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the photoactive compounds phloxine B and menadione are relatively safe food additives and possess efficient insecticidal properties against certain insect species. The two compounds were tested for toxicity to the peach fruit fly, Bactrocera Zonata (Saunders) under both dark and sunlight conditions. Both compounds had no significant toxic effect in dark and concentration-dependent toxicity when insect exposed to sunlight for 2 hrs. Phloxine B was approximately 1000 time more toxic to B. zonata than menadione with LC25 in the water of 0.0007 g% and 0.854 g% (w/v), respectively. Adding menadione to phloxine resulted in significant antagonistic effects on insect mortality, which could be due to menadione alteration of the pH of the feeding media and/or menadione induction of the ROS-detoxifying enzymes, particularly peroxidase. It is concluded that phloxine B and menadione are incompatible and their mixture is not likely to pose any significant addition to their control potential against B. zonata.

A fluorescent stain, calcofluor white (CFW), was used for detection of spores of the microsporidium Enterocytozoon hepatopenaei (EHP). EHP spores in suspension, in feces, or in the infected hepatopancreas of the shrimp Penaeus vannamei, can be easily stained with this chitin and cellulose binding dye to show distinct blue-white fluorescent oval walls. The dye does not stain the host tissues. EHP spores showed orange-red spots by staining with hematoxylin and phloxine (H&P) in the section. CFW staining provides a simple and rapid method for determining the presence of EHP spores in fecal or tissue samples.

The aim of this study was to determine the presence, number, and morphometrical characteristics of Paneth cells (PC) in the small intestine of guinea pigs during lactation. We used 48 pups from 0 to 15 days old. Samples from small intestine were fixed in 10% buffered formaldehyde (pH 7.4) and processed for histological and morphometrical studies using hematoxylin and eosin (HE), Phloxine tartrazine or Masson's Trichome staining, or immunohistochemistry for lysozyme. PC were morphologically identified at day 2 using Masson's Trichome or Phloxine tartrazine stainings, and at day 4 using HE, whereas using immunohistochemistry they were recognized from birth. Morphometrical differences were found between the intestinal sections at each age studied, and within each section during the first weeks of life. In all developmental stage, the highest number of PC was observed in the duodenum of 13 days old guinea pigs. Our results confirm the presence of PC in the small intestine of guinea pigs from birth.

Our previous study revealed that the photo-irradiation of rose bengal, erythrosine, and phloxine, xanthene photosensitizers, used as dental plaque disclosing agents, could exert bactericidal action on planktonic Streptococcus mutans via the singlet oxygen. In the present study, the photo-irradiation induced bactericidal activity of the three xanthene compounds against the experimental biofilm of S. mutans was investigated in combination with acid electrolyzed water (AcEW) and alkaline electrolyzed water (AlEW). As a result, only the photo-irradiated rose bengal in AlEW showed prominent bactericidal activity with a >3-log reduction of the viable bacterial count. Since our previous study showed that the affinity of rose bengal to bacterial cells was superior to that of erythrosine and phloxine, it was speculated that AlEW damaged the extracellular matrix of the experimental biofilm, which would let the rose bengal easily be bound to the bacterial cells. From these results, it is strongly suggested that rose bengal is a suitable photosensitizer for use as a plaque disclosing agent in photodynamic antimicrobial chemotherapy to treat dental plaque.

Phenotypic switching is characterized as a virulence factor of Candida spp. This study was carried out to evaluate the phenotypic switching ability of C. krusei ATCC 14243 and to determine its effect on the biological properties, adherence capacity and susceptibility towards chlorhexidine digluconate (CHX). To induce switched generations C. krusei was cultured under nitrogen-depleted growth conditions by adding phloxine B. These phenotypically switched colonies were designated as the 1st generation. Subsequent sub-culturing was performed to produce the 2nd, 3rd and 4th switched generations. The recovery of the 3rd generation was the highest at 85.7% while that of the 4th generation was lower at 70.8%, and the recovery of the 1st and 2nd generations gradually reduced to 46.6% and 36.4%, respectively. All generations of C. krusei were susceptible towards CHX. The unswitched C. krusei was the most susceptible but the least adherent to coated hard surfaces. The 2nd generation was the least susceptible, but with the highest adherent ability. The minimum inhibition concentration and minimal fungicidal concentration of C. krusei of all generations were determined at 0.4 mg mL(-1) . These observations suggest that the switching activity of C. krusei induces changes to its biological properties and susceptibility towards CHX.

Variability among microorganisms in barotolerance has been demonstrated at genus, species, and strain levels. Identification of conditions and additives that enhance the efficacy of ultrahigh pressure (UHP) against important foodborne microorganisms is crucial for maximizing product safety and stability. Preliminary work indicated that FD&C Red No. 3 (Red 3), a xanthene derivative, was bactericidal and acted synergistically with UHP against Lactobacillus spp. The objective of this study was to determine the antimicrobial efficacy of Red 3 and other xanthene derivatives, alone and combined with UHP, against spoilage and pathogenic bacteria in citrate-phosphate buffer (pH 7.0). Xanthene derivatives tested were fluorescein, Eosin Y, Erythrosin B, Phloxine B, Red 3, and Rose Bengal. Halogenated xanthene derivatives (10 ppm) were effective at reducing Listeria monocytogenes survivors but ineffective against Escherichia coli O157:H7. When combined with UHP (400 MPa, 3 min), the presence of derivatives enhanced inactivation. Because Red 3 was the only xanthene derivative to produce synergistic inactivation of both pathogens, further studies using this colorant were warranted. Efficacy of Red 3 against gram-positive bacteria (Lactobacillus plantarum and L. monocytogenes) was concentration dependent (1 to 10 ppm). E. coi O157: H7 strains were resistant to Red 3 concentrations up to 300 ppm. When Red 3 was combined with UHP, the lethality against gram-positive and gram-negative bacteria was dose dependent, with synergy being significant for most strains at > or = 3 ppm. Additional gram-positive and gram-negative bacteria showed lethalities similar to those observed for L. plantarum or L. monocytogenes, and E. coli O157:H7, respectively. Red 3 is a potentially useful additive to enhance the safety and stability of UHP-treated food products.

The hyphal sheath is a morphological feature of many kinds of fungi. Although the fine structures of the sheath have been studied in detail by a number of electron microscopy techniques, the function and physiology of the hyphal sheath are not yet clarified. One reason for this is that the hyphal sheath is a colorless, mucilaginous, and delicate material so that it is not easily identified. We developed a simple method to visualize and identify the hyphal sheath of the white-rot fungus Phanerochaete crassa WD1694. The small mycelial pellets in shaken liquid cultures of P. crassa WD1694 were stained directly with phloxine B. Both the hyphae and the hyphal sheath that filled the gaps between each of the hyphae were visualized and observed by light microscopy. The stained hyphae were further studied by transmission electron microscopy, atomic force microscopy, and fl uorescence microscopy. Based on these observations, we confirmed that the staining of the hyphae was also due to the presence of the hyphal sheath that closely covered the fungal cell wall. These results clearly showed that the hyphal sheath was selectively stained with phloxine B and could be observed and identified by conventional light microscopy.

Phloxine B (DC red no. 28) is a color additive for food, drugs, and cosmetics. It has been previously shown to have anti-Staphylococcus aureus activities. In this work, the effect of Phloxine B on various gram-negative bacteria and other gram-positive bacteria including Bacillus cereus, Bacillus thuringiensis, Bacillus mycoides, Bacillus subtilis, Bacillus aureus, Salmonella, Escherichia coli and Shigella was studied, along with the mechanism of anti-microbial activity. In the presence of fluorescent light, the viable count for gram-positive bacteria, (Bacillus spp. and S. aureus) decreased in a dose and time dependent manner when incubated with Phloxine B. The viability of gram-positive bacteria was reduced by 99.99% in 40 min, while there was no effect on gram-negative bacteria (Salmonella choleraesuis, E. coli and Shigella flexneri). However, the use of ethylenediaminetetraacetic acid (EDTA) expands the spectrum of activity for Phloxine B to include gram-negative bacteria. EDTA increased membrane-permeability by releasing lipopolysaccharide. Overall, in an Agar diffusion test the light-dependent bactericidal activity of 1 microg of Phloxine B had a potency of 0.64 units of chloramphenicol and 0.5 units of tetracycline when tested on B. cereus, and had a potency of 0.7 units of chloramphenicol and 0.2 units of tetracycline when tested on S. aureus. The data suggest that the dye may have some potential anti-microbial applications.

Studies were conducted to determine attraction and feeding propensity of Mediterranean fruit fly, Ceratitis capitata (Wiedemann), to different protein bait mixtures with and without the insecticides malathion, spinosad, and phloxine B. Protein baits were more attractive to females than to males. Protein-starved females responded more than protein-fed females. The type of protein (USB yeast hydrolysate enzymatic, MazofermE802, Nu-LureInsect Bait, or Provesta 621 autolyzed yeast extract) in the bait had a major influence on C. capitata attraction, which was strongest to fresh Provesta. Aged baits (four day-old) were not as attractive as fresh baits. In feeding propensity studies, highest response was observed for USB protein. On the basis of attraction and feeding responses Provesta (attraction and feeding) and USB (feeding) outperformed the standard Nu-Lure. Protein-starved flies were much more likely to feed on protein compared to protein-fed flies. For protein-starved flies, a mixture of Provesta and malathion repelled fruit flies, compared to a mixture of Provesta and spinosad or phloxine B. This was not the case with protein–fed flies. The wasp Fopius arisanus (Sonan), one of C. capitata’s primary natural enemies in Hawaii, would not consume protein baits. Our studies suggest that spinosad or phloxine B, with low contact toxicity, mixed with protein baits offers a more environmentally friendly choice for control of C. capitata and conservation of F. arisanus, whereby the nontarget effects of broad spectrum contact poisons such as malathion can be avoided. Presumably, due to greater selectivity with spinosad and phloxine B bait treatments, the host would be killed, but not the natural enemy.

In order to choose the best insecticide for fruit fly protein hydrolysate baits in field,laboratory tests were taken to assess the stomach toxicity of spinosad and phloxine B with torula yeast protein hydrolysate on oriental fruit flies,Bactrocera dorsalis (Hendel) female and male adults.Median lethal concentration values of spinosad and phloxine B on B.dorsalis male and female adults at 5 h were 12.757 0 mg/kg,13.953 5 mg/kg;779.937 8 mg/kg,826.625 9 mg/kg respectively.Median lethal time of spinosad was 4.190 4 h(females) and 4.973 0 h(males) at 16 mg/kg and 100 mg/kg;The median lethal time of spinosad was 2.487 7 h(females) and 2.675 5 h(males).For phloxine B,at 630 mg/kg the median lethal time was 5.350 7 h(females) and 4.946 2 h(males),and at 1,000 mg/kg the median lethal time was 4.765 0 h(females) and 4.571 5 h(males).

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. © 2001 Society of Chemical Industry

The effects of the fluoresceine derivative, phloxine B, on the Cl− current through the cystic fibrosis transmembrane conductance regulator (CFTR) were examined in Xenopus oocytes expressing human CFTR. In whole oocytes, the CFTR Cl− current (ICFTR) was activated by superfusion with isobutylmethylxanthine and forskolin. ICFTR was stable during activation and deactivated rapidly upon washout of the activation solution. Phloxine B slowed deactivation and, at high concentrations, inhibited ICFTR weakly. In excised inside-out macropatches, ICFTR was activated by the catalytic subunit of protein kinase A (cPKA) and MgATP. Phloxine B (0.01–3 μM), applied after activation, increased ICFTR within 30 s followed by a slow decrease which became dominant at high concentrations. Slowing of deactivation of the CFTR was observed at all concentrations. The effect of phloxine B after 30 s had a bell-shaped concentration-dependence with midpoints at 45 and 1600 nM for the stimulatory and the inhibitory limb, respectively; maximum stimulation was about 1.8 times. The slow inhibitory component, measured after 6 min, occurred with an IC50 value of ∼1 μM. In the absence of cPKA, phloxine B did not stimulate ICFTR. In the presence of cPKA and MgATP, the effects of phloxine B were more prominent at low (0.02 mM) than at high ATP (2 mM). The data show that phloxine B modulates ICFTR by increasing channel activity and slowing channel deactivation; at high concentrations inhibition dominates. The effects may be mediated by direct interactions with CFTR from the inside of the cell. British Journal of Pharmacology (2000) 131, 433–440; doi:10.1038/sj.bjp.0703600

Many photoactive dyes are relatively nontoxic to vertebrates despite their insecticidal properties. Several photoactive dyes known to be toxic to some groups of insects were evaluated at various concentrations for toxicity to American and migratory grasshoppers in laboratory and field studies. Rose bengal and phloxine B were effective at inducing mortality of grasshoppers when applied at 2 and 5% to bran bait, though erythrosin B and uranine were ineffective. Partial replacement of phloxine with uranine in dye mixtures resulted in no significant loss of efficacy. Some indication of feeding inhibition was observed at high dye concentrations, so minimum effective dosages, probably 2%, are optimal. Phloxine B and rose bengal appeared to be stable upon exposure to sunlight, and able to withstand at least 24 h of sunlight without significant degradation. Dyes such as phloxine B could be a viable grasshopper control agent for small or medium-sized grasshopper species because mortality can be induced by consumption of a single flake dusted with 5% dye, and yet pose little hazard to vertebrates. Large species such as American grasshopper must consume several flakes before mortality is induced.

Agrobacterium tumefaciens cells were killed upon illumination when treated with submicromolar concentrations of phloxine B. Phloxine B could be distributed systemically through a plant by amending the water supply or delivered directly to specific locations through topical applications in 0.1% adjuvant (Kinetic HV). Sweet basil (Ocimum basilicum L.), jimsonweed (Datura stramonium L.), and soybean (Glycine max L.) were infected with A. tumefaciens to establish a marker for a systemic pathogenic bacteria. There was a >90% reduction in the plants' population of A. tumefaciens when watered daily with a 1 micromolar phloxine B solution. Furthermore, the plants treated with phloxine B were asymptomatic to the pathogenic challenge. Leaf tissue, collected from basil and soybean, also expressed a reduction in the population of A. tumefaciens by >90% when treated under greenhouse conditions for 24 h with 10 μM phloxine B in 0.1% Kinetic HV. The possible use of phloxine B as an agent to control bacterial diseases in plants is suggested.

A method was developed for the analysis of phloxine B and uranine, photoactive dyes being evaluated as fruit fly toxicants, in coffee cherries and green and roasted beans. The analytes were measure...

The toxicity of a mixture of two photoactive dyes, Phloxine B and Uranine, was determined for six species of beneficial insects in the laboratory. The dye had no measurable contact toxicity to any test insect. The dye, when mixed in protein baits, was significantly more toxic than the bait alone to Chrysopa carnea Stephens, Cryptolaemus montrouzieri Mulsant, Delphastes pusillus Le Conte and Goniozus legneri (Gordh). The toxicity of all dye and bait mixtures was significantly less to all test insects than that of the malathion and bait mixtures. Substituting the dye mixture for malathion in protein bait sprays used to eradicate exotic fruit flies from the United States should significantly reduce the kill of non-target beneficial insects.

Spectrophotometric investigations of dye solutions in different media and of living stained cells from the upper epidermis of the scaleleaf of Allium cepa were carried out with the dyes K-fluorescein, rose Bengal and phloxine red to elucidate the mechanism of the accumulation of these dyes in the cytoplasm, the nucleus and the cell sap. Thin layer chromatography and paper electrophoresis indicate that the K-fluorescein used here contains no detectable contaminants. Besides the main component, rose Bengal contains two components in small quantities with Rf values of 0.64 and 0.57, plus three more components in traces. Besides the two main components (Rf values of 0.83 and 0.73), phloxine red also contains five more components in traces. Electrophoretic investigations reveal that in aqueous solution the fluorones rose Bengal and phloxine red from pH 2.0-11 show a migration toward the anode. K-fluorescein from pH 2.9-10.4 shows a migration toward the anode, but at pH 1.9 a migration toward the cathode. By shaking aqueous solutions of K-fluorescein, rose Bengal and phloxine red at different pH values with different organic solvents, the above used stainings show different spectral absorption curves according to the polarity of the solvent. The position of the absorption maxima and the shape of the absorption curves of these three anionic dyes lead to the conclusion that the staining of the living cytoplasm and nucleus is due to ion accumulation by means of the "ion trap mechanism" within the aqueous phase of the cytoplasm (cytosol) and the nucleus. Adsorption of dye particles in the protein phase of the cytoplasm cannot be excluded. There seems to be a fundamental difference in the vital staining of the protoplasm by anionic and cationic dyes, the latter apparently accumulating as neutral dye molecules in the lipid phase of the protoplasm. The concentration of the dyes used in the living cytoplasm (cytosol) is approximately 0.2-0.05%. During natural and artificial displacement of K-fluorescein from the cytoplasm to the vacuole, it appears that accumulation of the dye within the vacuole is performed through an ion trap mechanism in the form of bivalent ions. Along with natural displacement, it is possible that ion accumulation also occurs in metabolic products.

The authors investigated the applicability of an assay method for immediate allergic reaction in vitro to determine radioactivity of pre-incorporated [3H] serotonin from rat basophilic leukemia cells (RBL-2H3) stimulated with biotinylated IgE-avidin complex. Using the newly improved assay method, the effects of synthetic food colors on immediate allergic reaction were determined. The optimum ratio and amount of stimulator for the cell stimulation was found to be 3μg/ml (0.015μM) of biotinylated IgE and 1 μg/ml (0.015 μM) of avidin. Seven kinds of food colors including food red No. 2 (amaranth) and food yellow No. 4 (tartrazine) inhibited about 50% of [3H] serotonin release at 1 mM compared to that of the cells exposed to the stimulator alone. Food blue No. 2 (indigo carmine) inhibited 20% of the release at 1 mM, while food red No. 106 (acid red) had no effect. Food red No. 3 (erythrosine), food red No. 104 (phloxine) and food red No. 105 (rose bengale) promoted [3H] serotonin release from nonstimulated cells at lower concentration and showed strong lethal effect to the cells at higher concentration. Thus, it was suggested that food colors might modify immediate allergic response, and show cytotoxic action when ingested orally with foods.

Soil eco-toxicity testing was conducted in support of Canada’s Chemical Management Plan (CMP) to fill data gaps for organic chemicals known to primarily partition to soil, and of which the persistence and inherent toxicity are uncertain. Two compounds representative of specific classes of chemicals: non-chlorinated bisphenols containing an –OH group (4,4′-methylenebis(2,6-di- tert -butylphenol (Binox)) and xanthene dyes (2′,4′,5′,7′-tetrabromo-4,5,6,7-tetrachloro-3′,6′-dihydroxy-, disodium salt (Phloxine B), 2′,4′,5’,7′-tetrabromofluorescein (TBF), 4′,5′-dibromofluorescein (DBF), and 4,5,6,7-tetrachlorofluorescein (TCF)) were evaluated. The effect of these substances on plant growth ( Elymus lanceolatus and Trifolium pratense ) and soil invertebrate survival and reproduction ( Folsomia candida and Eisenia andrei ) were assessed using a field-collected sandy soil. Binox was persistent throughout testing (up to 63 d) with an average recovery of 77 ± 2.9% at test end. Binox was not toxic to plants (IC50s > 1076 mg kg −1 ) or E. andrei (IC50s > 2651 mg kg −1 ); however, a significant reduction in F. candida adult survival and reproduction (IC50 = 89 (44–149) mg kg −1 ) was evident. Phloxine B was also persistent throughout testing, with an average recovery of 82 ± 3.0% at test end. Phloxine B was significantly more toxic than Binox, with significant reductions in plant root growth (IC50s ⩾ 11 mg kg −1 ) and invertebrate reproduction (IC50s ⩾ 22 mg kg −1 ). DBF toxicity was not significantly different from that of Phloxine B for plant root growth (IC50s ⩾ 30 mg kg −1 ), but was significantly less toxic for shoot growth (IC50s ⩾ 1758 mg kg −1 ), and invertebrate adult survival (IC50s ⩾ 2291 mg kg −1 ) and reproduction (IC50s ⩾ 451 mg kg −1 ). A comparison between all four xanthene dyes was completed using F. candida , with the degree of toxicity in the order of Phloxine B ⩾ TBF ∼ DBF > TCF. The results from these studies will contribute to data gaps for poorly understood chemicals (and chemical groupings) under review for environmental risk assessments, and will aid in the validation of model predictions used to characterize the fate and effects of these substances in soil environments.

Spores of a yeast, Saccharomyces cerevisiae , and a bacterium, Bacillus subtilis , were exposed to high voltage electric pulses. The viabilities of spores and vegetative cells of the yeast were significantly decreased after the electric pulse treatment, and some of the spores and almost all of the cells were stained red with an agent, phloxine B. On the other hand, (endo) spores of the bacterium were highly resistant to the electric pulses and little decrease in viability was observed, although the viability of vegetative cells was sharply lowered. The results revealed marked structural and/or biochemical differences between eukaryotic and prokaryotic spores.

Following our observation that erythrosine B (FD&C Red No. 3) is a relatively potent inhibitor of the TNF-R-TNFα and CD40-CD154 protein-protein interactions, we investigated whether this inhibitory activity extends to any other protein-protein interactions (PPI) as well as whether any other approved food colors possess such inhibitory activity. We found erythrosine, a poly-iodinated xanthene dye, to be a non-specific promiscuous inhibitor of a number of PPIs within the tumor necrosis factor superfamily (TNF-R-TNFα, CD40-CD154, BAFF-R-BAFF, RANK-RANKL, OX40-OX40L, 4-1BB-4-1BBL) as well as outside of it (EGF-R-EGF) with a remarkably consistent median inhibitory concentration (IC(50)) in the 2-20 μM (approximately 2-20mg/L) range. In agreement with this, erythrosine also showed cellular effects including clear cytotoxic effects around this concentration range (IC₅₀≈50 μM). Among the seven FDA-approved food colorants, only erythrosine showed consistent PPI inhibitory activity in the sub-100 μM range, which might also explain (at least partially) why it also has the lowest approved acceptable daily intake (ADI) (0.1 mg/kg body weight/day). Among a number of xanthene structural analogs of erythrosine tested for activity, rose Bengal, a food colorant approved in Japan, showed similar, maybe even more pronounced, promiscuous inhibitory activity, whereas fluorescein was inactive and gallein, phloxine, and eosin were somewhat active in some of the assays.

Xanthene dyes have been tested as photoinsecticides on different dipteran species. Phloxine B ingested by Ceratitis capitata larvae showed low toxicity under dark conditions and acute light-dependent toxicity when the insects were exposed to light during the dispersion stage before pupariation. In this study we show that sub-lethal concentrations of Phloxine B generated subtle changes in weight gain and in the accurately regulated jumping behavior of metamorphosing larvae. These changes are correlated with a strong accumulation of glycogen in fat body, intestine and muscles, as well as with an inhibition of the glycogen phosphorylase activity. These results suggest that some of the non-phototoxic effects of Phloxine B might be caused by an alteration of the glycogen catabolism, which can eventually affect the viability of the flies. Fil: Berni, Jimena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Rabossi, Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Pujol Lereis, Luciana Mercedes. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Tolmasky, Diana Silvia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina Fil: Quesada Allue, Luis Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentina

Hydroxyxanthenes are commonly used as dyestuffs in the food, cosmetics, and textile industries. These compounds also have medical applications due to their unique staining and fluorescent properties. The halogenated hydroxyxanthenes exhibit antimicrobial properties that may be useful for reducing or eliminating bacterial pathogens from a variety of environments, including drinking water and food products. Antimicrobial characteristics of Eosin, Erythrosine, Phloxine, and Rose Bengal have been known for many years, but their application as antimicrobial agents has been limited primarily to selective agents in microbiological growth media. The primary mechanism of bacterial inactivation by hydroxyxanthenes is photooxidation. When halogenated hydroxyxanthenes are photooxidized, a variety of compounds are produced (e.g., singlet oxygen, superoxide anion and other radicals); these exhibit toxicity to the microbial cell. Gram-positive bacteria are particularly sensitive to inactivation by photooxidation of halogenated hydroxyxanthenes; however, different species vary in their sensitivity. Gram-negative bacteria are inherently resistant to inactivation by these compounds due to the barrier properties of the outer membrane that prevent the necessary localization. Treatments such as chelation and ultrahigh pressure may be used to destabilize the outer membrane leading to sensitivity of Gram-negative bacteria to halogenated hydroxyxanthenes.

The oriental fruit fly, Bactrocera dorsalis (Hendel), is a tephritid fruit fly that is a serious pest of a wide range of tropical fruits. Populations of oriental and other fruit fly species are often suppressed by utilizing aerial or ground sprays of hydrolysed protein bait mixed with malathion. Although proven to be efficacious, spraying food bait mixed with malathion in urban areas is strongly opposed by the public because of perceived public health and environmental risks. To suppress the population level of oriental fruit fly in a guava orchard in Hawaii, we used the light-activated xanthene dye, phloxine B (2',4',5',7'-tetrabromo-4,5,6,7-tetrachlorofluorescein, disodium salt), as an alternative to malathion in protein bait sprays. Because phloxine B must be ingested to be toxic, it is expected that phloxine B bait sprays will have less impact on non-target insects than the contact insecticide malathion. Evidence of suppression of oriental fruit fly population in the treatment orchard was provided by both protein bait trap catches and assessment of fruit infestation. This suppression was achieved even though there was an unsprayed guava orchard on two sides of the small (less than 2.0ha) sprayed orchard. These results, combined with results for other fruit fly species (presented elsewhere), indicate that phloxine B-protein bait sprays can be effective in suppressing populations of several different fruit fly species and that phloxine B is a potential replacement for malathion in bait sprays for tephritid fruit fly suppression/eradication programs.

Synthetic food dyes are xenobiotics, and, after ingestion, portions of these dyes may be absorbed and metabolized by phase I and II drug-metabolizing enzymes, and excreted by transporters of phase III enzymes. In the previous report, it was shown that inhibition of UDP-glucuronosyltrasnferase 1A6 occurred following ingestion of phloxine, erythrosine, and rose bengal present in 12 permitted synthetic food dyes. In this report, the influence of dyes was examined on CYP3A4, a major phase I drug-metabolizing enzyme, and P-glycoprotein, a major transporter by synthetic food dyes. Human cytochrome P-450 (CYP) 3A4 and P-glycoprotein were inhibited by xanthene food dyes. The IC(50) values of these dyes to inhibit CYP3A4 and P-glycoprotein were the same as the level of inhibition of UGT1A6 produced by three haloganated xanthene food dyes in the previous report, except acid red, which inhibited only CYP3A4. Data suggest that inhibition by dyes is not enzyme specific but may be in a membrane-specific or protein-specific manner, such as conformational changes in protein. In the previous study, it was suggested that inhibition by dyes depended upon light irradiation due to generation of (1)O2 from these dyes. In this study, the influence of superoxide dismutase and catalase on inhibition by dyes was examined. Superoxide dismutase but not catalase was effective in preventing the inhibition of UGT1A6 by the dyes. Data suggest that superoxide anions, originating from dyes via light irradiation, may attack drug-metabolizing enzymes. It is possible that red cosmetics containing phloxine, erythrosine, or rose bengal react with proteins in skin and may lead to skin damage.

The cystic fibrosis transmembrane conductance regulator (CFTR) protein contains a canonical ATP-binding cassette (ABC) signature motif, LSGGQ, in nucleotide binding domain 1 (NBD1) and a degenerate LSHGH in NBD2. Here, we studied the contribution of the conserved residues G551 and G1349 to the pharmacological modulation of CFTR chloride channels by phloxine B using iodide efflux and whole-cell patch clamp experiments performed on the following green fluorescent protein (GFP)-tagged CFTR: wild-type, delF508, G551D, G1349D, and G551D/G1349D double mutant. We found that phloxine B stimulates and inhibits channel activity of wild-type CFTR (Ks = 3.2 +/- 1.6 microM: , Ki = 38 +/- 1.4 microM: ) and delF508 CFTR (Ks = 3 +/- 1.8 microM: , Ki = 33 +/- 1 microM: ). However, CFTR channels with the LSGDQ mutated motif (mutation G551D) are activated (Ks = 2 +/- 1.13 microM: ) but not inhibited by phloxine B. Conversely, CFTR channels with the LSHDH mutated motif (mutation G1349D) are inhibited (Ki = 40 +/- 1.01 microM: ) but not activated by phloxine B. Finally, the double mutant G551D/G1349D CFTR failed to respond not only to phloxine B stimulation but also to phloxine B inhibition, confirming the importance of both amino acid locations. Similar results were obtained with genistein, and kinetic parameters were determined to compare the pharmacological effects of both agents. These data show that G551 and G1349 control the inhibition and activation of CFTR by these agents, suggesting functional nonequivalence of the signature motifs of NBD in the ABC transporter CFTR.

A laboratory bioassay was developed to determine both the chemical toxicity and the phototoxicity of the xanthene dye, phloxine B (D&C Red N°28), to the immature stages of the Mediterranean fruit fly, Ceratitis capitata (Wiedemann). An additional goal was to find out which main tissues are affected first. A low, but significant, level of toxicity was observed when the insects were maintained in the dark: at the point of adult ecdysis, the LC50 was 11.03 mM. As expected, after 8-h exposure of late larva III to light, a high level of mortality was produced (LC50 at ecdysis: 0.45 mM) as a dose-dependent function of dye concentration. At sublethal concentrations of the dye, the surviving insects showed a number of physiological abnormalities. Phloxine B appeared to mainly affect the larval longitudinal muscles as well as the abdominal muscles of ecdysing adults, giving rise to abnormal puparia and failed adult ecdysis, respectively. Moreover, a significant phloxine B-dependent delay in the jumping of surviving larvae for dispersal was documented. This could be attributed to a delay in attaining a threshold weight for jumping and/or to abnormalities in neuromuscular coordination, thus reinforcing the idea of pleiotropic effects of the dye.

Fluorescein dyes in which the benzoic acid moiety has been tetrachlorinated (50 to 100 μg/ml) inhibit in vitro Staphylococcus aureus growth (MIC, 25 μg/ml). Specifically, under standard room illumination, phloxine B at a concentration of 100 μg/ml killed 99% of the cultures (mid-log phase). It also reduced S. aureus CFU by 10 4 . Structure-activity analysis revealed that the activity against S. aureus increases with the increase in the number of the substituting halogens in the hydroxyxanthene moiety.

The fluorescein derivative phloxine B is a potent modulator of the cystic fibrosis transmembrane conductance regulator (CFTR). Low micromolar concentrations of phloxine B stimulate CFTR Cl(-) currents, whereas higher concentrations of the drug inhibit CFTR. In this study, we investigated the mechanism of action of phloxine B. Phloxine B (1 microm) stimulated wild-type CFTR and the most common cystic fibrosis mutation, DeltaF508, by increasing the open probability of phosphorylated CFTR Cl(-) channels. At each concentration of ATP tested, the drug slowed the rate of channel closure without altering the opening rate. Based on the effects of fluorescein derivatives on transport ATPases, these data suggest that phloxine B might stimulate CFTR by binding to the ATP-binding site of the second nucleotide-binding domain (NBD2) to slow the dissociation of ATP from NBD1. Channel block by phloxine B (40 microm) was voltage-dependent, enhanced when external Cl(-) concentration was reduced and unaffected by ATP (5 mm), suggesting that phloxine B inhibits CFTR by occluding the pore. We conclude that phloxine B interacts directly with CFTR at multiple sites to modulate channel activity. It or related agents might be of value in the development of new treatments for diseases caused by the malfunction of CFTR.

The xanthene dye phloxine B (D&C Red #28) bait was sprayed against fruit flies in mango orchards in 1996 and 1997. The flies used for testing were Mexican fruit fly, Anastrepha ludens (Loew), West Indian fruit fly, Anastrepha obliqua (Macquart), and Mediterranean fruit fly, Ceratitis capitata (Wiedemann). Results of the experiments indicate that the toxic efficacy of phloxine B against these fruit flies is as good as that of malathion-bait sprays. Results also indicate that type of protein used with phloxine B can dramatically influence its efficacy. Hydrolyzed proteins of corn origin, Mazoferm 802 and Nutriplus, and one from microbial origin, Coltec yeast broth, were best. Phloxine B-bait applications as complete coverage or alternate swaths reduced fly populations as well as 19.5 or 9.8% (AI) malathion-Captor 300. Applications of phloxine B bait at concentrations of 0.12% phloxine B reduced populations as well as those applied at 0.48% (AI). The fruit fly parasitoid Diachasmimorpha longicaudata was adversely affected when exposed to phloxine B-Nutriplus bait but not when exposed to the other proteins.

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.

The aim of this study was to examine the tumorigenicity of the food colouring phloxine (Food Red 104) in (C57BL/6N × C3H/HeN) F1 mice. Groups of male or female mice received 0, 0.1 and 0.4% phloxine in the diet starting at 6 wk of age for a maximum of 90 wk. Survivals of mice were more than 85% at 64 wk after the start of phloxine administration. Their body weights reached maximum at 48 wk and both male and female mice given 0.1 and 0.4% phloxine weighed significantly more than their respective controls. Tumours developed in 13 organs, the greatest incidence in males being of liver tumours (67.3%), followed by lung tumours (10.9%), lymphomas (9.7%), altered foci of liver (8.4%) and hepatic haemangioma (7.8%). In females, hepatic tumours (11.5%) had the greatest incidence followed by lymphomas (10.8%), pulmonary tumours (8.2%) and hepatic haemangioma (3.2%). In males, the incidence of altered foci and haemangioma in the liver was 19 and 14%, respectively, in the control groups, whereas only 2% occurred in the group given 0.4% phloxine (P < 0.05). It is concluded that average body weights increased significantly in female mice of groups given 0.1 or 0.4% phloxine, compared with body weights of the female controls, and that altered liver foci were significantly reduced in male mice given 0.4% phloxine. There was a significant increase in pituitary tumour incidence in the test groups compared with the control groups.

The colour additive phloxine was given in the diet to provide dietary levels of 0 (control), 0.1, 0.3 and 0.9%, from 5 wk of age of the F0 generation to 8 wk of age of the F1 generation in mice, and selected reproductive and neurobehavioural parameters were measured. There was little effect of phloxine on either litter size or weight, or sex ratio, whereas the body weight of the pups in the lactation period was significantly increased in all treatment groups. Among the neurobehavioural parameters measured, surface righting at postnatal day 4 of male pups was significantly reduced in all treatment groups. Some parameters of the motor activity of pups at 3 wk of age differed from those of the controls; in particular, the average speed of movement male pups was significantly reduced in all treatment groups. The dose levels of phloxine in this study produced a few adverse effects in reproductive and neurobehavioural parameters in mice.

The toxicity of xanthene dyes were studied by various interaction between the dyes and the components in vital body. (1) An increase in the amount of Rose Bengale adsorbed on the gill of fish was followed by the increase in red corpuscle number, and it was assumed that the death of fish in xanthene dye solution was due to anoxemia. (2) Binding capacity of xanthene dyes with bovine serum albumin decreased in the following; Rose Bengale, Phloxine, Erythrosine, Eosine and Uranine. This order was quite coincident with the toxicity compared by TLm values. (3) From the results of rec-assay test by use of Bacillus subtilis, it was confirmed that Phloxine and Rose Bengale had DNA-damaging capacity related to the mutagenecity.

Summary Disodium 9-(3′,4′,5′,6′-tetrachloro- o -carboxyphenyl)-6-hydroxy-2,4,5,7-tetrabromo-3-isoxanthone (phloxine), a red dye used as a food additive, was tested for its activity to induce 8-azaguanine (8AG) resistant mutations in cultured human embryonic cells. Phloxine had a severe cytotoxic effect on the cells at concentrations of 1 to 10 μ g/ml. At concentrations of more than 30 μ g/ml of phloxine no further decrease in cell survival was found. This cytotoxic effect of phloxine was not dependent on the duration of treatment. After treatment with phloxine for 2h division of cells in normal medium was inhibited for 120 h. When cells were treated with phloxine at various concentrations for 2 h, cultured in normal medium for 48 h, and then selected with 30 μg/ml of 8AG, an increase in the induced mutation frequency was found. This increase in mutation frequency was dependent on the concentration of phloxine used as a mutagen and treatment with 100 μg/ml of phloxine increased the frequency to six times that in untreated cultures.

An improved modification upon Ivic's method (1959) for differentially staining A- and B-cells in the pancreatic islets of Langerhans is described. In accordance with usual procedure, pancreas tissue is fixed in Bouin's fluid, washed in 70% alcohol, and then dehydrated and embedded in paraffin. After being mordanted in Bouin's fluid containing 3-5% chrome alum for 24hr or more at 37°C, sections are moderately oxidated in an equal mixture of 0.3% potassium permanganate and 0.3% sulphric acid for 3-5min.As proposed by Ivic, a Victoria blue solution is then used to exclusively stain the B-cells blue. In this case, however, the solution is allowed to ripen for at least 10 days after mixing before use. A-cells are then stained deep red with a 0.5% aqueous solution of phloxine for 30-120sec. without any differentiation. After treatment with a 5% aqueous solution of phosphotungstic acid for 1-2min, sections are stained in 0.5% aqueous solution of light green for 1-2min in order that the collagenous elements obtain a green color.This modified method produces a good differential staining of A- and B-cells more easily and more consistently than does Ivic's original method.

A histological investigation was made on the juxtaglomerular cell granules (JG granules), using the kidneys of rockfish, Sebastiscus marmoratus, rabbitfish, Chimaera monstrosa, and pig, Sus scrofa. JG granules in these three species were clearly stained with Bowie's method . In other staining methods, however, remarkable aifferences were observed in JG granules of these species. Only JG granules of rockfish were PAS-positive in PAS and Mayer's acid hemalum stain. In Heidenhain's iron-hematoxylin stain, JG granules of pig and rabbitfish were siderophilic. Furthermore, JG granules of porcine kidney were stained with phloxine in Gomori's chrome-hematoxylin and phloxine stain. On the other hand, in Mallory's triple stain, JG granules in porcine kidney were stained with acid fuchsin, whereas those in rockfish and rabbitfish with orange G.

Trypan blue has proved effective for demonstrating the presence of certain plant viruses within infected tissues. The amorphous and crystalline inclusions which constitute cytological evidence of viruses stain proportionately. The effects produced by different viruses react differently to the stain and those inclusions which do not absorb trypan blue tend to stain with phloxine. This selective staining is the basis for using trypan blue singly and in combination with phloxine as standardized procedures for demonstrating and differentiating cytological evidence of plant viruses. These tests are very rapid and are especially applicable to temporary mounts of living tissue but permanent mounts can be made from material fixed in formalin.

The secretory granules of the corpuscular cells of STANNIUS and the renal juxtaglomerular cells (JG cells) were examined histologically, using five species of teleosts. The granules in the corpuscular cells are stainable by the BOWIE'S method which is used exculsively far the staining of JG cell granules. However, remarkable differences were detected in the staining of both kinds of granules when the following stains were used: GOMORI'S chrome-hematoxylin and phloxine, HEIDENHAIN'S iron-hematoxylin, and PAS stain. Therefore, it may be concluded that the JG cells and the corpuscles of STANNIUS, histologically speaking, are composed of different kinds of cells.

The effect of some synthetic food dyes on RNA-synthesizing reaction was studied by employing the DNA-dependent RNA polymerase purified from Escherichia coli. And the modes of inhibition by ponceau 3R and rose bengale were discussed.Ponceau 3R, ponceau R and orange I strongly inhibited the polymerase reaction at the concentration of 1mM. Among them, ponceau 3R was assumed to prevent the polymerase activity by interacting with the initiating region of RNA-transcription on DNA. The dye, however, showed a weak affinity to DNA.Xanthene dyes which contained halogen atoms such as eosine, erythrosine, rose bengale and phloxine strongly inhibited the polymerase at 0.1mM. Since acid red showed no effect on the polymerase at higher concentrations, it was presumed that halogen molecule was essential to the inhibitory effect of xanthene dyes. The inhibition took place even in the dark, indicating no essential participation of photo-chemical reaction by these dyes. Rose bengale seemed to inhibit the RNA synthesis by binding with the polymerase molecule.Naphthol yellow S and indigo carmine gave on inhibition for the polymerase reaction.

Round or slightly oval inclusion bodies were found in low numbers in epidermal cells of spiked (expressing spike disease symptoms) leaves. They could not be demonstrated in epidermal tissue of apparently healthy leaves. The intracellular inclusions stained with phloxine and rose bengal. Their relationship to sandal spike virus is discussed.

Azure A, azure B and Nile blue in both neutral and alkaline solutions were found to be suitable for use as indicator dyes in the Sabin-Feldman test. Toluidine blue, brilliant cresyl blue and thionine were also suitable as neutral solutions. Azure C, phloxine, phloxine B, neutral red, methyl green, pyronine Y, eosin and celestine blue were found to be unsuitable. Azure B and toluidine blue in saline solutions were serviceable up to 5 months after preparation. The significance of these findings is discussed.

1. Neurosecretory materials within the corpora cardiaca of adult female roaches (Blaberus craniifer) were studied histologically, using chrome hematoxylin and phloxin or aldehyde fuchsin stains.2. Both the neurosecretory substances which are stained dark blue with chrome hematoxylin and those stained pink by phloxine are markedly depleted in the corpora cardiaca following administration of electric shocks for periods of 15 minutes or more to the heads or abdomens of the roaches.3. Forced hyperactivity of the roaches, when continued for periods of 15 minutes or longer, also causes a marked decrease in the same neurosecretory materials within the corpora cardiaca.4. Following periods of forced hyperactivity of the animals, there is also a loss of the potency of extracts prepared from their corpora cardiaca. when such extracts are assayed for their ability to depress spontaneous activity in roach central nerve cords in vitro.5. It is suggested that the release of neurosecretory substances from the corpora ca...

Photodynamic action of phloxine on mitochondrial respiration was examined, using rat liver mitochondria. Rat liver mitochondria were suspended in a medium containing phloxine, and irradiated with visible light in the presence of oxygen. The respiratory activities of the photooxidized mitochondria were assayed. The results are summarized as follows. (1) Phloxine-sensitized photooxidation of mitochondria inhibited 2, 4-dinitrophenol (2, 4-DNP)-released respiration. It can be deduced, from an approximate calculation, that the released respiration was inhibited by 10% as every 3 nmoles of oxygen per mg of mitochondrial protein was consumed by photooxidation. (2) Phloxine-sensitized photooxidation of mitochondria released the suppressed respiration. The supernatant obtained by centrifugation of photooxidized mitochondria also released the suppressed respiration of intact mitochondria. This suggests that certain uncoupling factors were released from photooxidized mitochondria. Uncoupling factor in question may be unsaturated fatty acids or U-factor, because bovine serum albumin abolished the respiration-releasing effect of the supernatant of photooxidized mitochondria.

SUMMARY Inclusion bodies in the cells of the leaf epidermis of dahlia plants infected with dahlia mosaic virus are spherical and highly refractive. They stain with trypan blue or phloxine, and this staining provides a quick and simple test for the detection of dahlia mosaic virus in dahlia plants, which is particularly useful for varieties that do not show external symptoms.

Lipid cytochemistry studied in 130 rat pineals demonstrates two common types of parenchymal cells which may be involved in an endocrine secretory activity. The first cell type is characterized by its content of abundant lipid droplets, composed primarily of ethanol-soluble carbonyl lipids coated with phospholipid. The second cell type is distinguished by its content of a phospholipid cytoplasmic matrix containing few droplets on vacuoles. This cell type appears to be identical with the cell staining with orange G or phloxine in a modified chrome alum hematoxylin technique described previously. Parenchymal cells containing lipid occur in seven human pineals of diverse ages; and in the older pineals interlobular cells containing lipid occur as well. The lipid in human parenchymal cells differs from that in the type 1 parenchymal cells of the rat by, (1) forming smaller droplets, (2) containing a greater amount of neutral lipid and less carbonyl lipid, and (3) being associated with pigment fomation in adults and older people.

In order to study the safety of food additives with regard to their effects on alimentary tract, we examined the effects of food colors on leucine aminopeptidase activity and amino acid uptake in rat small intestine.Leucine aminopeptidase activity in the homogenate of intestinal mucosa was estimated by use of leucine-β-naphthylamide as the substrate. Amino acid uptake was examined by the tissue accumulation method of α-aminoisobutyric-3-14C-acid in intestinal segment of rat. The results obtained are as follow;(1) The leucine aminopeptidase activity in mucosal homogenate was extremely decreased by the addition of xanthene or triphenylmethane colors to the incubation mixture (final concentration; 10-4M), although azo and indigoid colors gave no effect.(2) Decrease in the enzyme activity was not observed by sorbate, saccharin and other several food additives. Xanthene-related compounds, fluorescein and certain halogenated fluorescein derivatives, inhibited leucine aminopeptidase activity.(3) Lineweaver-Burk's plot showed that inhibition of xanthene colors was “uncompetitive type”.(4) Tissue accumulation of α-aminoisobutyric acid in intestine was inhibited by xanthene color, phloxine, in the same rate as the metabolic inhibitors, cyanide and 2, 4-dinitrophenol. However, other types of colors did not give the inhibitory effect.(5) The declines of leucine aminopeptidase activity and amino acid uptake were not observed with the rats administrated food color per os for three months.

The cultures of Lophotrichus antmpullus Benjamin and L. martinii Benjamin employed in this study were subcultures of the material upon which Benjamin based his descriptions. Material to be sectioned was cultured, killed and fixed, and stained by the same procedures outlined in an earlier paper on Chaetomium (Whiteside, 1961). Under the procedures followed, the Gram stain usually proved to be more satisfactory than Heidenhain's hematoxylin for staining sectioned material of this genus. For the observation of ascocarp initials, as well as for the study of the contents of crushed perithecia, a dilute aqueous phloxine solution was used. The nuclei of the ascogenous cells stained readily with the phloxine but only with difficulty when treated with the carmine stains.

In epidermal strips of pea plants with the Wisconsin strain of the pea streak virus, studied from about three weeks after inoculation, extensive parts of the cytoplasm stained red when treated with phloxine and methylene blue. Usually the stained areas were not sharply delimited. Sometimes more or less well-defined granular and often vacuolated inclusion bodies were observed. They were mainly located near the nucleus and along the cell wall.