In vitro interaction of posaconazole with calcineurin inhibitors and sirolimus against zygomycetes.

OBJECTIVES: Zygomycosis is an uncommon but devastating disease with few therapeutic options. Calcineurin inhibitors and sirolimus (mTOR inhibitor), commonly used in transplant patients as immunosuppressives, have antifungal activity. They are known to demonstrate synergy with triazoles against certain fungi, though limited data exist about their activity against zygomycetes. Our aim was to study the in vitro interaction of posaconazole with calcineurin inhibitors and sirolimus against zygomycetes. METHODS: Drug interactions were assessed with chequerboard dilution for posaconazole with calcineurin inhibitors and sirolimus according to the CLSI M38-A2 method for filamentous fungi. Twenty-eight clinical isolates were studied, including Rhizopus arrhizus, Rhizopus microsporus, Rhizomucor pusillus, Mucor sp., Cunninghamella bertholletiae, Myocladus corymbifera and Apophysomyces elegans. Combinations of posaconazole with tacrolimus, cyclosporin A or sirolimus were used. Experiments were performed in duplicate. Mean fractional inhibitory concentration indices were calculated. RESULTS: Posaconazole with calcineurin inhibitors demonstrated consistent synergy against C. bertholletiae, M. corymbifera and A. elegans, whereas synergy or no interaction was primarily observed against R. arrhizus, R. microsporus, R. pusillus and Mucor. Antagonism was seen with the combination of posaconazole and sirolimus. Strain variability was noted among the same species. CONCLUSIONS: The clinical significance of these findings is unclear, but further studies are warranted given the potential for concomitant use of these agents in transplant patients treated for zygomycosis.

DNA-mediated transformation of Chlamydomonas reinhardi cells: use of aminoglycoside 3'-phosphotransferase as a selectable marker.

Using a modified vector, we developed a method for DNA-mediated transformation of Chlamydomonas reinhardi with increased efficiency. The vector contained the yeast 2 microns origin of replication as a heterologous replicon. The aminoglycoside 3'-phosphotransferase (APH) gene linked to the simian virus 40 early promoter was used as an antibiotic selectable marker. The C. reinhardi transformants were resistant to 12 micrograms of G418 or 150 micrograms of kanamycin per ml. A quick-blot mRNA analysis demonstrated the presence of RNase-sensitive transcripts from the APH gene in the transformants, suggesting that the acquisition of antibiotic resistance was due to the expression of the APH gene. Southern blot analysis revealed the presence of free plasmid DNA in the transformant. The transforming vector was recovered by transforming recipient bacteria with the total DNA extracted from the C. reinhardi transformant.

Nucleotide sequence analysis and expression of a tetracycline-resistance gene from Campylobacter jejuni.

Resistance to tetracycline in the microaerophilic Gram-negative bacterium Campylobacter jejuni is plasmid-mediated. A 6.9-kb HindIII DNA fragment containing the tetracycline-resistance (TcR) gene (designated tetO) from the C. jejuni conjugative plasmid pUA466 was cloned into pUC8, and the resultant plasmid pUOA1 was used to transform Escherichia coli to Tc resistance. The tetO gene was localized at a 2.0-kb region comprising 0.2-kb and 1.8-kb HincII fragments, and the nucleotide sequences were determined. The protein coding region of tetO contained a 1911-bp open reading frame which corresponded to a 72.3-kDa protein. Upstream from the start codon were hexanucleotides that resembled the canonical sequences found at the -10 region, -35 region and the ribosome-binding site of the prokaryotic promoter. The tetO gene product was expressed utilizing an E. coli-derived in vitro transcription/translation system. The polypeptide had an apparent Mr of 68,000. Comparison of the amino acid sequences of TetO to those of TetM (derived from the Gram-positive Streptococcus pneumoniae) revealed 76% homology. Hydrophilicity plot analyses of TetO and TetM proteins provided almost identical profiles. These results clearly support our earlier [Taylor et al., J. Bacteriol. 169 (1987) 2984-2989] suggestion that TcR determinants found in Gram-positive bacteria and in C. jejuni may have a common ancestry.

Expression of herpes simplex virus thymidine kinase gene in aquatic filamentous fungus Achlya ambisexualis.

We have constructed a plasmid vector pSV2neo-MK alpha G in which the structural tk gene for Herpes simplex virus thymidine kinase (HSV-TK) was placed downstream from the metallothionein-I promoter. The vector also contained the selection marker aminoglycoside 3'-phosphotransferase (Km). This vector was able to transform the filamentous fungus Achlya ambisexualis and G-418-resistant colonies were obtained. Southern blot analyses revealed that multiple bands hybridizing to the HSV tk gene probe were present in the genomic DNA of the transformants. Upon analysis by gel electrophoresis, one of the transformants exhibited TK activity bearing electrophoretic mobility similar to that of the HSV-TK. An increase of approx. 40% of [3H]thymidine uptake and incorporation into cellular DNA was also observed in this transformant. This study suggested that the HSV tk gene can be expressed in the fungus A. ambisexualis that can be considered as a candidate host cell for further gene-expression studies.

A conformational and structure-activity relationship study of cytotoxic 3,5-bis(arylidene)-4-piperidones and related N-acryloyl analogues.

A series of 3,5-bis(arylidene)-4-piperidones 1 and related N-acryloyl analogues 2 were prepared as candidate cytotoxic agents with a view to discerning those structural features which contributed to bioactivity. A number of the compounds were markedly cytotoxic toward murine P388 and L1210 leukemic cells and also to human Molt 4/C8 and CEM neoplasms. Approximately 40% of the IC50 values generated were lower than the figures obtained for melphalan. In virtually all cases, the N-acyl compounds were significantly more bioactive than the analogues 1. In general, structure-activity relationships revealed that the cytotoxicity of series 1 was correlated positively with the size of the aryl substituents, while in series 2, a -sigma relationship was established. In particular, various angles and interatomic distances were obtained by molecular modeling, and the presence of an acryloyl group on the piperidyl nitrogen atom in series 2 affected the relative locations of the two aryl rings. This observation, along with some differences in distances between various atoms in series 1 and 2, may have contributed to the disparity in cytotoxicity between 1 and 2. The results obtained by X-ray crystallography of representative compounds were mainly in accordance with the observations noted by molecular modeling. Selected compounds interfered with the biosynthesis of DNA, RNA, and protein in murine L1210 cells, while others were shown to cause apoptosis in the human Jurkat leukemic cell line. This study has revealed the potential of these molecules for development as cytotoxic and anticancer agents.

Cytotoxic activities of Mannich bases of chalcones and related compounds.

Various Mannich bases of chalcones and related compounds displayed significant cytotoxicity toward murine P388 and L1210 leukemia cells as well as a number of human tumor cell lines. The most promising lead molecule was 21 that had the highest activity toward L1210 and human tumor cells. In addition, 21 exerted preferential toxicity to human tumor lines compared to transformed human T-lymphocytes. Other compounds of interest were 38, with a huge differential in cytotoxicity between P388 and L1210 cells, and 42, with a high therapeutic index when cytotoxicity to P388 cells and Molt 4/C8 T-lymphocytes were compared. In general, the Mannich bases were more cytotoxic than the corresponding chalcones toward L1210 but not P388 cells. A ClusCor analysis of the data obtained from the in vitro human tumor screen revealed that the mode of action of certain groups of compounds was similar. For some groups of compounds, cytotoxicity was correlated with the sigma, pi, or molar refractivity constants in the aryl ring attached to the olefinic group. In addition, the IC50 values in all three screens correlated with the redox potentials of a number of Mannich bases. X-ray crystallography and molecular modeling of representative compounds revealed various structural features which were considered to contribute to cytotoxicity. While a representative compound 15 was stable and unreactive toward glutathione (GSH) in buffer, the Mannich bases 15, 18, and 21 reacted with GSH in the presence of the pi isozyme of glutathione S-transferase, suggesting that thiol alkylation may be one mechanism by which cytotoxicity was exerted in vitro. Representative compounds were shown to be nonmutagenic in an intrachromosomal recombination assay in yeast, devoid of antimicrobial properties and possessing anticonvulsant and neurotoxic properties. Thus Mannich bases of chalcones represent a new group of cytotoxic agents of which 21 in particular serves as an useful prototypic molecule.

Aspergillus: rising frequency of clinical isolation and continued susceptibility to antifungal agents, 1994-1999.

We investigated the frequency of clinical isolation and the in vitro susceptibility to antifungal agents of Aspergillus species obtained from patients at the Detroit Medical Center from January 1994 to December 1999. During this period, 593 clinical isolates of Aspergillus species [406 A. fumigatus, 68%; 82 A. niger, 14%; 42 A. flavus, 7%; 63 Aspergillus spp., 11%] were recovered from hospitalized patients. From January 1996 to December 1999, approximately 2.5-4.5 fold yearly increase of the number of aspergillus isolates occurred compared to that of 1994. Conidial suspensions from clinical isolates were prepared and their in vitro susceptibility to amphotericin B and three azoles were determined. All four agents examined were extremely active. The minimum inhibitory concentrations (MIC(90)) (microg/mL) of amphotericin B, itraconazole, voriconazole and posaconazole for A. fumigatus (n = 406) were 0.5, 1.0, 0.5 and 0.25. Similar values were noted for non-A. fumigatus isolates. A year-to-year comparison of the MIC(90) of the four agents for A. fumigatus and non-A. fumigatus isolates over the 6-year study period showed no significant differences. Our study showed a steady increase in the frequency of clinical isolation of Aspergillus species; and the organism has remained susceptible to amphotericin B/triazoles without any change in susceptibility levels during the 6-year study period.

In vitro susceptibilities of Aspergillus species to voriconazole, itraconazole, and amphotericin B.

We studied the in vitro activity of voriconazole (VCZ) itraconazole (ITZ) and amphotericin B (AMB) against 216 clinical isolates of Aspergillus spp. (142 Aspergillus fumigatus and 74 nonfumigatus Aspergillus spp. isolates) using a broth macrodilution method. The MICs (microgram/mL) (mean, range) for A. fumigatus were: VCZ 0.88, 0.25-4; ITZ 0.54, 0.25-4; AMB 2.16, 0.5-8. MIC90s were: VCZ 2, ITZ 1, AMB 4. MICs for nonfumigatus Aspergillus spp. were: VCZ 1.57, 0.25-4; ITZ 1.74, 0.25-4; AMB 2.88, 0.5-8. MIC90s for this group were: VCZ 4, ITZ 4, AMB 4. We also studied the susceptibility to VCZ of 18 AMB-resistant (mean, MIC 6.0 micrograms/mL) and 28 ITZ-resistant (mean, MIC 13.28 micrograms/mL) A. fumigatus isolates selected in the laboratory. The mean MICs of VCZ were 0.59 microgram/mL for AMB-resistant and 1.32 micrograms/mL for ITZ-resistant isolates. Our study showed that VCZ and ITZ had comparable in vitro activity against the isolates studied, except against A. fumigatus, where the MIC of ITZ was lower. The azoles had better in vitro activity than AMB against A. fumigatus and non-fumigatus spp. The non-fumigatus Aspergillus spp. were less susceptible to all three antifungals evaluated. When tested against ITZ- or AMB-resistant A. fumigatus strains, VCZ retained good activity, showing only a modest rise in the MIC against ITZ-resistant strains.

Glutathione levels during thermal induction of the yeast-to-mycelial transition in Candida albicans.

Glutathione (GSH) levels were directly monitored by reverse phase HPLC during the thermal yeast-to-mycelial induction of Candida albicans. The GSH levels decreased approximately 100-fold within 120 min which corresponded to the time of maximal yeast-to-mold conversion. The yeast to mold conversion was inhibited by 1-p-chlorophenyl-4,4-dimethyl-5-diethylamino-1-penten-3-one (CDDP), a thiol-specific alkylator, which prevented the decline in GSH levels. These results are discussed with respect to the potential involvement of intracellular GSH levels in regulation of the yeast-to-mold dimorphism in Candida albicans.

Isolation and in vitro susceptibility to amphotericin B, itraconazole and posaconazole of voriconazole-resistant laboratory isolates of Aspergillus fumigatus.

OBJECTIVES: To select voriconazole-resistant mutants of Aspergillus fumigatus in the laboratory from drug-susceptible clinical isolates and examine their in vitro susceptibility to amphotericin B and investigational azoles, and to compare the intramycelial accumulation of voriconazole in the resistant isolates with that in the susceptible parent. METHODS: Voriconazole-resistant Aspergillus fumigatus isolates were selected in the laboratory from three highly susceptible (MIC < or = 0.5 mg/L) clinical isolates by stepwise selection on peptone yeast extract glucose (PYG) agar containing 0.5 mg and 4 mg voriconazole/L. Twenty-three colonies that grew in the presence of 4 mg voriconazole/L on PYG agar (frequency 1.9 x 10(-8)) were tested for their in vitro susceptibility to amphotericin B, itraconazole, voriconazole and posaconazole by a broth macrodilution technique. The accumulation of voriconazole in the mycelia of two representative resistant isolates (VCZ-W42 and VCZ-W45) was determined by a previously described bioassay. RESULTS: The geometric mean MICs (mg/L) of amphotericin B, itraconazole, voriconazole and posaconazole for these isolates were 0.45 +/- 0.19, 0.69 +/- 0.45, 5.24 +/- 3.74 and 0.27 +/- 0.18, respectively. A comparison of the geometric mean MICs of the antifungals obtained for the resistant isolates to those of the susceptible parents showed 1.15-, 2.76-, 16.90- and 1.42-fold increases, respectively, for amphotericin B, itraconazole, voriconazole and posaconazole, suggesting that low-level cross-resistance exists between the azole antifungals. The susceptible parent and the resistant isolates accumulated similar amounts of voriconazole. CONCLUSIONS: These results suggest that spontaneous mutants of Aspergillus fumigatus resistant to voriconazole could emerge among clinical isolates under selection pressure and that the observed reduced in vitro susceptibility to voriconazole may not be due to reduced accumulation of the drug in the mycelia.

Efficacy of voriconazole plus amphotericin B or micafungin in a guinea-pig model of invasive pulmonary aspergillosis.

The efficacy of voriconazole in combination with amphotericin B or micafungin was studied in a transiently neutropenic guinea-pig model of invasive pulmonary aspergillosis. Guinea-pigs treated with the antifungal drugs, alone or in two-drug combinations, had an improved survival rate and reduced fungal burden in the lungs compared to untreated control animals. The efficacy of monotherapy and combination therapy was similar; activity was neither enhanced nor reduced with the two-drug combinations. Further studies of efficacy, dosing and optimal regimens for antifungal combinations are warranted.

In-vitro activity of nikkomycin Z alone and in combination with polyenes, triazoles or echinocandins against Aspergillus fumigatus.

The in-vitro activity of nikkomycin Z was investigated in combination with polyenes, triazoles or echinocandins against 20 clinical isolates of Aspergillus fumigatus with the fractional inhibitory concentration index (FICI) method. The drug interactions were classified as synergic (FICI < or = 0.5), no interaction (FICI > 0.5, but FICI < or = 4) or antagonistic (FICI > 4). The fungicidal activity of nikkomycin Z alone and in combination with a representative echinocandin (caspofungin) or triazole (voriconazole) was also examined with time-kill experiments and fungal cell viability assays. Two-drug combinations of nikkomycin Z with amphotericin B (FICI 3.59 +/- 0.57), amphotericin B lipid complex (FICI 3.95 +/- 0.74), liposomal amphotericin B (FICI 3.62 +/- 0.98), itraconazole (FICI 2.0 +/- 0.0), voriconazole (FICI 1.07 +/- 0.37), posaconazole (FICI 2.20 +/- 0.44) or ravuconazole (FICI 1.76 +/- 0.44) showed no interactions, but the pairwise combination of nikkomycin Z with caspofungin (FICI 0.22 +/- 0.19) or micafungin (FICI 0.35 +/- 0.27) showed synergic activity against A. fumigatus. Time-kill studies and fungal cell viability assays showed that neither nikkomycin Z nor caspofungin alone possessed fungicidal activity against A. fumigatus, whereas a combination of these two drugs at concentrations > or = 2 mg/L (> or = 0.031 x the concentration of drug that produced no visible growth) killed germinated conidia within 24 h in a concentration-dependent manner. These data suggest that two-drug combinations of nikkomycin Z with echinocandins, but not with polyenes and triazoles, have a synergic effect against A. fumigatus.

Reduced susceptibility in laboratory-selected mutants of Aspergillus fumigatus to itraconazole due to decreased intracellular accumulation of the antifungal agent.

To study the mechanism of resistance of Aspergillus fumigatus to itraconazole, spontaneous mutants with reduced susceptibility were selected by spreading 2 x 10(8) conidia from a clinical isolate (W73355) susceptible to miconazole (MIC 2 mg/l), itraconazole (MIC 0.25 mg/l) and amphotericin B (MIC 0.5 mg/l) on 40 peptone yeast extract glucose agar plates containing miconazole (32 mg/l). The 19 colonies that grew (frequency 0.95 x 10(-7)) in the presence of miconazole were screened by broth macrodilution technique for their susceptibility to itraconazole. A total of two isolates (frequency 1 x 10(-8)) MCZ14 and MCZ15 had MICs of 16 and 8 mg/l, respectively, for itraconazole. Both MCZ14 and MCZ15 showed concomitant increases in MICs for ketoconazole and miconazole, but not for amphotericin B. Growth inhibition studies as well as kill curve experiments revealed that MCZ14 and MCZ15 were less susceptible to itraconazole compared to the parental strain. The intracellular accumulation of itraconazole in A. fumigatus was time and concentration dependent. Maximum accumulation was obtained within 30 min at 5 microM itraconazole. In MCZ14 and MCZ15 intracellular accumulation of [3H]itraconazole was reduced by approximately 80 and 60%, respectively, compared to the susceptible parent. The respiratory inhibitor carbonyl cyanide m-chlorophenyl hydrazone at 200 microM reduced the intracellular accumulation of itraconazole by approximately 36.2% (P < or = 0.05) in the parent and in the mutant strains. These results suggest that (i) the reduced accumulation of itraconazole in MCZ14 and MCZ15 is due to diminished permeability of the drug and perhaps not due to efflux, (ii) the uptake of itraconazole in A. fumigatus may be an energy dependent process, and (iii) decreased accumulation of itraconazole is at least in part responsible for the reduced susceptibility of the mutant isolates to itraconazole.

Proton translocating ATPase mediated fungicidal activity of a novel complex carbohydrate: CAN-296.

CAN-296 is a complex carbohydrate (approximately 4300 Da) isolated from the cell wall of Mucor rouxii. It exhibits excellent in vitro fungicidal activity against a wide spectrum of pathogenic yeasts, including isolates resistant to azoles and polyenes. The rapid irreversible action of CAN-296 on intact fungal cells and protoplasts suggested a membrane-located target for its action. The proton translocating ATPase (H+-ATPase) of fungi is an essential enzyme required for the regulation of intracellular pH and nutrient transport. Inhibition of H+-ATPase leads to intracellular acidification and cell death. We therefore investigated the effect of CAN-296 on H+-ATPase-mediated proton pumping by intact cells of Candida and Saccharomyces species by measuring the glucose-induced acidification of external medium. CAN-296 inhibited proton pumping of Candida albicans, Candida glabrata, Candida krusei, Candida guilliermondii and Saccharomyces cerevisiae at low concentrations (0.078-1.25 mg/l). Other commonly used antifungal agents such as amphotericin B, itraconazole and fluconazole had no effect on H+-ATPase-mediated proton pumping. A clinical isolate of C. glabrata with reduced in vitro susceptibility (MIC = 10 mg/l) to CAN-296 also showed resistance to CAN-296 inhibition of proton pumping. Purified membrane fractions rich in H+-ATPase activity were not inhibited by CAN-296 suggesting that the effect on the H+-ATPase-mediated proton pumping in intact yeast cells is an indirect effect, perhaps mediated by local or global disruption of the plasma membrane. These results suggest that the inhibition of fungal H+-ATPase is at least partly responsible for the antifungal activity of CAN-296.

In vitro antifungal activity of CAN-296: a naturally occurring complex carbohydrate.

The in vitro activity of a naturally occurring complex carbohydrate, CAN-296, was evaluated by testing 132 clinical and ATCC isolates of yeast and Aspergillus fumigatus, many of which were azole-resistant. The in vitro susceptibility tests were performed by standardized broth micro- and macrodilution methods and results were compared with those obtained for amphotericin B, fluconazole, ketoconazole, flucytosine and the pneumocandin L-733,560. All tested Candida species showed highly uniform susceptibility to CAN-296 at concentrations of 0.078 to 0.312 microgram/ml; non-albicans Candida were as susceptible to CAN-296 as the Candida albicans strains. Multi-azole-resistant Candida species were highly sensitive to CAN-296. Minimum inhibitory concentration measurements did not differ from minimum lethal concentrations by more than two-fold for all tested Candida species. Aspergillus fumigatus, on the other hand, showed only moderate susceptibility to CAN-296. The kinetics of the anti-Candida activity of CAN-296 was investigated by kill-curve experiments using C. albicans and C. glabrata and the results were compared with those obtain for amphotericin B. CAN-296 was found to be rapidly fungicidal in concentrations ranging from 4-16 fold the mean MIC value. The broad spectrum of anti-Candida activity together with the rapid fungicidal effect make this complex carbohydrate a promising agent for clinical use.

Amino acid variations of cytochrome P-450 lanosterol 14 alpha-demethylase (CYP51A1) from fluconazoleresistant clinical isolates of Candida albicans.

We studied six clinical isolates of Candida albicans. All six isolates showed high level resistance to fluconazole (minimum inhibitory concentrations 64 microg/ml) with varying degrees of cross-resistance to other azoles but not to amphotericin B. Neither higher dosage nor upregulation of the gene encoding the cytochrome P- 450 lanosterol 14 alpha-demethylase (CYP51A1 or P-450LDM) was responsible for fluconazole resistance. The resistant and the susceptible isolates accumulated similar amounts of azoles. To examine whether resistance to fluconazole in these clinical isolates of C. albicans is mediated by an altered target of azole action, we cloned the structural gene encoding P-450LDM from the fluconazole resistant isolates. The amino acid sequences of the P-450LDMs from the isolates were deduced from the gene sequences and compared to the P-450LDM sequence of the fluconazole-susceptible C. albicans B311. The enzymes from the clinical isolates showed 2 to 7 amino acid variations scattered across the molecules encompassing 10 different loci. One-half of the amino acid changes obtained were conserved substitutions (E116D, K143R, E266D, D278E, R287K) compared to the susceptible strain. Non-conserved substitutions were T128K, R267H, S405F, G450E and G464S, three of which are in and around the hemebinding region of the molecule. R287K is the only amino acid change that was found in all six clinical isolates. One or more of these mutational alterations may lead to the expression of an azole-resistant enzyme.

Effect of test medium on in vitro susceptibility testing results for Aspergillus fumigatus.

We examined the effect of peptone yeast extract glucose broth (PYG), RPMI 1640 and Antibiotic Medium 3 (M3) on the in vitro susceptibility of clinical isolates (n = 200) of Aspergillus fumigatusto amphotericin B (AMB), itraconazole (ITZ) and voriconazole (VCZ). The MICs (mug/mL) of various antifungal agents (geometric mean plus/minus standard deviation) obtained in PYG, RPMI 1640 and M3, respectively, were as follows; AMB: 1.64 plus/minus 0.92, 0.42 plus/minus 0.21, 0.33 plus/minus 0.16; ITZ: 0.44 plus/minus 0.54, 0.40 plus/minus 0.65, 0.17 plus or minus 0.32; VCZ: 0.70 plus/minus 0.58, 0.29 plus/minus 0.22, 0.36 plus/minus 0.21. Pairwise comparisons of the MICs of AMB, ITZ and VCZ obtained in PYG, RPMI 1640 and M3 showed no significant differences except for AMB in PYG broth compared to those obtained in RPMI 1640 and M3 (p

Activity of some Mannich bases of conjugated styryl ketones against Candida albicans.

A number of Mannich bases of conjugated styryl ketones displayed activity against Candida albicans strains 3153A and B311. Antifungal potency was influenced by the relative hydrophobicities of the molecules and on occasions by the electronic nature of the aryl substituents. The compounds inhibited one or more of the following enzymes in the glutathione metabolic pathway namely glutathione S-transferases, glutathione reductase, gamma-glutamyl transpeptidase and glutathione peroxidase. Nearly half of the compounds examined on the yeast-to-mycelium transition in C. albicans 3153A prevented this conversion from occurring.

Cytotoxic 4'-aminochalcones and related compounds.

A series of 4'-aminochalcones 1 and related maleamic acids 2 and Schiff bases 3 were designed and synthesized as candidate cytotoxic agents. The atomic charges on different atoms of representative compounds were calculated. Evaluation of the enones 1-3 against human Molt 4/C8 and CEM T-lymphocytes as well as murine P388 and L1210 leukemic cells revealed that approximately 40% of the IC50 values generated were less than 10 microM. In some cases cytotoxicity was correlated with the Hammett sigma values of the aryl substituents and less frequently with the aryl Hansch pi values. Evidence was obtained that in general these compounds displayed selective toxicity for certain malignant cells and were well tolerated in mice. This study has revealed various directions whereby the project may be amplified in the future with a view to finding compounds with increased cytotoxicity to tumour cells.

Chromosomal localization of phosphoglycerate kinase (PGK) gene in Candida albicans using a heterologous probe.

The glycolytic enzyme phosphoglycerate kinase (PGK) is highly conserved throughout the evolutionary ladder. Using a previously cloned Saccharomyces cerevisiae PGK gene as hybridization probe, chromosomal localization and strain-specific variation of PGK gene in C. albicans have been examined. Separation of chromosome-size DNA fragments by pulse field gel electrophoresis followed by DNA- DNA hybridization analysis revealed that the PGK is encoded by a single copy gene located on the large chromosome of the commonly used laboratory strain ATCC 32354, as well as of several clinical isolates of C. albicans. Restriction fragment length analysis indicated that the PGK gene locus is highly polymorphic, and this strain-specific variation could be exploited to study intra-specific relatedness of clinical isolates.