Calcineurin is essential for survival during membrane stress in Candida albicans

The immunosuppressants cyclosporin A (CsA) and FK506 inhibit the protein phosphatase calcineurin and block T-cell activation and transplant rejection. Calcineurin is conserved in microorganisms and plays a general role in stress survival. CsA and FK506 are toxic to several fungi, but the common human fungal pathogen Candida albicans is resistant. However, combination of either CsA or FK506 with the antifungal drug fluconazole that perturbs synthesis of the membrane lipid ergosterol results in potent, synergistic fungicidal activity. Here we show that the C.albicans FK506 binding protein FKBP12 homolog is required for FK506 synergistic action with fluconazole. A mutation in the calcineurin B regulatory subunit that confers dominant FK506 resistance (CNB1-1/CNB1) abolished FK506-fluconazole synergism. Candida albicans mutants lacking calcineurin B (cnb1/cnb1) were found to be viable and markedly hypersensitive to fluconazole or membrane perturbation with SDS. FK506 was synergistic with fluconazole against azole-resistant C.albicans mutants, against other Candida species, or when combined with different azoles. We propose that calcineurin is part of a membrane stress survival pathway that could be targeted for therapy.     

Probing backbone hydrogen bonds in the hydrophobic core of GCN4

Backbone amide hydrogen bonds play a central role in protein secondary and tertiary structure. Previous studies have shown that substitution of a backbone ester (-COO-) in place of a backbone amide (-CONH-) can selectively destabilize backbone hydrogen bonds in a protein while maintaining a similar conformation to the native backbone structure. The majority of these studies have focused on backbone substitutions that were accessible to solvent. The GCN4 coiled coil domain is an example of a stable alpha-helical dimer that possesses a well-packed hydrophobic core. Amino acids in the a and d positions of the GCN4 helix, which pack the hydrophobic core, were replaced with the corresponding alpha-hydroxy acids in the context of a chemoselectively ligated heterodimer. While the overall structure and oligomerization state of the heterodimer were maintained, the overall destabilization of the ester analogues was greater (average DeltaDeltaG of 3+ kcal mol(-1)) and more variable than previous studies. Since burial of the more hydrophobic ester should stabilize the backbone and reduce the DeltaDeltaG, the increased destabilization must come from another source. However, the observed destabilization is correlated with the protection factors for individual amide hydrogens from previous hydrogen exchange experiments. Therefore, our results suggest that backbone engineering through ester substitution is a useful approach for probing the relative strength of backbone hydrogen bonds.     

The structure of the FMO protein from Chlorobium tepidum at 2.2 Å resolution

The bacteriochlorophyll protein, or FMO protein, from Chlorobium tepidum, which serves as a light-harvesting complex and directs light energy from the chlorosomes attached to the cell membrane to the reaction center has been crystallized in a new space group. The crystals belong to the cubic space group P4₃32 and the structure has been refined to a resolution 2.2 Å with a R factor of 19.7%. The electron density maps show that the structure is composed of two sheets that surround seven bacteriochlorophylls as previously reported (Li et al. (1997) J Mol Biol 271: 456–471). The availability of the new data allows a more accurate refinement of the pigment–protein complex including identification of bound solvent molecules. Several structural differences probably contribute to the observed spectroscopic differences between the FMO proteins from Cb. tepidum and Prosthecochloris aestuarii, including differences in the planarity of corresponding tetrapyrroles. A citrate molecule is found on the surface of each protein subunit of the trimer from Cb. tepidum. However, the citrate molecule is over 15 Å from any bacteriochlorophyll. The presence of the citrate probably does not contribute to the function of the protein although it does contribute to the crystallization as it interacts with a crystallographically related trimer. Among the 236 water molecules found in the protein are four that appear to play a special role in the properties of bacteriochlorophyll 2, as this tetrapyrrole is coordinated by one of these water molecules and the waters form a hydrogen-bonded network that leads to the surface of the protein.This revised version was published online in October 2005 with corrections to the Cover Date.     

Mechanisms of TCDD-induced abnormalities and embryo lethality in white leghorn chickens

The toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds in birds has been well-established in laboratory and field studies. Observed effects of TCDD and related chemicals in birds include developmental deformities, reproductive failure, liver damage, wasting syndrome and death. The mechanism of action of TCDD at the cellular level is primarily mediated through the aryl hydrocarbon receptor (AhR). However, the mechanism of toxic action at the organism level is poorly understood. In this study, the role of radical oxygen species and mixed function oxidize (MFO; cytochrome P4501A) in the mechanism of TCDD-induced abnormalities and lethality were examined by co-injecting radical scavengers and an MFO inhibitor (piperonyl butoxide). Egg injection studies were conducted to determine if in ovo TCDD exposure can cause oxidative stress in white leghorn chicken eggs. Test agents were injected into the yolk prior to incubation. Treatments included TCDD (150 ng/kg), triolein (vehicle control), and various co-treatments including MnTBAP (a mimetic of superoxide dismutase), piperonyl butoxide, piroxicam, vitamin A acetate, and vitamin E succinate. Phenytoin, which is known to cause teratogenesis through oxidative stress was used as a positive control. Eggs were incubated until hatch and then the following parameters were assessed: mortality, hatching success, abnormalities, weights for whole body, liver, heart and brain, and biochemical endpoints for oxidative stress. As a measure of exposure, concentrations of TCDD and ethoxyresorufin-O-deethylase (EROD) activities were measured in tissues of hatchlings. While greater mortality and abnormalities were observed in the TCDD treatment groups, the number of the replicates were not great enough to detect statistically significant differences in abnormality rates for the co-treatments. Some of the observed developmental abnormalities included edema, liver necrosis and bill, eye and limb deformities with TCDD treatments, bill and brain deformities with phenytoin treatments, eye abnormalities with Vitamin E treatments, and abnormal feather pigmentation with piperonyl butoxide treatments.     

A thin-film electrochemical study of the "blue" copper proteins,auracyanin A and auracyanin B,from the photosynthetic bacterium <Emphasis Type="Italic">Chloroflexus</Emphasis><Emphasis Type="Italic">aurantiacus</Emphasis>: the reduction potential as a function of pH

The reversible formal potentials of auracyanin A and auracyanin B, two closely related "blue" copper proteins from the photosynthetic bacterium Chloroflexus aurantiacus, have been determined by protein film voltammetry in the range 4相似文献   

High-pressure and stark hole-burning studies of chlorosome antennas from Chlorobium tepidum

Results from high-pressure and Stark hole-burning experiments on isolated chlorosomes from the green sulfur bacterium Chlorobium tepidum are presented, as well as Stark hole-burning data for bacteriochlorophyll c (BChl c) monomers in a poly(vinyl butyral) copolymer film. Large linear pressure shift rates of -0.44 and -0.54 cm(-1)/MPa were observed for the chlorosome BChl c Q(y)-band at 100 K and the lowest Q(y)-exciton level at 12 K, respectively. It is argued that approximately half of the latter shift rate is due to electron exchange coupling between BChl c molecules. The similarity between the above shift rates and those observed for the B875 and B850 BChl a rings of the light-harvesting complexes of purple bacteria is emphasized. For BChl c monomer, fDeltamu++ = 0.35 D, where Deltamu+ is the dipole moment change for the Q(y) transition and f is the local field correction factor. The data establish that Deltamu+ is dominated by the matrix-induced contribution. The change in polarizability (Deltaalpha) for the Q(y) transition of the BChl c monomer is estimated at 19 A(3), which is essentially identical to that of the Chl a monomer. Interestingly, no Stark effects were observed for the lowest exciton level of the chlorosomes (maximum Stark field of 10(5) V/cm). Possible explanations for this are given, and these include consideration of structural models for the chlorosome BChl c aggregates.     

Effect of Inoculum Rate of Biological Control Agents on Preharvest Aflatoxin Contamination of Peanuts

Studies were conducted during 1994 and 1995 in the environmental control plot facility at the National Peanut Research Laboratory to determine the effect of different inoculum rates of biological control agents on preharvest aflatoxin contamination of Florunner peanuts. Biocontrol agents were nontoxigenic color mutants ofAspergillus flavusandAspergillus parasiticusthat were grown on rice for use as soil inoculum. Three replicate plots (4.0 × 5.5 m) were treated with 0, 2, 10, and 50 g/m of row (0, 20, 100, and 500 lb/acre, respectively) of an equal mixture of the color mutant-infested rice in 1994, and the same plots were retreated in 1995. Aflatoxin concentrations were determined by high performance liquid chromatographic analysis of all peanuts. Treatment means for total kernels in 1994 were 337.6, 73.7, 34.8, and 33.3 ppb for the 0, 2, 10, and 50 g/m treatments, respectively. Regression analysis indicated a trend toward lower aflatoxin concentrations with increasing rates of inoculum (R²= 0.40;P< 0.05). For the same repeated treatments in 1995 aflatoxin concentrations in total kernels averaged 718.3, 184.4, 35.9, and 0.4 ppb. Regression analysis revealed a stronger relationship between inoculum rate and aflatoxin concentrations (R²= 0.66;P< 0.05) in the second year of treatment. Compared with untreated controls, the 2, 10, and 50 g/m treatments produced respective reductions in aflatoxin of 74.3, 95.0, and 99.9% in the second year. The data indicated not only a treatment-related effect, but also that a higher degree of control might be achieved when plots or fields are retreated with biocontrol agents in subsequent years.     

Validated predictive modelling of the environmental resistome

Multi-drug-resistant bacteria pose a significant threat to public health. The role of the environment in the overall rise in antibiotic-resistant infections and risk to humans is largely unknown. This study aimed to evaluate drivers of antibiotic-resistance levels across the River Thames catchment, model key biotic, spatial and chemical variables and produce predictive models for future risk assessment. Sediment samples from 13 sites across the River Thames basin were taken at four time points across 2011 and 2012. Samples were analysed for class 1 integron prevalence and enumeration of third-generation cephalosporin-resistant bacteria. Class 1 integron prevalence was validated as a molecular marker of antibiotic resistance; levels of resistance showed significant geospatial and temporal variation. The main explanatory variables of resistance levels at each sample site were the number, proximity, size and type of surrounding wastewater-treatment plants. Model 1 revealed treatment plants accounted for 49.5% of the variance in resistance levels. Other contributing factors were extent of different surrounding land cover types (for example, Neutral Grassland), temporal patterns and prior rainfall; when modelling all variables the resulting model (Model 2) could explain 82.9% of variations in resistance levels in the whole catchment. Chemical analyses correlated with key indicators of treatment plant effluent and a model (Model 3) was generated based on water quality parameters (contaminant and macro- and micro-nutrient levels). Model 2 was beta tested on independent sites and explained over 78% of the variation in integron prevalence showing a significant predictive ability. We believe all models in this study are highly useful tools for informing and prioritising mitigation strategies to reduce the environmental resistome.