Estimation of the bioaccumulation potential of a nonchlorinated bisphenol and an ionogenic xanthene dye to Eisenia andrei in field-collected soils, in conjunction with predictive in silico profiling.

In silico-based model predictions, originating from structural and mechanistic (e.g., transport, bioavailability, reactivity, and binding potential) profiling, were compared against laboratory-derived data to estimate the bioaccumulation potential in earthworms of 2 organic substances (1 neutral, 1 ionogenic) known to primarily partition to soil. Two compounds representative of specific classes of chemicals were evaluated: a nonchlorinated bisphenol containing an -OH group (4,4'-methylenebis[2,6-di-tert-butylphenol] [Binox]), and an ionogenic xanthene dye (2',4',5',7'-tetrabromo-4,5,6,7-tetrachloro-3',6'-dihydroxy-, disodium salt [Phloxine B]). Soil bioaccumulation studies were conducted using Eisenia andrei and 2 field-collected soils (a clay loam and a sandy soil). In general, the in silico structural and mechanistic profiling was consistent with the observed soil bioaccumulation tests. Binox did not bioaccumulate to a significant extent in E. andrei in either soil type; however, Phloxine B not only accumulated within tissue, but was not depurated from the earthworms during the course of the elimination phase. Structural and mechanistic profiling demonstrated the binding and reactivity potential of Phloxine B; this would not be accounted for using traditional bioaccumulation metrics, which are founded on passive-based diffusion mechanisms. This illustrates the importance of profiling for reactive ionogenic substances; even limited bioavailability combined with reactivity can result in exposures to a hazardous substance not predictable by traditional in silico modeling methods.

Viability assays to monitor yeast autophagy.

In the yeast Saccharomyces cerevisiae, autophagy contributes to the sustaining of cell viability under starvation conditions, possibly through the supply of amino acids that is generated as a result of the degradation of cytosolic materials. Therefore, cellular viability is one of the best indexes for monitoring the completion of the entire autophagic process. In this chapter, several assays for monitoring yeast viability are presented. Along with the standard colony-formation assay, assays using the dye phloxine B are introduced.

Intra-strain variability of Cryptococcus neoformans can be detected on phloxin B medium.

A method was devised for easy detection of intra-strain variability of the human pathogenic yeast Cryptococcus neoformans. Cultivation of strains on a medium containing Phloxin B resulted in different coloured colonies. Generally, colonies were either pink or red; however there were also several colony-colour segregant in which both colours could be observed. A number of these segregants were isolated and analysed. Virulence factors such as the cell and capsule sizes were measured; further temperature sensitivity, growth rates, mating-types and melanin production were also studied. Segregants were examined by random amplified polymorphic DNA (RAPD) fingerprinting and electrophoretic karyotyping by pulsed-field gel electrophoresis (CHEF). They showed both phenotypic and genotypic differences. The main differences appeared in phenotypic characters and RAPD patterns; while the chromosomal patterns remained unchanged. Reversion frequency analysis revealed that the reason for this segregation could be due to phenotypic switching. The physiological reason for the colour changes was also investigated and was attributed to the differential ability of the cells to accumulate Phloxin B either into their capsules or into their cells. The method described here is potentially applicable for the detection of strain heterogeneity in both basic and clinical microbiology laboratories.

Efficacy and residues of phloxine B and uranine for the suppression of Mediterranean fruit fly in coffee fields.

The field efficacy of a bait containing phloxine B, uranine and Provesta 621 protein was tested against Mediterranean fruit fly (Ceratitis capitata; Medfly) by aerial and ground spraying in about 84 ha of coffee fields in Kauai, Hawaii, USA. Concurrently, soil and crop samples were collected from the aerially sprayed field and its unsprayed control field for residue studies. Efficacy of the sprays was assessed through trapping with both protein-baited and trimedlure-baited traps and through the infestation level of coffee cherries collected at least three-quarters ripe. The C capitata population was low at the start of the aerial and ground spray studies, but dramatically increased in the control fields. This increase coincided with initial ripening of coffee cherries. During times of peak population levels, C capitata populations were reduced by more than 91% in the ground-sprayed field and 99% in the aerial-sprayed field, relative to the populations in their respective control fields and based on protein-baited trap catches. Results of residue analyses indicated that uranine dissipated quickly compared with phloxine B on coffee and soil. Coffee samples collected at pre-spray periods had phloxine B residues of 7.2-25.5 ng g-1 on berries. Phloxine B concentrations were much higher on coffee leaves (163-1120 ng g-1). Lower concentrations of the dye were found from coffee samples collected during rainy days. Average phloxine B concentrations immediately after spraying were 56 and 2840 ng g-1 in coffee berries and leaves, respectively. Dissipation of phloxine B on berries was fast, with a half-life (t1/2) of 3 days. Dissipation of phloxine B on leaves was fitted to two linear phases: the initial (0-4 days) with a shorter t1/2 of 3 days and the later phase (4-28 days) with a longer t1/2 of 15 days. Average concentrations of phloxine B in the top soil ranged from 50 to 590 ng g-1 at pre-spray. Phloxine B initial concentration (770 ng g-1) reached a plateau immediately after the last spraying, but showed a steady decline over time with t1/2 of 16 days. Fast dissipation of the dyes in the field indicates that these chemicals may be environmentally compatible and therefore a promising alternative for fruit fly control.

Effect of a phloxine B-cucurbitacin bait on diabroticite beetles (Coleoptera: Chrysomelidae).

Cucurbitacin E glycoside, extracted from a bitter mutant of Hawkesbury watermelon [Citrulls lanatus (Thunb.) Matsum. & Nakai (Syn. Citrullus vulgaris Schrad)] is the active ingredient of a feeding stimulant for the corn rootworm complex. It is the primary component of a water-soluble bait that can be combined with toxins for adult diabroticite control. Studies were conducted using phloxine B (D&C Red 28), a xanthene dye, as the toxin. This dye was efficacious against Diabrotica undecimpunctata howardi Barber, spotted cucumber beetle, and Acalymma vittatum (F.), striped cucumber beetle, in cucumber plots and could be recovered from cucumber leaves for 8 d after treatment. The average amount of dye recovered per dead spotted cucumber beetle at 8 d after treatment was 0.173 microg. Concentrated and sugar-free fermented forms of the watermelon extract were developed and compared with the fresh juice in field applications on cucumber plants. There was no significant difference in mortality of beetles from phloxine B-bait prepared with fresh, fermented, or concentrated extract, although in laboratory studies, fermented juice had higher feeding stimulant activity.

Kinetics of all stages of electron transfer in nitrogenase in the presence of a photodonor.

A photodonor is considered as an alternative electron donor for nitrogenase. The kinetic mechanism of nitrogenase turnover is discussed. The turnover is initiated by the transfer of an electron to the enzyme and results in formation of a substrate molecule. The effective rate constant of concerted transfer of the first and the second electron from Av2 (Fe-protein) to Av1 (Mo-Fe-protein) and the rate constant of transfer of the second electron are 70 +/- 7 and 116 +/- 10 sec-1, respectively. The rate constant of the rate-limiting reaction--MgADP release during formation of the superreduced state of Av1 (*Av12-)--is 12 +/- 2 sec-1. Nitrogenase (E) states in complex E.N2 on binding and reduction of nitrogen are: E2, E4, E6 (2, 4, and 6 electrons).