James L. Cone

Transplantation in miniature swine. I. Fixation of the major histocompatibility complex.

Three strains of miniature swine, each homozygous for a different allele of the major histocompatibility locus (MHC), have been developed by a selective breeding scheme based on tissue typing of the offspring of each generation. Prior to breeding, the original parents were reciprocally immunized by skin grafts and lymphocyte injections to produce lymphocytotoxic antisera. These antisera were then used to assess the MHC genotype of the offspring by an analysis based on selective absorption of cytotoxicity. Offspring inheriting the same serologically determined genotype were then bred sequentially. Subsequent mixed lymphocyte cultures showed a pattern of reactivity consistent with the serological genotyping, further confirming the homology between the MHC of these miniature swine and those of man and mouse. In addition to their usefulness as a model for large animal surgical transplantation, these animals provide an abundant homozygous source of histocompatibility antigens and of antihistocompatibility antisera for use in chemical characterization of products of the MHC.

Inhibition of transforming growth factor-induced cell growth in soft agar by oxidized polyamines

An inhibitor of the transforming growth factor-induced growth in soft agar of normally anchorage-dependent rat kidney fibroblasts has been detected in the acid-ethanol extracts of human placenta, bovine lung and kidney, and human rhabdomyosarcoma cells (A673). The inhibitor has been purified from human placenta by gel-filtration and cation-exchange chromatography followed by acetylation and HPLC. Acetylation destroys inhibitory activity and deacetylation, by treatment with 6 N HCl at 110 degrees C for 16 h, restores full activity. The purified compound has been identified as spermine by mass spectral and NMR analyses and by cochromatography on HPLC of the acetylated material with acetylated spermine. Both the compound isolated from the placenta and spermine and spermidine show approximately equal activity in inhibiting the transforming growth factor-induced growth of cells in soft agar with an ED50 of 0.7-1.1 microM, while putrescine displays no inhibitory activity. Evidence suggests that the polyamines must first be oxidized by serum polyamine oxidase before inhibition will occur. Acrolein, a product of polyamine oxidation, will also inhibit cell growth in soft agar with an ED50 of 6.8 microM. It is concluded that an oxidation product of spermine is responsible for the previously reported inhibition of colony growth in soft agar following treatment of normal fibroblasts with transforming growth factors.

Guanethidine N-oxide formation as a measure of cellular flavin-containing monooxygenase activity

The usefulness of guanethidine N-oxide formation as a measure of cellular flavin-containing monooxygenase activity was assessed using the purified hog liver enzyme, rat liver microsomes and hepatocytes. The apparent Km and Vmax for this reaction in hepatocytes were 0.30 +/- 0.20 mM and 0.81 +/- 0.36 nmole per 10(6) cells min-1 respectively. The Km for the purified enzyme was 0.31 mM and the Vmax was 0.56 nmole per microgram enzyme min-1. 2-Diethylaminoethyl-2,2-diphenyl valerate (SKF-525A) at a concentration of 0.5 mM had no effect on guanethidine N-oxide formation by either rat liver microsomes or the purified enzyme. In contrast 2,4-dichloro-6-phenylphenoxyethylamine (DPEA) at the same concentration caused greater than a 100% increase in the microsomal production of guanethidine N-oxide. The tertiary amines imipramine, chloropromazine and methylpyrilene inhibited N-oxide formation by both hepatocytes and the purified enzyme. These data indicate that guanethidine N-oxide formation can be used as a measure of cellular flavin-containing monooxygenase activity.

Structural studies and two-dimensional gel electrophoresis of γ-glutamyl transpeptidase

Abstract The heterodimeric enzyme γ-glutamyl transpeptidase (EC 2.3.2.2) was isolated from adult rat kidney and purified to homogeneity for structural studies using papain solubilization and multiple chromatographies. Two-dimensional gel electrophoresis was found to resolve the active papain-purified enzyme into at least 18 components. Seven components with apparent molecular masses of 23,000–26,000 and isoelectric point range of 5.4–7.0 constitute the light subunit, and 11 components with apparent molecular mass of 51,000–53,000 and isoelectric point range of 5.8–7.1 constitute the heavy subunit. Immunoblot analysis of two-dimensional gels showed that all of these components are immunoreactive with a mixture of the two antibodies generated separately against the light and heavy subunits. Preparative subunit separation was achieved using reversephase HPLC under acidic but nonreducing conditions. N-Terminal amino acid sequencing of the separated subunits of the papain-purified enzyme yielded sequence information for the first 32 residues of the heavy chain with the N-terminal starting sequence Gly LysProAspHisValTyrSerArgAla, and for the first 36 residues of the light subunit with the N-terminal starting sequence ThrAlaHisLeuSerValValSerGluAsp.