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Culture-based methods of monitoring fecal pollution in recreational waters require 24 to 48 hours to obtain results. This delay leads to potentially inaccurate management decisions regarding beach safety. We evaluated the quantitative polymerase chain reaction (QPCR) as a faster method to assess recreational water quality and predict swimming-associated illnesses.

Indoor mold concentrations were measured in the dust of moldy homes (MH) and reference homes (RH) by quantitative PCR (QPCR) assays for 82 species or related groups of species (assay groups). About 70% of the species and groups were never or only rarely detected. The ratios (MH geometric mean RH geometric mean) for 6 commonly detected species (Aspergillus ochraceus, A. penicillioides, A. unguis, A. versicolor, Eurotiumgroup, and Cladosporium sphaerospermum) were >1 (Group I). Logistic regression analysis of the sum of the logs of the concentrations of Group I species resulted in a 95% probability for separating MH from RH. These results suggest that it may be possible to evaluate whether a home has an abnormal mold condition by quantifying a limited number of mold species in a dust sample. Also, four common species of Aspergilluswere quantified by standard culturing procedures and their concentrations compared to QPCR results. Culturing underestimated the concentrations of these four species by 2 to 3 orders of magnitude compared to QPCR.

We developed a sensitive fluorescence assay for the quantitation of proteins in solution using the NanoOrange®reagent, a merocyanine dye that produces a large increase in fluorescence quantum yield upon interaction with detergent-coated proteins. The NanoOrange assay allowed for the detection of 10 ng/mL to 10 μg/mL protein with a standard fluorometer, offering a broad, dynamic quantitation range and improved sensitivity relative to absorption-based protein solution assays. The protein-to-protein variability of the NanoOrange assay was comparable to those of standard assays, including Lowry, bicinchoninic acid, and Bradford procedures. We also found that the NanoOrange assay is useful for detecting relatively small proteins or large peptides, such as aprotinin and insulin. The assay was somewhat sensitive to the presence of several common contaminants found in protein preparations such as salts and detergents; however, it was insensitive to the presence of reducing agents, nucleic acids, and free amino acids. The simple assay protocol is suitable for automation. Samples are briefly heated in the presence of dye in a detergent-containing diluent, allowed to cool to room temperature, and fluorescence is measured using 485-nm excitation and 590-nm emission wavelengths. Therefore, the NanoOrange assay is well suited for use with standard fluorescence microplate readers, fluorometers, and some laser scanners.

Analyses of fungal spores or conidia in indoor dust samples can be useful for determining the contamination status of building interiors and in signaling instances where potentially harmful exposures of building occupants to these organisms may exist. A recently developed method for the quantification of Stachybotrys chartarum conidia, using real-time, fluorescence probe–based detection of PCR products (TaqMan™ system) was employed to analyze indoor dust samples for this toxigenic fungal species. Dust samples of up to 10 mg were found to be amenable to DNA extraction and analysis. Quantitative estimates of S. chartarum conidia in composite dust samples, containing a four-log range of these cells, were within 25–104% of the expected quantities in 95% of analyses performed by the method. Calibrator samples containing known numbers of S. chartarum conidia were used as standards for quantification. Conidia of an arbitrarily selected strain of Geotrichum candidum were added in equal numbers to both dust and calibrator samples before DNA extraction. Partial corrections for reductions in overall DNA yields from the dust samples compared to the calibrator samples were obtained by comparative analyses of rDNA sequence yields from these reference conidia in the two types of samples. Dust samples from two contaminated homes were determined to contain greater than 103S. chartarum conidia per milligram in collection areas near the sites of contamination and greater than 102conidia per milligram in several areas removed from these sites in analyses performed by the method. These measurements were within the predicted range of agreement with results obtained by direct microscopic enumeration of presumptive Stachybotrys conidia in the same samples.Journal of Exposure Analysis and Environmental Epidemiology (2001) 11, 12–20. 10.1038/sj.jea.7500147

ABSTRACTStachybotrys chartarumis a toxigenic fungus that has been associated with human health concerns, including pulmonary hemorrhage and hemosiderosis. This fungus produces a hemolysin, stachylysin, which in its apparent monomeric form has a molecular mass of 11,920 Da as determined by matrix-assisted laser desorption ionization–time of flight mass spectrometry. However, it appears to form polydispersed aggregates, which confounds understanding of the actual hemolytically active form. Exhaustive dialysis or heat treatment at 60°C for 30 min inactivated stachylysin. Stachylysin is composed of about 40% nonpolar amino acids and contains two cysteine residues. Purified stachylysin required more than 6 h to begin lysing sheep erythrocytes, but by 48 h, lysis was complete. Stachylysin also formed pores in sheep erythrocyte membranes.

AbstractMembers of the anamorphic fungal genus Memnoniellademonstrate morphological and biological similarities to species within the genus Stachybotrys, and the taxonomic distinctions between the genera have been the subject of controversy in the past. Sixteen strains representing described species of Memnoniellawere examined for morphology using light and scanning electron microscopy and for phylogenetic relationships using comparative sequence analysis of a segment of the nuclear ribosomal RNA gene operon (rDNA) including the internal transcribed spacer 1 and 2 regions (ITS1 and ITS2) and 5.8S gene. These analyses resolved the Memnoniellastrains into two highly divergent phylogenetic clades with morphologies generally consistent with the current descriptions of M. echinataand M. subsimplex.One strain, showing morphological features more similar to M. subsimplex, was placed in the M. echinataclade in the phylogenetic analysis and probably represents a new species. A second strain, showing a typical M. echinataDNA sequence, showed morphological features that were similar to Stachybotrysspecies when grown on certain culture media. The evolutionary relationships between the genera were evaluated by phylogenetic analyses of sequence data from the 18S, 28S, 5.8S rDNA genes and ITS1 and ITS2 regions. Results of several different analyses were in agreement in indicating that Memnoniellais paraphyletic to Stachybotrys.

5-(Pentafluorobenzoylamino)fluorescein (PFB-F), a new thiol-reactive molecule was synthesized to improve the detection limits and specificity of the assays for glutathioneS-transferase (GST) activity and glutathione (GSH). A rapid assay method to measure GSH concentration or GST activity and the simultaneous analysis of multiple samples is possible because the glutathione adduct, GS-TFB-F, is separated from PFB-F by thin-layer chromatography (TLC) and can be quantitated by a fluorescence scanner. The detection limits for GSH and for GST activity using TLC were found to be as low as 10 pmol/μl and 1 ng/μl using equine liver GST, respectively. Determination of GSH concentration or GST activity in bovine pulmonary artery endothelial (BPAE) cell lysates gave a linear response for samples corresponding to 500–2500 cells. PFB-F could also measure GST activities of GST fusion proteins and prove to be a suitable substrate for determining the activities of human GST isozymes and other sources of mammalian GST. The selectivity of PFB-F with GSH was proven by comparing trace amount of the adducts that formed with cysteine and β-galactosidase to that formed with GSH. The HPLC profile of a reaction mixture where cell lysate was used in place of purified GST, also shows only two main peaks, corresponding to GS-TFB-F and unreacted PFB-F. The selectivity of PFB-F for GSH was further confirmed by exposing BPAE cells todl-buthionine-[S,R]-sulfoximine (BSO). Our results of GS-TFB-F determination indicate that 12-, 24-, or 36-h incubations with BSO caused 2-, 6-, or 7.6-fold reductions in GSH levels, respectively.

Fluorogenic substrates based on 4-methylumbelliferone (4-MU) have been widely used for the detection of phosphatase and glycosidase activities. One disadvantage of these substrates, however, is that maximum fluorescence of the reaction product requires an alkaline pH, since 4-MU has a pKa≈ 8. In an initial screening of five phosphatase substrates based on fluorinated derivatives of 4-MU, all with pKavalues lower than that of 4-MU, we found that one substrate, 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP), was much improved for the detection of acid phosphatase activity. When measured at the preferred acid phosphatase reaction pH (5.0), DiFMUP yielded fluorescence signals that were more than 10-fold higher than those of 4-methylumbelliferyl phosphate (MUP). DiFMUP was also superior to MUP for the detection of protein phosphatase 1 activity at pH 7 and was just as sensitive as MUP for the detection of alkaline phosphatase activity at pH 10. A β-galactosidase substrate was also prepared based on 6,8-difluoro-4-methylumbelliferone. This substrate, 6,8-difluoro-4-methylumbelliferyl β-d-galactopyranoside (DiFMUG), was found to be considerably more sensitive than the commonly used substrate 4-methylumbelliferyl β-d-galactopyranoside (MUG), for the detection of β-galactosidase activity at pH 7. DiFMUP and DiFMUG should have great utility for the continuous assay of phosphatase and β-galactosidase activity, respectively, at neutral and acid pH.

Current methods for detecting lacZexpression in transformed cells are limited because they require such harsh conditions that viability of the cells after detection is drastically reduced. To overcome this problem, we developed a series of new substrates for detection of lacZexpression in living cells under standard culture or physiological conditions. After incubation with these fluorogenic substrates, cultured lacZ‐positive mammalian cells appear morphologically normal, continue to divide, and retain the fluorescent product. Because the product is so well retained, fluorescence intensity can be quantitatively related to the level of gene expression. We have demonstrated this correlation using transformed yeast cells bearing various plasmids, each containing the lacZgene and a unique promoter sequence with known capabilities for promoting gene expression in yeast.— Zhang, Y.‐z.; Naleway, J. J.; Larison, K. D.; Huang, Z.; Haugland, R. P. Detecting lacZgene expression in living cells with new lipophilic, fluorogenic β‐galactosidase substrates. FASEB J.5: 3108‐3113; 1991.

Characteristics of fluorogenic substrates designed for detection of enzyme activity in living cells are reviewed. Improved retention of the fluorescent products in the cell of origin can be achieved by structural modifications to the substrate that result in association with membrane lipids or conjugation to intracellular glutathione. Newly-developed substrates that yield fluorescent precipitates provide the additional advantage of allowing subcellular localization of sites of enzymatic activity. Improved detection sensitivity can also be achieved by targeted delivery of substrates for processing by specific organelles. Substrates designed for monitoring oxidative activity and lipid metabolism provide examples of this approach.

The enzymatic determination of hydrogen peroxide can be accomplished with high sensitivity and specificity usingN-acetyl-3,7-dihydroxyphenoxazine (Amplex Red), a highly sensitive and chemically stable fluorogenic probe for the enzymatic determination of H2O2. Enzyme-catalyzed oxidation of Amplex Red, which is a colorless and nonfluorescent derivative of dihydroresorufin, produces highly fluorescent resorufin, which has an excitation maximum at 563 nm and emission maximum at 587 nm. The reaction stoichiometry of Amplex Red and H2O2was determined to be 1:1. This probe allows detection of 5 pmol H2O2in a 96-well fluorescence microplate assay. When applied to the measurement of NADPH oxidase activation, the Amplex Red assay can detect H2O2release from as few as 2000 phorbol myristate acetate-stimulated neutrophils with a sensitivity 5- to 20-fold greater than that attained in the scopoletin assay under the same experimental conditions. Furthermore, the oxidase-catalyzed assay using Amplex Red results in anincreasein fluorescence on oxidation rather than adecreasein fluorescence as in the scopoletin assay. In comparison with other fluorometric and spectrophotometric assays for the detection of monoamine oxidase and glucose oxidase, this probe is also found to be more sensitive. Given its high sensitivity and specificity, Amplex Red should have a broad application for the measurement of H2O2in a variety of oxidase-mediated reactions and very low levels of H2O2in food, environmental waters, and consumer products.

Two strains of the soybean endosymbiont Bradyrhizobium japonicum, USDA 110 and 61 A101 C, were mutagenized with transposon Tn5. After plant infection tests of a total of 6,926 kanamycin and streptomycin resistant transconjugants, 25 mutants were identified that are defective in nodule formation (Nod-) or nitrogen fixation (Fix-). Seven Nod- mutants were isolated from strain USDA 110 and from strain 61 A101 C, 4 Nod- mutants and 14 Fix- mutants were identified. Subsequent auxotrophic tests on these symbiotically defective mutants identified 4 His- Nod- mutants of USDA 110. Genomic Southern analysis of the 25 mutants revealed that each of them carried a single copy of Tn5 integrated in the genome. Three 61 A101 C Fix- mutants were found to have vector DNA co-integrated along with Tn5 in the genome. Two independent DNA regions flanking Tn5 were cloned from the three nonauxotrophic Nod- mutants and one His-Nod- mutant of USDA 110. Homogenotization of the cloned fragments into wild-type strain USDA 110 and subsequent nodulation assay of the resulting homogenotes confirmed that the Tn5 insertion was responsible for the Nod- phenotype. Partial EcoR1 restriction enzyme maps around the Tn5 insertion sites were generated. Hybridization of these cloned regions to the previously cloned nod regions of R. meliloti and nif and nod regions of B. japonicum USDA 110 showed no homology, suggesting that these regions represent new symbiotic clusters of B. japonicum.

Several lines of evidence were obtained that the previously identified, repeated sequence RS 1100 of Pseudomonas cepacia strain AC1100 undergoes transposition events. DNA sequences flanking the chlorohydroxy hydroquinone (CHQ) degradative genes of this organism were examined from sources, including several independently isolated cosmid clones from an AC1100 genomic library and genomic DNAs of two independently maintained wild-type AC1100 isolates. Hybridization and restriction endonuclease mapping studies revealed these sequences to be similar except for their numbers and distributions of RS1100 copies. A recombinant plasmid containing the immediate chq gene region and excluding any copies of RS1100 was conjugated into AC1100 mutant RHA5 which was shown to have undergone a deletion of its corresponding DNA. Hybridization and restriction mapping analyses of several reisolated plasmids revealed the presence of RS1100 sequences at different positions within either the vector or insert portions. One such plasmid contained tandem copies of RS1100 with an intervening DNA sequence also of AC1100 origin. Similar experiments involving introduction of the promoter probe plasmid pKT240 into wild-type AC1100 cells resulted in the acquisition of high-concentration streptomycin resistance by a number of recipients. The reisolated plasmids in most cases also conferred streptomycin resistance to Escherichia coli transformants and in each case were found to contain insertions close to the upstream portion of the aphC structural gene. These insertions alternatively contained RS1100 sequences or a newly identified 3400 by repeated sequence from AC1100. Based on these results, RS1100 has been redesignated as insertion sequence IS931 and the 3400 bp repeated sequence has been designated as IS932.[/ab]

A gene bank of Rhizobium japonicumDNA was constructed by using the broad host range conjugative cosmid pLAFR1. Eighty-three percent of the clones in the bank contained cosmids with insert DNA averaging 22.6 kilobase pairs in length. A series of cosmids containing a hydrogen uptake (hup) gene was identified by transferring the gene bank into a H2uptake-negative (Hup-) R. japonicumpoint mutant (PJ17nal) and screening tetracycline-resistant colonies for the ability to grow chemolithotrophically and to reduce methylene blue in a recently devised colony assay. Hup+transconjugants arose at a frequency of approximately 6 × 10-3. Plasmid DNAs from II of the Hup+transconjugants were isolated and used to transform Escherichia coli. EcoRI digests of all plasmids isolated from Hup+transconjugants had three DNA fragments in common. Eight of the E. colitransformants containing hupgene cosmids were conjugated with PJ17nal and another Hup-point mutant, PJ18nal. All PJ17nal transconjugants were Hup+. The frequency of Hup+transconjugants with PJ18nal was approximately 10-3. The results indicate that the hupgene cosmids may contain one gene and a portion of another.