Retrospective study of Gen-Probe rapid diagnostic system for detection of legionellae in frozen clinical respiratory tract samples.

The Gen-Probe Rapid Diagnostic System for legionellae, which uses 125I-labeled cDNA directed against the rRNAs of legionellae, was evaluated for its ability to detect members of the genus by using clinical specimens which had been frozen at -70 degrees C for 2 to 8 years. Culture and direct immunofluorescence (DFA) results obtained at the time of specimen collection were used to categorize samples. The specimens tested were 112 samples culture positive for legionellae and 230 samples negative on culture and DFA tests. They were tested in a blinded and randomized fashion. Results were expressed in terms of the ratio of counts per minute of the sample to the counts per minute of the provided negative control. A ratio of greater than or equal to 4.0 was picked for optimal specificity. Of the 112 previously positive specimens, 63 (57%) were positive by the probe assay, and of the 230 previously negative samples, 228 (99.1%) were negative. The 51 discrepant specimens were reexamined by culture and DFA testing if adequate amounts remained; this was possible for 34 specimens. On repeat culture, 22 of 33 previously culture-positive samples yielded legionellae and 11 were negative. Ten of the positive repeat cultures yielded two or fewer colonies per plate. One probe-positive but previously culture-negative sample was overgrown by contaminants on repeat culture. Reanalysis of data after exclusion of the 17 unavailable, 11 repeat culture-negative, and 1 unevaluable specimen gave a probe sensitivity of 74% and specificity of 100%. The Gen-Probe test is therefore specific and is of useful sensitivity.

Isolation of human factor VIII:C by preparative high-performance size-exclusion chromatography.

Human Factor VIII procoagulant protein (VIII:C) is a plasma protein that participates in the cascade of events leading to blood coagulation. It is absent or defective in patients with hemophilia A. In vivo Factor VIII:C associates with Von Willibrand factor and its multimers to form a high-molecular-weight particle that can be dissociated into a lower-molecular-weight form in the presence of high concentrations of salt. We have been able to purify rapidly Factor VIII:C on a large scale by sequential high-performance size-exclusion chromatography (HPSEC) under conditions of first low salt and then high salt concentration. Reconstituted commercial Factor VIII:C concentrate was purified by chromatography on a preparative HPSEC column (Toyo Soda, 60 X 2.5 cm, 300 ml) in 0.05 M imidazole buffer, (pH 7.0), containing 0.15 M sodium chloride. Factor VIII:C activity was eluted in the void volume in less than 20 min as a high-molecular-weight particle, well separated from low-molecular-weight contaminants. Purification was 20-fold, with a yield of 80%. Up to 4 g of Factor VIII concentrate could be purified at one time in this manner. This material was then concentrated and made 0.35 M in calcium chloride prior to re-chromatography on the same column in a buffer containing 0.30 M calcium chloride. Under these conditions, Factor VIII:C activity was eluted in the inner volume of the column at a position corresponding to a molecular weight of several hundred thousand in less than 1 h. It was well separated from both larger proteins and smaller peptide fragments.(ABSTRACT TRUNCATED AT 250 WORDS)

A comparison of 2,4-dichlorophenoxyacetic Acid metabolism in cultured soybean cells and in embryogenic carrot cells.

The removal or reduction in concentration of auxin is often a successful method for obtaining morphogenesis in cell cultures of higher plants, such as carrot, but not for soybean. For this reason, the metabolism of one auxin, 2,4-dichlorophenoxyacetic acid (2,4-D), was compared in both carrot and soybean cells. Whereas soybean cells conjugated a high percentage of their 2,4-D to amino acids, carrot cells contained primarily free 2,4-D. Moreover, after long-term exposure to 2,4-D, carrot cells released much more 2,4-D upon transfer to 2,4-D-free (embryogenic) medium than did soybean cells. It appears that the retention of 2,4-D by soybean cells might interfere with subsequent morphogenesis. Because no impairment of 2,4-D efflux was found with short-term exposure to radiolabeled 2,4-D, it was concluded that 2,4-D retention in soybean cells might be due to a time-dependent, metabolic process. The conjugation of 2,4-D to amino acids was shown to be one such time-dependent process. Additionally, the release of 2,4-D from the cells was shown to be due primarily to a loss of free 2,4-D and not 2,4-D-amino acid conjugates. It seems that the greater retention of 2,4-D by soybean cells upon transfer to 2,4-D-free medium is due to greater formation of 2,4-D-amino acid conjugates.

Inhibition of 2,4-dichlorophenoxyacetic Acid conjugation to amino acids by treatment of cultured soybean cells with cytokinins.

Kinetin, and all other cytokinins tested, inhibited the conjugation of [(14)C]2,4-dichlorophenoxyacetic acid (2, 4-D) to amino acids when supplied simultaneously with the 2,4-D to cultured soybean cells. Upon transfer to hormone-free medium, the cytokinin-treated cells released more of their [(14)C]2,4-D than did the control cells. Initial exposure to low 2,4-D and high kinetin levels resulted in the greatest release of 2,4-D upon subsequent transfer. The observed alteration in 2,4-D metabolism did not seem to be correlated with growth rate. Appropriate treatment of soybean cells with kinetin resulted in 2,4-D metabolism that resembled the 2,4-D metabolism of embryogenic carrot cells. However, no new morphological structures were observed in these soybean cultures, indicating that other factors are related to the failure of soybean cells to regenerate in culture.

Soluble polyvinylpyrrolidine and bovine serum albumin adsorb polyphenols from soybean suspension cultures.

Soybean (Glycine max [L.] Merrill, cv. Dare) suspension cultures grown in Gamborg B5 medium became discolored and the cells began aggregating after 1 week in culture, especially in the absence of 2,4-dichlorophenoxyacetic acid (2,4-D). The addition of either soluble polyvinylpyrrolidine (PVP) or bovine serum albumin (BSA) to cultures grown in Gamborg B5 medium with 2,4-D prevents discoloration and cell aggregation by adsorbing excess polyphenols from the cells. Transfer of the PVP-treated cultures to fresh medium without 2,4-D stimulated the recurrence of excess polyphenols. Cultures pretreated with BSA did not develop excess polyphenols when transferred to fresh 2,4-D-free medium. Addition of either PVP or BSA to cultures grown in the absence of 2,4-D was found to inhibit growth.