Ghrelin O -acyltransferase (GOAT) attaches octanoate to proghrelin, which is processed to ghrelin, an octanoylated peptide hormone that stimulates release of growth hormone (GH) from pituitary cells. Elimination of the gene encoding ghrelin or its receptor produces only mild phenotypes in mice. Thus, the essential function of ghrelin is obscure. Here, we eliminate the Goat gene in mice, thereby eliminating all octanoylated ghrelin from blood. On normal or high fat diets, Goat −/− mice grew and maintained the same weights as wild-type (WT) littermates. When subjected to 60% calorie restriction, WT and Goat −/− mice both lost 30% of body weight and 75% of body fat within 4 days. In both lines, fasting blood glucose initially declined equally. After 4 days, glucose stabilized in WT mice at 58–76 mg/dL. In Goat −/− mice, glucose continued to decline, reaching 12–36 mg/dL on day 7. At this point, WT mice showed normal physical activity, whereas Goat −/− mice were moribund. GH rose progressively in calorie-restricted WT mice and less in Goat −/− mice. Infusion of either ghrelin or GH normalized blood glucose in Goat −/− mice and prevented death. Thus, an essential function of ghrelin in mice is elevation of GH levels during severe calorie restriction, thereby preserving blood glucose and preventing death.

The discovery of ghrelin O-acyltransferase (GOAT) opens the way to the design of drugs that block the attachment of an octanoyl group to the appetite-stimulating peptide hormone ghrelin, potentially preventing obesity. Here, we develop a biochemical assay that uses membranes from insect cells infected with baculovirus encoding mouse GOAT. The GOAT-containing membranes transferred the [ 3 H]octanoyl group from [ 3 H]octanoyl CoA to recombinant proghrelin in vitro. Transfer depended on the serine at residue 3 of proghrelin, which is the known site of acylation. GOAT also transferred [ 3 H]octanoyl to a pentapeptide containing only the N-terminal five amino acids of proghrelin. GOAT activity could be inhibited by an octanoylated ghrelin pentapeptide, and its potency was enhanced 45-fold when the octanoylated serine-3 was replaced by octanoylated diaminopropionic acid. The data suggest that GOAT is subjected to end-product inhibition and this inhibition is better achieved with substrates having the octanoyl group attached through an amide linkage rather than the corresponding ester. These insights may facilitate the future design of useful inhibitors of GOAT.appetite control ͉ in vitro ͉ inhibitors ͉ obesity ͉ octanoylation of proghrelin

Ghrelin, an octanoylated peptide hormone produced in the stomach, rises dramatically in mouse plasma during chronic severe calorie deprivation, an event that is essential to maintain life. The mechanism for this increase is not understood. Here, we study the control of ghrelin secretion in tissue culture cells derived from mice bearing ghrelinomas induced by a tissue-specific SV40 T-antigen transgene. We found that the ghrelin-secreting cells express high levels of mRNA encoding β 1 -adrenergic receptors. Addition of norepinephrine or epinephrine to the culture medium stimulated ghrelin secretion, and this effect was blocked by atenolol, a selective β 1 -adrenergic antagonist. When WT mice were treated with reserpine to deplete adrenergic neurotransmitters from sympathetic neurons, the fasting-induced increase in plasma ghrelin was blocked. Inhibition was also seen following atenolol administration. We conclude that ghrelin secretion during fasting is induced by adrenergic agents released by sympathetic neurons and acting directly on β 1 receptors on the ghrelinsecreting cells of the stomach.

Between November 1992 and February 1993, a large outbreak of Escherichia coli O157:H7 infections occurred in the western USA and was associated with eating ground beef patties at restaurants of one fast-food chain. Restaurants that were epidemiologically linked with cases served patties produced on two consecutive dates; cultures of recalled ground beef patties produced on those dates yielded E. coli O157:H7 strains indistinguishable from those isolated from patients, confirming the vehicle of illness. Seventy-six ground beef patty samples were cultured quantitatively for E. coli O157:H7. The median most probable number of organisms was 1.5 per gram (range, < 0.3-15) or 67.5 organisms per patty (range, < 13.5-675). Correlation of the presence of E. coli O157:H7 with other bacterial indicators yielded a significant association between coliform count and the presence of E. coli O157:H7 (P = 0.04). A meat traceback to investigate possible sources of contamination revealed cattle were probably initially colonized with E. coli O157:H7, and that their slaughter caused surface contamination of meat, which once combined with meat from other sources, resulted in a large number of contaminated ground beef patties. Microbiological testing of meat from lots consumed by persons who became ill was suggestive of an infectious dose for E. coli O157:H7 of fewer than 700 organisms. These findings present a strong argument for enforcing zero tolerance for this organism in processed food and for markedly decreasing contamination of raw ground beef. Process controls that incorporate microbiological testing of meat may assist these efforts.

The prevalence of Escherichia coli O157:H7 in dairy herds is poorly understood, even though young dairy animals have been reported to be a host. From February to May 1993, 662 fecal samples from 50 control herds in 14 states, and from June to August 1993, 303 fecal samples from 14 case herds in 11 states were collected for isolation of E. coli O157:H7. Case herds were those in which E. coli O157:H7 was isolated from preweaned calves in a previous U.S. Department of Agriculture study, whereas control herds from which E. coli O157:H7 had not been isolated previously were randomly selected from the same states as case herds. Among the control herds, E. coli O157:H7 was isolated from 6 of 399 calves (1.5%) that were between 24 h old and the age of weaning and from 13 of 263 calves (4.9%) that were between the ages of weaning and 4 months. Eleven of 50 control herds (22%) were positive. Among the case herds, E. coli O157:H7 was isolated from 5 of 171 calves (2.9%) that were between 24 h old and the age of weaning and from 7 of 132 calves (5.3%) that were between the ages of weaning and 4 months. Seven of 14 case herds (50%) were positive. Sixteen of 31 isolates were obtained by direct plating, with populations ranging from 10 3 to 10 5 CFU/g. Fifteen of 31 isolates were isolated by enrichment only. Nineteen of the isolates produced both verocytotoxin 1 (VT-1) and VT-2, whereas 12 produced VT-2 only.

Dairy cattle have been identified as a principal reservoir of Escherichia coli O157:H7. The fate of this pathogen in bovine feces at 5, 22, and 37؇C was determined. Two levels of inocula (10 3 and 10 5 CFU/g) of a mixture of five nalidixic acid-resistant E. coli O157:H7 strains were used. E. coli O157:H7 survived at 37؇C for 42 and 49 days with low and high inocula, respectively, and at 22؇C for 49 and 56 days with low and high inocula, respectively. Fecal samples at both temperatures had low moisture contents (about 10%) and water activities (<0.5) near the end of the study. E. coli O157:H7 at 5؇C survived for 63 to 70 days, with the moisture content (74%) of feces remaining high through the study. Chromosomal DNA fingerprinting of E. coli O157:H7 isolates surviving near the completion of the study revealed that the human isolate strain 932 was the only surviving strain at 22 or 37؇C. All five strains were isolated near the end of incubation from feces held at 5؇C. Isolates at each temperature were still capable of producing both verotoxin 1 and verotoxin 2. Results indicate that E. coli O157:H7 can survive in feces for a long period of time and retain its ability to produce verotoxins. Hence, bovine feces are a potential vehicle for transmitting E. coli O157:H7 to cattle, food, and the environment. Appropriate handling of bovine feces is important to control the spread of this pathogen.

The fate of enterohemorrhagic Escherichia coli O157:H7 was determined in three different lots of commercial mayonnaise, including four different samples from a lot implicated in an outbreak of E. coli O157:H7 infection. The initial pH of the products ranged from 3.6 to 3.9. Products were inoculated with 6.5 × 103 E. coli O157:H7/g and incubated at 5 or 20°C. Escherichia coli O157:H7 did not grow at either temperature but survived for 34 to 55 days at 5°C and for 8 to 21 days at 20°C, depending on the lot. Survival was greatest in real mayonnaise purchased at retail among six mayonnaise samples which included a reduced calorie mayonnaise. Escherichia coli O157:H7 populations decreased between 2- and 100-fold by 3 weeks at 5°C, and between 10- and 1,000-fold by 7 days at 20°C. There was little or no change in pH (&lt;0.1 unit), aerobic plate count, mold and yeast count or Lactobacillus count (&lt; 1 log10 CFU/g) for the duration of the study. Commercial mayonnaise manufactured under good manufacturing practices is not a public health concern. Abusive handling of mayonnaise resulting in cross-contamination with E. coli O157:H7-contaminated food or contamination by an infected foodhandler is the principal basis for concern.

A strain of enterohemorrhagic Escherichia coli serotype 0157:H7 isolated from a patient in an apple cider-related outbreak was used to study the fate ofE. coli 0157:H7 in six different lots of unpasteurized apple cider. In addition, the efficacy of two preservatives, 0.1% sodium benzoate and 0.1% potassium sorbate, used separately and in combination was evaluated for antimicrobial effects on the bacterium. Studies were done at 8 or 25°C with ciders having pH values of 3.6 to 4.0. The results revealed that E. coli 0157:H7 populations