Preparation of radioactive iodinated cholylhistamine for use in the radioimmunoassay of cholic acid

A major handicap in the development of simple and accurate radioimmunoassay procedures for bile acids has been the lack of a radioactive standard of high specific activity. To provide such a compound, we first synthesized cholylhistamine using the carbodiimide reaction. The hypothesized structure was confirmed by elemental analysis, thin-layer chromatography, infrared and mass spectral analysis. The cholylhistamine was then iodinated with 125I, using the choloramine-T method. The 125I-cholylhistamine was bound by antisera raised against a cholic acid-bovine serum albumin conjugate. This procedure should prove useful in preparing radioactive conjugates for all of the bile acids.     

Progesterone interaction with estrogen and antiestrogen in the rat uterus--receptor effects.

Daily injections of estradiol or the antiestrogen tamoxifen initially stimulate uterine weight increase and progesterone receptor synthesis, though continued tamoxifen fails to maintain the increased weight. The stimulatory actions of both estradiol and tamoxifen are inhibited or reversed by a single injection of progesterone. It has been hypothesized that progesterone antagonizes estrogen action by reducing estrogen receptor levels, but in the present experiments neither cytoplasmic nor nuclear estrogen receptor was affected. We conclude that progesterone acts at a point beyond estrogen receptor availability or translocation to antagonize estrogen action.     

Identification of mouse chromosomes required for murine leukemia virus replication.

The replication patterns of five ecotropic and two amphotropic strains of murine leukemia virus (MuLV) were studied by infecting 41 Chinese hamster x mounse hybrid primary clones segregating mouse (Mus musculus) chromosomes. Ecotropic and amphotropic strains replicated in mouse and some hybrid cells, but not in hamster cells, indicating that replication of exogenous virus requires dominantly expressed mouse cellular genes. The patterns of replication of the five ecotropic strains in hybrid clones were similar; the patterns of replication of the two amphotropic strains were also similar. When compared to each other, however, the replication patterns of ecotropic and amphotropic viruses were dissimilar, indicating that these two classes of MuLV require different mouse chromosomes for replication. Chromosome and isozyme analyses assigned a gene, Rec-1 (replication of ecotropic virus), to mouse chromosome 5 that is necessary and may be sufficient for ecotropic virus replication. Because of preferential retention of mouse chromosomes 15 and 17 in the hybrid clones, however, the possibility that these chromosomes carry genes that are necessary but not sufficient for ecotropic virus replication cannot be excluded. Similarly, the data indicate that mouse chromosome 8 (or possibly 19) carried a gene we have designated Ram-1 (replication of amphotropic virus) which is necessary and may be sufficient for amphotropic virus replication. Because chromosomes 8 and 19 tended to segregate together and two of the three clones excluding 19 have chromosome reaggrangements, we cannot exclude 19 as being independent of amphotropic virus replication. In addition, because of preferential retention, chromosomes 7, 12, 15, 16 and 17 cannot be excluded as being necessary but not sufficient. Hybrid cell genetic studies confirm the assignment of the Fv-1 locus to chromosome 4 previously made by sexual genetics. In addition, our results demonstrate that hybrid cells which have segregated mouse chromosome 4 but have retained 5 become permissive for replication of both N and B tropic strains of MuLV.     

Effects of Cycloate on Development of Heterodera schachtii and Growth of Three Beta Species

Greenhouse tests were set up to evaluate the effects of the herbicide, cycloate (S-ethyl cydohexylethylthiocarbamate), oil development of Heterodera schachtii and growth of three Beta species. Cycloate added to infested soil enhanced cyst development/gm root on B. vulgaris and larvae/gm of root in B. patellaris and B. procumbens at 4, 16, and 16 μg(a.i.)/gm of soil, respectively. Total numbers of nematodes/individual root system decreased because of poor root growth of seedlings in cycloate-amended soil. Penetration and larval development through stage three did occur in the wild Beta species in any treatment. Thus, resistance of B. patellaris and B. pocumbens to development of H. schachtii was not altered by cycloate. Cycloate also retarded growth (P = 0.05) of the sugarbeet cultivars and B. patellaris at 4 μg(a.i.)/gm and B. procumbens at 16 μg(a.i.)/gm of soil. Higher concentrations of nematodes/gm root in plants growing in cycloate-amended soil may be attributed to factors such as fewer roots available for penetration, possible effects of cycloate on egg hatch, greater attraction of nematodes to roots, and increased susceptibility of roots to larval penetration. Suppression of seedling growth in cycloate-amended soil may be attributed in part to higher nematode density and in part to direct root damage from cycloate.     

The detection of mutation in human diploid fibroblasts after mutagen treatment using non-selective cloning and enzyme electrophoresis

In an attempt to study mutagenesis in human diploid fibroblasts, clones derived from mass cultures treated with mutagen have been examined by starch-gel electrophoresis for 43 different enzyme loci. A technique of mutagen treatment was devised which facilitated the cloning and which enabled the cells to be exposed to very high doses of EMS and MNNG. Two alterations in phenotype, presumably the result of mutation, were observed, one involving peptidase D (PEPD) and the other phosphoglucomutase (PGM1).     

The heterocytotoxicity of human serum. II. Role of natural antibody and of the classical and alternative complement pathways.

Mouse lymphoid cells and tumor cell lines were employed as target cells for the investigation of the mechanism of heterocytotoxicity in human serum samples. It was shown that the heterocytotoxic effects were due to two differing mechanisms. Cytotoxicity was mediated in part, by activation of the alternative complement pathway on target cell membrane, a process which was antibody-independent. A second mechanism of cytotoxicity was induced by natural antibodies to a target cell, which probably mediated activation of the classical complement pathway. These data may shed light on the frequently observed cytotoxicity in mammalian sera for various target cells.     

Purification of glycopeptides of human ceruloplasmin and immunoglobulin D by high-pressure liquid chromatography

To facilitate structural studies of glycoproteins, reverse-phase high-pressure liquid chromatography (HPLC) methods have been developed for preparative isolation of glycopeptides and have been applied to human ceruloplasmin as an example of glycopeptides containing glucosamine (GlcN) and to human immunoglobulin D (IgD) for glycopeptides containing galactosamine (GalN). The use of RP-P columns and of trifluoroacetic acid and heptafluorobutyric acid as counterions was investigated. Various elution systems (both isocratic and programmed gradient) were used with n-propanol to assess the relative hydrophilicity of the peptides. The procedure developed for the GlcN glycopeptides of ceruloplasmin enabled purification of nine major chymotryptic peptides (ranging in size from 15 to 29 residues) and also of many minor peaks. These were characterized by amino acid and endgroup analysis, and the complete sequence of five was determined. These represent three different sites of GlcN attachment in the amino-terminal half of the ceruloplasmin chain. The procedures developed have enabled isolation of glycopeptides from ceruloplasmin having a single GlcN oligosaccharide attached; the latter are valuable for study of the structure and function of the carbohydrate groups. Separation of GalN glycopeptides from IgD was more difficult because of the high content of GalN in the hinge. Purification and sequence analysis was aided by partial removal of sugar by treatment with HF and by other methods. Four (or five) GalN oligosaccharides are attached to serine or threonine residues in the IgD hinge region, and all but one are in close proximity in the repeating sequence Ala-Thr-Thr-Ala-Pro-Ala-Thr-Thr.     

Binding, internalization and intracellular processing of 125I-epidermal growth factor purified by isoelectric focusing

When epidermal growth factor (EGF) which had been extensively purified by HPLC was subjected to iodination with sodium ¹²⁵iodide, 5 major species of differing isoelectric points were produced. Some of these species bound to rat fibroblasts with different affinities but were internalized with equal efficiency. Examination of the internalized ¹²⁵I-labelled molecules revealed processing of all the ¹²⁵I-EGF species to macromolecules with more acidic isoelectric points. The ¹²⁵I-EGF species with a pI of 4.5 corresponded in electrofocusing behavior with intact non-iodinated EGF. Other EGF species probably represented molecules which were covalently modified as a result of the iodination procedure.