Modified eggs are compatible with a diet that reduces serum cholesterol concentrations in humans.

The National Cholesterol Education Program (NCEP) guidelines recommend dietary restriction of fat and cholesterol to reduce high circulating cholesterol concentrations in adult Americans. Thus, diet counselors recommend consumption of fewer than four egg yolks per week. The present protocol was designed to determine whether the efficacy of an NCEP diet would be reduced by the incorporation of 12 modified eggs per week, and whether the resulting low fat, high cholesterol diet would increase serum lipid concentrations in adults with initial undesirably high (5.17-7.76 mmol/L) concentrations of serum total cholesterol. Feeding a controlled ration to laying hens produced modified eggs that consistently contained more vitamin E and iodine, and more unsaturated fat, than generic eggs. Subjects were randomly assigned to and NCEP diet including either no whole eggs or 12 whole study eggs a week. Ninety-eight subjects completed the parallel study. Subjects in both groups significantly reduced their serum total, LDL and HDL cholesterol (P < 0.001 for total and LDL cholesterol, P < 0.02 for HDL cholesterol) over the 6 wk of study. No significant differences were found between diet groups. We conclude that the study eggs did not adversely affect measured lipid concentrations when added to a low fat diet that favorably alters lipid profiles in hypercholesterolemic subjects.

Purification and partial sequence analysis of a 37-kDa protein that inhibits phospholipase A2 activity from rat peritoneal exudates.

We have purified from rat peritoneal exudates a 37-kDa protein that inhibits phospholipase A2 activity. It is the predominant phospholipase inhibitor protein in these preparations and also is detected in a wide variety of cell lines. Levels of expression range from 0 to 0.5% of total protein. In the peritoneal preparations, the inhibitor is partially proteolyzed into a series of lower mass forms, including species at 30, 24, and 15 kDa. These fragments all are immunoreactive with an antibody raised against the 37-kDa protein. The rat protein also is immunoreactive with an antibody developed against a 6-kDa phospholipase inhibitor protein from snake venom. The primary structure of more than half of the rat inhibitor has been deduced by protein microsequence analysis. These sequences are closely related to sequences from its human analogue, which we recently cloned and expressed (Wallner, B. P., Mattaliano, R. J., Hession, C., Cate, R. L., Tizard, R., Sinclair, L. K., Foeller, C., Chow, E. P., Browning, J. L., Ramachandran, K. L., and Pepinsky, R. B. (1986) Nature, in press), and thus we infer that the inhibitor is highly conserved evolutionarily. Properties of the molecule suggest that it is a member of a family of steroid-induced anti-inflammatory proteins collectively referred to as lipocortin.

D-ribose metabolism in Escherichia coli K-12: genetics, regulation, and transport.

We have isolated mutants defective in high-affinity D-ribose transport. The mutations map in rbsT or rbsB , the structural gene for ribose binding protein. rbsT consists of at least one gene coding for a protein required for high-affinity transport. The high-affinity transport-defective mutants were able to utilize D-ribose, indicating that at least a second, low-affinity transport system for D-ribose is present in Escherichia coli K-12. rbsT and rbsB are located at min 84 on the E. coli genetic map and, together with rbsK , the gene coding for ribokinase , constitute an rbs operon. The order of genes is rbsP /O rbsT rbsB rbsK . The rbs operon is subject to negative control by the product of the rbsR gene. rbsR is located distal to the rbs operon and appears to form a separate transcriptional unit.

The use of extragenic suppressors to define genes involved in protein export in Escherichia coli.

The secA gene codes for a membrane component involved in protein export in E. coli. In order to define other genes whose products play such a role, we have characterized extragenic suppressors of a secA(Ts) mutation. These suppressors fall into at least three genetic loci. One such locus is the prlA gene, previously identified by mutations which suppress signal sequence mutants. Thus, this approach may allow the identification of new genes involved in the export process.

Genetic and biochemical analyses of Escherichia coli mutants altered in the temperature-dependent regulation of membrane lipid composition.

We have previously studied two mutants of Escherichia coli altered in the regulation of membrane lipid composition by temperature. One class (represented by the fabFl allele) fails to regulate upon temperature shift and is defective in cis-vaccenic acid synthesis owing to the lack of the fatty acid elongation enzyme beta-ketoacyl-acyl carrier protein synthase II(EC 2.3.1.41). A second class of mutant, given the phenotypic designation Vtr, overproduces cis-vaccenic acid at all temperatures and hence is altered in temperature regulation. In this paper we report evidence for the following conclusions. (i) The Vtr and fabFl mutations show very tight genetic linkage. (ii) The Vtr lesion is allelic to the fabFl mutation since the presence of the fabFl mutation in merodiploid strains carrying the Vtr or fabF(+) alleles results in fatty acid compositions intermediate between those of the two monoploid strains. Merodiploids carrying both the fabF(+) and Vtr alleles likewise show an intermediate composition. These results indicate intra-allelic complementation. (iii) The two E. coli proteins recently discovered by Rock (J. Bacteriol. 152:1298-1300, 1982) that form mixed disulfide cross-links to acyl carrier protein are directly demonstrated to be beta-ketoacyl-acyl carrier protein synthases I and II. (iv) The fabFl strains produce a synthase II band of altered electrophoretic mobility, indicating that the fabF locus is the structural gene for synthase II. (v) The synthase II of Vtr strains is abnormally sensitive to cerulenin, an antibiotic that specifically inhibits synthases I and II. This increased sensitivity is readily demonstrated in vivo, but in vitro we failed to detect an increased sensitivity of the Vtr synthase II to cerulenin, nor have we detected any other kinetic or structural alteration in the enzyme. We interpret these results in terms of specific interactions of synthase II with other cellular components which occur in vivo but are not duplicated in vitro.

Overproduction of cis-vaccenic acid and altered temperature control of fatty acid synthesis in a mutant of Escherichia coli.

A mutant of Escherichia coli has been characterized which overproduces cis-vaccenic acid in the temperature range of 30 to 42 degrees C. The mutational lesion acts within the fatty acid biosynthetic pathway rather than at the level of fatty acid incorporation into phospholipid.

Secretion and processing of ribose-binding protein in Escherichia coli.

The periplasmic D-ribose-binding protein of Escherichia coli K-12 is made initially as a larger precursor form. This precursor was observed in wild-type cells and more stably in cells inhibited for protein secretion. The precursor could be processed to the mature D-ribose-binding protein either co-or posttranslationally. The secretion pathway of the D-ribose-binding protein and that of the maltose-binding secretion have many characteristics in common.

Structural, enzymatic, and genetic studies of beta-ketoacyl-acyl carrier protein synthases I and II of Escherichia coli.

Beta-Ketoacyl-acyl carrier protein synthases I and II of Escherichia coli were purified and characterized. Synthase I was shown to have a molecular weight of 80,000 +/- 5,000 and to be composed of two similarly sized subunits. Synthase II had a molecular weight of 85,000 +/- 5,000 and also was apparently homodimeric. Gel electrophoresis of partial proteolytic digests demonstrated that synthases I and II share few if any common peptides. Synthases I and II also were shown to be unrelated by immunological criteria. An improved assay for beta-ketoacyl-acyl carrier protein synthase activity gave kinetic parameters for synthases I and II at both 27 degrees C and 37 degrees C using five long chain acyl-acyl carrier protein substrates. The properties of synthase II are consistent with the proposed role of this enzyme in the modulation of fatty acid synthesis by temperature. fabF mutants of E. coli lack synthase II. The fabF locus was mapped at min 24.5 of the E. coli genetic map and the clockwise map order was found to be pyrC, fabD, fabF, purB.

Beta-ketoacyl-acyl carrier protein synthase II of Escherichia coli. Evidence for function in the thermal regulation of fatty acid synthesis.

Cvc- mutants of Escherichia coli are deficient in the synthesis of cis-vaccenic acid and in the temperature control of fatty acid synthesis. In this communication, it is demonstrated that these mutants lack beta-ketoacyl-acyl carrier protein synthase II. The deficiencies in cis-vaccenate synthesis and synthase II are shown to be due to a lesion in the same gene, fabF. Lesions in the fabF gene are found to affect growth only when the strain also carries a lesion in the fabB gene, the structural gene for beta-ketoacyl-acyl carrier protein synthase I.

Thermal modulation of fatty acid synthesis in Escherichia coli does not involve de novo enzyme synthesis.

An increased ratio of unsaturated to saturated fatty acids was synthesized within 30 s after shift of Escherichia coli K-12 from 42 degrees C to 24 degrees C. This was more than 10-fold faster than the induction of beta-galactosidase. Inhibition of ribonucleic acid or protein synthesis had no effect on the response of fatty acid synthesis to temperature shift.

Preparative enzymatic synthesis and hydrophobic chromatography of acyl-acyl carrier protein.

We have used purified preparations of acyl-acyl carrier protein synthetase to prepare pure, native acyl-acyl carrier proteins (acyl-ACP) ranging in chain lengths from C10:0 to C delta 9 18:1. Factors affecting yield are explored and reaction conditions are presented that yield 0.8 to 0.9 mg of C16:0-ACP/ml of reaction mix. Ohter acyl groups, such as C10:0 and C delta 9 18:1 are poorer substrates and gave correspondingly lower yields. Acyl-Acp synthetase may be recovered from the reaction mixture using blue-Sepharose CL-6B and recycled. ACP and acyl-ACP are separated by hydrophobic chromatography on octyl-Sepharose CL-4B. Mixtures of acyl-ACPs could be resolved according to acyl chain length using octyl-Sepharose CL-4B columns eluted with a 2-propanol gradient. The high resolution obtained using 2-propanol gradients to separate acyl-ACP species suggests that similar techniques would be applicable to the chromatography of protein mixtures on hydrophobic supports.