Macrophage catabolism of lipid A is regulated by endotoxin stimulation

Lipopolysaccharide (LPS) is a Gram-negative bacterial glycolipid that is believed to cause, by virtue of its stimulatory actions on macrophages and other eukaryotic cells, the life-threatening symptoms associated with Gram-negative infections. Macrophages both respond to and catabolically deactivate LPS. The lipid A moiety of LPS is responsible for the stimulatory actions of LPS on macrophages. We have previously developed methods employing a radiolabeled bioactive lipid A precursor, 4'-32P-lipid IVA, to study the interaction of this class of lipids with animal cells (Hampton, R. Y., Golenbock, D. T., and Raetz, C. R. H. (1988). J. Biol. Chem. 263, 14802-14807). In the current work, we have examined the uptake and catabolism of 4'-32P-lipid IVA by the RAW 264.7 cell line in serum-containing medium at physiological temperatures and have studied the effect of LPS stimulation on the ability of these cells to catabolize lipid IVA. RAW 264.7 macrophage-like cells avidly take up 4'-32P-lipid IVA under cell culture conditions at nanomolar concentrations. Uptake of lipid IVA was accompanied by lysosomal dephosphorylation of a fraction of the lipid to yield 4'-monophosphoryl lipid IVA. Chemically generated 4'-monophosphoryl lipid IVA was found to be substantially less bioactive than lipid IVA in the RAW cell, indicating that this catabolic dephosphorylation results in detoxification. In uptake experiments of 3-4 h duration, all metabolism of lipid IVA is blocked by ligands of the macrophage scavenger receptor. In longer experiments (24 h), both scavenger receptor-dependent and -independent uptake are responsible for the lysosomal catabolism of lipid IVA. Preincubation of RAW 264.7 cells with LPS caused dose-dependent inhibition of lipid IVA dephosphorylation. Sufficient LPS stimulation resulted in essentially complete inhibition of lipid IVA catabolism in both short- and long-term uptake experiments. This effect occurred at physiologically relevant concentrations of LPS (IC50 less than 1 ng/ml), and our data indicate that LPS-induced blockade of lipid IVA catabolism was due to the resultant physiological stimulation of the cells, and not inhibition of dephosphorylation by competition for uptake or enzymatic sites or by simple sequestration of labeled lipid IVA by LPS aggregates. We suggest that in the macrophage, LPS can modulate its own catabolism by virtue of its pharmacological properties. This effect of LPS could play a role in LPS pathophysiology as well as in macrophage biology.     

Substratum preference of the caddisfly Helicopsyche borealis (Hagen) (Trichoptera: Helicopsychidae)

The position of Helicopsyche borealis (Hagen) (Trichoptera: Helicopsychidae) larvae on the substratum surface is dependent on the current regime but varies with larval size. All size classes of larvae chose significantly different positions on the substratum under high versus low current velocities. All size classes preferred exposed surfaces under low current velocities. Small larvae preferred the upper surfaces of substrata under low current velocities and were physically displaced under high current velocities. Larger larvae also occurred on upper surfaces, but were more evenly dispersed over all surfaces than smaller larvae, and tended to aggregate on down-stream faces of rocks during high flow.     

Explanation for the apparent inefficiency of reduced nicotinamide adenine dinucleotide in energizing amino acid transport in membrane vesicles

Lineweaver-Burk plots of reduced nicotinamide adenine dinucleotide (NADH) oxidation by membrane preparations from Bacillus subtilis are biphasic, with two K(m) values for NADH. The higher K(m) corresponds to the only K(m) observed for NADH oxidation by whole cells, whereas the lower K(m) corresponds to that observed with open cell envelopes. Membrane preparations apparently contain a small fraction of open or inverted vesicles which is responsible for the low K(m) reaction, whereas entry of NADH into the larger portion of closed, normally oriented vesicles is rate limiting and responsible for the high K(m) reaction. In contrast, the oxidation of l-alpha-glycerol-phosphate (glycerol-P) by membrane preparations shows only one K(m) that corresponds to that of glycerol-P oxidation by whole cells or lysates. Since glycerol-P dehydrogenase (NAD independent) has the same K(m), this enzyme reaction rather than entry of glycerol-P into vesicles represents the rate-limiting step for glycerol-phosphate oxidation. The K(m) for amino acid uptake by vesicles in the presence of NADH corresponds to the high K(m) for NADH oxidation, indicating that NADH energizes transport only if it enters closed, normally oriented vesicles. Studies with rotenone and proteolytic enzymes support this interpretation. The apparent efficiency of NADH in energizing uptake seems to be lower than that of glycerol-P because, under the experimental conditions usually employed, open or inverted vesicles that do not participate in amino acid uptake are responsible for the major portion of NADH oxidation. When the results are corrected for this effect, the efficiency of NADH is essentially the same as that of l-alpha-glycerol-P.     

Identification of a conjugative plasmid carrying antibiotic resistance and salmonella plasmid virulence (spv) genes in epidemic strains of Salmonella typhimurium phage type 193

A plasmid pDEP34 that codes for resistance to ampicillin, streptomycin, sulphonamides and tetracyclines has been identified in strains of Salmonella typhimurium phage type 193 which have become increasingly common in England and Wales since 1988. pDEP34 is also self-conjugative, carries the genes responsible for the virulence of host strains for BALB/c mice ( spv genes) and is closely related to the Salm. typhimurium 'serotype-specific' plasmid pSLT.     

The effects of warming by active and passive means on the subsequent responses to cold water immersion

Two experiments were undertaken to investigate the effects of warming the body upon the responses during a subsequent cold water immersion (CWI). In both experiments the subjects, wearing swimming costumes, undertook two 45-min CWIs in water at 15° C. In experiment 1, 12 subjects exercised on a cycle ergometer until their rectal temperatures (T _re) rose by an average of 0.73°C. They were then immediately immersed in the cold water. Before their other CWI they rested seated on a cycle ergometer (control condition). In experiment 2, 16 different subjects were immersed in a hot bath (40° C) until their T _re rose by an average of 0.9° C; they were then immediately immersed in the cold water. Before their other CWI they were immersed in thermoneutral water (35° C; control condition). Heart rate in both experiments and respiratory frequency in experiment 1 were significantly (P < 0.05) higher during the first 30 s of CWI following active warming. In experiment 1, the rate of fall of T _re during the final 15 min of CWI was significantly (P < 0.01) faster when CWI followed active warming (2.46° C · h^–1) compared with the control condition (1.68°C · h^–1). However, this rate was observed when absolute T _re was still above that seen in the control CWIs. It is possible, therefore, that if longer CWIs had been undertaken, the two temperature curves may have converged and thereafter fallen at similar rates; this was the case with the aural temperature (T _au) seen in experiment 1 and the T _au and T _re in experiment 2. It is concluded that pre-warming is neither beneficial nor detrimental to survival prospects during a subsequent CWI.