The effects of N-6 polyunsaturated fatty acid supplementation on the lipid composition and atherogenesis in mouse models of atherosclerosis.

Despite numerous studies, the precise role of dietary n-6 polyunsaturated fatty acids in the pathogenesis of atherosclerosis remains controversial. It has been shown that feeding an n-6-enriched diet resulted in decreased atherosclerosis in African green monkeys and was associated with a reduction in LDL levels. However, other authors reported that n-6 supplementation increased the oxidative stress and the susceptibility of LDL to undergo in vitro oxidation, thus potentially enhancing atherosclerosis. The present study was designed to investigate the effect of dietary supplementation of n-6 polyunsaturated fats (safflower oil), as compared with a saturated fat-rich diet (Paigen), on the blood lipid profile and atherosclerosis in two mouse models. In the first experiment, female C57BL/6 mice (n=23-30 per group) were fed a cholate containing Paigen diet, a safflower oil-rich diet (with cholate), or normal chow for 15 weeks. No significant differences between the high fat diet groups were evident with respect to total cholesterol, LDL, HDL or triglyceride levels. The extent of aortic sinus fatty streaks did not differ significantly between the two groups. In the second experiment, LDL-receptor-deficient (LDL-RD) mice (n=20-30 per group) were randomized into similar dietary regimens. Mice consuming a safflower oil-enriched diet developed significantly less atherosclerosis, in comparison with Paigen diet-fed mice. A reduction in LDL levels, although not of a similar magnitude as the reduction in atherosclerosis, was evident in the safflower oil-fed mice when compared to the Paigen diet-fed littermates. In both mouse models of atherosclerosis, LDL isolated from the plasma of mice on the n-6 polyunsaturated diet was rendered slightly more susceptible to oxidation in vitro, as indicated by a shorter lag period for diene formation. Thus, the effects of n-6 fatty acids on the lipoprotein composition and other potential influences may have contributed to the anti-atherogenic effect in the LDL-RD mouse model.

Induction of p34cdc2 in mouse parotid glands upon activation of beta1-adrenergic receptors.

Dramatic morphological, biochemical and cytological changes occur in parotid glands of rats and mice which have been treated with the beta-adrenergic receptor agonist isoproterenol (Ipr). Changes include glandular hypertrophy, induction of tissue-specific proline-rich proteins (PRPs), increases in cAMP, and occurrence of polyploidy. Similar changes also occur after feeding mice polyphenolic compounds commonly referred to as tannins. Data are presented which show that changes in cell cycle proteins are due to stimulation of the beta-adrenergic receptor by either isoproterenol or tannin treatment of mice. Both p34cdc2 mRNA and protein levels were elevated dramatically after mice were treated. Induction of p34cdc2 occurred within 24 hrs. and was transient during treatment. The beta1-adrenergic receptor antagonist atenolol blocked both tissue-specific expression of proline-rich proteins and induction of p34cdc2. Coincident with the increase in p34cdc2, cyclin-dependent kinase complexes containing cyclins A and B increased forty- and ten-fold, respectively. These results show that in mouse parotid glands activation of the beta1-adrenergic receptor by either the administration of isoproterenol or ingestion of dietary tannins induces synthesis of p34cdc2 and most likely contributes to the occurrence of polyploidy.

A 1.1-Mb transcript map of the hereditary hemochromatosis locus.

In the process of positionally cloning a candidate gene responsible for hereditary hemochromatosis (HH), we constructed a 1.1-Mb transcript map of the region of human chromosome 6p that lies 4.5 Mb telomeric to HLA-A. A combination of three gene-finding techniques, direct cDNA selection, exon trapping, and sample sequencing, were used initially for a saturation screening of the 1.1-Mb region for expressed sequence fragments. As genetic analysis further narrowed the HH candidate locus, we sequenced completely 0.25 Mb of genomic DNA as a final measure to identify all genes. Besides the novel MHC class 1-like HH candidate gene HLA-H, we identified a family of five butyrophilin-related sequences, two genes with structural similarity to a type 1 sodium phosphate transporter, 12 novel histone genes, and a gene we named RoRet based on its strong similarity to the 52-kD Ro/SSA lupus and Sjogren's syndrome auto-antigen and the RET finger protein. Several members of the butyrophilin family and the RoRet gene share an exon of common evolutionary origin called B30-2. The B30-2 exon was originally isolated from the HLA class 1 region, yet has apparently "shuffled" into several genes along the chromosome telomeric to the MHC. The conservation of the B30-2 exon in several novel genes and the previously described amino acid homology of HLA-H to MHC class 1 molecules provide further support that this gene-rich region of 6p21.3 is related to the MHC. Finally, we performed an analysis of the four approaches for gene finding and conclude that direct selection provides the most effective probes for cDNA screening, and that as much as 30% of ESTs in this 1.1-Mb region may be derived from noncoding genomic DNA.

Six amino acids from the retroviral gene gag greatly enhance the transforming potential of the oncogene v-erb-B.

Avian erythroblastosis virus (AEV) induces both erythroblastosis and fibrosarcomas in susceptible birds and transforms the corresponding cells in culture. Neoplastic transformation by AEV is mediated principally by an oncogene known as v-erb-B. We have explored the means by which this gene is expressed from the genome of AEV and uncovered an important structural determinant for the potency of the oncogene. In order to define the boundaries of v-erb-B and the supplementary oncogene, v-erb-A, we sequenced all but a small portion of the genome of the ES4 strain of AEV. We then demonstrated that, during expression in infected cells, splicing fuses the first six amino acids of the retroviral gene gag to the body of the v-erb-B protein. In order to explore the impact of this fusion on the function of v-erb-B, we constructed vectors with Murine Leukemia Virus that express the oncogene either with or without the fusion to gag. Viruses generated from these two vectors differed greatly in their abilities to transform cells: fusion of v-erb-B with gag enhanced its transforming ability 50 to 100-fold as determined by focus transformation assays and growth in soft agar. Our data suggest that the difference in transforming ability is not due to alterations in transcription or translation but, rather, may result from changes in post-translational modification.