The effect of inhibitors on the oxygen kinetics of terminal oxidases of Acanthamoeba castellanii.

1. Respiration of growing cultures of Acanthamoeba castellanii is inhibited less than 60% by azide (35 mM); the respiration of early-exponential-phase cultures differs from that of late-exponential-phase cultures in being stimulated by up to 120% by low concentrations (less than 1 mM) of this inhibitor. Azide (0.5 mM) plus 1 mM-salicylhydroxamic acid gives 80% inhibition of respiration in early- or late-exponential-phase cultures. 2. Lineweaver-Burk plots of 1/v against 1/[O2] for growing and stationary-phase cultures give values of less than 1 muM for the apparent Km for oxygen. 3. These values are not significantly altered when determined in the presence of 1 mM-salicylhydroxamic acid. 4. Higher values (greater than 7 muM) for apparent Km values for oxygen were obtained in the presence of azide, which gives non-linear Lineweaver-Burk plots. 5. Competitive inhibition of respiration by CO occurs with Ki 2.4 muM. 6. The results are discussed in terms of the presence of three terminal oxidases in this organism, namely two oxidases with high affinities for oxygen (cytochrome c oxidase of the main phosphorylating electron-transport chain and the salicylhydroxamic acid-sensitive oxidase) and a third oxidase with a low affinity for oxygen, sensitive to inhibition by cyanide but not by azide or salicylhydroxamic acid. The relative contributions to oxygen utilization by these oxidases change during the growth of a batch culture.     

Bleomycin fragmentation of duplex DNA occurs as staggered single-strand scissions.

Electron microscopy of purified full-length linear duplex molecules produced by bleomycin reaction with PM2 DNA revealed low frequencies of closed circular duplex molecules as well as linear duplex molecules with opposed ends (cyclized molecules which have dissociated to yield a gap between the termini). The occurrence of these latter forms indicates that double-strand scissions produced by bleomycin reaction consist of two single-strand scissions which are physically staggered on the complementary strands. Analysis of the temperature dependence for cyclization led to the estimate that an average of 1.7 +/- 0.44 base-pairs (2.6 +/- 0.5 base pairs without base-stacking energies) occur between the staggered breaks. The reassociated termini cannot be ligated with T4 ligase. When PM2 DNA was fragmented at several sites within each molecule, circular duplexes and linear duplexes with opposed ends with a range of sizes from 350 base pairs up to full-length PM2 DNA were observed. Analysis of the frequency distribution of lengths of these fragments indicates that most, if not all, of the specific sites for bleomycin-directed double-strand scissions in PM2 DNA contain representatives of the same two base single-stranded termini.     

The synthesis of 6-deoxy-6-fluoro-α,α-trehalose and related analogues

Selective acid-catalysed methanolysis of 2,3,2′,3′-tetra-O-benzyl-4,6:4′,6′-di-O-benzylidene-α,α-trehalose yielded the monobenzylidene derivative, which was converted into the 4,6-dimesylate. Selective nucleophilic displacement of the primary sulphonyloxy group then gave 2,3-di-O-benzyl-6-deoxy-6-fluoro-4-O-mesyl-α-d-glucopyranosyl 2,3-di-O-benzyl-4,6-O-benzylidene-α-d-glucopyranoside. Removal of the protecting groups then yielded 6-deoxy-6-fluoro-α,α-trehalose. In addition, 6-deoxy-6-fluoro-4-O-mesyl-α,α-trehalose and a derivative of 4-chloro-4,6-dideoxy-6-fluoro-α-d-galactopyranosyl α-d-glucopyranoside were also prepared from the same substrate. Iodide displacement of 2,3-di-O-benzyl-4,6-di-O-mesyl-α-d-glucopyranosyl 2,3-di-O-benzyl-4,6-di-O-mesyl-α-d-glucopyranoside afforded the 6-iodide and 6,6′-di-iodide in yields of 31 and 36%, respectively. Similarly, the 6-azide and 6,6′-diazide were isolated in yields of 17 and 21%, respectively.     

The synthesis of 1,6-anhydro-β-d-glucopyranose and d-glucosyl oligosaccharides from maltose by a fungal glucosyltransferase

The formation of 1,6-anhydro-β-d-glucopyranose and several d-glucosyl oligosaccharides has been observed during the action of a purified, fungal glucosyltransferase (EC 2.4.1.24) on maltose. Such products are synthesized by a transglucosylation mechanism involving the formation of a d-glucosyl-enzyme complex and the displacement of the d-glucosyl group by appropriate acceptor-substrates. The formation of the 1,6-anhydro bond is a novel type of transfer reaction and occurs by displacement of the enzyme from the d-glucosyl-enzyme complex by the proton of the primary hydroxyl group of the same glucosyl group. This reaction is characterized by inversion of configuration at the position of glucosidic bond-cleavage of the substrate. Synthesis of the d-glucosyl oligosaccharides occurs by displacement of the d-glucosyl groups from the enzyme by suitable acceptor-substrates. In these cases, the reactions are characterized by retention of configuration of the d-glucosidic bonds of the substrate. The list of oligosaccharides produced from maltose includes nigerose, kojibiose, isomaltose, maltotriose, panose, isomaltotriose, and 6-O-d-glucosyl-panose. The identity of these compounds has been established by methylation analysis and enzymic hydrolysis. d-Glucose is also a product of the reaction and arises from both the reducing and the non-reducing groups of maltose.     

Immunity to primary and challenge infections of Trichinella spiralis in mice: a re-examination of conventional parameters.

In young (6- to 8-week-old) NIH strain inbred mice expulsion of a primary infection of Trichinella spiralis began on day 8 and was virtually complete by day 11-5. In older mice expulsion occurred 1 or 2 days earlier. Experience of a primary infection elicited strong immunity to challenge, whether the challenge was given immediately after worm expulsion (day 14) or delayed (day 42). Challenge infections were expelled rapidly the majority of worms being lost during the first day. Immunity to challenge was elicited by low-level primary infections and was effective against large ventionally accepted parameters of immunity to T. spiralis in mice which, it is considered, are applicable only to mice with a genetically determined low-level of responsiveness to the parasite.     

Effects of prostaglandins and other drugs on the cyclic AMP content of cultured bone cells.

Prostaglandins of the E-series (PGE1 and PGE2) may be involved in disease-related, localized loss of bone. E-prostaglandins increase the cyclic AMP content of many cells; and, to determine if their effects on bone are mediated by cyclic AMP, we examined the effects of E-prostaglandins and of other agents on the cyclic AMP content of cultured bone cells. PGE2 produced a rapid, marked and dose-related increase in the cyclic AMP content of confluent monolayers of bone cells isolated from newborn rat calvaria. At 2.8 X 10(-6) M, PGE1 and PGE2 had approximately the same effect, while the effect of PGF2alpha was much less pronounced. In the presence of theophylline, PGE2 had a more marked effect than parathyroid hormone (PTH) and the combination of PGE2 and PTH had a synergistic effect. The divalent, cationic, ionophore, A23187, produced an increase in cellular cyclic AMP and had an additive effect in combination with PGE2. Synthetic salmon calcitonin (CT), which inhibits the bone resorptive effect of PGE2, increased cellular cyclic AMP and had an additive effect in combination with PGE2. A prostaglandin antagonist, SC-19220, partially inhibited the resorptive effect of PGE2 and reduced its effect on cellular cyclic AMP. The calcium antagonist, D600, inhibited the bone resorptive effects of PGE2 but had no effect on increased cellular cyclic AMP produced by PGE2. The marked effect of PGE2 on bone cell cyclic AMP suggests that this action is involved in the mechanism of PGE2-related bone loss. The fact that agents with different effects on PGE2-induced increases in cellular cyclic AMP can inhibit its resorptive actions, suggests that PGE2-induced changes in cyclic AMP may be related less to its resorptive actions than to its inhibitory effect on bone formation.     

Biosynthesis of candicidin by phosphate-limited resting cells ofStreptomyces griseus

Summary A phosphate-limited resting cell system ofStreptomyces griseus in a synthetic medium has been developed in which biosynthesis of the polyene macrolide, candicidin, is linear for at least 36 h without cell growth. Glucose and to a lesser degree sucrose, but not lactose, support antibiotic synthesis. Glucose is utilized at a constant rate for antibiotic synthesis without affecting mycelial dry weight. Acetate and propionate, the building units of the macrolide aglycone, stimulate candicidin biosynthesis in cultures supplemented with glucose but do not support its synthesis in the absence of glucose. Maximal stimulation of candicidin biosynthesis was produced by 40 mM propionate or 250 mM acetate. The biosynthetic intermediate, methyl malonate, and the analog, 1-propanol, were more stimulatory than propionate at the same concentration.     

The agar-type polysaccharide from the red alga Ceramium rubrum

Aqueous extraction of the red alga C. rubrum gave a galactan sulphate and, possibly, a separate glucan and xylan. The galactan sulphate has an alternating structure of the agar-type with D-galactose or 6-O-methyl-D-galactose as one alternating unit, and L-galactose, 3,6-anhydro-L-galactose; and their respective 2-methyl ethers as the other unit. Sulphate hemi-ester groups are present on position 6 of both D- and L-galactose residues, with smaller amounts on positions 2 and 4 of, probably, D-galactose residues. The polysaccharide differs from others previously examined in that most of the L-galactose residues are non-sulphated.     

The reaction of phosphorus pentachloride with amides,in particular 2-acetamido-2-deoxyaldohexopyranoses

2-acetamido-2-deoxyaldohexopyranose polyacetates are transformed by the action of phosphorus pentachloride into 2-tetrachloroethylideneamino derivatives, the trans-2-acetamido-l-acetate system reacting more rapidly than the cis. A 1-acetamido-pyranosyl polyacetate afforded the 1-tetrachloroethylideneamino derivative and 2-O-trichloroacetyl-β-D-glucopyranosyl chloride. The latter was also observed, amongst other products, from the reaction of β-D-glucopyranosyl azide tetra-acetate with phosphorus pentachloride. Similar reactions on acetamidocyclohexane and its 2-acetoxy derivative afforded dichloroacetamido, trichloroacetamido, and tetrachloroethylideneamino derivatives. Likewise, 1-acetamido-2-acetoxyethane gave the 1-dichloroacetamido derivative.