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Washing bacteria by centrifugation through a water-immiscible layer of silicones. J. Bacteriol. 90:1692-1695.1965.-A method is described which enables the separation of cells from aqueous suspension without altering the internal aqueous environment of the cells. The method consists of centrifuging the cells from the aqueous environment through a more dense, immiscible solvent consisting of a mixture of two silicones. A thin film of the aqueous environment equal to 7 X 10-13 ml per cell remains with the bacteria during the separation procedure. The method by which this volume was determined is described. The procedure itself has no measurable effect on viability or permeability of the cells and permits recovery of about 90% of the cells from the aqueous environment. With this method, it has been found that the intracellular water volume of Escherichia coli ML 35 accessible to sucrose or inositol is 1.96 X 10-12 ml i 6%, or 85% of the total volume of the cell determined by visual measurement.

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We have previously shown that serum levels of Clq, unbound to Clr X Cls, are elevated in rheumatoid arthritis. We have also shown that RHP, a newly described serum protein which affects the Clq-anti Clq precipitin reaction, is also present at elevated levels in rheumatoid arthritis. We now show that RHP inhibits the hemolytic activity of Clq, disaggregates Cl, and inhibits the ability of Clq bound to latex beads or to aggregated IgG to enhance the oxidative metabolism of neutrophils.

Pseudomonas sp. strain Kim has previously been reported to be the only known naturally occurring organism lacking spermidine. We now show that it synthesizes this polyamine. The apparent lack of intracellular levels of spermidine results from an efficient conversion of spermidine to putrescine and hydroxyputrescine.Pseudomonas sp. strain Kim has been shown to contain putrescine and S-(+)-2-hydroxyputrescine as its only polyamines (2-4, 7) and has been considered to be the only known naturally occurring life form to lack spermidine. We now show that this organism does synthesize spermidine and that the reconversion of spermidine to putrescine and hydroxyputrescine appears to prevent its accumulation in the cells to detectable levels.Pseudomonas sp. strain Kim cells were grown at 30'C in minimal salts medium (1) containing 0.5% glucose. Cells were collected by centrifugation for preparation of cell extracts in a French pressure cell at 10,000 lb/in2 or for extraction of polyamines with 5% trichloracetic acid. The extracted polyamines were dansylated by the procedure of Seiler and Knodgen (9) and were then resolved by highpressure liquid chromatography in a Whatman Partisil 10 PAC column (chart speed, 1 cm/min). The elution mixture (30% ethanol, 70% hexane) was passed through the column at 1 ml/min, and the optical density of the eluate was read at 254 nm (Fig. 1). We quantitated the resolved polyamines by measuring the areas of recorded peaks. To measure extracellular polyamines, the supernatant growth medium was concentrated by flash evaporation at 35°C to 1.5 ml, and 0.5 ml was removed for dansylation. Analysis of polyamines added to bacterial extracts showed that their elution patterns and recovery were unaffected by the presence of the biological materials in the extracts. Verification of the presence of the polyamines in the acid extracts and supernatants of the cells was obtained by paper electrophoresis (4,7). The presence of excreted acetylspermidine was verified by recovery of spermidine after acid hydrolysis.["4C]spermidine was obtained from Du Pont, NEN Research Products. S-adenosylmethionine (AdoMet) and methylglyoxal bis(guanylhydrazone) (MGBG) were purchased from Sigma Chemical Co. Adodato (S-adenosyl-3-thiol-1,8-diaminooctane) was a gift from James Coward. MDL 72527 [N,N-1-bis(2,3-butadienyl)-1,4-butanediamine] was a gift from Merrill-Dow Research Institute. MDL 72527 is an inhibitor of oxidation of N-1-acetylspermidine by polyamine oxidase (7).A cell extract of Pseudomonas sp. strain Kim was shown to synthesize spermidine from putrescine in the presence of added AdoMet, and the synthesis was inhibited 30% by 100 [iM MGBG, an amine transferase, the enzymes required for synthesis of spermidine from putrescine, but raised the question of why the organism lacked detectable levels of spermidine.That Pseudomonas sp. strain Kim can convert spermidine to putrescine and hydroxyputrescine was shown by adding ['4C]spermidine at 12.5 ILM to growing log-phase cells for 6 h with and without added MGBG at 10...