Apolipoprotein E and diets: a case of gene-nutrient interaction?

Apolipoprotein E has key functions in lipoprotein metabolism, and polymorphisms in the apolipoprotein E gene are associated with distinct lipoprotein patterns. The possibility of gene-nutrient interactions for apolipoprotein E has been addressed in many studies. Although results have generally been mixed, the indications for such an interaction have been more common in studies employing a metabolic challenge. Studies directly designed to examine apolipoprotein E gene-nutrient interactions are needed.     

Role of the 2-amino group of deoxyguanosine in sequence recognition by EcoRI restriction and modification enzymes.

The dG residues within the EcoRI recognition sequence of ColE1 DNA have been selectively replaced with dI. Methylation of the altered sequence by the EcoRI modification enzyme is extremely slow as compared with methyl transfer to the natural recognition site. Since the affinity of the modification enzyme for the dI-containing sequence is considerably less than that for the natural sequence, we have concluded that the 2-amino group of dG has an important role in DNA site recognition by this enzyme. In contrast, the altered site is subject to cleavage by EcoRI endonuclease at rates essentially identical with those observed with the natural sequence. These results strongly suggest that the two enzymes utilize different contacts within the EcoRI site and are consisted with our conclusion (Rubin, R. A., and Modrich, P. (1977) J. Biol. Chem. 252, 7265-7272) that the two proteins interact with their common recognition sequence in different ways.     

The differential protection by WR2721 of skin versus growing cartilage following irradiation in weanling rats

The potential for radioprotection of growing cartilage by the thiophosphate WR2721 was evaluated in weanling rats using single fractions of irradiation. Protection of acute skin toxicity was monitored simultaneously. Single doses of 600, 1200, 1800, or 2400 cGy were administered to the left tibia of CrL:CD(SD)BR female rats in groups of 12. Identically treated groups were injected with 310 mg/kg WR2721 (2/3 the determined LD50/30) in a concentration of 26 mg/ml intraperitoneally 15 min prior to irradiation. Rats untreated or given WR2721 without radiation served as control groups. Radiographs of the irradiated and unirradiated tibiae for each animal were obtained weekly to the date of sacrifice at 80 days following the initial treatment. Skin toxicity was assessed weekly starting on the second week using Moulder's scale (J.E. Moulder, J.J. Fischer, and A. Casey, Radiology 115, 465-470 (1975]. No significant difference in bone growth as measured by tibial lengths for the WR2721-treated or untreated animals was observed. Skin toxicity including moist desquamation occurred in irradiated limbs and was substantially less in rats treated with WR2721. As opposed to previous work with cysteamine, WR2721 as administered had no significant radioprotective effect on tibial growth in weanling rats but substantially reduced the accompanying skin toxicity.     

The effects of fatty acids on phosphoinositide synthesis and myo-inositol accumulation in exocrine pancreas

The effects of arachidonic acid (20:4) on phosphoinositide turnover were examined in rat pancreatic acinar cells prelabeled with myo-[3H]inositol. Arachidonic acid (50 microM) increased the accumulation of myo-[3H]inositol, but not that of [3H]inositol monophosphate, [3H]inositol bisphosphate, or [3H]inositol trisphosphate. By contrast, 10 microM carbamoylcholine increased the accumulation of all four compounds. A combination of arachidonic acid plus carbamoylcholine caused a selective and marked accumulation of myo-[3H]inositol, which was abolished by 10 mM LiCl. Arachidonic acid (10-100 microM) produced a concentration-dependent inhibition of myo-[3H]inositol incorporation into phosphoinositides and markedly depressed carbamoylcholine-induced increases in myo-[3H]inositol incorporation into inositol phospholipids. Several other unsaturated and saturated fatty acids failed to elicit a synergistic response with carbamoylcholine in stimulating myo-[3H]inositol accumulation and did not retard the incorporation of myo-[3H]inositol into phosphoinositides. The fact that eicosapentaenoic acid (20:5), but not arachidic acid (20:0), mimicked the depressant effect of arachidonate on phosphoinositide labeling suggests that the degree of unsaturation of the fatty acid, rather than chain length, is important for inhibition of phosphoinositide synthesis. The arachidonate-induced decrease in myo-[3H]inositol incorporation was accompanied by a reduction in the steady state level of [32P]phosphatidylinositol 4,5-bisphosphate. The mass of arachidonic acid liberated in response to carbamoylcholine was measured by gas chromatography-mass spectrometry, and the time course of stimulated arachidonate accumulation paralleled that of inositol phosphate accumulation and amylase release. These observations suggest that in exocrine pancreas, endogenous arachidonic acid serves as a negative feedback regulator of phosphoinositide turnover.     

A direct evidence for the involvement of poly(A) in protein synthesis

A radioactive polyadenylated globin mRNA was translated in either rabbit reticulocyte lysate or wheat germ extract under various conditions. When globin mRNA was translated, globin synthesis was directly proportional to the rate of loss in A units from the poly(A) tail. On the other hand, when globin poly(A) mRNA was incubated under non-translated conditions, no loss of A units was detected. The presence of ribonuclease inhibitor in the reaction mixture did not alter either the rate of globin synthesis or the loss in A units from the poly(A) tail. The present data suggests a correlation between protein synthesis and loss in A units from the poly(A) tail.     

Developmental regulation of the human zeta globin gene in transgenic mice.

We have characterized the expression of the human zeta (zeta) gene, which encodes an embryonic alpha-like globin, in transgenic mice. We find that a 777 base pair fragment spanning erythroid specific hypersensitive site II (HSII) from the distal 5. region of the human beta globin gene cluster potentiates expression of the zeta globin gene. In the absence of the HSII fragment, no zeta expression is observed. Expression of the human zeta gene in mice parallels expression of a murine embryonic alpha-like globin gene (x). Thus, expression of the human zeta gene in mice requires linkage to an erythroid-specific enhancer sequence, but the presence of the enhancer does not affect the developmental regulation of the transgene. Our results indicate that the factors involved in switching from embryonic to adult alpha globin gene expression during development are evolutionarily conserved, and suggest that the transgenic mouse is an in vivo system in which the requirements for the developmental switch in alpha globin gene expression can be analyzed in detail.     

Gene duplication in the evolution of the two complementing domains of gram-negative bacterial tetracycline efflux proteins

The resistance of Gram- bacteria to the broad-spectrum antibiotic tetracycline (Tc) results from energy-dependent drug efflux mediated by the tet gene product, the cytoplasmic membrane Tet protein. Amino acid (aa) sequences deduced from total tet nucleotide sequences of three different resistance determinants (classes A, B and C) indicate that the protein products [Tet(A), Tet(B), and Tet(C)] share a common ancestor. Hydropathic analysis of Tet sequences predicts twelve transmembrane segments in each protein, with six occurring in each half of the molecule. More importantly, the linear distributions of these segments in the N- and C-terminal halves are nearly identical, suggesting that the two halves of each Tet protein are related by a process of tandem gene duplication and divergence. Indeed, a variable but significant conservation of sequence was detected among the N- and C-terminal halves for all possible comparisons of the three proteins. Such conservation was not observed within other prokaryotic integral membrane proteins or when other prokaryotic proteins were compared to Tet halves. Similarity, both in sequence and in predicted transmembrane structural organization, strongly suggests that a common ancestor of Tet(A), Tet(B), and Tet(C) arose by duplication of a gene reading frame specifying a transmembrane protein of approximately 200 aa residues. The two halves of Tet proteins correspond to the two domains, alpha and beta, which have distinct, complementary roles in Tc efflux. Nevertheless, selective constraints to function in the cytoplasmic membrane have apparently led to maintenance of similar patterns of secondary structural organization in these complementary domains.