Discrimination between forms of vitamin E by humans with and without genetic abnormalities of lipoprotein metabolism.

To study the mechanisms of discrimination between various forms of vitamin E, four normal subjects, one patient with lipoprotein lipase deficiency, and three patients with abnormal apolipoprotein B-100 production were given an oral dose containing three tocopherols labeled with differing amounts of deuterium (2R,4'R,8'R-alpha-(5,7-(C2H3)2)tocopheryl acetate (d6-RRR-alpha-tocopheryl acetate), 2S,4'R,8'R-alpha-5-(C2H3)tocopheryl acetate (d3-SRR-alpha-tocopheryl acetate), and 2R,4'R,8'R-gamma-(3,4-2H)tocopherol (d2-RRR-gamma-tocopherol). The tocopherol contents of plasma, red cells, and lipoproteins were measured up to 76 h after the dose. In normal subjects all three tocopherols were absorbed and secreted in chylomicrons with equal efficiencies. Both d2-gamma- and d3-SRR-alpha-tocopherols peaked at similar concentrations in the other lipoprotein fractions, then decreased similarly, but 2-4 times more rapidly than did d6-RRR-alpha-tocopherol. A lipoprotein lipase-deficient patient and a patient with prolonged production of chylomicrons with absent apolipoprotein B-100 also demonstrated the lack of discrimination between tocopherols during absorption. Despite abnormal apolipoprotein B-100 production in two patients, the "VLDL" was preferentially enriched in d6-RRR-alpha-tocopherol. Our results show that there is no discrimination between the three tocopherols during absorption and secretion in chylomicrons, but subsequently there is a preferential enrichment of very low density lipoprotein (VLDL) with RRR-alpha-tocopherol. Catabolism of this VLDL results in the maintenance of plasma RRR-alpha-tocopherol concentrations.     

Ovulation and embryo recovery rates following immunization of mares against an inhibin alpha-subunit fragment

Six normally cycling mares were immunized 5 times at 3-week intervals with a synthetic porcine inhibin alpha-subunit fragment which had been conjugated to bovine serum albumin and emulsified in Freund's incomplete adjuvant. Immunized mares ovulated a significantly larger (P < 0.01) number of follicles per estrous cycle (2.8 +/- 1.1; range 1 to 8 ovulations) than 14 nonimmunized control mares (1.1 +/- 0.1; range 1 to 2 ovulations). Day-7 embryo recovery rates tended to be higher (P < 0.1) in immunized mares (1.6 +/- 0.5 embryos per flush) than in control mares (0.7 +/- 0.2 embryos per flush). No differences in interovulatory intervals were found between the 2 groups. These results indicate that immunization against inhibin may be useful in inducing development and ovulation of multiple follicles for embryo transfer in the mare.     

Selection of Dictyostelium discoideum transformants and analysis of vector maintenance using live bacteria resistant to G418.

A protocol that allows the rapid isolation and growth of large numbers of independent G418-resistant Dictyostelium discoideum transformant colonies on the surface of agar media with live bacteria was developed. Transformants grown under these conditions form normal fruiting bodies. Discovery that aggregation of nontransformants was inhibited at a nonselective level of G418 (25 to 35 micrograms/ml) led to the development of a vector maintenance assay. Using this assay we examined the stability of recombinant plasmids derived from the D. discoideum native plasmids Ddp1 and Ddp2. We conclude that the origin of replication of plasmid Ddp1 does not alone confer stable maintenance and thus, Ddp1 must bear additional sequences required for its own maintenance. Analysis of the maintenance of vectors derived from Ddp2 showed that autonomously replicating shuttle vectors that contained bacterial plasmid DNA and from which one element of the Ddp2 inverted repeat was removed were much less stable than vectors that contained a complete inverted repeat or that did not carry a bacterial plasmid. Sequences between the 3' end of the rep gene and the inverted repeat appear to play a role in plasmid maintenance. An intact rep gene and one copy of the inverted repeat element were required for extrachromosomal replication. Maintenance of extrachromosomal vectors was found to be strain dependent. Four traits distinguishing integrating vectors from those capable of autonomous replication were identified.     

Increases in p11 and annexin II proteins correlate with differentiation in the PC12 pheochromocytoma.

Regulation of p11 and annexin II by nerve growth factor, staurosporine, and epidermal growth factor was examined in PC12 rat adrenal pheochromocytoma cells using immunoblot analysis. Nerve growth factor, which is known to induce neurite outgrowth in PC12 cells, stimulated a five-fold increase in p11 and the higher levels of p11 were characteristic of PC12 cells exposed to nerve growth factor for up to ten days. Nerve growth factor induced an even greater increase (13.6-fold) in annexin II. Staurosporine, a protein kinase inhibitor that at high concentrations induces neurite formation, was as effective as nerve growth factor in increasing the intracellular levels of p11 and annexin II. Epidermal growth factor was less effective than nerve growth factor and staurosporine, producing only a two-fold increase in p11 and a three-fold increase in annexin II. The ineffectiveness of epidermal growth factor in increasing intracellular levels of p11 and annexin II is consistent with the fact that epidermal growth factor does not stimulate neurite outgrowth in PC12 cells. Evidence presented here suggests that p11 and/or annexin II may play a role in PC12 cell differentiation.     

A sectored colony assay for monitoring mutagenesis by specific carcinogen-DNA adducts in Escherichia coli.

To study the mutagenicity of various carcinogen-DNA adducts in Escherichia coli, a cassette plasmid was developed that permits positioning of specific carcinogen-modified bases within the ATG initiation codon of the lacZ' alpha-complementation gene. Adduct-induced mutations inactivate the gene and lead to formation of blue and white sectored colonies when transformants from an alpha-complementing version of E. coli strain AB1157 are grown on media containing 5-bromo-4-chloro-3-indolyl beta-D-galactoside. In the absence of mutation, blue colonies are produced. This system has been used to measure the mutagenicity of O6-methyl-, O6-ethyl-, and O6-benzyl-2'-deoxyguanosine residues incorporated in place of the normal 2'-deoxyguanosine of the ATG initiation codon. Although a low percentage of sectored colonies was produced in this repair-proficient strain, pretreatment of the bacteria with N-methyl-N'-nitro-N-nitrosoguanidine to disable DNA repair led to a dose-dependent increase in the percentage of sectored colonies. This percentage increased as a function of modified guanine in the order O6-benzyl- less than O6-methyl- less than O6-ethyl-2'-deoxy-guanosine. The only mutations detected at the site of incorporation of these O6-substituted guanines were G-to-A transitions. This sectored colony assay system permits convenient screening of large numbers of colonies and simplifies quantification of modified-base-induced mutations whether they be single-base changes, frameshifts, insertions, or deletions.     

Investigation of the mechanism of biosynthesis of 8-hydroxyeicosatetraenoic acid in mouse skin

One of the many changes induced by topical application of phorbol ester or calcium ionophore A23187 to mouse skin is the appearance of an enzymic activity which will convert arachidonic acid to its 8-hydroxyeicosatetraenoic acid metabolite (8-HETE) (Gschwendt, M., et al (1986) Carcinogenesis 7, 449-455). Induction of this activity is lower in strains of mice with a weak inflammatory response to TPA, and the 8-HETE may be involved in the inflammation or hyperplasia. To further characterize the activity, we first measured the chirality of the product; it is almost exclusively the 8DS)-hydroxy enantiomer (8S-HETE). The 8(S)-HETE is formed from octadeuterated arachidonic acid with complete retention of deuterium labels, indicating that a keto intermediate is not involved in the biosynthesis. Using arachidonic acids labeled with a prochiral tritium in either the 10DR or 10LS positions, we found that the biosynthesis of 8S-HETE is associated with the stereoselective abstraction of the 10DR hydrogen from the 10-carbon of the substrate. This stereoselective hydrogen removal conforms to the properties of an 8S-lipoxygenase. This is the only lipoxygenase known to catalyze solely 8S-oxygenation of arachidonic acid. The recent characterization of stereoselective biological effects for other HETEs serve as strong precedents to suggest that 8S-HETE has a specific role in the cellular tissue response to TPA.     

Evolutionary origin and diversification of the mammalian CD1 antigen genes.

CD1 antigens are cell-surface glycoproteins which have a molecular structure which is similar (consisting of extracellular domains alpha 1, alpha 2, and alpha 3, a transmembrane portion, and a cytoplasmic tail) to that of class I MHC molecules. Phylogenetic analysis of mammalian CD1 DNA sequences revealed that these genes are more closely related to the class I major histocompatibility complex (MHC) than to the class II MHC and that mammalian genes are more closely related to avian class I MHC genes than they are to mammalian class I MHC genes. The CD1 genes form a multigene family with different numbers of genes in different species (five in human, eight in rabbit, and two in mouse). Known CD1 genes are grouped into the following three families, on the basis of evolutionary relationship: (1) the human HCD1B gene and a partial sequence from the domestic rabbit, (2) the human HCD1A and HCD1C genes, and (3) the human HCD1D and HCD1E genes plus the two mouse genes and a sequence from the cottontail rabbit. The alpha 1 and alpha 2 domains of CD1 are much less conserved at the amino acid level than are the corresponding domains of class I MHC molecules, but the alpha 3 domain of CD1 seems to be still more conserved than the well-conserved alpha 3 domain of class I MHC molecules. Furthermore, in the human CD1 gene family, interlocus exon exchange has homogenized alpha 3 domains of all CD1 genes except HCD1C.     

MAP1B is encoded as a polyprotein that is processed to form a complex N-terminal microtubule-binding domain.

Microtubule-associated protein 1B (MAP1B), an abundant developmentally regulated neuronal protein, is a stoichiometric complex of a heavy chain and two light chains (light chain 1 and light chain 3). We find that light chain 1 is encoded within the 3' end of a previously reported MAP1B heavy chain cDNA. Amino acid sequencing, epitope mapping, Northern blotting, and Southern blotting indicate that the light chain and heavy chain are encoded by the same mRNA within the same open reading frame. In addition, amino acid sequencing of a 120 kd microtubule-binding and light chain-binding fragment of the heavy chain reveals that light chain 1 binds near the heavy chain N-terminus. Together these data indicate that the heavy chain and light chain 1 are produced by proteolytic processing of a MAP1B polyprotein and form a complex microtubule-binding domain.