkylated with bromoacetylcholine (BAC). Aromatic trivalent arsenicals form stable complexes with pairs of appropriately-spaced thiols, but not single thiols. Furthermore, once complexed in proteins, trivalent arsenicals can be removed with dimercaptans, such as 2,3-dimercaptopropanesulfonic acid (DMPS). In an effort to develop reagents that will covalently, yet reversibly label AChRs, we investigated the effects of two model arsenicals, p-aminophenyldichloroarsine (APA) and 4-bromoacetyl-aminophenylarsenoxide (BAPA) on two types of nicotinic receptors: AChRs from Torpedo electroplax and neuronal receptors from chick retina. APA and BAPA significantly decrease the number of 125I-alpha-bungarotoxin binding sites in reduced Torpedo AChRs. Furthermore, arsenylation of neuronal and Torpedo receptors with APA or BAPA (1) prevents reoxidation with DTNB, (2) is reversible with DMPS, and (3) protects against irreversible alkylation by BAC. In Torpedo receptors, the EC50 of protection against BAC alkylation with APA or BAPA is approximately 30 nM. APA arsenylation of Torpedo receptors persists up to 20 h, but can be reversed at any time with DMPS. These results suggest that heterobifunctional arsenicals could anchor labeling groups in the agonist binding site in order to map the agonist binding site, quantitate receptors, or purify and reconstitute functional receptors.