Two novel Streptomyces protein protease inhibitors. Purification, activity, cloning, and expression.

In contrast to the Gram-negative bacteria, Gram-positive bacteria such as Streptomyces lack a mucopolysaccharide cell wall which allows them to produce and secrete a variety of proteins directly into their environment. In an effort to understand and eventually exploit the synthesis and secretion of proteins by Streptomyces, we identified and characterized two naturally occurring abundantly produced proteins in culture supernatants of Streptomyces lividans and Streptomyces longisporus. We purified these 10-kDa proteins and obtained partial amino acid sequence information which was then used to design oligonucleotide probes in order to clone their genes. Analysis of the sequence data indicated that these proteins were related to each other and to several other previously characterized Streptomyces protein protease inhibitors. We demonstrate that both proteins are protein protease inhibitors with specificity for trypsin-like enzymes. The presumptive signal peptidase cleavage sites and subsequent aminopeptidase products of each protein are characterized. Finally, we show that the cloned genes contain all of the information necessary to direct synthesis and secretion of the proteins by Streptomyces spp. or Escherichia coli.     

Genetic and biochemical evidence for the lack of significant hydrolysis of soman by a Flavobacterium parathion hydrolase.

Pure recombinant Flavobacterium parathion hydrolase (an organophosphorus acid anhydrase) from Streptomyces lividans was found to hydrolyze the toxic nerve agent soman at only 0.1% of the rate observed with parathion as substrate. Studies with wild-type and recombinant strains of S. lividans support the lack of significant soman breakdown by the hydrolase and also indicate the presence in S. lividans of other significant hydrolytic enzymatic activity towards soman.     

Developmental changes to gut microflora metabolism in mice

W.E. BRENNAN-CRADDOCK, A.K. MALLETT, I.R. ROWLAND AND S. NEALE. 1992. Developmental changes in the activities of bacterial nitrate reductase, nitroreductase and β-glucuronidase and their response to fermentable dietary fibre, were investigated in caecal contents from suckling mice (2-week-old) and in mice aged 4–24 weeks fed either a purified fibre-free diet or that diet supplemented with 5% (w/w) pectin. There was no apparent age-related trend common to the three enzymes studied. Nitrate reductase activity in the mice fed the fibre-free diet did not markedly alter with age. Pectin administration, however, was associated with a significant increase in nitrate reductase activity, particularly in 4-week-old mice. Nitroreductase activity exhibited an overall upward trend in mice from 2 to 12 weeks and thereafter decreased. Caecal β-glucuronidase activity in mice increased sharply between 2 weeks and 4 weeks of age, thereafter not changing significantly until the 24th week. Pectin feeding had no consistent effect on activities either of nitroreductase or β-glucuronidase. The changes in enzyme activities with age were not related to the concentration of bacteria in the caecum, which was highest in the 2-week-old mice.
We conclude that the weaning is a period in which marked changes in caecal bacterial enzyme activities can occur.     

Influence of cold acclimation on membrane injury in frozen plant tissue

Cold-acclimated twigs of Amelanchier alnifolia Nutt. released less HCN at −4.5 C than nonacclimated twigs following slow freezing to −25 C or rapid freezing to −78 C. Cold-acclimated twigs frozen slowly to −25 C released more HCN than cold-acclimated twigs frozen only to −4.5 C. Cold-acclimated twigs frozen slowly to −25 C and then rapidly to −78 C released less HCN at −4.5 C than cold-acclimated twigs frozen rapidly to −78 C. In general, K⁺ efflux and the inability to reduce triphenyl tetrazolium chloride following freezing and thawing paralleled HCN release at −4.5 C. Because low K⁺ efflux and high triphenyl tetrazolium chloride reduction are known to depend upon membrane integrity, the increased K⁺ efflux and the decreased triphenyl tetrazolium chloride reduction following freezing and thawing provide indirect evidence that HCN release at −4.5 C is a measure of membrane damage in frozen cells.     

Substrates and inhibitors of hepatic amine oxidase inhibit dimethylnitrosamine-induced mutagenesis in Salmonella typhimurium

The mutagenic effect of dimethylnitrosamine in Salmonella typhimurium TA100, in the presence of a rat-liver homogenate derived from animals treated with Aroclor 1254, was inhibited by substrates and inhibitors of monoamine oxidase. Substrates of diamine oxidase did not inhibit dimethylnitrosamine mutagenesis and, furthermore, monoamine oxidase inhibitors had no effects on mutagenesis by benzo[a]pyrene or aflatoxin B₁. The results suggest that monoamine oxidase participates in the activation of dimethylnitrosamine to a mustagen.     

Current "corrected" calcium concept challenged.

There is wide individual variation in the number of millimoles of calcium bound per gram of albumin in the serum. Individual regression coefficients for serum calcium concentration on serum albumin concentration have been determined in 62 people (25 of our own patients and 37 reported by others). The 95 percentile range was 0-007-0-053 mmol/g, with a median value of 0-025 mmol/g. Accordingly, it is not valid to "correct" a person''s measured serum total calcium concentration for variations in serum albumin concentration using an average regression coefficient. Rather, the individual''s own regression coefficient must be used. A tourniquet test seems to be the simplest technique for determining this value. Even then, precise interpretation of an individual''s corrected serum calcium concentration is possible only when an appropriate reference range for corrected serum calcium concentration has been established. Such an appropriate reference range must be determined from an adequate number of normal people using the individual''s own correction factor in each case.