Selective inhibition of monoamine oxidase B by aminoethyl substituted benzyl ethers.

Aminoethyl 3-chlorobenzyl ether was shown previously (Ding, C.Z. and Silverman, R.B. (1993). Bioorg. Med. Chem. Lett., 3, 2077-2078) to be a potent and selective time-dependent, but reversible inhibitor of monoamine oxidase B (MAO B). Based on this result, a series of novel aminoethyl substituted benzyl ethers was synthesized and the compounds were examined as potential inhibitors of both isozymic forms of MAO. Each compound in the series inhibits both MAO A and MAO B competitively, and IC50 values for each compound were determined. In general, the B isozyme is much more sensitive to these inhibitors than the A isozyme (except for the o- and p-substituted nitro analogues), in some cases by more than two orders of magnitude. The selectivity in favor of MAO B inhibition is relatively high for all of the meta-substituted analogues and quite low for all of the ortho-substituted analogues. Having the substituent at the ortho-position is most favorable for MAO A inhibition. With MAO B the meta-analogues were, in general, more potent than the corresponding ortho- and para-analogues with respect to their reversible binding constants. The meta-iodo analogue is the most potent analogue.

Quantitative comparison of DNA looping in vitro and in vivo: chromatin increases effective DNA flexibility at short distances.

The probability that two sites on a linear DNA molecule will contact each other by looping depends on DNA flexibility. Although the flexibility of naked DNA in vitro is well characterized, looping in chromatin is poorly understood. By extending existing theory, we present a single equation that describes DNA looping over all distances. We also show that DNA looping in vitro can be measured accurately by FLP recombination between sites from 74 bp to 15 kb apart. In agreement with previous work, a persistence length of 50 nm was determined. FLP recombination of the same substrates in mammalian cells showed that chromatin increases the flexibility of DNA at short distances, giving an apparent persistence length of 27 nm.

Inducible expression based on regulated recombination: a single vector strategy for stable expression in cultured cells.

When fused to the ligand binding domain (LBD) of steroid hormone nuclear receptors, site-specific recombinases (SSRs) acquire a ligand-dependent activity. Here, we describe the use of SSR-LBD fusion proteins in an inducible expression system, introduced into cells in a single step. A single transgene contains a constitutively active, bi-directional enhancer/promoter, which directs expression, on one side, of an SSR-LBD fusion protein gene and, on the other, a selectable marker/inducible gene cassette. The selectable marker, the puromycin acetyltransferase (pac) gene, is used for stable genomic integration of the transgene and is flanked by recombination target sites. The inducible gene is not expressed because the pac gene lies between it and the promoter. Activation of the SSR-LBD by a ligand induces recombination and the pac gene is excised. The inducible gene is thus positioned next to the promoter and so is expressed. This describes a ligand-inducible expression strategy that relies on regulated recombination rather than regulated transcription. By inducible expression of diptheria toxin, evidence that this system permits inducible expression of very toxic proteins is presented. The combination of the complete regulatory circuit and inducible gene in one transgene relates expression of the selectable marker gene to expression from the bi-directional enhancer/promoter. We exploit this relationship to show that graded increases in selection pressure can be used to select for clones with different induction properties.

Inducible formation of liver tumors in transgenic mice.

Transgenic mice have been generated with an inducible SV40 t/T antigen construct with the aim of analysing the early changes that take place in the course of liver tumorigenesis. The strictly liver-specific human C-reactive protein (CRP) gene promoter was chosen for the control of the transgene expression because this promoter can be turned on transiently by injection of bacterial lipopolysaccharide. Among 10 independently derived CRP-Tag mouse lines five showed inducible expression of the CRP-Tag transgene in liver. However, only one had a tight control of the transgene with virtually no expression under physiological conditions and high levels of Tag expression after stimulation. Females of this line were used to analyse the progression of liver alterations upon repeated induction of the t/T antigen for different lengths of time. The first signs of transgene-induced liver alterations could be monitored by the activation of the marker enzyme gamma-glutamyltranspeptidase 30 days after the start of the induction program. After 90 days hepatocellular carcinomas were already detectable. Thus, CRP-Tag mice constitute an excellent system to analyse the sequential events that take place during liver carcinogenesis.

Long-term consequences of interleukin-6 overexpression in transgenic mice.

With the aim of using interleukin-6 (IL-6)-inducible promoters to express transgenes, we investigated the long-term consequences of high levels of IL-6 in mice. As a first step, we generated transgenic mice constitutively expressing the murine IL-6 at a level sufficient to induce IL-6-responsive genes. These mice were analyzed with respect to the indirect and direct consequences of elevated IL-6 expression over a time period of about 2 years. Although biologically active IL-6 was expressed from the transgene and different alterations could be documented (less immature B cells in bone marrow, expression of IL-6-inducible liver genes), the mice appeared healthy and could easily be used for breeding. Only in mice older than 18 months did we find a high incidence of lymphomas associated with different tissues. These results indicate that the side effects of long-term treatment with IL-6 are relatively moderate, and that IL-6 might be used to mediate the expression of heterologous genes in the context of functional studies.

Lack of interleukin-2 (IL-2) dependent growth of TAC positive T-ALL/NHL cells is due to the expression of only low affinity receptors for IL-2.

Binding of interleukin-2 (IL-2) to high affinity receptors on activated normal T cells was shown to be the essential step in induction of proliferation of such cells. The finding of abundant IL-2 receptors on malignant T cells in adult T cell leukemia suggested a deregulation of the IL-2/IL-2 receptor system and was assumed to account for aberrant growth in malignant disorders of T cells. In this study we use malignant T cells from nine patients with the clinical diagnosis of T-ALL or T-NHL and did not detect IL-2 dependent growth under conditions in which normal T cells responded to IL-2. IL-2 receptors comparable in numbers to activated T cells were found on T-ALL/T-NHL cells stimulated with PHA and PMA. However, binding studies using radiolabeled IL-2 indicated that the receptors present on malignant T cells were not able to bind to IL-2 with high affinity. Therefore, if IL-2 is involved in the proliferation of malignant T cells, its mechanism of growth regulation may be different from the one for normal T cells. Alternatively, IL-2 may not play a role in the regulation of growth of malignant T cells in vitro.