Expression of hemopoietic histocompatibility antigens on H-2-loss variants of F1 hybrid lymphoma cells: evidence consistent with trans gene regulation

H-2 heterozygous marrow stem cells, lymphoid progenitor cells, and leukemia/lymphoma cells do not express hemopoietic or hybrid histocompatibility (Hh) antigens, which are important transplantation antigens recognized during the rejection of normal or neoplastic hemopoietic cells. The Hh-1b determinant of the H-2b haplotype maps to the D region of H-2. We have tested the hypothesis that gene(s) at or near H-2D of the H-2d haplotype down-regulate the expression of Hh-1b in the trans configuration. We used Abelson leukemia virus-transformed pre-B lymphoma cells (ACCb) of BALB/c X BALB.B (H-2d X H-2b) origin, as well as variant lines of ACCb, which were selected for resistance to monoclonal anti-H-2 antibodies plus complement. B6D2F1 (H-2b X H-2d), C3B6F1 (H-2k X H-2b), or B6 (H-2b) mice were infused with inocula of 5 X 10(6) B6 bone marrow cells (BMC). Proliferation of donor-derived marrow cells was judged in terms of DNA synthesis by measuring the splenic incorporation of 5-iodo(125I)-2'-deoxyuridine (IUdR) 5 days after cell transfer. B6 BMC grew much better in B6 than in F1 hybrid host mice, an expression of "hybrid resistance". As observed previously, the injection of EL-4 (H-2b, Hh-1b) tumor cells prior to infusion of B6 (H-2b, Hh-1b) BMC enhanced the growth of B6 BMC in F1 hybrid mice. Therefore, this in vivo "cold target cell competition" type of assay can be used to detect the expression of Hh-1b antigens. Unlike EL-4 (H-2b) cells, hybrid resistance was not affected by prior infusion of (H-2b X H-2d) heterozygous ACCb cells. In contrast, three ACCb variant cell lines, H-2d-, Ld-Dd-, and Dd-, enhanced the growth of B6 BMC in F1 hosts. The ACCb H-2b- cell line did not affect hybrid resistance to B6 BMC. The loss of gene expression on the H-2d chromosome at or very near the H-2Dd locus is correlated with the appearance Hh-1b, as determined by the in vivo cold target competition assay. These results support the hypothesis that heterozygous cells possess trans-acting, dominant, down-regulatory genes mapping near H-2D that control the Hh-1 phenotype of lymphoid tumor cells.     

Immunochemical specificity of antisera raised against the synthetic encephalitogenic peptide SH624, residues 59–74 of the myelin basic protein

Synthetic peptide SH624 (SHHPARTAHYGSLPQK), residues 59–74 of human myelin basic protein (MBP) was found to be encephalitogenic in the rabbit. Four antisera raised, against the peptide were employed in a liquid-phase equilibrium competitive radioimmunoassay with a series of synthetic peptide analogs of the region to probe the structural requirements of the B-cell determinant subsumed within SH624. The cross-reactivities of the four antisera with intact MBP were also examined. Immunochemical analyses of the four antisera suggested specificities directed against a conformational determinant dependent upon residues from the more phylogenetically conserved carboxyl C-terminal region, residues 65–74 (TAHYGSLPQK) of the synthetic immunogen. Peptide analogs shorter than SH624 from the C-terminal end showed no cross-reactivity with any of the reagent antisera while analogs shorter from the N-terminal end and including the encephalitogenic sequence TTHYGSLPQK, as well as, HYGSLPQK were reactive under equilibrium competitive conditions. SH624-reactive antibodies, cross-reactive with purified heterologous MBPs from 10 different species were also identified in all four reagent antisera. The results of these experiments support previous investigations demonstrating the accessibility of the encephalitogenic 65–74 region in intact MBP. They also underscore the importance of B-cell recognition of organ specific antigenic determinants with respect to MBP immunology and, in particular, the recognition of autoreactive determinants in the neighborhood of encephalitogenic centers.     

A peptidyl alpha-amidation activity in chromaffin granules of bovine adrenal medulla

Peptidyl alpha-amidation activity in bovine adrenal medulla has been localized in chromaffin granules by density gradient centrifugation. The activity was found to be both soluble and membrane-associated. Both enzymatic activities were stimulated by the addition of Cu2+ and ascorbate. The pH maximum for alpha-amidation in the chromaffin granules in pH 8.0-8.5. By gel filtration, the soluble enzyme activity appeared as a protein of approx. 40 kDa. It is suggested that this enzyme is involved in the carboxyl-terminal amidation of metorphamide, amidorphin and neuropeptide Y.     

Analysis of pyridine dinucleotides in cultured rat hepatocytes by high-performance liquid chromatography

An isocratic reverse-phase high-performance liquid chromatography method for the separation and quantitation of total pyridine dinucleotides in hepatocyte cultures is described. Cells are extracted with cold 3 M perchloric acid or 0.5 N sodium hydroxide containing 50% (v/v) ethanol and 35% cesium chloride for the determination of the oxidized or reduced pyridine dinucleotides, respectively. Pyridine dinucleotides in the neutralized extracts were separated on an Excellopak ODS C18 (4.6 X 150 mm) column with 0.1 M potassium phosphate, pH 6.0, containing 3.75% methanol as the mobile phase. NAD+ and NADP+ were detected spectrophotometrically at 254 nm. The response was linear from 5 to 4000 pmol with recoveries of NAD+ and NADP+ of 98 and 101.1%, respectively. NADH and NADPH were monitored fluorometrically by activation at 370 nm and emission in the 400-700 nm range. The reduced pyridine dinucleotides had a linear response from 7.5 to 60 pmol with recoveries of NADH and NADPH of 99.4 and 101.3%, respectively. The coefficients of variation for all of the pyridine dinucleotide standards were less than 3.5%.     

Affinity labeling of spinach phosphoribulokinase subsequent toS-methylation at Cys16

The chloroplast enzyme phosphoribulokinase is reversibly deactivated by oxidation of Cys16 and Cys55 to a disulfide. Although not required for catalysis, Cys16 is an active-site residue positioned at the nucleotide-binding domain (Porter and Hartman, 1988). The hyperreactivity of Cys16 has heretofore limited further active-site characterization by chemical modification. To overcome this limitation, the partially active enzyme,S-methylated at Cys16, has been probed with a potential affinity reagent. Treatment of methylated enzyme with bromoacetylethanolamine phosphate results in essentially complete loss of catalytic activity. Inactivation follows pseudo-first-order kinetics and exhibits a rate saturation with an apparentK _d of 3–4 mM. ATP, but not ribulose 5-phosphate, affords substantial protection. Complete inactivation correlates with incorporation of 1 mol of [¹⁴C]reagent per mole of enzyme subunit. Amino acid analysis of the [¹⁴C]-labeled enzyme demonstrates that only cysteine is modified, and mapping of tryptic digests shows that Cys55 is a major site of alkylation. These results indicate that Cys55 is also located in the ATP-binding domain of the active-site.     

Mapping of the bcl-2 oncogene on mouse chromosome 1

Two bcl-2 alleles have been identified in inbred strains of mice by restriction fragment length polymorphism (RFLP). Analysis of a bcl-2 RFLP in a series of bilineal congenic strains (C.D2), developed as a tool for chromosomal mapping studies, revealed linkage of bcl-2 to the Idh-1/Pep-3 region of murine chromosome 1. The co-segregation of bcl-2 alleles with allelic forms of two other chromosome 1 loci, Ren-1,2 and Spna-1, in a set of back-cross progeny, positions bcl-2 7.8 cM centromeric from Ren-1,2.     

Immunochemical identification of the serine protease inhibitor alpha 1-antichymotrypsin in the brain amyloid deposits of Alzheimer's disease

Two approaches--molecular cloning and immunochemical analysis--have identified one of the components of Alzheimer's disease amyloid deposits as the serine protease inhibitor alpha 1-antichymotrypsin. An antiserum against isolated Alzheimer amyloid deposits detected immunoreactivity in normal liver. The antiserum was then used to screen a liver cDNA expression library, yielding three related clones. DNA sequence analysis showed that these clones code for alpha 1-antichymotrypsin. Antisera against purified alpha 1-antichymotrypsin stained Alzheimer amyloid deposits, both in situ and after detergent extraction from brain. The anti-amyloid antiserum recognizes at least two distinct epitopes in alpha 1-antichymotrypsin, further supporting the presence of this protein in Alzheimer amyloid deposits. In addition to being produced in the liver and released into the serum, alpha 1-antichymotrypsin is expressed in Alzheimer brain, particularly in areas that develop amyloid lesions. Models by which alpha 1-antichymotrypsin could contribute to the development of Alzheimer amyloid deposits are discussed.     

Do holly leaf spines really deter herbivory?

Summary Although spinose teeth of holly leaves have been widely cited as an example of a physical defense against herbivores, this assumption is based largely on circumstantial evidence and on general misinterpretation of a single, earlier experiment. We studied the response of third and fifth instar larvae of the fall webworm, Hyphantria cunea Drury, a generalist, edge-feeding caterpillar, to intact American holly leaves and to leaves that had been modified by blunting the spines, by removing sections of leaf margin between the spines, or by removing the entire leaf margin. The results suggest that the thick glabrous cuticle and tough leaf margin of Ilex opaca are more important than the spinose teeth in deterring edge-feeding caterpillars. Microscopic examination of mature leaves revealed that the epidermis is thickened at the leaf margin, and that the leaf is cirucumscribed by a pair of fibrous veins. In simple choice tests neither domesticated rabbits nor captive whitetailed deer discriminated between spinescent holly foliage and foliage from which spines were removed. Nevertheless, we found little evidence of herbivory by mammals in the field, either on small experimental trees or in the forest understory. While it is possible that spinose teeth contribute to defense by reducing acceptibility of holly relative to other palatable plant species, we suggest that the high concentrations of saponins and poor nutritional quality of holly foliage may be more important than spines in deterring vertebrate herbivores. The degree of leaf spinescence and herbivory was compared at different heights with the tree canopy to test the prediction that lower leaves should be more spinescent as a deterrent to browsers. Leaves on lower branches of mature forest trees were slightly more spinescent than were upper leaves, and juvenile trees were slightly more spinescent than were mature trees. However, there was no relationship between degree of spinescence and feeding damage. The greater spinescence of holly leaves low in the canopy is probably an ontogenetic phenomenon rather than a facultative defense against browsers.The investigation reported in this paper (No. 87-7-8-77) is in connection with a project of the Kentucky Agricultural Experiment Station and is published with the approval of the Dirctor     

Isolation and partial characterisation of a Chinese hamster O6-alkylguanine-DNA alkyltransferase cDNA.

The cDNA encoding Chinese hamster O6-alkylguanine-DNA-alkyltransferase (ATase) has been isolated from a library prepared from RNA isolated from V79 lung fibroblasts which had an upregulated level of this repair activity following stepwise selection with a chloroethylating agent (1, 2). Expression of the cDNA in E. coli produced functionally active ATase at levels of 2.5% of total cellular protein as determined by in vitro assay. The recombinant hamster protein has a molecular weight of 28 kDa as estimated by SDS-PAGE and fluorography and this was identical to that in the upregulated cells. The characteristic PCHRV pentapeptide of the alkyl acceptor site has been identified and there is a 68 amino acid residue region which is 90% conserved across all the mammalian proteins so far analysed: in contrast, the N- and C-terminal domains diverge by as much as 50% between species. Polyclonal antibodies to the human and rat ATases hybridised to the hamster protein on western analysis suggesting at least one common epitope shared across species. However, in antibody inhibition experiments neither of the antisera cross reacted with the hamster ATase in a way which interfered with functional activity whereas the anti-human antibodies inhibited the human ATase and the anti-rat antibodies inhibited the rat and mouse ATases. There may therefore be significant tertiary structural differences between the hamster protein and the other mammalian ATases.