Structural analysis of covalently labeled estrogen receptors by limited proteolysis and monoclonal antibody reactivity

We have used limited proteolysis of affinity-labeled estrogen receptors (ER), coupled with antireceptor antibody immunoreactivity, to assess structural features of ER and the relatedness of ER from MCF-7 human breast cancer and rat uterine cells. MCF-7 ER preparations covalently labeled with [3H]tamoxifen aziridine [( 3H]TAZ) were treated with trypsin (T), alpha-chymotrypsin (C), or Staphylococcus aureus V8 protease prior to electrophoresis on sodium dodecyl sulfate gels. Fluorography revealed a distinctive ladder of ER fragments containing TAZ for each protease generated from the Mr 66,000 ER: for T, fragments of 50K, 38K, 36K, 31K, 29K, and 28K that with longer exposure generated a 6K fragment; for C, fragments of 50K, 38K, 35K, 33K, 31K, 19K, and 18K that with longer exposure generated 14K and 6K fragments; and for V8, ca. 10 fragments between 62K and 28K. Two-dimensional gels revealed charge heterogeneity (two to three spots between pI 5.5 and 6.2) of the 66K ER and the T-generated 28K meroreceptor form. Immunoblot detection with the primate-specific antibody D75P3 gamma revealed that all immunoreactive fragments corresponded to TAZ-labeled fragments but that some small TAZ-labeled fragments (V8-generated forms less than 47K and T-generated forms less than 31K) were no longer immunoreactive. In contrast, use of the antibody H222Sp gamma revealed a correspondence between TAZ-labeled and immunoreactive fragments down to the smallest fragments generated, ca. 6K for T and C and 28K for V8. MCF-7 nuclear and cytosol ER showed very similar digest patterns, and there was a remarkable similarity in the TAZ-labeled and H222-immunoreactive fragments generated by proteolysis of both MCF-7 and rat uterine ER. These findings reveal great structural similarities between the human (breast cancer) and rat (uterine) ER and between nuclear and cytosol ER, indicate charge heterogeneity of ER, and allow a comparison of the immunoreactive and hormone attachment site domains of the ER. The observation that T and C generate a ca. 6K TAZ-labeled fragment that is also detectable with the H222 antibody should be of interest in studies determining the hormone binding domain of the ER and in amino acid sequencing of this region.     

Short-term delay in blood processing does not alter basal or isoproterenol-stimulated cyclic AMP accumulation in human lymphocytes

Isoproterenol-stimulated cyclic AMP (cAMP) accumulation in lymphocytes is an index of beta-adrenergic receptor functioning. It is not known if a delay between blood collection and subsequent cell preparation influences cAMP accumulation. We compared cAMP levels in lymphocytes processed from whole blood immediately after collection and following a 3.5 hour delay. There were no significant changes in either basal or stimulated levels of cAMP. This information may alleviate time constraints in certain experimental designs where immediate isolation of lymphocytes from whole blood may not be practical.     

Analysis of the fine specificity and cross-reactivity of monoclonal anti-lipid A antibodies

We have investigated the fine specificity of anti-lipid A antibodies to identify conserved lipid A antigens. Because lipid A derived from many different Gram-negative bacteria has similar biologic activities, the conserved regions may be of particular importance for the immunostimulatory and toxic properties of lipid A. We found that five of nine antibodies bound to a wide variety of Gram-negative bacteria. All these widely cross-reactive antibodies bound to the same antigenic site within lipid A. Polymyxin B, an inhibitor of lipid A activity, bound to this site as well. The widely cross-reactive antibodies bound to native and base-hydrolyzed lipid A equally well, and also bound to the monosaccharide precursor lipid X. The less cross-reactive antibodies recognized base-hydrolyzed lipid A poorly, and did not recognize lipid X at all. Other investigators have shown that lipid X has some of the activities of lipid A in vitro and can inhibit the lethal toxicity of LPS in vivo. On the basis of this study, we suggest that lipid X contains a conserved lipid A epitope as well.     

Functional and morphological characteristics of isolated pancreatic islets in tissue culture

Islets were isolated from the pancreas of female rats by using the collagenase technique. After culturing for 4 days, the islets were taken for measurement of insulin release biosynthesis as well as glucose utilization in subsequent short-time incubations. A low glucose concentration was insufficient to maintain a glucose-stimulated insulin release in vitro. A high glucose concentration had a protecting and restoring effect on the insulin release: ultrastructurally, such islets showed signs of an active biosynthesis in the electron micrograph. The enhancement of Mg++ in the culture medium resulted in an improvement of insulin storage in the islets, accompanied by a well-preserved action of glucose in a subsequent incubation.     

Binding studies on internal immunodeterminants: synthesis of beta-(1----6)-linked oligosaccharide methyl glycosides having one to four internal D-galactopyranosyl residues flanked by gentiobiose residues.

The oligosaccharide glycosides beta-D-Glcp-(1----6)-beta-D-Glcp-(1----6)-[beta-D-Galp-(1----6)]n-beta-D - Glcp-(1----6)-beta-D-Glcp-1----OMe (n = 1-4) were prepared by a convergent block synthesis. Haloacetyl, tert-butyldiphenylsilyl, and dimethylthexylsilyl groups were used as temporary protective groups for the preparation of the intermediate glycosyl donors and acceptors. The deoxygenated trisaccharide glycosides beta-D-Glcp-(1----6)-beta-D-Galp-(1----6)-4-deoxy-beta-D-xylo-Hexp -1----OMe and beta-D-Glcp-(1----6)-4-deoxy-beta-D-xylo-Hexp-(1----6)-beta-D-Galp -1----OMe were also synthesized. The binding of each glycoside to the monoclonal antigalactan antibody IgA J539 was studied and the results support the previous finding that J539 can bind to internal antigenic epitopes. The data are consistent with the interpretation that subsite C of that antibody binds glucose with a Ka of approximately 6 (cf. 10.9 for galactose).     

Reversal of diabetes by isogeneic transplantation of cultured pancreatic islets

Pancreatic islets were isolated by collagenase digestion from female Wistar rats and cultured at 20 mmol/l glucose. The enhancement of Mg++ concentration from 0.8 mmol/l up to 5.3 mmol/l had a protecting effect on the glucose-induced insulin release in the subsequent short-time incubation and prevented the age-depending decrease of B-cell function. About 1,000 cultured islets injected into portal vein normalized the plasma glucose of streptozotocin-diabetic rats. The plasma glucose patterns during the glucose load were nearly identical to healthy controls. These findings suggest that the cultured islets maintain the ability to secrete insulin in response to glucose in vitro as well as in vitro and that such islets can reverse an experimentally induced diabetes.     

Preparation and characterization of insulin of carp (Cyprinus carpio).

Insulin of carp (Cyprinus carpio) was isolated and crystallized. The insulin was biologically active in two tests; it decreased the blood glucose level and stimulated 14CO2-formation from glucose. The chemical properties are similar to those of insulins from other species. The insulins of carp and of mammals differ greatly immunologically. Antibodies against carp insulin crossreact with carp proinsulin.