No abstract

The influence of estrogen in provoking nuclear recompartmentation of lysosomal components in hormone-sensitive cells was investigated by immunological analyses of isolated nuclei from the preputial glands of ovariectomized rats. Fixed smears were prepared from ultrapurified nuclei freed of outer membrane, 2-30 min after iv injection of placebo-control solution or submicrogram amounts of estradiol-17 beta. Cytoplasmic contamination was negligible in such preparations, as monitored by vital staining with acridine orange. On challenge with immunoglobulin G directed toward a group of lysosomal high density lipoproteins which have been shown to bind estradiol-17 beta specifically, control preparations exhibited minimal indirect immunofluorescence that was essentially confined to the nuclear periphery. In contrast, a high proportion of the nuclei exposed for as little as 2 min to estradiol-17 beta but not to the relatively inert 17 alpha-congener, displayed generally more intense immunofluorescence which was distributed over the entire organellar area. Thus, the immunoglobulin becomes accessible to the nuclear interior in vitro as a function of pretreatment in vivo with active hormone. Nuclei from estrogen-pretreated rats were more structurally labile than corresponding controls, as judged by morphological criteria, even when isolated by gentle teasing rather than subjection to the rigorous ultrapurification process. By either method, integrity of the specimens was enhanced somewhat when they were prepared from rats ovariectomized before experiencing even a single estrous cycle. The observations verify and extend independent biochemical and ultrastructural evidence that structural labilization of cellular organelles and enhanced accessibility of limited amounts of lysosomal constituents to the nuclear compartment of specific target cells are early correlates of estrogen action.

The influence of estrogen on the subcellular localization and distribution of lysosomal components of preputial gland was investigated in the ovariectomized rat. Antisera of high titer and specificity toward high-density lysosomal lipoproteins of this organ were raised in rabbits. The immunologic effectiveness of the IgG fraction so obtained was confirmed by microcomplement fixation, immunodiffusion, and immunoelectrophoresis. By both direct and indirect immunofluorescence techniques, cryostat sections of preputial gland from the control animals exhibited pinpoint cytoplasmic fluorescence, of dimensions corresponding to those of lysosomes. In contrast, specific immunoreactive material in corresponding target cells from animals receiving 0.1 /zg of estradiol-17fl/100 g body wt only 2 min earlier was distributed more homogeneously, indicating release of antigen from the membrane-bounded organelles. Moreover, specific immunofluorescence became evident at cell surfaces and in peri-and supranuclear localization, sites essentially negative in the controls. These effects were intensified at 15 min, as well as by maximal physiologic dose (0.5 /~g/100 g body wt) of hormone. The relatively less active epimer, estradiol-17a, exhibited only very limited effectiveness by some of these criteria. These observations, taken together with independent biochemical and ultrastructural evidence, lead to the conclusion that structural labilization of lysosomal constituents and their translocation to the nuclear compartment are early correlates of estrogen action.Available biochemical and morphologic evidence indicates that reduced structural latency of components of lysosomes is a rapid response of specific target cells to certain tropic hormones (see reference 25 for review). This process is accompanied by redistribution of constituents so released to the cell surface (14, 15) as well as their access to the nucleus (27,29,31). To extend these observations further, we have analyzed this phenomenon in an estrogen-responsive tissue by immunocytologic methods. By means of specific immunofluorescent probes, the present work demonstrates that mobilization of lysosomes and redistribution of their antigenic components in preputial gland are early manifestations of the administration of physiologic doses of estradiol-17fl to the ovariectomized rat. MATERIALS AND METHODS Animals and Tissue SourcesSprague-Dawley rats were ovariectomized at approx. 6 wk of age and 150 g body wt. They were then maintained under controlled conditions of light and temperature in a low-steroid environment for 3 wk, as previously

Bei der Behandlung des Dichloramins (I) mit AlCl3 entsteht primär ein Umlagerungsprodukt, das bei Zugabe von wäßriger Säure n‐Butylamin (II) neben dem Keton (III) in Ausbeuten von 84 bzw. 94% ergibt.

Rearrangement of a series of iV,iV-dichlorocarbinamines (RWR'CNCU) at low temperatures with aluminum chloride in methylene chloride, followed by acid hydrolysis, produced carbonyl and amine products in moderate to high yields. The following relative migratory aptitudes were determined: phenyl, 18; sec-butyl, 2.4; benzyl, 1.8; «-butyl, 1.0; hydrogen, 0.09; methyl, 0.05. These values compare reasonably well with migratory aptitudes observed in the Schmidt and Baeyer-Villiger rearrangements which appear to proceed by concerted processes. Further support for synchronous loss of chloride ion and 1,2 alkyl shift is provided by the low degree of hydrogen migration, indicating the importance of a trans migratory requirement.Most of the previous work on rearrangement of N,Ndihaloamines by aluminum chloride comprised bi-5 or tricyclic systems.6•7 We recently reported8 the rearrangement of IV , -dichIor otri-n-butylcarbinamine, which yielded, after acid hydrolysis, di-n-butyl ketone and n-butylamine. Migration appeared to involve electron-deficient nitrogen. The present study was concerned with obtaining relative migratory aptitudes for various types of alkyl groups, hydrogen, and phenyl, with the aim of elucidating additional aspects of the reaction mechanism. Useful comparisons are made with related systems entailing 1,2 shifts from carbon to nitrogen, carbon to oxygen, and carbon to carbon. Results and DiscussionPreparation of Starting Materials.-All the amines except di-n-butylcarbinamine were prepared by means of the Ritter reaction or Hofmann degradation as described in the earlier paper.8 (n-Bu)2CHNH2 was obtained from reduction of di-n-butylketoxime with sodium in ethanol. Acetylation of tri-n-butylcarbinamine, followed by LiAlH4 reduction, provided jV-ethyltri-n-butylcarbinamine. N ,N-Dichloroamines 2-8 were synthesized by the previous procedure.8 Treatment of the amine with iV-chlorosuccinimide was used to generate A-chloro-N-ethyltri-n-butylcarbinamine. Yields of products are calculated8 on the basis of N-monochloroamine as the impurity in crude N,AT-dichloroamine.Rearrangement.-Rearrangement of the N,N-dichlorocarbinamines by aluminum chloride, followed by acid hydrolysis, generally yielded a mixture of two carbonyl compounds, two alkyl amines, recovered parent amine, and intractable material (Table I). The best procedure developed in the previous work8 was employed in the majority of runs.Rearrangement of (n-Bu)2(sec-Bu) CNCl2 (2) was studied under a variety of conditions. Little change in yield was noted for (1) Paper XX. Chemistry of .V-Haloamines.