The octadecylrhodamine B chloride (R18) membrane marker was incorporated into PR8 influenza viruses and virus receptor (GD1a-) containing small unilamellar vesicles (SUV). Both were tested in a fusion/lipid transfer assay [Wunderli-Allenspach, H., & Ott, S. (1990) Biochemistry 29, 1990-1997] to find out whether incorporation into artificial and biological membranes yields equivalent results. The R18 assay is based on incorporation of quenched concentrations of the label into donor membranes and monitoring of the dequenching upon its dilution into unlabeled acceptors. With PR8 viruses and R18-labeled SUV, a fast, hemagglutinin-specific fusion takes place at pH 5.3, independently of the initial quenching. At neutral pH, a slow, nonspecific R18 transfer occurs. Both processes follow second-order kinetics. Upon incubation of R18-labeled PR8 viruses with unlabeled SUV or LUV in neutral buffer, transfer is also found. At pH 5.3, a complex dequenching curve best described with superposition of two second-order functions was encountered: a fast, hemagglutinin-specific component and a slow, nonspecific component. A decreasing proportion of the fast fusion was found with increasing initial quenching of labeled virus. Extrapolation showed that full fusion activity is obtained only with low initial quenching (20-30%). The gradual inactivation of the virus by increasing amounts of R18 was confirmed with biological assays (e.g., infectivity). The R18 surface density is much higher in viruses than in liposomes to obtain the same initial quenching. Analysis with the Stern-Volmer plot revealed that R18 monomers and dimers contribute to the quenching in labeled PR8 viruses, whereas only dimers determine the quench curve in liposomes.

Octadecylrhodamine B chloride (R18) and ganglioside GD1a (virus receptor) were incorporated into small unilamellar liposomes [Hoekstra et al. (1984) Biochemistry 23, 5675-5681]. Upon interaction of these liposomes with PR8 influenza viruses without prebinding, two types of dequenching were observed at 37 degrees C, both second-order processes: a fast reaction at pH 5.3, 2k = 17.53 x 10(-3) (Q.s)-1, and a slow reaction at pH 7.4, 2k = 0.335 x 10(-3) (Q.s)-1. The maximal level of dequenching was the same for both. Upon prebinding of liposomes to PR8 viruses (30 min, 0 degrees C, pH 7.4) at high concentrations, a very fast dequenching occurred when the prebinding mixture was diluted into prewarmed (37 degrees C) 10 mM PBS, pH 5.3. For the initial phase, a first-order rate constant of 0.5 s-1 could be extrapolated. After a quick drop in velocity during the first 30 s, the reaction was kinetically indistinguishable from the one found without prebinding. A second-order process with 2k = 16.52 x 10(-3) (Q.s)-1 became rate-limiting. The fast reactions at pH 5.3 can be abolished by inactivation or removal of the virus hemagglutinin. We conclude that the reaction at pH 5.3 reflects the hemagglutinin-dependent fusion process known to occur between influenza viruses and partner membranes at low pH; however, second-order kinetics indicate that specific binding rather than fusion is the rate-limiting step. For the slow dequenching, which is not affected by prebinding, the rate constant is 20 times lower than for the fast reaction, and the process is independent of viral hemagglutinin.(ABSTRACT TRUNCATED AT 250 WORDS)

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In 92 patients who underwent abdominal and goiter surgery endotoxin and endotoxin-neutralizing capacity (ENC) were determined in plasma preoperatively and daily postoperatively. Endotoxin plasma levels started to increase on day 1 in patients who were laparotomized. Correspondingly ENC was reduced during the 1st postoperative week. Even on the 1st postoperative day determination of ENC made the differentiation between patients possible who showed an uneventful course and patients who developed pneumonia, pulmonary failure, or mental disorders. Furthermore the need for diuretics in order to maintain sufficient renal function was associated with lower ENC during days 1–3. Endotoxin plasma levels were significantly elevated in patients who developed pneumonia during days 1–4, whereas the occurrence of pulmonary failure was only correlated with elevated endotoxin levels on day 3. Endotoxemia was pronounced in patients needing diuretics on days 1, 4, and 5. In patients who underwent goiter surgery ENC changed significantly as found for endotoxin plasma values during the postoperative course. Determination of endotoxin and especially ENC with the use of the limulus amebocyte lysate test turned out to be a reliable method correlating with impending complications at least in postsurgical patients.

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