Soils and decayed plant litter contain significant quantities of chlorinated aromatic polymers that have a natural but largely unknown origin. We used cupric oxide ligninolysis coupled with gas chromatography/mass spectrometry to show that Curvularia inaequalis, a widely distributed litter ascomycete, chlorinated the aromatic rings of lignin in wood that it was degrading. In aspen wood decayed for 24 weeks, two chlorolignin fragments, 5-chlorovanillin and 2-chlorosyringaldehyde, were each found at Ϸ10 g/g of wood (dry weight). These levels resemble those of similar structures generally found in unpolluted environmental samples. Fractionation of the extractable proteins followed by tandem mass spectrometric analysis showed that the colonized wood contained a previously described C. inaequalis chloroperoxidase that very likely catalyzed lignin chlorination. Chlorolignin produced by this route and humus derived from it are probably significant components of the global chlorine cycle because chloroperoxidase-producing fungi are ubiquitous in decaying lignocellulose and lignin is the earth's most abundant aromatic substance.chloroperoxidase ͉ humus ͉ hypochlorous acid ͉ Pleosporales ͉ soil

Two fungal chloroperoxidases (CPOs), the heme enzyme from Caldariomyces fumago and the vanadium enzyme from Curvularia inaequalis, chlorinated 1-(4-ethoxy-3-methoxyphenyl)-2-(2-methoxyphenoxy)-1,3-dihydroxypropane, a dimeric model compound that represents the major nonphenolic structure in lignin. Both enzymes also cleaved this dimer to give 1-chloro-4-ethoxy-3-methoxybenzene and 1,2-dichloro-4-ethoxy-5-methoxybenzene, and they depolymerized a synthetic guaiacyl lignin. Since fungal CPOs occur in soils and the fungi that produce them are common inhabitants of plant debris, CPOs may have roles in the natural production of high-molecular-weight chloroaromatics and in lignin breakdown.Natural organochlorine compounds are widely distributed in the environment, but the processes that generate them are not completely understood. It is clear that many natural organochlorines are microbial secondary metabolites, whereas others are generated abiotically (22), but little attention has been given to the possibility that biological chlorinations of lignin, the most abundant terrestrial aromatic substance, may produce chloroaromatic compounds. If microbial mechanisms exist to oxidize Cl Ϫ in plant material, the resulting electrophilic chlorine species are likely to react with the electron-rich aromatic rings of lignin (4). This chemistry could explain, in part, why field samples of soil, litter, and decayed wood have all been shown to contain high-molecular-weight chloroaromatics (5,14).Certain plant-pathogenic ascomycetes that are cosmopolitan inhabitants of plant debris provide a possible route for Cl Ϫ oxidation in the vicinity of lignin (6,8,20). These fungi produce extracellular chloroperoxidases (CPOs), heme-or vanadium-containing enzymes that oxidize Cl Ϫ to hypochlorous acid (HOCl) or a similarly reactive chlorine electrophile (18,21). CPOs chlorinate a variety of aromatic substrates (2,15,19,23), but their reactivity with lignin is unknown. We report here that the major structures in lignin are not only chlorinated but also cleaved by the heme CPO of Caldariomyces fumago and the vanadium CPO of Curvularia inaequalis.Chlorination and cleavage of a lignin model dimer. Compound I, 14 C]phenyl)-2-(2-methoxyphenoxy)-1,3-dihydroxypropane (1.0 mCi/mmol), a model of the major nonphenolic arylglycerol-␤-aryl ether structure in lignin (Fig. 1A), was prepared as described previously (9, 12). The model was treated with either commercially available Caldariomyces fumago heme CPO (Sigma) or partially purified Curvularia inaequalis vanadium CPO. The latter enzyme was obtained by DEAE-Sephacel chromatography of the extracellular medium from Curvularia inaequalis cultures (ATCC 10713) as described earlier (17). The reaction mixtures (1.0 ml) contained compound I (3.4 ϫ 10 5 dpm, 160 M), KCl (20 mM), and buffer (25 mM). The buffers were potassium phosphate, pH 3.0, for heme CPO reactions and sodium acetate, pH 4.0, for vanadium CPO reactions. Enzyme was added (35 monochlorodimedone units of heme CPO or 1 monochlorodimedone unit of va...

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Recently, liquid crystalline elastomers (LCEs) have been proposed as active substrates for cell culture due to their potential to attach and orient cells, and impose dynamic mechanical signals through the application of external stimuli. In this report, the preparation of anisotropic and oriented nematic magnetic-sensitized LCEs with iron oxide nanoparticles, and the evaluation of the effect of particle addition at low concentrations on the resultant structural, thermal, thermo-mechanical, and mechanical properties is presented. Phase transformations produced by heating in alternating magnetic fields were investigated in LCEs in contact with air, water, and a common liquid cell culture medium was also evaluated. The inclusion of nanoparticles into the elastomers displaced the nematic-to-isotropic phase transition, without affecting the nematic structure as evidenced by similar values of the order parameter, while reducing the maximum thermomechanical deformations. Remote and reversible deformations of the magnetic LCEs were achieved through the application of alternating magnetic fields, which induces the nematic-isotropic phase transition through nanoparticle heat generation. Formulation parameters can be modified to allow for remote actuation at values closer to the human physiological temperature range and within the range of deformations that can affect the cellular behavior of fibroblasts. Finally, a collagen surface treatment was performed to improve compatibility with NIH-3T3 fibroblast cultures, which enabled the attachment and proliferation of fibroblasts on substrates with and without magnetic particles under quiescent conditions. The LCEs developed in this work, which are able to deform and experience stress changes by remote contact-less magnetic stimulation, may allow for further studies on the effect of substrate morphology changes and dynamic mechanical properties during in vitro cell culture.

For over 3 decades, sexual development in the human fungal pathogen Cryptococcus neoformans and other fungi has been initiated by growing compatible mating partners on V8 juice medium. Although this medium is an efficient inducer of sexual development, the mechanism by which it promotes the process is unknown. To understand how V8 juice medium induces sexual development, we attempted to purify inducing factors from V8 juice, and we carried out a complete compositional analysis of V8 juice. We discovered that no single factor is responsible for the effects of V8 juice medium. Rather, the unique composition of V8 juice medium provides the proper nutrient composition for inducing and sustaining complete sexual development. Utilizing these findings, we developed a defined V8 (DV8) medium that mimics V8 juice medium in sexual development assays. Then, using DV8 as a tool, we explored the roles that specific molecules play in enhancing sexual development. Surprisingly, we discovered that copper is a key factor, leading to an upregulation of the mating pheromone genes MFa and MF␣, both required for the initial steps in sexual development. The utilization of DV8 to investigate the effects of copper on sexual development presented here is an example of how defining the conditions that induce sexual development will advance the study of C. neoformans.