Mutations in Aspergillus fumigatus resulting in reduced susceptibility to posaconazole appear to be restricted to a single amino acid in the cytochrome P450 14alpha-demethylase.

To better understand the molecular basis of posaconazole (POS) resistance in Aspergillus fumigatus, resistant laboratory isolates were selected. Spontaneous mutants arose at a frequency of 1 in 10(8) and fell into two susceptibility groups, moderately resistant and highly resistant. Azole resistance in A. fumigatus was previously associated with decreased drug accumulation. We therefore analyzed the mutants for changes in levels of transcripts of genes encoding efflux pumps (mdr1 and mdr2) and/or alterations in accumulation of [(14)C]POS. No changes in either pump expression or drug accumulation were detected. Similarly, there was no change in expression of cyp51A or cyp51B, which encode the presumed target site for POS, cytochrome P450 14alpha-demethylase. DNA sequencing revealed that each resistant isolate carried a single point mutation in residue 54 of cyp51A. Mutations at the same locus were identified in three clinical A. fumigatus isolates exhibiting reduced POS susceptibility but not in susceptible clinical strains. To verify that these mutations were responsible for the resistance phenotype, we introduced them into the chromosome of a POS-susceptible A. fumigatus strain under the control of the glyceraldehyde phosphate dehydrogenase promoter. The transformants exhibited reductions in susceptibility to POS comparable to those exhibited by the original mutants, confirming that point mutations in the cyp51A gene in A. fumigatus can confer reduced susceptibility to POS.

Synthesis and antifungal activity of the 2,2,5-tetrahydrofuran regioisomers of SCH 51048.

The four 2,2,5-regioisomer counterparts of SCH 51048 were synthesized and evaluated. As with the parent series, only the two cis isomers possessed any in vitro activity, and only the activity of the isomer with the R-configuration at the tetrahydrofuran 2-carbon was significant. The activity data suggests that oxygen at only one of the two possible ring positions benzylic to the difluorobenzene participates usefully in active site binding.

An extract from cultured human keratinocytes that contains the major autoantigens related to autoimmune bullous skin diseases.

Autoantibodies characteristic of autoimmune bullous skin diseases (AIBDs) can be detected by immunoblotting on epidermal, dermal, or bovine muzzle extracts. However, none of those substrates contain all the autoantigens involved in AIBDs, and the diagnosis requires the use of various substrates. Human keratinocytes were cultured under such conditions that they expressed the major autoantigens associated with AIBDs. Forty-two sera with antiepidermal antibodies were immunoblotted on the keratinocyte extract. Bands corresponding to desmoglein III, desmoglein I, BPAg2, BPAg1, and type VII collagen were found in 38 sera. Desmoplakins I and II were revealed by specific monoclonal antibodies. A review of the patients' charts showed a perfect correlation between the blots and the diagnoses of pemphigus vulgaris, pemphigus foliaceus, bullous pemphigoid, cicatricial pemphigoid, and epidermolysis bullosa acquisita. Four sera revealing no band typical of AIBD were from patients with no autoimmune skin disease. Therefore, a single extract of keratinocytes can be used for the differential diagnosis of AIBDs.