Antimicrobial activity of lysozyme against bacteria involved in food spoilage and food-borne disease.

Egg white lysozyme was demonstrated to have antibacterial activity against organisms of concern in food safety, including Listeria monocytogenes and certain strains of Clostridium botulinum. We also found that the food spoilage thermophile Clostridium thermosaccharolyticum was highly susceptible to lysozyme and confirmed that the spoilage organisms Bacillus stearothermophilus and Clostridium tyrobutyricum were also extremely sensitive. Several gram-positive and gram-negative pathogens isolated from food poisoning outbreaks, including Bacillus cereus, Clostridium perfringens, Staphylococcus aureus, Campylobacter jejuni, Escherichia coli O157:H7, Salmonella typhimurium, and Yersinia enterocolitica, were all resistant. The results of this study suggest that lysozyme may have selected applications in food preservation, especially when thermophilic sporeformers are problems, and as a safeguard against food poisoning caused by C. botulinum and L. monocytogenes.     

Characteristics of alcohol/polyol dehydrogenases. The zinc-containing long-chain alcohol dehydrogenases

Sixteen characterized alcohol dehydrogenases and one sorbitol dehydrogenase have been aligned. The proteins represent two formally different enzyme activities (EC 1.1.1.1 and EC 1.1.1.14), three different types of molecule (dimeric alcohol dehydrogenase, tetrameric alcohol dehydrogenase, tetrameric sorbitol dehydrogenase), metalloproteins with different zinc contents (1 or 2 atoms per subunit), and polypeptide chains from different kingdoms and orders (mammals, higher plants, fungus, yeasts). Present comparisons utilizing all 17 forms reveal extensive variations in alcohol dehydrogenase, but with evolutionary changes that are of the same order in different branches and at different times. They emphasize the general importance of particular residues, suggesting related overall functional constraints in the molecules. The comparisons also define a few coincidences between intron positions in the genes and gap positions in the gene products. Only 22 residues are strictly conserved; half of these are Gly, and most of the remaining ones are Pro or acidic residues. No basic residue, no straight-chain hydrophobic residues, no aromatic residues, and essentially no branched-chain or polar neutral residues are invariable. Tentative consensus sequences were calculated, defining 13 additional residues likely to be typical of but not invariant among the alcohol dehydrogenases. These show a predominance of Val, charged residues, and Gly. Combined, the comparisons, which are particularly relevant to the data base for protein engineering, illustrate the requirements for functionally important binding interactions, and the extent of space restrictions in proteins with related overall conformations and functions.     

Use of acivicin in the determination of rate constants for turnover of rat renal gamma-glutamyltranspeptidase

The specific enzymatic activity of renal gamma-glutamyltranspeptidase is decreased from control levels (0.86 unit-1 mg-1) to minimal values within 2 h postinjection of 100-g rats with acivicin, an irreversible inhibitor of the enzyme. The recovery of transpeptidase specific activity was followed from 2 to 24 h postinjection and the data were used to calculate the absolute rate constants for degradation (kd = 0.47 +/- 0.03 day-1) and synthesis (ks = 0.41 +/- 0.04 unit-1 mg-1 day-1). This corresponds to a half-life for the renal transpeptidase of 1.46 +/- 0.09 days and 99% recovery of the specific activity by 10 days postinjection. Recovery was followed for 14 days and closely approximates this theoretical curve. The data from control experiments designed to test for secondary effects of the drug, acivicin, show that neither the relative rate of synthesis nor apparent rate of degradation for either total protein or gamma-glutamyltranspeptidase is significantly altered by acivicin treatment of rats. The results also show that the acivicin-inhibited transpeptidase is not degraded differently than enzymatically active enzyme. The individual heterodimer subunits also exhibit similar apparent half-lives in both control and treated animals. Thus, recovery of renal gamma-glutamyltranspeptidase specific activity after acivicin treatment can be used in vivo to determine absolute values of ks and kd for this enzyme. These values have not been reported for any other constituent of the renal brush-border membrane.     

Yeasts in molecular biology. Spheroplast preparation withCanadida utilis,Schizosaccharomyces pombe andSaccharomyces cerevisiae

Efficient preparation of spheroplasts fromCandida utilis, Saccharomyces cerevisiae, andSchizosaccharomyces pombe, using a purified mixture of enzymes fromTrichoderma harzianum, is described. Limitations of other methods, and differences between yeasts are demonstrated.     

Kidney transplantation in Canada, 1981-84.

The Canadian Renal Failure Register was established in 1980. Data have been collected annually for all Canadian patients in whom irreversible kidney failure developed and who required dialysis or transplantation. The authors present actuarial patient and graft survival rates for 1981-84. In 1984, patients with a functioning renal graft accounted for 43.9% of the patients with end-stage renal disease. The number of transplants performed increased from 482 in 1981 to 662 in 1984; however, 1,022 patients undergoing dialysis (25.2%) were on an active waiting list for a transplant at the end of 1984. Greater effort is needed to increase the transplantation rate.     

Some metabolites of 1-bromobutane in the rabbit and the rat

1. Rabbits and rats dosed with 1-bromobutane excrete in urine, in addition to butylmercapturic acid, (2-hydroxybutyl)mercapturic acid, (3-hydroxybutyl)mercapturic acid and 3-(butylthio)lactic acid. 2. Although both species excrete both the hydroxybutylmercapturic acids, only traces of the 2-isomer are excreted by the rabbit. The 3-isomer has been isolated from rabbit urine as the dicyclohexylammonium salt. 3. 3-(Butylthio)lactic acid is formed more readily in the rabbit; only traces are excreted by the rat. 4. Traces of the sulphoxide of butylmercapturic acid have been found in rat urine but not in rabbit urine. 5. In the rabbit about 14% and in the rat about 22% of the dose of 1-bromobutane is excreted in the form of the hydroxymercapturic acids. 6. Slices of rat liver incubated with S-butylcysteine or butylmercapturic acid form both (2-hydroxybutyl)mercapturic acid and (3-hydroxybutyl)mercapturic acid, but only the 3-hydroxy acid is formed by slices of rabbit liver. 7. S-Butylglutathione, S-butylcysteinylglycine and S-butylcysteine are excreted in bile by rats dosed with 1-bromobutane. 8. Rabbits and rats dosed with 1,2-epoxybutane excrete (2-hydroxybutyl)mercapturic acid to the extent of about 4% and 11% of the dose respectively. 9. The following have been synthesized: N-acetyl-S-(2-hydroxybutyl)-l-cysteine [(2-hydroxybutyl)mercapturic acid] and N-acetyl-S-(3-hydroxybutyl)-l-cysteine [(3-hydroxybutyl)mercapturic acid] isolated as dicyclohexylammonium salts, N-toluene-p-sulphonyl-S-(2-hydroxybutyl)-l-cysteine, S-butylglutathione and N-acetyl-S-butylcysteinyl-glycine ethyl ester.     

O-linked glycosylation of rat renal gamma-glutamyltranspeptidase adjacent to its membrane anchor domain

Large domains rich in serine and threonine, that are likely to exhibit clusters of O-linked oligosaccharides, have been reported adjacent to the anchor of several cell surface proteins. No such domain is evident in the primary sequence of rat renal gamma-glutamyltranspeptidase. However, papain treatment of the amphipathic enzyme (Triton-purified gamma-glutamyltranspeptidase, T gamma GT), pretreated with galactose oxidase and NaB3H4 (Frielle, T., and Curthoys, N. P. (1983) Biochemistry 22, 5709-5714), yields the hydrophilic enzyme (papain-treated Triton-purified gamma-glutamyltranspeptidase, PT gamma GT) and a labeled peptide which contains both the amino-terminal membrane anchor and the sequence Pro27-Thr28-Thr29-Ser30. Since [3H]galactose was identified in this peptide, the presence of O-linked oligosaccharides was investigated. Carbohydrate analysis is consistent with the presence of two simple O-linked oligosaccharides on T gamma GT and one on PT gamma GT. Lectin blot analysis of T gamma GT and PT gamma GT was carried out after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The small subunits of both T gamma GT and PT gamma GT and the large amphipathic subunit of T gamma GT all react with the peanut agglutinin lectin, but the large subunit of PT gamma GT exhibits no such reactivity. The reactivity with PNA is consistent with the presence of one oligosaccharide with the structure galactose beta 1-3N-acetylgalactosamine alpha 1-Ser/Thr attached to each subunit of T gamma GT. The papain-sensitivity of the oligosaccharide from the larger subunit is consistent with O-glycosylation at the Thr28-Thr29-Ser30 sequence. The results of lectin blot analysis with wheat germ agglutinin imply that the content of N-linked oligosaccharides is unaffected by papain treatment of the transpeptidase. These data represent the first direct evidence for O-glycosylation of a microvillar hydrolase at a site immediately adjacent to the membrane anchor and indicates that even small clusters of Thr and Ser can be O-glycosylated. Isolated O-linked oligosaccharides may have functional significance since single Ser and Thr residues are consistently found near the membrane anchor of many cell surface proteins.