A whole-cell C. albicans screen was designed to identify novel inhibitors interacting with the synthesis, assembly and regulation of the fungal cell wall. C. albicans was grown in a paired broth assay in 96-well plates with natural product extracts or pure chemical compoundsin the presence and absence of the osmotic stabilizer, sorbitol. Growthwas visually examinedover a 7-day period and scored into different growth categories. Positives from the sorbitol rescue were then examined under the microscope for morphological alterations and grouped into several morphological classes. Sorbitol protection and cell morphology were indicators of novel antifungal agents from natural product extracts and pure compounds.

A systematic evaluation of the in vitro (1,3)-P-glucan synthase assay parameters was performed using microsomes prepared from Candida albicans from either yeast or mycelial phase cells. Enzyme activities of both yeast and mycelial phase microsomes depended on the presence of guanosine-5'-0-(3-thiophosphate) and either bovine serum albumin or a detergent [W-I (polyoxyethylene ether detergent) or Brij-35 (polyoxyethylene ether, 23 lauryl ether)]. Brij-35 was included in standard assays as it was compatible with the permeabilized whole-cell assay. Microsomes derived from both the yeast and mycelial phases generally yielded similar glucan synthase activities under a range of different assay conditions. Brij-35 significantly stabilized the enzyme, yielding a half-life of 5.6 d at 4 "C, compared with 0 9 d without detergent. The addition of detergent during mechanical breakage of yeast cells dramatically improved glucan synthase stability and activity. Enzyme catalysis was linear for at least 75 min with 100 pg protein from microsomes of yeast cells grown to mid-exponential phase, with an apparent K, for UDP-glucose of 1.1 mM. The pH and temperature optima were 7-75 and 30 "C, respectively. Glucan synthase activity was highest in cells derived from early mid-exponential phase and declined t o a basal level by stationary phase. A permeabilization-based in situ assay for glucan synthase was developed. Cells were permeabilized with 2 % (v/v) solution of toluene/methanol (1 : 1) and assayed for glucan synthase activity using standard reaction mixtures. Reactions were linear for 30 min and were inhibited by known inhibitors of glucan synthesis. This study represents the first characterization of glucan synthase using yeast and mycelial phase microsomes and adaptation of a permeabilized system using a single C. albicans strain with standardized assay conditions.

The fusacandins, antifungal agents of the papulacandin class, are produced by a strain of Fusarium sambucinum. Fermentation yielded 60mg/liter of fusacandin A and minor amounts of fusacandin B. As expected, the fusacandins inhibit (l ,3)-/?-glucan synthesis. Fusacandin A is slightly less active than papulacandin B against Candida albicans and, like papulacandin, loses activity in the presence of serum.

Conjugates formed by reaction of monoclonal antibody B72.3 with benzyl isothiocyanate derivatives of four amino polycarboxylate chelators (NTA, EGTA, EDTA, DTPA) were labeled with indium-111 and administered iv to athymic mice bearing antigen-positive (LS174T) and antigen-negative (A375) human tumor xenografts. Conjugate immunoreactivities, antibody dose, and xenograft size were controlled, so that the effects of varying chelate structure could be evaluated under conditions where immunological and physiological factors were effectively held constant. Tissue distribution and excretion of the radiometal at 24 and 48 h postinjection were shown to correlate directly with chelate thermodynamic stability (NTA less than EGTA less than EDTA less than DPTA). Radioactivity levels in the blood and the LS174T xenograft increased, while kidney levels and excretion levels decreased, with increasing chelate stability. The kidney was the only normal organ that accumulated non-antibody-bound 111In, uptake of radioactivity into all other tissues, and in particular the liver, being unaffected by changes in chelate structure. Mean transferrin saturation in the tumor-bearing athymic mice was found to be 65%. It is proposed that uptake of free 111In by serum transferrin is precluded in this model, leading to the observed renal localization of unbound label. Kidney:blood and kidney:LS174T activity ratios at 48 h postinjection provided the most sensitive indices of conjugate instability in vivo, spanning 50- and 20-fold ranges, respectively, between the least stable and the most stable conjugate. It is concluded that this antigen/antibody system and mouse model are well-suited to structure-function studies of immunoglobulin labels.