Fungal biodegradation of dibutyl phthalate and toxicity of its breakdown products on the basis of fungal and bacterial growth.

Phthalates are esters of phthalic acid that give flexibility to polyvinyl chloride. Diverse studies have reported that these compounds might be carcinogenic, mutagenic and/or teratogenic. Radial growth rate, biomass, hyphal thickness of Neurospora sitophyla, Trichoderma harzianum and Aspergillus niger, grown in two different concentrations of dibutyl phthalate (DBP) (500 and 1,000 mg/l) in agar and in submerged fermentation were studied. The inhibitory concentration (IC50) and the constant of biodegradation of dibutyl phthalate in Escherichia coli cultures were used to evaluate toxicity. The radial growth rate and thickness of the hypha were positively correlated with the concentration of phthalate. The pH of the cultures decreased as the fermentation proceeded. It is shown that these fungi are able to degrade DBP to non-toxic compounds and that these can be used as sole carbon and energy sources by this bacterium. It is demonstrated that the biodegradation of the DBP is directly correlated with the IC50. This is the first study that reports a method to determine the biodegradation of DBP on the basis of the IC50 and fungal growth, and the effect of this phthalate on the growth and thickness of hyphae of filamentous fungi in agar and in submerged fermentation.

Molecular characterization of two ammonium transporters from the ectomycorrhizal fungus Hebeloma cylindrosporum.

Heterologous expression of the yeast triple Mep mutant has enabled the first molecular characterization of AMT/MEP family members in an ectomycorrhizal fungus. External hyphae, which play a key role in nitrogen nutrition of trees, are considered as the absorbing structure of the ectomycorrhizal symbiosis and therefore molecular studies on ammonium transport in hyphae are urgently needed. The kinetic properties of AMT2 and AMT3 from Hebeloma cylindrosporum were studied in Saccharomyces cerevisiae. Expression of HcAmts in the yeast triple Mep mutant restored ammonium retention within cells. The HcAmts did not complement the ammonium sensing defect phenotype of Mep2Delta cells during pseudohyphal differentiation. Northern blot analysis in H. cylindrosporum showed that the HcAMTs were up-regulated upon nitrogen deprivation and down-regulated by ammonium.

Fourier transform infrared studies of secondary structure and orientation of pulmonary surfactant SP-C and its effect on the dynamic surface properties of phospholipids.

SP-C, a highly hydrophobic, 3.7-kDa protein constituent of lung surfactant, has been isolated from bovine lung lavage, purified, and reconstituted into binary lipid mixtures of 1,2-dipalmitoyl-phosphatidylcholine (DPPC) and 1,2-dipalmitoylphosphatidylglycerol (DPPG). Fourier transform infrared (FT-IR) spectroscopy has been applied to examine SP-C secondary structure, the average orientation of alpha-helical segments relative to the bilayer normal in membrane films, and the effect of protein on the thermotropic properties of the phospholipid acyl chains. In addition, dynamic surface measurements were made on phospholipid films at the A/W interface in the presence and absence of SP-C. SP-C (0.5 mol %) was found to possess about 60% alpha-helical secondary structure in lipid vesicles. Higher levels (1.5 mol %) of SP-C resulted in a slight increase of beta-forms, possibly resulting from protein aggregation. The helical segments exhibited an average angle of orientation of about 24 degrees with respect to the bilayer normal, suggesting a trans-bilayer orientation of the peptide. The observation that 70% of the peptide bond hydrogens are hard to exchange in D2O further reflects the hydrophobic nature of the molecule. SP-C produced little effect on the thermotropic properties of the binary lipid mixture, as measured from acyl chain C-H and C-D stretching frequencies. However, the presence of 1 mol % protein markedly reduced the viscance and increased the elasticity of surface films suggesting a mechanism by which SP-C facilitates the spreading of phospholipids on an aqueous surface. The possible physiological consequences of these observations are discussed.

Mechanism of inhibition of glycolysis by vanadate.

Vanadate is known to inhibit several phosphatases including Na+, K+-ATPase, alkaline phosphatase, and glyceraldehyde-3-P dehydrogenase. Inhibition presumably results because vanadium adopts a stable structure which resembles the transition state of phosphate during the reactions involving these enzymes. We performed experiments to further examine the effects of vanadate (VO3-4) on erythrocyte (red blood cells (RBC] glycolytic intermediates. RBC obtained from human subjects were centrifuged and washed with lactated Ringer's 5% dextrose. 31P nuclear magnetic resonance analysis of the RBC revealed the characteristic peaks for the 3-phosphate and 2-phosphate of 2,3-diphosphoglycerate (DPG), inorganic phosphate (Pi), and ATP. Incubation of RBC with 10(-6) M VO3-4 led to a disappearance of ATP and 2,3-DPG while the peak for Pi increased. By the end of 4 h over 90% of the VO3-4 had been reduced to VO2+ (vanadyl) in the RBC. The effects of 10(-4) M iodoacetamide and 10(-5) M ethacrynic acid, known inhibitors of glyceraldehyde-3-P dehydrogenase that act by interactions with sulfhydryl groups (-SH) of the enzyme, were similar to those of VO3-4. Incubation with vanadyl did not affect the peaks for Pi, 2-DPG, or 3-DPG. Furthermore, using electron spin resonance we demonstrated that in the presence of glyceraldehyde-3-P dehydrogenase, VO3-4 is reduced to VO2+. The findings demonstrate that VO3-4 inhibits glycolysis at micromolar concentrations and that the ion is reduced to VO2+ in the cell. The similarity of the effect of VO3-4 to those of iodoacetamide and ethacrynic acid suggests that interactions with -SH groups is its mechanism of inhibition. Since under physiological conditions intracellular VO3-4 concentrations are in the micromolar range and may exist in oxidized and/or reduced forms, VO3-4 could regulate the activity of glyceraldehyde-3-P dehydrogenase through changes in the redox state of the enzyme rather than by substituting for the PO3-4 ion.