However, including 100% of this time as time at risk of transmitting HIV sexually would only have doubled the estimates and thereby not change our results significantly. Wilson et al. [10] have suggested that the risk of transmitting HIV sexually may be higher than assumed by the the Swiss Federal Commission for HIV/AIDS. Their statistical model is, however, based on the presumption ACP-196 concentration that the relationship between VL and risk of HIV transmission is linear. Although there is little

evidence supporting the idea of an infectious threshold, our study (like the recommendations of the Swiss Federal Commission for HIV/AIDS) assumes that there is a VL threshold below which the risk of HIV transmission is negligible. Also, it is still a matter of debate

whether the relationship between VL and risk of HIV transmission is linear or based on a threshold mechanism. However, the findings of Quinn et al. [1] and Melo et al. [2] suggest a low risk of transmission in patients Selleck X-396 with VL<1500 copies/mL. Concerning the choice of cut-off value, our test for robustness showed that the cut-off value of 1000 copies/mL is reasonable. The median period between VL tests was 3 months, and 81.2% of the VL tests were taken within 4 months of the previous test. Although a VL increase may have passed undetected, we presume that most viraemias above 1000 copies/mL are captured in the present study design. We only had access to self-reported civil status and sexual behaviour for 37.7% and 37.4%, respectively, of the included patients. However, our data indicate that neither patients living in stable partnerships nor patients practising safe sex differ from other patients in risk of transmitting HIV. In terms of compliance among patients in stable partnerships, this is consistent with Fludarabine cost previous findings [11]. Our results indicate that injecting drug users on successful HAART have a higher risk of transmitting HIV, which is probably mainly a result of compliance problems [11]. Stratification by year of HAART initiation

did not change our estimates, but when stratified by the duration of periods of suppressed VL, the risk of viral rebound was highest among patients with periods of suppressed VL of less than 6 months. Smith et al. [12] found similar results with regard to viral rebound among highly experienced HIV-infected patients. Among patients with periods of suppressed VL of more than 5 years, the risk of transmission of HIV was very low. This group of patients has been able to maintain a suppressed VL through times of less efficient and user-friendly regimens and is a selected group with high adherence. Our results therefore indicate that there would be a substantial gain in reducing the risk of infecting the sexual partner, if the time limit recommended by the Swiss Federal Commission for HIV/AIDS of undetectable VL was extended from 6 months to at least 12 months.

Mineralization of [14C]phenanthrene to 14CO2 was measured in contaminated soils at temperatures down to 0 °C and sizable naphthalene-, undecane-, biphenyl- and phenanthrene-degrading check details populations were measured by microplate-based most-probable-number analysis. Cloning and 16S rRNA gene sequencing, focused on the dominant phenanthrene-degrading bacteria, revealed strains related to bacteria previously found in cold and contaminated environments. Overall, we provide evidence

for the presence and potential activity of phenanthrene-degrading bacteria in polluted St. Nord soils and this study is the first to indicate an intrinsic bioremediation potential in hydrocarbon-contaminated soils from the Greenland High Arctic. The Arctic warming and the reduction in the polar ice sheet during the last few years (Graversen et al., 2008) will boost human activity in the High Arctic regions of Greenland. This will inevitably lead to increased inputs of anthropogenic compounds and the issue arises as to whether an intrinsic attenuation

potential is present in these areas. Intrinsic remediation of petroleum hydrocarbons under cold conditions have been indicated before (Bradley & Chapelle, 1995; Aislabie et al., 1998; Rike et al., 2005) and contaminated alpine, Antarctic and Arctic soils may harbour hydrocarbon-degrading populations (Margesin & Schinner, http://www.selleckchem.com/products/FK-506-(Tacrolimus).html 2001; Rike et al., 2003; Saul et al., 2005; Aislabie et al., 2006). In some cases, however, the natural attenuation potential present in these cold environments is insufficient to clean up soils within a reasonable time and active bioremediation approaches have been suggested (Filler et al., 2001; Margesin & Schinner, 2001). Studies on contaminant degradation in High Arctic regions have until now addressed areas in Alaska (Bradley & Chapelle, 1995; Filler et al., 2001), Canada (Whyte et al., 2001) and Svalbard (Rike et al., 2003, 2005), but no studies have focused on the Greenland

High Arctic. Previous studies have mainly focused on the fate of easily biodegradable oil fractions, whereas knowledge on the biodegradation Progesterone of polycyclic aromatic hydrocarbons (PAHs) in Arctic regions is lacking. Station Nord (St. Nord) is a military base operated by the Danish army located at 81°36′N and 16°40′W approximately 500 miles from the geographical North Pole. The area is an Arctic desert with an average annual air temperature of −14 °C and <100 mm annual precipitation. The temperature can reach 16 °C during the summer period and down to −50 °C during the winter. St. Nord is a gateway to the national park in the northern part of Greenland as well as the North Pole and the storage and handling of fuels have led to accidental spillage. In addition, St.

Mineralization of [14C]phenanthrene to 14CO2 was measured in contaminated soils at temperatures down to 0 °C and sizable naphthalene-, undecane-, biphenyl- and phenanthrene-degrading www.selleckchem.com/HSP-90.html populations were measured by microplate-based most-probable-number analysis. Cloning and 16S rRNA gene sequencing, focused on the dominant phenanthrene-degrading bacteria, revealed strains related to bacteria previously found in cold and contaminated environments. Overall, we provide evidence

for the presence and potential activity of phenanthrene-degrading bacteria in polluted St. Nord soils and this study is the first to indicate an intrinsic bioremediation potential in hydrocarbon-contaminated soils from the Greenland High Arctic. The Arctic warming and the reduction in the polar ice sheet during the last few years (Graversen et al., 2008) will boost human activity in the High Arctic regions of Greenland. This will inevitably lead to increased inputs of anthropogenic compounds and the issue arises as to whether an intrinsic attenuation

potential is present in these areas. Intrinsic remediation of petroleum hydrocarbons under cold conditions have been indicated before (Bradley & Chapelle, 1995; Aislabie et al., 1998; Rike et al., 2005) and contaminated alpine, Antarctic and Arctic soils may harbour hydrocarbon-degrading populations (Margesin & Schinner, Crizotinib in vivo 2001; Rike et al., 2003; Saul et al., 2005; Aislabie et al., 2006). In some cases, however, the natural attenuation potential present in these cold environments is insufficient to clean up soils within a reasonable time and active bioremediation approaches have been suggested (Filler et al., 2001; Margesin & Schinner, 2001). Studies on contaminant degradation in High Arctic regions have until now addressed areas in Alaska (Bradley & Chapelle, 1995; Filler et al., 2001), Canada (Whyte et al., 2001) and Svalbard (Rike et al., 2003, 2005), but no studies have focused on the Greenland

High Arctic. Previous studies have mainly focused on the fate of easily biodegradable oil fractions, whereas knowledge on the biodegradation either of polycyclic aromatic hydrocarbons (PAHs) in Arctic regions is lacking. Station Nord (St. Nord) is a military base operated by the Danish army located at 81°36′N and 16°40′W approximately 500 miles from the geographical North Pole. The area is an Arctic desert with an average annual air temperature of −14 °C and <100 mm annual precipitation. The temperature can reach 16 °C during the summer period and down to −50 °C during the winter. St. Nord is a gateway to the national park in the northern part of Greenland as well as the North Pole and the storage and handling of fuels have led to accidental spillage. In addition, St.

1% between F. aquatile and Flavobacterium reichenbachii to 94.9% between F. xanthum and F. omnivorum. The phylogenetic trees based on the gyrB sequences (Figs 2 and S2) show that the groups found in the 16S rRNA gene dendrogram (Figs 1 and S1) were confirmed. The

Antarctic Flavobacterium groups generally showed lower gyrB gene sequence similarity to neighbouring groups and species, which confirmed their status as potentially new species. Flavobacterium sp. 13 and sp. 5, which, in the 16S rRNA gene phylogeny, were closely related to F. micromati and F. gelidilacus, respectively, also group with these species in the gyrB phylogeny. Both groupings are well supported; however, the gyrB similarity of Flavobacterium sp. 13 to F. micromati LMG 21919 (97.0%) is higher than that of Flavobacterium sp. 5 to F. gelidilacus LMG 21477 (91.9%). Flavobacterium sp. 13 probably belongs to F. micromati that was originally selleck isolated from microbial mats in Antarctic lakes (Van Trappen et al., 2004) as were the isolates of Flavobacterium sp. 13 (Table 1). Flavobacterium sp. 5 probably represents a new species in view of the rather low gyrB gene sequence

similarity to F. gelidilacus in comparison with the higher similarity values obtained between some type strains. Nevertheless, the precise relation to F. gelidilacus, Daporinad cell line another species from Antarctic microbial mats (Van Trappen et al., 2003), remains to be investigated further. The similarities within the delineated Flavobacterium groups are generally very high for the 16S rRNA gene sequences (Table 3). The gyrB sequences were mostly also very similar within groups and ranged from 97.2% to 100% (Table 3). In Flavobacterium sp. 2, sp. 8 and sp. 13 (Figs 2 and S2) subclusters

were observed with 97.2–99.0% sequence similarity. In other genera, comparable high intraspecies gyrB gene sequence similarities were observed, for example 98.5–100%gyrB gene sequence similarity within the genus Streptomyces (Actinobacteria) (Hatano et al., 2003), 97.4–100% within the genus Aeromonas Diflunisal (Gammaproteobacteria) (Yanez et al., 2003), 95.0–100% within the genus Bacillus (Firmicutes) (Wang et al., 2007) and 94.6–100% within the genus Helicobacter (Epsilonproteobacteria) (Hannula & Hanninen, 2007). It should be noted that all Flavobacterium groups studied here comprised several rep-types (Peeters et al., submitted) and the strains were chosen to represent this diversity. The topologies of the neighbour-joining and the maximum likelihood dendrogram were slightly different for the 16S rRNA gene compared with the gyrB gene (Figs 1, 2, S1 and S2), as has also been observed for other groups (Yamamoto & Harayama, 1996). However, overall, the phylogenies of the 16S rRNA (Figs 1 and S1) and gyrB (Figs 2 and S2) gene were similar and confirmed the division of the Antarctic strains into 15 groups, one probably belonging to F. micromati and one close to F. gelidilacus.

1% between F. aquatile and Flavobacterium reichenbachii to 94.9% between F. xanthum and F. omnivorum. The phylogenetic trees based on the gyrB sequences (Figs 2 and S2) show that the groups found in the 16S rRNA gene dendrogram (Figs 1 and S1) were confirmed. The

Antarctic Flavobacterium groups generally showed lower gyrB gene sequence similarity to neighbouring groups and species, which confirmed their status as potentially new species. Flavobacterium sp. 13 and sp. 5, which, in the 16S rRNA gene phylogeny, were closely related to F. micromati and F. gelidilacus, respectively, also group with these species in the gyrB phylogeny. Both groupings are well supported; however, the gyrB similarity of Flavobacterium sp. 13 to F. micromati LMG 21919 (97.0%) is higher than that of Flavobacterium sp. 5 to F. gelidilacus LMG 21477 (91.9%). Flavobacterium sp. 13 probably belongs to F. micromati that was originally Mitomycin C nmr isolated from microbial mats in Antarctic lakes (Van Trappen et al., 2004) as were the isolates of Flavobacterium sp. 13 (Table 1). Flavobacterium sp. 5 probably represents a new species in view of the rather low gyrB gene sequence

similarity to F. gelidilacus in comparison with the higher similarity values obtained between some type strains. Nevertheless, the precise relation to F. gelidilacus, click here another species from Antarctic microbial mats (Van Trappen et al., 2003), remains to be investigated further. The similarities within the delineated Flavobacterium groups are generally very high for the 16S rRNA gene sequences (Table 3). The gyrB sequences were mostly also very similar within groups and ranged from 97.2% to 100% (Table 3). In Flavobacterium sp. 2, sp. 8 and sp. 13 (Figs 2 and S2) subclusters

were observed with 97.2–99.0% sequence similarity. In other genera, comparable high intraspecies gyrB gene sequence similarities were observed, for example 98.5–100%gyrB gene sequence similarity within the genus Streptomyces (Actinobacteria) (Hatano et al., 2003), 97.4–100% within the genus Aeromonas MRIP (Gammaproteobacteria) (Yanez et al., 2003), 95.0–100% within the genus Bacillus (Firmicutes) (Wang et al., 2007) and 94.6–100% within the genus Helicobacter (Epsilonproteobacteria) (Hannula & Hanninen, 2007). It should be noted that all Flavobacterium groups studied here comprised several rep-types (Peeters et al., submitted) and the strains were chosen to represent this diversity. The topologies of the neighbour-joining and the maximum likelihood dendrogram were slightly different for the 16S rRNA gene compared with the gyrB gene (Figs 1, 2, S1 and S2), as has also been observed for other groups (Yamamoto & Harayama, 1996). However, overall, the phylogenies of the 16S rRNA (Figs 1 and S1) and gyrB (Figs 2 and S2) gene were similar and confirmed the division of the Antarctic strains into 15 groups, one probably belonging to F. micromati and one close to F. gelidilacus.

e. the extent to which they are encoded with respect to the external environment or the anatomical frame of reference provided by the body). This research was supported by an award from the European Research Council under the European Community’s Seventh Framework Programme (FP7/2007-2013) (ERC Grant agreement no. 241242) to A.J.B. We acknowledge the kind assistance of the Centre for Brain http://www.selleckchem.com/products/BMS-777607.html and Cognitive Development, Birkbeck

College, and Leslie Tucker in facilitating this research. We also extend our thanks to Elisa Carrus for her assistance in preparing Fig. 5. Abbreviations ERPs event-related potentials fMRI functional magnetic resonance imaging SEPs somatosensory evoked potentials “
“Slc4a10 was originally identified as a Na+-driven Cl−/HCO3− exchanger NCBE that transports extracellular Na+ and HCO3− in exchange for intracellular Cl−, whereas other studies argue against a Cl−-dependence for Na+–HCO3− transport, and thus named it the electroneutral Na+/HCO3− cotransporter NBCn2. Here we investigated Slc4a10 expression in adult mouse brains by in situ hybridization and immunohistochemistry. Slc4a10 mRNA was widely expressed, with higher levels HM781-36B mw in pyramidal cells in the hippocampus and cerebral cortex, parvalbumin-positive interneurons in the hippocampus, and Purkinje cells (PCs) in the cerebellum. Immunohistochemistry revealed an uneven distribution

of Slc4a10 within the somatodendritic compartment of cerebellar neurons. In the cerebellar molecular layer, stellate cells and their innervation targets (i.e. PC dendrites in the superficial molecular layer) showed significantly higher labeling than basket cells and their targets (PC dendrites in the basal molecular layer and PC somata). Moreover, the distal dendritic trees of PCs (i.e. parallel fiber-targeted dendrites) had significantly greater labeling than the proximal dendrites (climbing fiber-targeted dendrites). These observations suggest

that Slc4a10 expression is regulated in neuron type- and input pathway-dependent manners. Because such an elaborate regulation is also found for K+–Cl− cotransporter KCC2, a major neuronal Cl− extruder, we compared their expression. Slc4a10 and KCC2 overlapped in most somatodendritic elements. However, relative abundance was largely complementary in the Tolmetin cerebellar cortex, with particular enrichments of Slc4a10 in PC dendrites and KCC2 in molecular layer interneurons, granule cells and PC somata. These properties might reflect functional redundancy and distinction of these transporters, and their differential requirements by individual neurons and respective input domains. “
“There is growing agreement that genetic factors play an important role in the risk to develop heroin addiction, and comparisons of heroin addiction vulnerability in inbred strains of mice could provide useful information on the question of individual vulnerability to heroin addiction.

2. Autolysis assays were performed as described previously (Singh et al., 2008). Briefly, wild-type and the lytM mutant cultures of S. aureus were grown to an OD600 nm of 0.7 at 37 °C in PYK medium (0.5% Bacto peptone, 0.5% yeast extract, 0.3% K2HPO4, pH 7.2). After one wash with cold water (8500 g, 4 °C, 15 min), cells were suspended in 0.05 M Tris-HCl buffer, pH 7.2, containing 0.05% Triton X-100 to an OD600 nm of 1.0. selleck products Cell suspension was incubated in flasks at 37 °C with shaking (125 r.p.m.) and autolysis was determined by measuring decline in the turbidity spectrophotometrically at 600 nm every 30 min. Autolysis was also analyzed using a zymographic procedure

as described previously (Singh et al., 2008). The total autolysins were extracted after bead beating bacterial cells in 0.25 M phosphate buffer (pH 7.2) using a BioSpec Mini-Beadbeater after growth in PYK to an OD600 nm=0.7. Purified His6–LytM, extracts from E. coli cells overexpressing selleck chemicals llc His6–LytM and an S. aureus bead-beated cell-free extract was analyzed for the presence of autolysins in a zymographic method using autoclaved S. aureus 8325-4 cells as described previously (Singh et al., 2008). To construct a mutation, lytM upstream and downstream flanking regions were PCR amplified and sandwiched with a tetracycline resistance cassette in plasmid

pTZ18R. This construct was used to replace the wild-type lytM gene in the S. aureus chromosome by double homologous recombination. This mutant represents a deletion of 706 nt of the 966 nt lytM gene. In PCR assays, primers P9 and P10 amplified an ∼1.0 kb lytM region when the genomic DNA from the wild-type S. aureus was used as the template (Fig. 1, lane 1) as compared with an ∼2.5 kb amplicon when genomic DNA from the lytM mutant strain was used as a template (Fig. 1, lane 2). The mutation in the lytM gene was also confirmed by Southern blot analysis (data not shown). The deletion of LytM was investigated for any impact on the growth of S. aureus in TSB or in modified TSB to

impose stresses such as acidic stress (pH 5.5), alkaline stress (pH 9.0) or salt stress (TSB added with additional 1.5 M NaCl). No growth defect was observed whether the lytM mutants used were in S. aureus strain SH1000 or 8325-4 (data not shown). Surprisingly, the presence of oxacillin led to increased Clostridium perfringens alpha toxin lysis of mid-log-phase lytM mutant cells compared with a culture of wild-type S. aureus 8325-4 cells under identical conditions (Fig. 2). To verify whether it was indeed the lack of a functional LytM that is responsible for oxacillin-induced lysis, the mutant was complemented with the lytM gene under its own promoter in trans on plasmid pCU1. As evident in Fig. 2, the level of resistance to oxacillin-induced lysis was restored in the complemented strain. Expression of lytM was monitored using the lytM promoter–lacZ fusion in S. aureus SH1000.

, 2008). The difference between both ZrSod2-22 and ZrNha1 transporters in their substrate preferences (sodium vs. potassium) and physiological functions (sodium detoxification vs. maintenance of potassium homeostasis) has been demonstrated directly in Z. rouxii cells lacking Cobimetinib research buy or overexpressing the two antiporters (Pribylova et al., 2008). In general, the three sodium-specific antiporters (SpSod2, YlNha2 and

ZrSod2-22) possess shorter C-terminal hydrophilic parts than their potassium-transporting paralogues, and YlNha2 and ZrSod2-22 antiporters have an extremely high capacity to export sodium cations (Kinclova et al., 2001b; Papouskova & Sychrova, 2006), much higher than ScNha1 or other yeast antiporters with broad substrate specificities described below. One plasma-membrane antiporter with a broad substrate specificity for at least four alkali cations (K+, Na+, Li+, Rb+) has been characterized in two osmotolerant yeast species, D. hansenii (Velkova & Sychrova, 2006) and P. sorbitophila (Banuelos et al., 2002) and in five members of the Candida genus –C. albicans, C. dubliniensis, C. parapsilosis, C. glabrata and C. tropicalis (Kinclova et al., 2001a; Kamauchi et al.,

2002; Krauke & Sychrova, 2008, 2011). All of these transporters have been characterized upon heterologous expression in S. cerevisiae. find more Phenotypes of increased salt tolerance as well as direct measurements of cation efflux showed that the individual transporters, though having the same large substrate specificity, differ in their capacity to transport cations, for example C. parapsilosis and C. albicans antiporters being the most and those of C. dubliniensis and C. glabrata being the least

efficient (Krauke & Sychrova, 2008, 2011). Candida albicans and C. glabrata deletion mutants lacking the genes encoding Na+/H+ antiporters have been constructed (Soong et al., 2000; Kinclova-Zimmermannova & Sychrova, about 2007; Krauke & Sychrova, 2011) and characterization of their phenotype and transport capacity revealed that though these two antiporters are able to transport both potassium and sodium cations when expressed in S. cerevisiae, their absence in Candida cells only results in an increased sensitivity to high external potassium concentrations and did not alter their tolerance to NaCl. Detailed measurements of alkali–metal–cation efflux in wild-type cells, deletion and reintegration mutants confirmed that the two transporters play only a marginal role in sodium detoxification, but are highly important for cell survival in the presence of high external potassium concentrations. Thus these antiporters of C. albicans and C. glabrata are the very first known examples of the plasma-membrane Na+/H+ antiporter family from prokaryotes and lower eukaryotes, whose primary function is not the elimination of toxic sodium cations, but contribution to the optimal intracellular potassium concentration, and thereby to cell volume, turgor and membrane potential.

4d). A close examination showed narrow hyphae with an average diameter of 2.8 μm. The number of layers that composes the interface fungal structure affects the oxygenation of the microorganism, especially for the hyphae close to the substrate (Rahardjo, 2005). In this sense, the oxygenation of the hyphae from C. unicolor was expected to be higher than those shown by the other fungal strains because almost the entire fungal structure was on a single layer, making favorable oxygen diffusion click here possible. Trametes pubescens and T. versicolor exhibited a similar number of layers in

the interface structure, suggesting a similar behavior between members of the same genus. Compared with the other fungal strains tested, the oxygenation of both Trametes can be described as just medium, higher than the one exhibited by P. ostreatus, but lower than that exhibited by C. unicolor. Finally, P. ostreatus exhibited about four layers in its interface structure, making this structure extremely dense and limiting the oxygen transport; thus, the oxygenation

of the inner layers of this fungus was low. Our results are in agreement with those found by Dynesen & Nielsen (2003) when culturing eight strains of filamentous fungi with hypha diameters ranging from 1.82 to 6.70 μm. Also, Aime et al. (2003) studied some species from Guyana and found hypha diameters from 3 to 7 μm, while Lecault et al. (2007) determined the hypha diameter of the filamentous fungus Trichorderma reesei to be about 2–2.5 μm. The four fungi studied also presented considerable differences in the distribution of their hyphae find more and the size of the clumps. The narrow hyphae of T. pubescens created clumps in a very random distribution (Fig. 3a). Thus, clumps produced by two hyphae varied in size from 3.8 to 4.5 μm, while clumps produced by three hyphae ranged from 6 to 8 μm (numbers 1 and 2 in Fig. 5a). Trametes versicolor had a defined network structure where thick hyphae intercrossed, creating large clumps in a radial distribution,

whereas the small hyphae covered the rest of the surface area in a transversal orientation with respect to the thick hyphae (Fig. 3b). Large clumps created by T. versicolor varied Edoxaban between 9 and 12 μm (number 1 in Fig. 5b), which represents the intercross of four or five hyphae. This fungus showed a more organized growing structure than that found for T. pubescens. Cerrena unicolor clearly had two types of clumps: the ones formed by two hyphae with an average size of 8 μm and the ones formed by three hyphae with an average size of 12 μm (number 1 in Fig. 5c). Cerrena unicolor had a network structure that covered most of the substrate, but it did not present a clear geometry like the one seen with T. versicolor (Fig. 3c). Pleurotus ostreatus presented many clumps of about 11.5 μm (number 1 in Fig. 5d), comprised of about four hyphae (Fig. 4d). The network structure of P.

neoformans electron transport chain and suggest that the effect of microplusin on the growth of the fungi may be related to the damage of the classical respiratory chain, probably at the copper-containing complex IV. Although we cannot entirely discard the effects of Fe2+ on microplusin, our assumption that microplusin is preferentially a copper chelator is based on the fact that the four respiratory complexes that have iron as prosthetic groups or bound to the heme check details group remained functional, whereas complex IV, the only complex that has copper as a prosthetic group, was affected by microplusin. We also show that microplusin stimulated the alternative respiratory

pathway in C. neoformans, likely this website to compensate for the damaged classical electron transport chain. The alternative pathway is not coupled to oxidative phosphorylation and ATP synthesis, and hence, energy production in microplusin-treated yeasts is likely to be deficient. However, uncoupled respiration helps the cells to manage reactive oxygen species production under stress conditions. Similar to complex IV, the assembly and functioning of other copper proteins, such as the antioxidant enzyme Cu-Zn superoxide dismutase (SOD1), might also be compromised in microplusin-treated C. neoformans. Microplusin

at concentrations ≥3.12 μM clearly inhibited C. neoformans melanization as well as reduced laccase activity. This further suggests that the copper-chelating ability of microplusin may affect the loading of copper ions to laccase apoenzyme. In addition, we observed that copper supplementation of the medium prevented the inhibition of melanization by microplusin, according to 1 : 1 binding ratio

(Silva et al., 2009). A correct laccase metallation is reportedly crucial for its biological activity, as shown for the laccase produced by the avirulent Δvph1 mutant of C. neoformans. PAK6 Defective vesicular acidification disrupts the insertion of copper cofactors into proteins, resulting in the inability of Δvph1 laccase to catalyze phenolic compounds to melanin (Erickson et al., 2001). As expected, addition of 1 mM of the copper chelator BCS to the medium abolished laccase activity not only in the Δvph1 mutant but also in the wild-type strain and copper supplementation, restored laccase activity as well as induced its transcription (Zhu et al., 2003). Therefore, these data support the hypothesis that microplusin sequesters copper and may affect the availability of this metal to copper-dependent enzymes, such as laccase. The microplusin concentrations that inhibited melanization (≥3.12 μM) also increased the autopolymerization of l-dopa. l-dopa autopolymerization is a process that occurs spontaneously by exposure to light (Mason, 1955). Microplusin probably stimulates the spontaneous autopolymerization of the products derived from l-dopa oxidation; however, this possible action did not interfere with its inhibitory effect on melanization of C.