Phthalates are ubiquitous environmental pollutants associated with endocrine disruption and peroxisome proliferation in experimental animals. In yeasts exposed to environmental chemicals, including phthalates, alterations in cell growth, cellular morphology, and H2O2 detoxification occur. Nutrient availability also influences diverse cellular processes. Differences in responses to environmental stress between Candida albicans and the model yeast, Saccharomyces cerevesiae, have been reported. In this study, we chose C. albicans as an alternate model for testing estrogen-like chemicals because of its high affinity estrogen-binding protein and, in contrast to S. cerevesiae, estrogens are not growth inhibitory for C. albicans. Cultures were grown in either yeast nitrogen dextrose (YND; phosphate limiting) or YNDP (YND plus 100 mmol/L inorganic phosphate). For chemical testing, 0.5% dibutylphthalate (DBP), 0.05% Tween 80, or a combination of the two (DBPT) were incorporated in growth media to investigate the effects of these estrogenic agents on cell proliferation, morphology, and catalase demonstration. We observed significant differences in cell growth related to DBP and changes in cell wall thickness related to both Tween 80 and phosphate. We describe ultrastructural changes including detachment of the outer yeast cell wall layer and presence of putative peroxisomes. Our findings support the proposal that C. albicans may be particularly suitable for use in studies involving cellular responses associated with exposure to estrogenic chemicals contained in complex mixtures.

Mobiluncus spp. are gram variable anaerobic rods found associated with bacterial vaginosis. Recent molecular analyses of Falcivibrio spp. has identified these species as misclassified Mobiluncus [1]. We therefore will use Mobiluncus for further description of these organisms.Clinical isolates of M. mulieris (ATCC) were maintained on Columbia CNA Blood Agar under anaerobic conditions [2]. For growth of cultures for electron microscopic analyses, cells were grown anaerobically in thioglycolate broth for 24 hr at 37 o C, collected by centrifugation and transferred immediately to 4% paraformaldehyde, 2.5% glutaraldehyde in either sodium cacodylate or Sorenson's phosphate buffer. Cells were fixed for 24 hr, rinsed in appropriate buffer, and post-fixed in 1% OsO 4. The cells were dehydrated in an ascending ethanol series and transferred to propylene oxide as a transition fluid. The cells were then infiltrated in Epon-Araldite (substituted; Electron Microscopy Sciences) and embedded in BEEM capsules. The resin was cured 24 hr at 60 o C, semithick sections were obtained (Leica Ultracut UCT) for light microscopic analysis. Thin sections were obtained using a Diatome diamond knife, picked up on copper grids, stained in uranyl acetate and a modified Sato's lead stain [3]. Sections were imaged with a Philips CM100 and digital images collected (SIA-7C CCD camera; Scientific Instruments and Applications, Inc.; Duluth GA).

Mobiluncus spp. are gram variable anaerobic rods found associated with bacterial vaginosis. A common finding in bacterial vaginosis is the presence of excessive numbers of bacteria attached to detached vaginal epithelial cells giving rise to the "clue cell." In the ultrastructural study of vaginosis involving Mobiluncus species, De Boer and Plantema observed adhesion of microorganisms to epithelial cells, however no specialized surface structures were noted [1]. A polysaccharide glycocalyx, demonstrated by ruthenium red staining, which could contribute to adhesive properties of the organism was reported by these authors [1]. Hernández et al. report cultured M. mulieris with four subpolar flagella with multiple origins, but no pili were reported [2]. We wanted to investigate whether this species does exhibit pili, which could be a method employed for colonization. Clinical isolates of M. mulieris (ATCC) were grown in Columbia Blood Broth (CBB), one of five media that promote growth of Mobiluncus [2]. Cultures were grown anaerobically for 48 hr in CBB at 37˚C.

Inhibition of outer segment (OS) processing by retinal pigment epithelium (RPE) has been linked to photoreceptor injury and retinopathy onset. Sub-retinal clearance by RPE is facilitated by specialized phagocytosis featuring both RPE-specific and traditional FCγR mediated signaling cascades. As a result of this combinatory approach, RPE are capable of internalizing both specific and non-specific external targets alike. The discovery that lack of c-Met signaling results in impairment of phagocytosis in alveolar and hepatocyte macrophages [1] suggests cMet's role as modulator of this activity in post-mitotic cells secreting HGF. Since activated PI3K has been identified as an activator of Rac1during FCγR mediated phagocytosis, we hypothesize that c-Met activation by HGF and subsequent PI3K activation is capable of mediating OS clearance by RPE.To test our hypotheses, cultured ARPE-19 cells were grown to 70% confluence, then serum starved for 24 hr. Post starvation, cells were exposed to various concentrations of HGF for 24hr before fixation with 2.5% paraformaldehyde and .5% glutaraldehyde. Cells were then prepared for immunohistochemistry for receptor expression (non-phosphorylated and phosphorylated forms), focal adhesion kinase (FAK) and binding of fluorescently-labeled E. coli. Intensity values suggest that ARPE-19 respond maximally to concentrations of 25 ng/ml of HGF when compared to controls (Fig 1). While phosphorylated c-Met was not significantly altered (Fig 2) at 24 hr, this may be attributed to the transient expression of phospho-c-Met following activation by HGF. Our findings suggest that RPE respond to increases of exogenous HGF concentrations by up-regulating its receptor and subsequent second messengers systems. In addition, our data show a significant increase of fluorescently labeled E. coli (Fig 3). Taken together, these findings suggest that RTK cross-talk initiated by c-Met activation may be sufficient in mediating general uptake of external debris by RPE. Future studies including RPE challenge with fluorescently labeled OS during peak c-Met phosphorylation evoked by increased HGF exposure will provide evidence for HGF's role as a mediator of specialized phagocytosis of OS.