Candida albicans

Mycoses, 2000 Oct, 43(9-10), 349 - 53
The frequency of Candida parapsilosis in onychomycosis . An epidemiological survey in Israel; Segal R et al.; Candida albicans is regarded as the major pathogen in yeast-induced onychomycosis . Based on our impression of an increasing prevalence of Candida parapsilosis in this disease, we examined the data of two mycology laboratories in the same geographic location, from 1994 to 1996 in one (centre A) and for 1995 (6 months) in the other (centre B) . A total of 954 and 230 toenails and 621 and 190 fingernails, respectively, underwent KOH microscopy and culture studies in each centre . Positive findings were noted in 45 and 65% of the toenails and 44 and 72% of the fingernails, respectively . In the toenails, Candida spp . were found in 22 and 15%, respectively, and in the fingernails, in 77 and 63%, respectively . The most frequent Candida species was C . parapsilosis (39.5% in toenails, 36.7% in fingernails), followed by C . albicans (19.5% in toenails, and 34.4% in fingernails) . These results demonstrate a higher frequency of isolation of C . parapsilosis compared with C . albicans in onychomycosis . This might have important therapeutic implications.

Ned Tijdschr Geneeskd . 2000 Oct 28;144(44):2103.
{Diagnostic image (9) . Chronic mucocutaneous candidiasis}; van der Meer JW et al.; An 18-year-old man had lifelong mucocutaneous lesions from which Candida albicans was persistently cultured . Upon fluconazole treatment the lesions diminished notably.

J Antimicrob Chemother, 2000 Dec, 46(6), 1001 - 8
Impact of fluconazole prophylaxis on fungal colonization and infection rates in neutropenic patients . The Canadian Fluconazole Study; Laverdiere M et al.; Fungal colonization profiles from four different anatomical sites were evaluated in 266 neutropenic cancer patients receiving intensive cytotoxic therapy for acute leukaemia or for autologous marrow transplantation . At the beginning of chemotherapy patients were allocated randomly to receive oral fluconazole 400 mg daily or an identical placebo until prophylaxis failure or marrow recovery . Candida albicans colonization was reduced from 30 to 10% in the fluconazole recipients while it increased from 32 to 57% in the placebo patients (P<0.001) . By the end of prophylaxis, colonization with non-albicans Candida species increased from 7 to 21% and 8 to 18% in the fluconazole and placebo patients, respectively (P = 0.396) . Although Candida glabrata was isolated more frequently at the end of the prophylactic period in the fluconazole patients than in the placebo patients (16 versus 7%), only one definite invasive C . glabrata infection was noted . Overall, definite invasive fungal infections were documented in 26 patients {four fluconazole versus 22 placebo patients (P< or =0.001)} . In 23 (92%) patients the infections were caused by persistently colonizing or newly acquired organisms . While probable invasive fungal infections were noted in five fluconazole patients, 10 placebo patients were also affected (P = 0.19) . An end-of-prophylaxis colonization index >0.25 was 76% sensitive but only 69% specific for invasive fungal infection . However, a colonization index < or =0.25 at baseline had a negative predictive value of 88% for development of invasive fungal infection . Fluconazole prophylaxis decreased colonization by fungi and subsequent invasive fungal infections in neutropenic cancer patients.

J Clin Microbiol, 2000 Dec, 38(12), 4626 - 8
Tween 80 opacity test responses of various Candida species; Slifkin M; A total of 111 Candida isolates representing 11 species were examined for their respective responses to a Tween 80 opacity test . The strains of Candida albicans and C . tropicalis that were examined produced an opacity response around their colonies at 2 to 3 days postinoculation . A second group of Candida species yielded a halo around their colonies at 8 to 10 days postinoculation . The remaining Candida species did not produce a positive test response through 10 days postinoculation . The strains of C . dubliniensis were easily differentiated from strains of C . albicans by this test . The Tween 80 opacity test is simple and economical to prepare and is easy to interpret.

J Clin Microbiol, 2000 Dec, 38(12), 4554 - 9
Comparative genotyping of Candida albicans bloodstream and nonbloodstream isolates at a polymorphic microsatellite locus; Dalle F et al.; Molecular typing studies have shown that the predominant form of reproduction of Candida albicans is clonal and that, in a majority of situations, persistent or recurrent infections are due to a unique strain . Characterization of distinct subpopulations and correlation with clinical features may thus be important to understanding the pathogenesis of candidiasis . In a clonal model, a unique polymorphic marker may identify populations with different biological properties . We therefore compared 48 bloodstream isolates and 48 nonbloodstream matched strains of C . albicans at the elongation factor 3-encoding gene (CEF3) polymorphic microsatellite locus of C . albicans . Sizing of the alleles was performed by automated capillary electrophoresis . A new, 137-bp allele was characterized, and seven nondescribed combinations were observed, resulting in 15 and 11 distinct CEF3 profiles in bloodstream and control strains, respectively . Genotypes 126-135, 130-136, and 131-131 accounted for 60.4% of both bloodstream and control strains . Four bloodstream isolates but no control strains displayed the 135-135 combination . None of the other genotypes was present at an increased frequency in bloodstream isolates . Bloodstream and nonbloodstream strains of C . albicans thus have a heterogeneous structure at the CEF3 locus, with three major and multiple minor allelic combinations.

J Clin Microbiol, 2000 Dec, 38(12), 4503 - 10
Identification and phylogenetic relationship of the most common pathogenic Candida species inferred from mitochondrial cytochrome b gene sequences; Yokoyama K et al.; We sequenced a 396-bp region of the mitochondrial cytochrome b gene of the most common clinically important Candida species: Candida albicans, C . glabrata, C . parapsilosis, C . tropicalis, C . krusei, and C . lusitaniae . The recently described species of Candida, C . dubliniensis, associated with mucosal candidiasis in human immunodeficiency virus-infected individuals, was also included . Two to five strains of each species were examined . Some species represented intraspecies variation, which was not more than 1.8% (DNA) . However, interspecies variations were more than 10 and 7%, respectively, for DNA and amino acid sequences . Multiple alignments of nucleotide and deduced amino acid sequences revealed species-specific nucleotides and amino acids . Nucleotide- and amino acid-based phylogenetic trees were constructed and are discussed . Using the database, it is possible to identify presumptive Candida species within a working day.

J Antibiot (Tokyo), 2000 Sep, 53(9), 920 - 7
FR901469, a novel antifungal antibiotic from an unidentified fungus No.11243 . II . In vitro and in vivo activities; Fujie A et al.; FR901469 is a water-soluble macrocyclic lipopeptidolactone (C71H116N14O23) that has inhibitory activity against 1,3-beta-glucan synthase and exhibits in vitro and in vivo antifungal activity against both Candida albicans and Aspergillus fumigatus . The MICs of FR901469 against Candida albicans FP633 and Aspergillus fumigatus FP1305 in a micro-broth dilution test were 0.63 and 0.16 microg/ml, respectively . FR901469 showed excellent efficacy by subcutaneous injection against both Candida albicans and Aspergillus fumigatus in a murine systemic infection mode, with ED50s of 0.32 and 0.2 mg/kg, respectively . This compound also showed potent anti-Pneumocystis activity in the nude mice model with experimental Pneumocystis pneumonia . The hemolytic activity of FR901469 towards mouse red blood cells, is about 30-fold weaker than that of amphotericin B.

J Antibiot (Tokyo), 2000 Sep, 53(9), 912 - 9
FR901469, a novel antifungal antibiotic from an unidentified fungus No.11243 . I . Taxonomy, fermentation, isolation, physico-chemical properties and biological properties; Fujie A et al.; FR901469 is a novel antifungal antibiotic produced by an unidentified fungus No.11243 . This compound was isolated from the culture broth by solvent extraction, HP-20 and YMC ODS gel column chromatography, and lyophilization . FR901469 is a white powder which melts at 182 approximately 187 degrees C and possesses the molecular formula C71H116N14O23 . This compound has good water solubility . FR901469 inhibited the activity of 1,3-beta-glucan synthase from Candida albicans with an IC50 value of 0.05 microg/ml, and displayed greater inhibitory activity than other 1,3-beta-glucan synthase inhibitors such as, WF11899A, echinocandin B, aculeacin A, and papulacandin B.

Z Naturforsch {C}, 2000 Sep-Oct, 55(9-10), 785 - 9
Chemical composition and biological activity of propolis from Brazilian meliponinae; Velikova M et al.; Twenty-one propolis samples produced by 12 different Meliponinae species were analyzed by GC-MS . Several chemical types of stingless bees' propolis could be grouped, according to the prevailing type of compounds like: 'gallic acid", "diterpenic" and "triterpenic" types . The results confirm that neither the bee species nor the geographical location determine the chemical composition of Meliponinae propolis and the choice of its plant source, respectively . This could be explained by the fact that Meliponinae forage over short distances (maximum 500 m) and thus use as propolis source the first plant exudate they encounter during their flights . The antibacterial, antifungal and cytotoxic activities of the samples were also investigated . Most samples had weak or no activity against E . coli, weak action against Candida albicans . Some of them showed significant activity against St . aureus., presumably connected to the high concentration of diterpenic acids . Samples rich in diterpenic acids possessed also high cytotoxic activity (Artemia salina test).

Mycoses, 2000, 43 Suppl 1, 29 - 35
{Asteroid bodies: host-pathogen reactions in mycoses}; Muller J; A review of the literature on asteroid bodies (Splendore-Hoeppli phenomenon) as well as an immunoelectronmicroscopic study on asteroid bodies with Candida albicans and electronmicroscopic observations on asteroid bodies with Aspergillus are presented . The following definitions are proposed: Asteroid bodies with Candida albicans: precipitate consisting of fungal antigen and antibodies of host origin in light microscopically visible dimension deposited on the cell wall surface of Candida cells in parasitic condition . Asteroid bodies with Aspergillus: precipitate consisting of fungal antigen, antibodies of host origin and necrotic cellular detritus originated from cells of host defense, in light microscopically visible dimension, deposited on the cell wall surface of abortive Aspergillus cells, reduced in longitudinal growth, in parasitic condition.

J Pept Res, 2000 Nov, 56(5), 275 - 82
Divergent solid-phase synthesis and candidacidal activity of MUC7 D1, a 51-residue histidine-rich N-terminal domain of human salivary mucin MUC7; Satyanarayana J et al.; Domain 1 of the low-molecular-weight human salivary mucin, designated MUC7 D1, spans the 51 N-terminal amino acid residues . This domain contains a 15-residue basic histidine-rich subdomain (R3-Q17) which has 53% sequence similarity to histatin 5 (Hsn-5), a salivary molecule known to exert potent in vitro cidal activity against Candida albicans and many other medically important fungi . The MUC7 D1-15mer and its derivatives have previously been synthesized in our laboratory and their candidacidal activities have been found to be inferior to that of Hsn-5 . We were therefore intrigued to explore the candidacidal potency of the full-length MUC7 D1 (51-mer) . Linear solid-phase synthesis of this domain has been accomplished following standard Fmoc chemistry . The problems of partial coupling, owing to the peptide chain length, at several stages of the solid-phase step-by-step synthesis were circumvented either by double-coupling techniques or efficient coupling procedures . The MUC7 D1 peptide was purified to homogeneity by conventional reverse-phase HPLC using two columns connected in series . Secondary structure of the purified peptide was assessed by circular dichroism (CD) spectroscopy in phosphate buffer and trifluoroethanol and compared to that of MUC7 D1-15mer and Hsn-5 . The MUC7 D1 candidacidal activity was assessed against azole-sensitive and azole-resistant C . albicans strains and was found, unlike that of the MUC7 D1-15mer, to be comparable with that of Hsn-5, indicating that in addition to Hsn-5, MUC7 D1 could provide an attractive alternative to the classical antifungal agents . The candidacidal potency of MUC7 D1, like that of MUC7 D1-15mer, and of Hsn-5, appears to be largely dependent on peptide charge, irrespective of alpha-helical structure.

Med Mycol, 2000 Oct, 38(5), 363 - 9
Effect of pH, carbon source and K+ on the Na+-inhibited germ tube formation of Candida albicans; Biswas SK et al.; The effect of pH, carbon source and K+ on the Na+ -inhibited germ tube formation of the pathogenic fungus Candida albicans was examined in the arginine-phosphate modified (APM) medium . All C . albicans cells formed germ tubes in APM medium at pH 5.0-9.0 . Na+ inhibited germ tube formation in a concentration dependent manner ranging from 0.2 to 1.0 M, and was further influenced by the pH of the medium . The inhibitory effect of Na+ was lowest at pH 8.0, and germ tube formation ceased at 1.0 M Na+ for any pH (4.0-9.0) . At pH > or = 6.0, non-germ tube-forming cells did not show yeast growth; whereas at pH < or = 5.0, Na+ inhibited only germ tube formation but did not inhibit yeast growth . The inhibitory effect of Na+ was stronger in glucose medium than in galactose medium as carbon source . K+, at 0-0.8 M, had almost no effect on germ tube formation . However, in the presence of Na+, a very low concentration of K+ (0.5 mM) was able to release the cells from Na+ arrest and produced an increase in the rate as well as the percentage of germ tube formation . Intracellular Na+/K+ ratios increased with the increase in extracellular Na+ concentration, whereas the ratios decreased and remained within nontoxic levels when the extracellular K+ concentration was increased.

Med Mycol, 2000 Oct, 38(5), 355 - 62
Melaleuca alternifolia (tea tree) oil inhibits germ tube formation by Candida albicans; Hammer KA et al.; The effect of tea tree oil (TTO) on the formation of germ tubes by Candida albicans was examined . Two isolates were tested for germ tube formation (GTF) in the presence of TTO concentrations (% v/v) ranging from 0.25% (1/2 minimum inhibitory concentration {MIC}) to 0.004% (1/128 MIC) . GTF at 4 h in the presence of 0.004 and 0.008% (both isolates) and 0.016% (one isolate) TTO did not differ significantly (P > 0.05) from controls . At all other concentrations at 4 h, GTF differed significantly from controls (P < 0.01) . A further eight isolates were tested for GTF in the presence of 0.031% TTO, and at 4h the mean GTF for all 10 isolates ranged 10.0-68.5% . Two isolates were examined for their ability to form germ tubes after 1 h of pre-exposure to several concentrations of TTO, prior to induction of germ tubes in horse serum . Cells pre-exposed to 0.125 and 0.25% TTO formed significantly fewer germ tubes than control cells at 1 h (P < 0.05), but only those cells pre-exposed to 0.25% differed significantly from control cells at later time points (P < 0.01) . GTF by C . albicans is affected by the presence of, or pre-exposure to, sub-inhibitory concentrations of TTO . This may have therapeutic implications.

Int J Antimicrob Agents, 2000 Nov, 16(3), 205 - 9
Activity of voriconazole against Candida albicans and Candida krusei isolated since 1984; Lee JK et al.; With the recent dramatic rise in fluconazole use, there has been an increase in Candida species resistant to that agent . This has led to the clinical development of newer triazoles such as voriconazole that have greater potency and a broader spectrum of activity . We therefore hypothesized that fluconazole-resistant Candida albicans and Candida krusei would be susceptible to voriconazole . Susceptibility testing was performed on 205 isolates of C . albicans collected from 1984 to 1995, and on C . albicans and C . krusei that were identified as fluconazole resistant since 1995 . The anti fungal agents used were amphotericin B, 5-flucytosine, itraconazole, ketoconazole, fluconazole and voriconazole . Three C . albicans and 26 C . krusei isolates had a minimum inhibitory concentration (MIC) >/=20 mg/l and were defined as fluconazole resistant . Of these, 28 isolates were susceptible to </=2.5 mg/l voriconazole, with a mean MIC of 0.78 mg/l . The mean amphotericin B MIC for these same strains was 0.98 mg/l . Only one isolate of C . krusei was relatively resistant to voriconazole with a MIC of 5 mg/l . Of these 29 isolates, there were ten amphotericin-resistant strains of C . krusei (MIC>2 mg/l) but all were susceptible to </=2.5 mg/l voriconazole . The higher potency of voriconazole may be useful in the treatment of fluconazole- and amphotericin-resistant C . albicans and C . krusei.

J Adolesc Health, 2000 Dec, 27(6), 384 - 90
The prevalence of anergy in human immunodeficiency virus-infected adolescents and the association of delayed-type hypersensitivity with subject characteristics; Smith Rogers A et al.; PURPOSE: To examine the prevalence of anergy in HIV-infected adolescents and factors associated with its occurrence . METHODS: Anergy was defined as less than 2mm induration to each of three intradermally applied antigens (Candida albicans, tetanus toxoid, and mumps) between 24 and 96 hours in a population of HIV-infected adolescents aged 12-18 at entry in a national multicenter study of HIV disease progression . CD4(+) T-cell counts and plasma HIV-1 RNA were measured in quality controlled laboratories . Factors associated with the probability of anergy were examined with contingency table comparisons, tree-structured classification, and logistic regression analyses . RESULTS: Overall prevalence of anergy in this clinic-based population of 167 was 11% {7% in males and 12% in females (p = 0.57)} . The sole significant predictor of anergy was decreased CD4(+) T-cell count (p = 0.005) . CONCLUSION: The prevalence of anergy is low in this HIV-infected population compared to older infected cohorts . The occurrence of differential rates of anergy in particular age and sex groupings that may be related to intrinsic immunologic differences requires further study.

Proc Natl Acad Sci U S A, 2000 Dec 19, 97(26), 14433 - 7
Prevalence of small inversions in yeast gene order evolution; Seoighe C et al.; Gene order evolution in two eukaryotes was studied by comparing the Saccharomyces cerevisiae genome sequence to extensive new data from whole-genome shotgun and cosmid sequencing of Candida albicans . Gene order is substantially different between these two yeasts, with only 9% of gene pairs that are adjacent in one species being conserved as adjacent in the other . Inversion of small segments of DNA, less than 10 genes long, has been a major cause of rearrangement, which means that even where a pair of genes has been conserved as adjacent, the transcriptional orientations of the two genes relative to one another are often different . We estimate that about 1,100 single-gene inversions have occurred since the divergence between these species . Other genes that are adjacent in one species are in the same neighborhood in the other, but their precise arrangement has been disrupted, probably by multiple successive multigene inversions . We estimate that gene adjacencies have been broken as frequently by local rearrangements as by chromosomal translocations or long-distance transpositions . A bias toward small inversions has been suggested by other studies on animals and plants and may be general among eukaryotes.

Rev Esp Quimioter, 2000 Sep, 13(3), 314 - 7
{Azole resistance in Candida albicans}; Perea S; The molecular mechanisms of azole resistance in Candida albicans include alterations in the target enzyme (lanosterol 14-demethylase) and overexpression of efflux transporters that decrease the intracellular concentration of the drug . Although the rate of azole resistance in systemic isolates of C . albicans remains very low, resistance to fluconazole appears as an important issue in the management of oropharyngeal candidiasis (OPC) in patients with AIDS . In order to establish the prevalence of resistance to azole antifungal agents in this setting, we investigated the molecular mechanisms of resistance to azoles in highly resistant C . albicans isolates (fluconazole MIC 64 mg/l) from HIV-infected patients with OPC . Antifungal susceptibility testing of serial C . albicans isolates was performed by NCCLS methodology . Strain identity was investigated by DNA-typing techniques . Overexpression of genes encoding lanosterol 14-demethylase (erg11) and efflux transporters (mdr1 and cdr) implicated in the development of resistance was monitored in matched sets of susceptible and resistant isolates . In addition, erg11 genes were PCR-amplified and their nucleotide sequences determined in order to detect point mutations . A combination of different mechanisms of resistance contributed to the development of resistance to fluconazole . The multifactorial character of the azole resistance in C . albicans makes necessary the development of approaches to overcome the problem . Accordingly, new triazoles have been developed; new classes of antifungals are being investigated; and combinations with inhibitors of efflux transporters are being studied.

Infect Immun, 2000 Dec, 68(12), 7159 - 61
Repression of hyphal proteinase expression by the mitogen-activated protein (MAP) kinase phosphatase Cpp1p of Candida albicans is independent of the MAP kinase Cek1p; Schroppel K et al.; Cpp1p is a putative mitogen-activated protein (MAP) kinase phosphatase that suppresses Candida albicans hyphal formation at 25 degrees C through its probable substrate, the Cek1p filamentation MAP kinase . Here we report that expression of the serum-induced genes SAP4-6 and HYR1 increased several fold in hyphal forms of a cpp1/cpp1 null mutant, while the rate and extent of hyphal development up to 5 h were normal . Therefore, we provide evidence that Cpp1p represses hyphal gene expression by acting through a Cek1p-independent mechanism . SAP4-6 and HYR1 transcripts were undetectable in a null mutant of another key regulator of filamentation, Efg1p; thus, Efg1p and Cpp1p oppose each other during the expression of these genes in hyphal forms.

Infect Immun, 2000 Dec, 68(12), 6848 - 56
Released ATP is an extracellular cytotoxic mediator in salivary histatin 5-induced killing of Candida albicans; Koshlukova SE et al.; Salivary histatins (Hsts) are antifungal peptides with promise as therapeutic agents against candidiasis . Hst 5 kills the fungal pathogen Candida albicans via a mechanism that involves release of cellular ATP in the absence of cytolysis . Here we demonstrate that released ATP has a further role in Hst 5 killing . Incubation of the cells with ATP analogues induced cell death, and addition of the ATP scavenger apyrase to remove extracellular ATP released during Hst 5 treatment resulted in a reduction in cell killing . Experiments using anaerobically grown C . albicans with decreased susceptibility to Hst 5 confirmed that depletion of cellular ATP as a result of ATP efflux was not sufficient to cause cell death . In contrast to Hst-susceptible aerobic cultures, anaerobically grown cells were not killed by exogenously applied ATP . These findings established that Hst binding, subsequent entry into the cells, and ATP release precede the signal for cytotoxicity, which is mediated by extracellular ATP . In a higher-eukaryote paradigm, released ATP acts as a cytotoxic mediator by binding to membrane nucleotide P2X receptors . Based on a pharmacological profile and detection of a C . albicans 60-kDa membrane protein immunoreactive with antibody to P2X(7) receptor, we propose that released ATP in response to Hst 5 activates candidal P2X(7)-like receptors to cause cell death.

Infect Immun, 2000 Dec, 68(12), 6833 - 9
Correlation of susceptibility of immature mice to fungal infection (blastomycosis) and effector cell function; Ganer A et al.; Immature mice are highly susceptible to blastomycosis, which is similar to other mycoses and has parallels in humans . The murine susceptibility is noteworthy in that it persists beyond the development of resistance to other, nonfungal pathogens and the maturation of most immune functions . As the susceptibility to blastomycosis appeared to be related to an early event after infection, primary effector cell function was studied . We found that peritoneal inflammatory cells, enriched for neutrophils, from immature (3-week-old) mice killed nonphagocytizable Blastomyces dermatitidis cells less (25%) than did cells from mature (8-week) mice (70%) (P<0.01), a defect intrinsic to the neutrophils . This correlated with an impaired immature cell oxidative burst . Killing of phagocytizable Candida albicans was not significantly different, 73 versus 87% . Thioglycolate-elicited cells were more impaired; killing of B . dermatitidis was insignificant, and killing of C . albicans was more impaired in immature (16% killing) than in mature (45%) cells (P<0.02) . Peripheral blood neutrophils from mature animals killed B . dermatitidis (41%) more than did those from immature animals (10%) (P<0.02); C . albicans was killed efficiently by both . Resting or activated peritoneal macrophages from both types of animals showed no differences in B . dermatitidis killing . These results suggest that the susceptibility of immature mice is related at least in part to the depressed capacity of their neutrophils to kill B . dermatitidis.

Infect Immun, 2000 Dec, 68(12), 6777 - 84
Generation of a recombinant 65-kilodalton mannoprotein, a major antigen target of cell-mediated immune response to Candida albicans; La Valle R et al.; A 65-kDa mannoprotein (CaMp65) has long been studied as a major, immunodominant antigen of the human opportunistic pathogen Candida albicans . An expression library of C . albicans was screened with serum from mice immunized with ScMp65 (ScW10), a Saccharomyces cerevisiae recombinant protein of about 48 kDa . This serum recognized the CaMp65 from a cell wall extract of C . albicans . After cloning and sequencing of the relevant C . albicans cDNA, an open reading frame encoding a protein of 379 amino acids was identified . Its deduced amino acid sequence showed regions of identity with all previously characterized tryptic fragments of CaMp65, as well as with the corresponding regions of ScMp65 . A prepeptide of 32 amino acids with signal peptidase and Kex2 cleavage sites as well as a high number of potential O-glycosylation sites but no N-glycosylation sites or GPI anchor were observed in sequence studies of CaMp65 . A putative adhesin RGD sequence was also present in the C-terminal region of the molecule . This triplet was absent in the ScMp65 . The relevant gene (designated CaMP65) was localized to chromosome R of C . albicans as determined by pulse-field gel electrophoresis . Northern blot analysis demonstrated that gene transcription was heat inducible and associated with germ-tube formation by the fungus . A recombinant, His(6)-tagged protein (rCaMp65) was expressed in Escherichia coli under an inducible promoter . After purification by nickel-chelate affinity chromatography, the recombinant product was detected as a 47-kDa protein band in immunoblots with the anti-ScMp65 serum, as well as with CaMp65-specific monoclonal antibodies . Both ScMp65 and CaMp65 were assayed for antigenic stimulation in cultures of peripheral blood mononuclear cells (PBMC) from 10 unselected human donors . While ScMp65 was substantially nonstimulatory, both rCaMp65 and the native CaMp65 were equally able to induce lymphoproliferation of the PBMC from all the donors . In addition, a number of CD4(+) T-cell clones were generated using a C . albicans mannoprotein fraction as an antigenic stimulant . Several of these clones specifically responded to both the native and the recombinant C . albicans Mp65 but not to ScMp65 . Thus, the recombinant Mp65 of C . albicans retains antigenicity and, as such, could be a valid, standardized reagent for serodiagnostic and immunological studies.

Infect Immun, 2000 Dec, 68(12), 6712 - 9
Tetracycline-regulatable system to tightly control gene expression in the pathogenic fungus Candida albicans; Nakayama H et al.; Conventional tools for elucidating gene function are relatively scarce in Candida albicans, the most prevalent human fungal pathogen . To this end, we developed a convenient system to control gene expression in C . albicans by the tetracycline-regulatable (TR) promoters . When the sea pansy Renilla reniformis luciferase gene (RLUC1) was placed under the control of this system, doxycycline (DOX) inhibited the luciferase activity almost completely . In the absence of DOX, the RLUC1 gene was induced to express luciferase at a level 400- to 1,000-fold higher than that in the presence of DOX . The same results were obtained in hypha-forming cells . The replacement of N-myristoyltransferase or translation elongation factor 3 promoters with TR promoters conferred a DOX-dependent growth defect in culture media . Furthermore, all the mice infected with these mutants, which are still virulent, survived following DOX administration . Consistently, we observed that the number of these mutant cells recovered from the mouse kidneys was significantly reduced following DOX administration . Thus, this system is useful for investigating gene functions, since this system is able to function in both in vitro and in vivo settings.

Antimicrob Agents Chemother, 2000 Dec, 44(12), 3389 - 94
Activities of sordarins in experimental models of candidiasis, aspergillosis, and pneumocystosis; Martinez A et al.; Sordarin derivatives represent a new class of antifungal agents that act as potent inhibitors of fungal protein synthesis and possess a broad spectrum of activity . The in vivo activity of GM193663 and GM237354 was studied in mouse models of disseminated candidiasis and aspergillosis and in a rat model of pneumocystosis . The pharmacokinetic behavior of both sordarin derivatives was studied in mice and rats . In all studies, compounds were administered by the subcutaneous route . After a subcutaneous dose of 50 mg/kg of body weight to mice, the maximum level in serum, area under the concentration-time curve, half-life, and clearance for GM193663 and GM237354 were 51.8 and 23 microg/ml, 79.5 and 46 microg . h/ml, 0.8 and 0.85 h, and 21 and 25 ml/h, respectively . Systemic candidiasis and aspergillosis were established in CD-1 male mice infected with Candida albicans or Aspergillus fumigatus . For systemic candidiasis, compounds were given three times per day for seven consecutive days at 15, 30, 60, or 120 mg/kg/day . GM193663 and GM237354 showed dose-related efficacy against C . albicans, with 50% effective doses, 1 month after infection, of 25.2 and 10.7 mg/kg/dose, respectively . In experimental infections with A . fumigatus, GM237354 was given three times per day at 30, 60, or 120 mg/kg/day for five consecutive days . Animals treated with GM237354 demonstrated irregular responses . The survival of animals treated with GM237354 was 0, 30, and 0% at 30, 60, and 120 mg/kg/day, respectively . The therapeutic efficacy of GM193663 and GM237354 against Pneumocystis carinii was studied in an experimental P . carinii pneumonia (PCP) rat model . After a subcutaneous dose of 10 mg/kg given to rats, the maximum level in serum, area under the concentration-time curve, half-life, and clearance for GM193663 and GM237354 were 6.6 and 7.2 microg/ml, 8.5 and 11.8 microg . h/ml, 0.7 and 0.8 h, and 230 and 133 ml/h, respectively . To induce spontaneous PCP, rats were chronically immunosuppressed with dexamethasone . Infected animals were treated twice daily for 10 days at 0.2, 2, or 10 mg/kg/day . The therapeutic effect was estimated by the reduction in the number of cysts in the lungs of treated versus untreated animals . GM193663 and GM237354 significantly reduced the mean (+/- standard deviation) log number of cysts from 7.6 +/- 0.2 in the untreated group to 4.7 +/- 0.2 and 4.6 +/- 0.1, respectively, when the drugs were administered at a dose of 2 mg/kg/day . The log number of cysts was also reduced in infected animals given lower doses of the compounds (0.2 mg/kg/day) . In summary, GM193663 and GM237354 are new sordarin derivatives that may potentially play a major role in the treatment of candidiasis and PCP . Further testing with Aspergillus in other animal models is warranted.

Antimicrob Agents Chemother, 2000 Dec, 44(12), 3310 - 6
Salivary histatin 5 and human neutrophil defensin 1 kill Candida albicans via shared pathways; Edgerton M et al.; Salivary histatins are a family of basic histidine-rich proteins in which therapeutic potential as drugs against oral candidiasis is apparent, considering their potent in vitro antifungal activity and lack of toxicity to humans . Histatin 5 (Hst 5) kills the fungal pathogen Candida albicans via a mechanism that involves binding to specific sites on the yeast cell membrane and subsequent release of cellular ATP in the absence of cytolysis . We explored the killing pathway activated by Hst 5 and compared it to those activated by other antifungal agents . The candidacidal activity of human neutrophil defensin 1 (HNP-1) shared very similar features to Hst 5 cytotoxic action with respect to active concentrations and magnitude of induction of nonlytic ATP efflux, depletion of intracellular ATP pools, and inhibitor profile . Hst 5 and HNP-1 are basic proteins of about 3 kDa; however, they have unique primary sequences and solution structures that cannot explain how these two molecules act so similarly on C . albicans to induce cell death . Our finding that HNP-1 prevented Hst 5 binding to the candidal Hst 5 binding protein suggests that the basis for the overlapping actions of these two naturally occurring antimicrobial proteins may involve interactions with shared yeast components.

Antimicrob Agents Chemother, 2000 Dec, 44(12), 3257 - 63
Candidacidal activities of human lactoferrin peptides derived from the N terminus; Lupetti A et al.; In light of the need for new antifungal agents, the candidacidal activities of human lactoferrin (hLF) and synthetic peptides representing the first, hLF(1-11), and second, hLF(21-31), cationic domains of its N terminus were compared . The results revealed that hLF(1-11) was more effective in killing fluconazole-resistant Candida albicans than hLF(21-31) and much more effective than lactoferrin, as determined microbiologically and by propidium iodide (PI) staining . By using hLF(1-11) and various derivatives, it was found that the second and third residues of the N terminus of hLF(1-11) were critical for its candidacidal activity . Detailed investigation to elucidate the mechanism of action of hLF(1-11) revealed a dose-dependent release of ATP by Candida upon exposure to hLF(1-11) . Our observations that sodium azide reduced the PI uptake and candidacidal activity of hLF(1-11) and that, upon exposure to hLF(1-11), the fluorescent dye rhodamine 123 first accumulated inside the mitochondria and later was released into the cytoplasm indicate that the peptide triggers the energized mitochondrion . Furthermore, oxidized ATP, which interferes with the interaction of ATP with its extracellular receptors, blocked the candidacidal action of hLF(1-11), as measured microbiologically and by PI staining . Addition of ATP (or analogues) was not a sufficient stimulus to kill C . albicans or to act synergistically with suboptimal concentrations of the peptide . The main conclusions are that the first two arginines at the N terminus of hLF are critical in the candidacidal activity of hLF(1-11) and that extracellular ATP is essential but not sufficient for the peptide to exert its candidacidal activity.

Ginekol Pol, 2000 Sep, 71(9), 971 - 4
{In vitro assessment of the sensitivity of Candida albicans strains isolated from the vagina on basis antimycotics}; Lisiak M et al.; OBJECTIVE: The purpose of the study was to analyses the sensitivity of 73 randomly selected Candida albicans strains isolated from the vagina of pregnant and delivering women against seven basic antimycotics . MATERIALS AND METHODS: The microtest FUNGITEST (Sanofi Diagnostics Pasteur) was applied in assessing the sensitivity of 5-fluorocytosin, amphotericin B, ketoconazol, fluconazol, itraconazol and miconazol and the disk-diffusion method with the use of a Casitone base for nystatin . RESULTS AND CONCLUSIONS: Variations in the sensitivity against drugs have been noted between individual strains of Candida albicans species . The largest number of strains was resistant against ketoconazol--56.16%, and only 10.96% was resistant against nystatin.

Ginekol Pol, 2000 Sep, 71(9), 959 - 63
{Yeast species identification in vulvovaginal candidiasis: susceptibility to nystatin}; Lisiak M et al.; OBJECTIVE: The rates of Candida species and susceptibility to nystatin were evaluated . MATERIALS AND METHODS: In the period from January 1, 1998 to December 31, 1998 mycological tests have been carried out for identification of yeast species in the group of pregnant and delivering women hospitalized in the Obstetrics-Gynecological Departments of the Municipal Hospital in Bydgoszcz . We used two commercial media: Albicans ID and CHROMagar Candida . Noted have been 389 positive inoculation results for Candida from vaginal secretions . RESULTS AND CONCLUSIONS: From the total number of 416 differentiated fungus strains decidedly dominant was the species Candida albicans--constituting 81.97% of all strains . The second frequently occurring fungus species was Candida glabrata--11.06% . Further species were C . krusei--2.16%, C . tropicalis--1.20% and C . guilliermondii--1.20% . In 10 cases (2.41%)--in spite of carrying out the laboratory activities that are necessary in such a situation--the species type of tested strains could not be determined . The simultaneous occurrence of two Candida species has been noted in material originating from 27 women (6.94% of cases) . Defining the drug-resistance of 93 Candida species strains against nystatin by means of the disk-diffusion method--it has been started that this drug is highly effective--81.72% of sensitive strains.

Curr Microbiol, 2000 Dec, 41(6), 384 - 7
Cloning and characterization of a Candida albicans gene homologous to fructose-1,6-bisphosphatase genes; De la Rosa JM et al.; By sequencing of the DNA adjacent to the Candida albicans SEC61 gene, an open reading frame encoding a polypeptide of 331 amino acids was found . The predicted protein showed a strong homology with the fructose-1,6-bisphosphatase {FbPase} from other organisms, and conserved regions included the catalytic motif found in all known FbPases . Although the cloned gene did not complement the growth failure of a Saccharomyces cerevisiae fbp1 mutant in media with gluconeogenic carbon sources, it was transcribed in the transformants in a fashion that indicates a partial repression by glucose . A similar control on the transcription of this gene and on FbPase activity was found in wild-type C . albicans, where the cloned gene (CaFBP1) was shown to be localized in a single chromosomal locus in the genome.

Oral Surg Oral Med Oral Pathol Oral Radiol Endod, 2000 Nov, 90(5), 651 - 5
The effect of ethylenediamine-tetraacetic acid on Candida albicans; Sen BH et al.; OBJECTIVES: The aim of this study was to evaluate the antifungal effect of ethylenediamine-tetraacetic acid (EDTA) on Candida albicans, comparing it with that of various disinfectants and common antifungal agents . STUDY DESIGN: Two clinical oral isolates and 1 standard strain of C albicans were included in this study . Main contents of the test solutions were sodium hypochlorite, EDTA, chlorhexidine, hexetidine, benzalkonium chloride, povidone-iodine, nystatin, and ketoconazole . The agar diffusion method was used to determine the antifungal effects of the solutions . Zones of inhibition were recorded and the results were analyzed statistically by using a 2-way analysis of variance . RESULTS: EDTA demonstrated the highest antifungal activity in comparison with routine antifungal drugs and all other solutions (P <.0001) . Oral cavity isolate was more resistant to the test solutions (P <.0001) . CONCLUSION: The selection of irrigating and disinfecting solution in root canals of patients with a particularly high incidence of oral candidiasis gains extreme importance . EDTA may be strongly recommended during endodontic therapy of these patients.

Fitoterapia, 2000 Dec, 71(6), 690 - 2
Cassine, an antimicrobial alkaloid from Senna racemosa; Sansores-Peraza P et al.; The leaves of Senna racemosa yielded the piperidine alkaloid cassine and an inositol methyl ether . Antimicrobial screening of the compounds revealed antibacterial activity of cassine with minimum inhibitory concentrations of 2.5 mg/ml for Staphylococcus aureus and Bacillus subtilis and 5.0 mg/ml for Candida albicans.

Eur J Med Res, 2000 Oct 30, 5(10), 455 - 9
Influence of fluconazole on phagocytosis, oxidative burst and killing activity of human phagocytes . Using a flow cytometric method with whole blood; Baldauf C et al.; OBJECTIVES: The aim of this study was to investigate the influence of fluconazole, an antimycotic on phagocytosis, oxidative burst and killing activity of phagocytes in human whole blood with Candida albicans as a test strain using a flow cytometric method . METHODS: Candida albicans was stained with Calcein AM, a greenfluorescent dye from Bioprobes (Molecular Probes, Inc., P.O . Box 22010, Eugene, OR 97402-0469, USA) . To measure phagocytosis and burst activity diluted monoclonal antibody (CD-13-R-PE, Molecular Probes, Inc., P.O . Box 22010, Eugene, OR 97402-0469, USA) attaching at the surface of granulocytes and monocytes was added as well as Dihydroethidium solution (Molecular Probes, Inc., P.O . Box 22010, Eugene, OR 97402-0469, USA) which changes into red fluorescent Ethidium by oxidation when killing activity takes place . With Ethidium-Homodimer-1-solution (Molecular Probes, Inc., P.O . Box 22010, Eugene, OR 97402-0469, USA) killing activity can be observed . Three different tests, one incubating the Candida for 1- 4 hrs in advance, another incubating whole blood for 1 h, and the third incubating neither yeast nor blood, and a combined main test were carried out . Measurement of phagocytosis, burst- and killing activtiy was performed with a flow cytometric method (Coulter Company, type: Epics-Profile II) . RESULTS: Three different concentrations of fluconazole (5, 20 and 100 microg/ml) show neither decreasing nor increasing influence on phagocytosis and burst activity, irrespective of whether yeasts or phagocytes had been incubated with fluconazole in advance or not . Also after incubating the drug with phagocytes for 1 h, neither an increase nor a decrease of killing activity was observed . A significant increase was, however, found with increasing incubation time of yeasts and fluconazole . - The minimum concentration of fluconazole, just enough to show a significant increase of the killing rate was 1 microg/ml after 3hrs of incubation . No further significant increase was detected when the concentration exceeded 5 microg/ml . CONCLUSION: 1 h incubation of human phagocytes with fluconazole does not have any significant influence on cellular activities . After advanced incubation of Candida a corresponding increase of the intracellular killing rate in phagocytes occurs, probably due to changes of the cytomorphology of yeasts.

J Nat Prod, 2000 Oct, 63(10), 1447 - 8
CR377, a new pentaketide antifungal agent isolated from an endophytic fungus; Brady SF et al.; Cultures of endophytic fungi collected in the Guanacaste Conservation Area of Costa Rica were screened for antifungal activity . CR377, a new pentaketide antifungal agent, was isolated from the culture broth of a fungus, CR377 (Fusarium sp.), that showed potent activity against Candida albicans in this assay . The structure of CR377 was established using 1- and 2-D NMR and HRFABMS.

J Nat Prod, 2000 Oct, 63(10), 1440 - 3
Lyngbyabellin B, a toxic and antifungal secondary metabolite from the marine cyanobacterium Lyngbya majuscula; Milligan KE et al.; Lyngbyabellin B (1) was isolated from a marine cyanobacterium, Lyngbya majuscula, collected near the Dry Tortugas National Park, Florida . This new cyclic depsipeptide displayed potent toxicity toward brine shrimp and the fungus Candida albicans . The planar structure was deduced using 1D and 2D NMR spectroscopic methods, and the stereochemistry is proposed through a combination of NMR and chiral GC/MS analysis.

Mol Cell Biol, 2000 Dec, 20(23), 8696 - 708
Crk1, a novel Cdc2-related protein kinase, is required for hyphal development and virulence in Candida albicans; Chen J et al.; Both mitogen-activated protein kinases and cyclin-dependent kinases play a role in hyphal development in Candida albicans . Using an oligonucleotide probe-based screen, we have isolated a new member of the Cdc2 kinase subfamily, designated Crk1 (Cdc2-related kinase) . The protein sequence of Crk1 is most similar to those of Saccharomyces cerevisiae Sgv1 and human Pkl1/Cdk9 . In S . cerevisiae, CRK1 suppresses some, but not all, of the defects associated with an sgv1 mutant . Deleting both copies of CRK1 in C . albicans slows growth slightly but leads to a profound defect in hyphal development under all conditions examined . crk1/crk1 mutants are impaired in the induction of hypha-specific genes and are avirulent in mice . Consistent with this, ectopic expression of the Crk1 kinase domain (CRK1N) promotes filamentous or invasive growth in S . cerevisiae and hyphal development in C . albicans . The activity of Crk1 in S . cerevisiae requires Flo8 but is independent of Ste12 and Phd1 . Similarly, Crk1 promotes filamentation through a route independent of Cph1 and Efg1 in C . albicans . RAS1(V13) can also activate filamentation in a cph1/cph1 efg1/efg1 double mutant . Interestingly, CRK1N produces florid hyphae in ras1/ras1 strains, while RAS1(V13) generates feeble hyphae in crk1/crk1 strains.

J Med Microbiol, 2000 Nov, 49(11), 985 - 91
Heterogeneity of oral isolates of Candida albicans in HIV-positive patients: correlation between candidal carriage, karyotype and disease stage; Capoluongo E et al.; Opportunist infections involving Candida albicans often develop in HIV-positive patients and oral lesions tend to become more frequent as the disease progresses . Previous studies have shown contrasting results concerning the variability of the pulsed-field gel electrophoresis (PFGE) subtypes of C . albicans observed in HIV-positive patients . Carriage of C . albicans was determined by an oral rinse technique; 41 strains of C . albicans (78% serotype A and 22% serotype B) were isolated . There was a direct correlation between candidal load (cfu/ml) and the blood HIV load, whereas there was an inverse correlation with the stage of disease and the CD4 cell counts . The PFGE patterns of isolates were variable with regard to the number and positions of bands . The variability of the band sizes in some run positions showed a Gaussian distribution . Generally, the most frequent size variants were associated with the strains with the highest cfu/ml and lowest CD4 counts (< or =200 cells/microl) . These findings suggest a possible strain selection over time during disease progression, especially in HIV-positive subjects with low CD4 counts.

Mol Microbiol, 2000 Nov, 38(3), 435 - 45
The TEA/ATTS transcription factor CaTec1p regulates hyphal development and virulence in Candida albicans; Schweizer A et al.; The temporal and spatial expression of stage-specific genes during morphological development of fungi and higher eukaryotes is controlled by transcription factors . In this study, we report the cloning and functional analysis of the Candida albicans TEC1 (CaTEC1) gene, a new member of the TEA/ATTS family of transcription factors that regulates C . albicans virulence . The promoters of the type 4, 5 and 6 proteinase isogenes (SAP4-6) contain repetitive TEA/ATTS consensus sequence motifs . This finding suggests a possible role for a homologue of Saccharomyces cerevisiae TEC1 during the activation of proteinase gene expression in C . albicans . CaTEC1 is predominantly expressed in the hyphal form of C . albicans . In vitro, serum-induced hyphal formation as well as evasion from MPhi after phagocytosis is suppressed in catec1/catec1 mutant cells . Furthermore, expression of the proteinase isogenes SAP4-6 is no longer inducible in these mutant cells . The deletion of the CaTEC1 gene attenuates virulence of C . albicans in a systemic model of murine candidiasis, although both mutant and revertant cells that were prepared from infected tissues or the vaginal mucosa grew in a hyphal morphology in vivo . CaTEC1 complements the pseudohyphal and invasive growth defect of haploid and diploid S . cerevisiae tec1/tec1 mutant cells and strongly activates the promoter of FLO11, a gene required for pseudohyphal growth . This study provides the first evidence pointing to an essential role for a member of the TEA/ATTS transcription factor family that had so far only been ascribed to function during development as a virulence regulator in microbial pathogenesis.

Microbiology, 2000 Nov, 146 ( Pt 11), 2755 - 64
A phosphatidylinositol 3-kinase of Candida albicans influences adhesion, filamentous growth and virulence; Bruckmann A et al.; To determine if cellular functions of the phosphatidylinositol 3-kinase CaVps34p are related to processes governing Candida albicans pathogenicity, both copies of the gene were sequentially disrupted . Homozygous deletion of C . albicans VPS34 resulted in a mutant strain which exhibited defects not only in intracellular vesicle transport processes but also in morphogenesis . The CaVPS34 null mutant was unable to form hyphae on different solid media whilst showing a significantly delayed yeast-to-hyphae transition in liquid media . In addition, the mutant was rendered hypersensitive to temperature and osmotic stresses and had a strongly decreased ability to adhere to mouse fibroblast cells compared to the wild-type strain SC5314 . Finally, evidence was obtained that CaVPS34 is essential for pathogenicity of C . albicans as the CaVPS34 null mutant was shown to be avirulent in a mouse model of systemic infection . C . albicans pathogenicity was restored to a near wild-type degree upon reintroduction of CaVPS34 into the chromosome of the null mutant, demonstrating that the observed avirulence corresponded to the loss of CaVPS34 . Thus, the results suggest that CaVPS34 may serve as a potential target for antifungal drugs.

Microbiology, 2000 Nov, 146 ( Pt 11), 2743 - 54
A novel multidrug efflux transporter gene of the major facilitator superfamily from Candida albicans (FLU1) conferring resistance to fluconazole; Calabrese D et al.; Azole resistance in Candida albicans can be mediated by several resistance mechanisms . Among these, alterations of the azole target enzyme and the overexpression of multidrug efflux transporter genes are the most frequent . To identify additional putative azole resistance genes in C . albicans, a genomic library from this organism was screened for complementation of fluconazole hypersusceptibility in Saccharomyces cerevisiae YKKB-13 lacking the ABC (ATP-binding cassette) transporter gene PDR5 . Among the C . albicans genes obtained, a new gene was isolated and named FLU1 (fluconazole resistance) . The deduced amino acid sequence of FLU1 showed similarity to CaMDR1 (formerly BEN(r)), a member of the major facilitator superfamily of multidrug efflux transporters . The expression of FLU1 in YKKB-13 mediated not only resistance to fluconazole but also to cycloheximide among the different drugs tested . The disruption of FLU1 in C . albicans had only a slight effect on fluconazole susceptibility; however, it resulted in hypersusceptibility to mycophenolic acid, thus suggesting that this compound could be a substrate for the protein encoded by FLU1 . Disruption of FLU1 in a background of C . albicans mutants with deletions in several multidrug efflux transporter genes, including CDR1, CDR2 and CaMDR1, resulted in enhanced susceptibility to several azole derivatives . FLU1 expression did not vary significantly between several pairs of azole-susceptible and azole-resistant C . albicans clinical isolates . Therefore, FLU1 seems not to be required for the development of azole resistance in clinical isolates.

Nippon Ishinkin Gakkai Zasshi . 2000;41(4):219.
Protective antigens and mechanisms of anti-Candida immunity; Antonio C; Life threatening fungal diseases are now frequent in a substantial fraction of the immunocompromised host population . The toxicity and the relative scarcity of efficacious antifungal drugs highlight the need for developing alternative or integrative immunoprophylactic and therapeutic tools; among them the need to develop prophylactic or therapeutic vaccines against candidiasis, a widespread mucosal or deep-seated infection caused primarily by the fungus Candida albicans, are of clear priority . Vaccination is a highly beneficial medical practice, and probably the most cost-effective measure against disease onset and progression . It is based on the use of microbial antigens capable of conferring protection in a susceptible target host . To date, only a handful of Candida albicans antigens have been produced and very few of them have been thoroughly investigated for immunogenicity and protection in experimental models of candidiasis . Thus, approaches to the molecular, biochemical and functional characterization of novel C . albicans encoded molecules are most welcome to improve the perspective of developing in the near future an effective vaccine against C . albicans . Identification of anti-Candida vaccine candidates must take into account the diversity of Candida diseases, the various underlying mechanisms of protection as well as the major immune dysfunctions observed as predisposing factors for disease . Antigens to be considered possible vaccine candidates include members of the aspartyl proteinase (Sap2) family and the 65kDa mannoprotein (MP65) antigen . An additional molecule of C . albicans which has not yet been identified but deserves great consideration as a vaccine candidate is the yeast-killer toxin receptor (KTR) . Initial experimental evidence strongly suggests that the above antigens are able to elicit protective immunity against mucosal and/or systemic candidiasis . A series of molecular, biochemical and immunological studies aimed at validating and strengthening this initial evidence are in progress, with the ultimate goal of producing recombinant or natural antigens that can be assessed for their ability to elicit a protective immunity in animal models and the mechanisms whereby protection is achieved, with emphasis on determination of immune correlates of protection.

Nippon Ishinkin Gakkai Zasshi, 2000, 41(4), 211 - 7
{Structural and biochemical characteristics of pathogenic fungus: cell walls, lipids and dimorphism, and action modes of antifungal agents}; Kitajma Y; Cell walls (0.1-0.5 microm in thickness) of dermatophytes, at least Trichophyton mentagrophytes and Epidermophyton floccosum, are built of microfibrils (20 nm in diameter) and matrix embedding the fibrils . These fibrils are composed of chitin (70-80%) and a small amount of glucans, and the matrix is composed of beta-1-3, beta1-6 glucan, glucomannan, galactomannan and peptides . Another characteristic structure is the outermost layer (20-50 nm in thickness) of the cell wall, which consists of hydrophobic protein rodlets . Lipids are thought to play important roles in the regulation of dimorphism and virulence in pathogenic fungus . Generally, the ratio of phospholipid/ergosterol is less than 1 in yeast form and 2-20 in mycelial form cells in Candida albicans and Sporothrix schenckii . During the transition from yeast to mycelial forms, phosphatidylinositol and phosphatidylserine are reduced, whereas phosphatidylcholine increases . Phospho-lipase D is activated on this transition . Phospholipase B is now known to be a virulence factor in C . albicans . Polyene antifungal agents bind to ergosterol in membrane to form complexes, which generate pores and destroy the structures and functions of membrane . Azole antifungal agents inhibit the synthesis of ergosterol leading to deficiency in ergosterol content in membrane, and impair the function of membranes in fungal cells . We show the effects of polyenes on the ultrastructure of fungal plasma membrane and impairment of ionomycin-induced calcium influx in T . mentagrophytes, so that we can compare the differences in mode of actions between these two groups of agents.

FEMS Microbiol Lett, 2000 Nov 15, 192(2), 159 - 62
Bactericidal and inhibitory effects of azole antifungal compounds on Mycobacterium smegmatis; Jackson CJ et al.; Azole antifungals are central to therapy and act by inhibiting a cytochrome P450, sterol 14-demethylase and blocking normal sterol synthesis . Our recent identification of a mycobacterial sterol biosynthetic pathway led us to probe the efficacy of a range of these compounds against Mycobacterium smegmatis . Several showed equivalent or greater inhibitory effects to those against Candida albicans, and bactericidal activity was demonstrated for four compounds, clotrimazole, econazole, miconazole and tebuconazole . The major drug used clinically, fluconazole, was ineffective . The results are discussed in the light of the world-wide spread of tuberculosis, including drug-resistant forms and the requirement for new drugs.

Pediatrics . 2000 Nov;106(5):E63.
Should central venous catheters be removed as soon as candidemia is detected in neonates?
Karlowicz MG, Hashimoto LN, Kelly RE Jr, Buescher ES.
BACKGROUND: Controversy exists regarding the most appropriate acute management of central venous catheters (CVCs) in neonates with candidemia, with up to two thirds of neonatologists preferring to attempt antifungal therapy without removing CVCs . OBJECTIVE: To determine whether CVCs should be removed as soon as candidemia is detected in neonates . Methods . A cohort study of candidemia and CVC was conducted in infants in a neonatal intensive care unit (NICU) over a 5-year period (1994-1998) . RESULTS: Fifty infants had early-removal CVC (ER-CVC) within 3 days and 54 infants had late-removal CVC (LR-CVC) >3 days after the first positive blood culture for Candida species . All infants were treated with amphotericin B . There was no significant difference between infants in the ER-CVC and LR-CVC groups in terms of gender, ethnicity, birth weight, gestational age, age at candidemia, severity-of-illness scores, distribution of types of CVC, or in the distribution of Candida species causing candidemia . The ER-CVC group had significantly shorter duration of candidemia (median: 3 days; range: 1-14 days), compared with the LR-CVC group (median: 6 days; range: 1-24 days) . The case fatality rate of Candida albicans candidemia was significantly affected by the timing of CVC removal: 0 of 21 (95% confidence interval {CI}: 0-14) infants died in the ER-CVC group in contrast to 9 of 23 (39%; 95% CI: 19-59) in the LR-CVC group . CONCLUSION: Failure to remove CVC as soon as candidemia was detected in neonates was associated with significantly increased mortality in C albicans candidemia and prolonged duration of candidemia regardless of Candida species.

J Clin Microbiol, 2000 Nov, 38(11), 4272 - 3
Fungal contamination of food in hematology units; Bouakline A et al.; The prevalence of thermotolerant fungi on non-heat-sterilizable food was determined . Aspergillus spp . were noted in 100% of pepper and regular tea samples, 12 to 66% of fruits, 27% of herbal teas, and 20% of freeze-dried soup samples . All soft cheese samples were contaminated by Geotrichum and yeast (Candida norvegensis) but Candida albicans was never identified.

Bioorg Med Chem, 2000 Oct, 8(10), 2487 - 99
Understanding the antifungal activity of terbinafine analogues using quantitative structure-activity relationship (QSAR) models; Gokhale VM et al.; Terbinafine and its analogues, which are a major class of non-azole antifungal agents, are known to act by inhibition of squalene epoxidase enzyme in fungal cells . We have performed a quantitative structure-activity relationship (QSAR) study on a series of 92 molecules using different types of physicochemical descriptors . Inhibitors were divided into five classes depending upon chemical structure . QSAR models were generated for correlation between antifungal activity against Candida albicans using genetic function approximation (GFA) technique . Equations were evaluated using internal as well as external test set predictions . Models generated for all these classes show that steric properties and conformational rigidity of side chains play an important role for the activity . The present QSAR analysis agrees with the results of the previously reported CoMFA study.

Eur J Clin Microbiol Infect Dis, 2000 Sep, 19(9), 663 - 70
Epidemiology of yeast colonization in the intensive care unit; Hedderwick SA et al.; In order to investigate the epidemiology of colonization and possible transmission of yeasts among patients and healthcare workers in adult intensive care units (ICUs), 194 patients were followed for a mean of 9 +/- 11 days and 63 healthcare workers were followed for a mean of 132 +/- 52 days . Among the patients, 142 (73%) were colonized by yeast, with Candida albicans being the species most commonly recovered . Most patients (65%) were already colonized with yeast upon admission to the intensive care unit; only 17% became colonized after admission . Persistent colonization occurred in 51 (55%) of 92 patients who had more than three cultures performed; in 75% of them, colonization persisted with the same strain of Candida albicans or Candida glabrata . Bacterial infection in the month preceding entry into the ICU was the only risk factor significantly associated with yeast colonization . Among the healthcare workers, yeasts were isolated from 42 (67%) . Candida albicans was most frequently recovered from the oropharynx (19% of occasions), and Candida parapsilosis was most frequently found on hands (8% of occasions) . Persistent colonization of the oropharynx occurred in only six healthcare workers, and none had persistence of yeasts on hands . In this non-outbreak setting, 5 (4%) of 123 patient/healthcare worker interactions that were linked epidemiologically yielded the same strain of Candida albicans, providing evidence for possible cross-transmission . No similar link was found between healthcare worker-patient interactions and colonization with Candida glabrata or Candida parapsilosis.

Yeast, 2000 Nov, 16(15), 1413 - 9
Molecular cloning and characterization of the Candida albicans UBI3 gene coding for a ubiquitin-hybrid protein; Roig P et al.; Using a polyubiquitin cDNA as a probe, we have isolated a clone (pPR3, a pEMBLYe23 derivative plasmid) containing the Candida albicans UBI3 gene coding for a fusion protein . This protein is formed by one ubiquitin subunit fused, at its C-terminus, to an unrelated peptide which is similar to the ribosomal protein encoded by the 3' tail of the Saccharomyces cerevisiae UBI3 gene . Southern blot analysis of chromosomal DNA probed with the 3' non-ubiquitin tail of UBI3 indicated that only one homologous gene is present in the C . albicans genome . Heterelogous expression of pPR3 in a S . cerevisiae ubi3 mutant strain complements the mutant phenotype (slow growth) conferred by the ubi3 defect; this provides direct evidence indicating that the clone contains the C . albicans UBI3 gene Northern blot analysis showed that UBI3 gene is expressed in yeast and germ-tube cells of C . albicans, although the UBI3 mRNA levels in starved yeast cells are below the detection limit; UBI3 mRNA drops to undetectable levels on shifting the temperature of growing yeast cells from 28 degrees C to 42 degrees C . When Northern blot analysis was performed using a specific probe for the polyubiquitin (UBI4) gene, no drop in the mRNA levels was detected following thermal upshift or in starved cells . These results indicate that stress conditions (starvation or thermal upshift) negatively regulate UBI3 expression (transcriptional arrest and/or enhanced mRNA decay), and suggest that UBI4 gene provides ubiquitin during the stress response . In addition, we failed to obtain C . albicans UBI3 null mutant cells by sequential disruption of both alleles using the hisG::URA3::hisG ('ura-blaster') cassette, suggesting that null mutants cells may be unable to grow on selective media after transformation.

Chemotherapy, 2000 Nov-Dec, 46(6), 395 - 401
In vitro susceptibility of Candida dubliniensis to current and new antifungal agents; Quindos G et al.; BACKGROUND: Candida dubliniensis is a recently described Candida species closely related to Candida albicans, which has been associated with oral candidiasis in HIV-infected patients . Fluconazole-resistant strains of C . dubliniensis are easily obtained in vitro and this fact could be a complication if this resistance develops during treatment with this drug . METHODS: In the present study, the in vitro antifungal susceptibilities of 36 C . dubliniensis clinical isolates and culture strains to current and new antifungal agents, such as amphotericin B (AMB), amphotericin B lipid complex (ABLC), amphotericin B colloidal dispersion (ABCD), 5-fluorocytosine (5FC), fluconazole (FLC), itraconazole (ITC), ketoconazole (KTC), liposomal amphoteri- cin B (LAMB), liposomal nystatin (LNYT), LY303366 (LY), SCH56592 (SCH), and voriconazole (VRC), were determined according to the National Committee for Clinical Laboratory Standards M27-A broth microdilution method for yeasts . RESULTS: Most isolates of C . dubliniensis were susceptible to both new and current antifungal drugs, with 75.9% isolates susceptible to KTC, 86.2% to FLC and to ITC, and approximately 100% to the other antifungal agents tested . The cross-resistance phenotypes are detailed . Four isolates were resistant (MIC > or =64 microg/ml) to FLC . These 4 isolates were also resistant to KTC, and 3 of them were also resistant to ITC (MIC > or =1 microg/ml for both agents) . However, these isolates were highly susceptible to 5FC and all polyene formulations (AMB, ABLC, ABCD, LAMB, and LNYT), triazole (SCH and VRC) and echinocandin (LY) antifungal agents . CONCLUSION: The new liposomal and lipidic formulations of AMB, LNYT, and the new triazoles and echinocandins may provide new alternatives to FLC for the treatment of infections by C . dubliniensis .

Acta Biochim Pol, 2000, 47(2), 481 - 6
QSARs of some novel isosteric heterocyclics with antifungal activity; Yalcin I et al.; QSAR analysis of a set of previously synthesized 2,5,6-trisubstituted benzoxazole, benzimidazole and 2-substituted oxazolo(4,5-b)pyridine derivatives tested for growth inhibitory activity against Candida albicans, was performed by using the computer-assisted multiple regression procedure . The activity contributions for either heterocyclic ring systems or substituent effects of these compounds were determined from the correlation equation and the predictions for the lead optimization were described . The resulting QSAR revealed that the oxazolo(4,5-b)pyridine ring system with the substitution of a benzyl moiety at position 2 was the most favourable structure among the heterocyclic nuclei . Moreover, the fifth position in the fused ring system is found more significant than the other positions in improving the activity.

Br J Biomed Sci, 2000, 57(3), 241 - 9
Adherence mechanisms of Candida albicans; Cotter G et al.; The yeast Candida albicans is an opportunistic fungal pathogen that is capable of inducing a range of superficial and systemic diseases in the immunocompromised host . Although it displays a variety of virulence factors, one--the ability to adhere to host tissue--is considered essential in the early stages of colonisation and tissue invasion . Adherence is achieved by a combination of specific (ligand-receptor interactions) and non-specific (electrostatic charge, van der Waals forces) mechanisms which allow the yeast to attach to a wide range of tissue types and inanimate surfaces . Conventional methods for treating disease cause by C . albicans rely upon the use of antifungal drugs designed to kill the yeast or arrest its growth . An alternative approach, aimed at disrupting the adherence of the yeast to host tissue in cases of superficial infection, may have potential for controlling disease, particularly in situations where the unattached fungal cell can be removed from the affected site, either by the flushing action of the oropharynx or by the production of mucus in the vagina.

Hosp Med, 2000 Sep, 61(9), 605 - 9
Epidemiology; Ellis M et al.; Invasive fungal infections have emerged as important causes of hospital related morbidity and mortality . They are increasingly seen in patients not previously considered at risk, e.g . patients on an intensive care unit . Candida albicans and Aspergillus spp . are the most common pathogens, posing challenges in epidemiology, control and treatment.

Ned Tijdschr Geneeskd . 2000 Oct 7;144(41):1953.
{Diagnostic image (6) (Pustulous skin lesions)}; van der Meer JW et al.; During chemotherapy a 35-year-old woman developed pustulous skin lesions . Biopsies of these lesions showed Candida albicans . Blood cultures were positive for C . albicans . The patient died.

Life Sci, 2000 Sep 22, 67(18), 2247 - 56
Candida albicans and Saccharomyces cerevisiae cell wall mannans produce fever in rats: role of nitric oxide and cytokines; Ataoglu H et al.; Mannan components of C . albicans (5 mg/kg, i.p.) and S . cerevisiae (2.5 mg/kg, i.p.) cell walls produced pyrogenic responses which were completely inhibited by indomethacin (5 mg/kg, s.c.) pretreatment in rats . A non-selective NOS inhibitor, L-NAME (10 mg/kg, s.c.), also inhibited the pyrogenic effectiveness of C . albicans mannan, whereas it was ineffective on the fever induced by S . cerevisiae mannan . A selective elevation in the serum TNF-alpha levels was observed at the initial phase of the fever due to S . cerevisiae mannan, whereas there was no significant change on the serum levels of TNF-alpha, IL-1beta and IFN-gamma during the latent period or at the initial phase of the fever induced by C . albicans mannan . Injections of N-linked and/or O-linked oligomannosides of the either mannan did not cause any significant change in the body temperature and serum cytokine levels . These data suggest that the mannan components of C . albicans and S . cerevisiae cell walls produce a prostaglandin-dependent fever in rats . The initial signal for fever seems to be different for each mannan . Data also indicate that integrity of the mannans is necessary for the pyrogenic response.

Int J Med Microbiol, 2000 Mar, 290(1), 97 - 104
Assessment of genetic relatedness of vaginal isolates of Candida albicans from different geographical origins; Pinto de Andrade M et al.; PCR fingerprinting with single non-specific primers was used to type vaginal isolates of C . albicans from Portugal, Angola, Madagascar, and two regions of Germany (Berlin and Munich) . In addition to analysing isolates that exhibited the normal biotype of C . albicans, the study included atypical strains that failed to assimilate glucosamine and N-acetylglucosamine, which were isolated from women in Angola and Madagascar . A total of 212 strains of C . albicans were studied, representing 87 different multi-locus genotypes . The genotypes of strains from each geographical population were highly similar but not identical . There was one exception: a strain from Portugal grouped with the typical strains from Angola . The typical and especially the atypical populations from Africa displayed less genotype variation than the populations from Europe . The Portuguese samples exhibited the greatest genotypic heterogeneity . Distance analysis (UPGMA) revealed a statistically weak correlation between genotype and geographical origin of the C . albicans isolates.

Mycopathologia, 1999, 147(3), 117 - 20
New antifungal agents that inhibit the growth of Candida species: dichlorinated 8-quinolinols; Lentz DL et al.; Five dichlorinated 8-quinolinols (2,5- 5,6-, 3,5-, 3,7-, and 4,5-dichloro-8-quinolinol) were tested against Candida albicans and C . tropicalis in Sabouraud dextrose broth with and without bovine serum . The 5,6-, 3,5-, and 3,7-dichloro-8-quinolinols proved to be more effective than the control, 5-fluorocytosine . In cytotoxicity tests employing baby hamster kidney (BHK) cells, all test agents proved to be more cytotoxic than the control . However, the minimum inhibitory concentration (MIC) of 3,5-dichloro-8-quinolinol to both fungi was only one tenth the cytotoxic dose, suggesting that the compound may be useful as a topical or systemic antifungal agent.

FEMS Microbiol Lett, 2000 Nov 1, 192(1), 27 - 31
Complement activation in SCID and nude mice is related to severity of tissue inflammation in the Candida mastitis model; Guhad FA et al.; A small animal model of localised candidiasis is needed for the evaluation of new antifungal compounds . Mammary glands of immunocompetent (BALB/cJ) and immunodeficient (SCID and athymic nude) mice were infected with a wild-type of Candida albicans . Some of the animals were treated with amphotericin B (AmB) while others were treated with saline and acted as controls . The histologic changes of infected mammary gland tissues and a number of other organs were evaluated . Complement (C) activation was analysed by immunoelectrophoretic quantification of molecules with C3c epitopes (C3, C3b, iC3b, and C3c) in serum . In all animals the organisms were confined to the mammary glands . Serum C3c levels were significantly higher (P<0.05) in infected untreated BALB/cJ and SCID mice, which also had severe mammary gland tissue inflammation, compared with control mice treated with AmB (4 mg kg(-1) i.p . once daily for 4 days) . Both treated and control nude mice showed less tissue inflammation compared to BALB/cJ and SCID mice, and revealed insignificant activation of the complement system . It is concluded that innate immune response is important in the control of candidiasis and that the murine mastitis model is useful for immunopathological studies as well as evaluation of potential antifungal compounds.

Mycoses, 2000 Sep, 43(7-8), 273 - 9
Changes of virulence factors accompanying the phenomenon of induced fluconazole resistance in Candida albicans; Fekete-Forgacs K et al.; We investigated a fluconazole-sensitive (MICflu = 5 micrograms ml-1) clinical isolate and a fluconazole-resistant (MICflu > 80 micrograms ml-1) laboratory mutant Candida albicans strain developed from the sensitive one . We studied putative virulence factors including germination, adherence ability to either buccal epithelial cells or acrylate surface, the secreted aspartic proteinase, and the extracellular phospholipase activity of the two strains as well as their growth . The fluconazole-resistant strain proved to be superior to the original strain in all the virulence traits tested . The higher virulence of the fluconazole-resistant strain was also supported by a mouse model . These results suggest that the development of fluconazole resistance can be accompanied by serious morphological and physiological changes: several putative virulence traits, moreover the in vivo virulence can increase simultaneously.

Antimicrob Agents Chemother, 2000 Nov, 44(11), 2985 - 90
Genetic analysis of azole resistance in the Darlington strain of Candida albicans; Kakeya H et al.; High-level azole resistance in the Darlington strain of Candida albicans was investigated by gene replacement in C . albicans and expression in Saccharomyces cerevisiae . We sequenced the ERG11 gene, which encodes the sterol C(14)alpha-demethylase, from our copy of the Darlington strain . Both alleles contained the histidine for tyrosine substitution at position 132 (Y132H) reported in Darlington by others, but we also found a threonine-for-isoleucine substitution (I471T) not previously reported in the C . albicans ERG11 . The encoded I471T change in amino acids conferred azole resistance when overexpressed alone and increased azole resistance when added to the Y132H amino acid sequence in an S . cerevisiae expression system . Replacement of one copy of ERG11 in an azole-susceptible strain of C . albicans with a single copy of the Darlington ERG11 resulted in expression of the integrated copy and a modest increase in azole resistance . The profound azole resistance of the Darlington strain is the result of multiple mutations.

Antimicrob Agents Chemother, 2000 Nov, 44(11), 2932 - 8
Fluconazole plus cyclosporine: a fungicidal combination effective against experimental endocarditis due to Candida albicans; Marchetti O et al.; Recent observations demonstrated that fluconazole plus cyclosporine (Cy) synergistically killed Candida albicans in vitro . This combination was tested in rats with C . albicans experimental endocarditis . The MICs of fluconazole and Cy for the test organism were 0.25 and >10 mg/liter, respectively . Rats were treated for 5 days with either Cy, amphotericin B, fluconazole, or fluconazole-Cy . Although used at high doses, the peak concentrations of fluconazole in the serum of rats (up to 4.5 mg/liter) were compatible with high-dose fluconazole therapy in humans . On the other hand, Cy concentrations in serum (up to 4.5 mg/liter) were greater than recommended therapeutic levels . Untreated rats demonstrated massive pseudohyphal growth in both the vegetations and the kidneys . However, only the kidneys displayed concomitant polymorphonuclear infiltration . The therapeutic results reflected this dissociation . In the vegetations, only the fungicidal fluconazole-Cy combination significantly decreased fungal densities compared to all groups, including amphotericin B (P < 0.0001) . In the kidneys, all regimens except the Cy regimen were effective, but fluconazole-Cy remained superior to amphotericin B and fluconazole alone in sterilizing the organs (P < 0.0001) . While the mechanism responsible for the fluconazole-Cy interaction is hypothetical, this observation opens new perspectives for fungicidal combinations between azoles and other drugs.

Curr Microbiol, 2000 Mar, 40(3), 145 - 8
Inhibition of germ tube formation by Candida albicans by local anesthetics: an effect related to ionic channel blockade; Rodrigues AA et al.; Formation of germ tubes by Candida albicans has been assumed as a putative virulence factor . Local anesthetics (LAs), e.g., lidocaine and bupivacaine, are known to inhibit germ tube formation . The study confirmed this observation for the novel drug ropivacaine, although it was less potent than the former two drugs . Hypothesizing that the effect is due to blockading ionic channels, we exposed Candida albicans to selective calcium blockers, i.e., nifedipine and verapamil, and to a general blocker of ionic channels, i.e., lanthanum . All blockers inhibited germ tube formation . The effect was dose-dependent and pH-independent . Addition of calcium reverted the effect of the blockers as well as the effect of lidocaine and ropivacaine . The study suggests that the inhibitory effect of LAs on germ tube formation by C . albicans is due to blockade of ionic channels, particularly calcium channels . Therefore, LAs can affect morphology and probably also the pathogenesis of C . albicans.

Microbios, 2000, 103(404), 53 - 7
The effect of azidothymidine on germ tube formation in Candida albicans; Monno R et al.; Candida albicans have a marked propensity to cause infections in AIDS patients . A virulent trait of C . albicans is the yeast-hypha transition (Y-->H) which is influenced, in vitro and in vivo, by several factors . Since azidothymidine (AZT) is used in HIV-positive patients, the effect, in vitro, of different concentrations of AZT on C . albicans Y-->H transition was evaluated . C . albicans isolated from HIV-negative and HIV-positive patients were used and strains of C . tropicalis isolated from HIV-positive patients were also tested . AZT concentrations from 0.01 microg/ml to 10 microg/ml did not have any influence on the Y-->H transition, whereas 100 microg/ml AZT significantly inhibited the germ tube formation . AZT did not influence the formation of pseudohyphae in C . tropicalis . It is suggested that C . albicans infection observed in HIV-positive patients was not influenced by AZT therapy, because at currently used dosages, the Y-->H transition was not expected to increase.

J Biol Chem, 2001 Jan 19, 276(3), 1857 - 64 Epub 2000 Oct 16.
Characterization of the mRNA capping apparatus of Candida albicans; Schwer B et al.; The mRNA capping apparatus of the pathogenic fungus Candida albicans consists of three components: a 520- amino acid RNA triphosphatase (CaCet1p), a 449-amino acid RNA guanylyltransferase (Cgt1p), and a 474-amino acid RNA (guanine-N7-)-methyltransferase (Ccm1p) . The fungal guanylyltransferase and methyltransferase are structurally similar to their mammalian counterparts, whereas the fungal triphosphatase is mechanistically and structurally unrelated to the triphosphatase of mammals . Hence, the triphosphatase is an attractive antifungal target . Here we identify a biologically active C-terminal domain of CaCet1p from residues 202 to 520 . We find that CaCet1p function in vivo requires the segment from residues 202 to 256 immediately flanking the catalytic domain from 257 to 520 . Genetic suppression data implicate the essential flanking segment in the binding of CaCet1p to the fungal guanylyltransferase . Deletion analysis of the Candida guanylyltransferase demarcates an N-terminal domain, Cgt1(1-387)p, that suffices for catalytic activity in vitro and for cell growth . An even smaller domain, Cgt1(1-367)p, suffices for binding to the guanylyltransferase docking site on yeast RNA triphosphatase . Deletion analysis of the cap methyltransferase identifies a C-terminal domain, Ccm1(137-474)p, as being sufficient for cap methyltransferase function in vivo and in vitro . Ccm1(137-474)p binds in vitro to synthetic peptides comprising the phosphorylated C-terminal domain of the largest subunit of RNA polymerase II . Binding is enhanced when the C-terminal domain is phosphorylated on both Ser-2 and Ser-5 of the YSPTSPS heptad repeat . We show that the entire three-component Saccharomyces cerevisiae capping apparatus can be replaced by C . albicans enzymes . Isogenic yeast cells expressing "all-Candida" versus "all-mammalian" capping components can be used to screen for cytotoxic agents that specifically target the fungal capping enzymes.

J Enzyme Inhib, 2000, 15(5), 429 - 41
N3-oxoacyl derivatives of L-2,3-diaminopropanoic acid and their peptides; novel inhibitors of glucosamine-6-phosphate synthase; Andruszkiewicz R et al.; Novel inhibitors 1-4 of glucosamine-6-phosphate synthase from Candida albicans have been designed based on acylation of the N3 amino group of L-2,3-diaminopropanoic acid with the corresponding ketoacids . These inhibitors have been shown to alkylate the fungal enzyme in a time-dependent manner . Compound 3 containing trans-beta-benzoyl acrylic acid as an acyl residue was found to be the most potent inhibitor in the series . Dipeptides composed of the active inhibitors and norvaline demonstrated potent antifungal activity against selected strains of Candida spp . and Saccharomyces cerevisiae . Their activity was reversed upon addition of N-acetylglucosamine to the medium.

Shock, 2000 Sep, 14(3), 278 - 83
Phagocytosis of Candida albicans induces apoptosis of human neutrophils; Rotstein D et al.; Neutrophil-mediated inflammation is terminated through the programmed cell death or apoptosis of the neutrophil, a process that can be inhibited by soluble mediators released during an inflammatory response . It has been reported, however, that the phagocytosis of intact bacteria can accelerate apoptosis . We evaluated the effects of the phagocytosis of a common nosocomial pathogen, Candida albicans, on the expression of apoptosis . Phagocytosis of killed Candida induced a dose-dependent increase in the apoptosis of normal neutrophils after 18 h of in vitro culture, from 40.7+/-9.1% to 81.7+/-4.5%, while supernatants from neutrophil:Candida co-cultures actually inhibited apoptosis . Induction of apoptosis was not dependent on phagocytosis, since opsonization of yeast with serum failed to increase apoptosis, while inhibition of phagocytosis with latrunculin B resulted in a slightly increased apoptotic rate . Increased apoptosis induced by Candida was associated with increased activity of the membrane-associated apoptotic enzyme, caspase 8, and with increased expression of the active form of the key executioner caspase, caspase 3 . Increased apoptosis was associated with depletion of intracellular glutathione (GSH), and could be inhibited by the addition of exogenous GSH . These data demonstrate an important physiologic role for host-pathogen interactions in the resolution of inflammation and suggest that the response to an invading pathogen is an important stimulus to the restoration of normal immunologic homeostasis.

Biochem Biophys Res Commun, 2000 Oct 5, 276(3), 1278 - 85
Solution structure of a designed amphipathic antimicrobial synthetic peptide, PGAa; Hong E et al.; A designed peptide, PGAa showed an excellent antifungal activity as well as an efficient bactericidal activity toward gram-positive, especially in the pathogenic yeast Candida albicans 28838 . The solution structures of PGAa have been determined both in 40% TFE/water solution and DPC micelle by CD and NMR spectroscopy . Based on NOEs, vicinal coupling constants, backbone amide exchange rates, and chemical shift indices, PGAa formed a long amphipathic alpha-helical conformation in both TFE and DPC micelle environments, spanning the residues Ile(2)-Ala(19) in TFE and Lys(5)-Ala(19) in DPC micelle, respectively . Solution structures suggested that the hydrophobic residues would interact with the fatty acyl chains of the lipid bilayer, while the positively charged side-chains exposed to aqueous environments . Therefore, we conclude that the alpha-helical structure as well as the highly amphiphatic nature of PGAa peptide may play a critical role in its antimicrobial activity as well as selectivities in different species .

Diagn Microbiol Infect Dis, 2000 Sep, 38(1), 17 - 20
Treatment with oxoglaucine can enhance host resistance to Candida albicans infection of mice with adjuvant arthritis; Ivanovska N et al.; The alkaloid oxoglaucine reduced CD4+ cell clones in adult mice and decreased CD4+, CD8+ and Ig+ levels in newborn mice . It prevented the increase of CD8+ and Ig+ clones induced by Candida albicans (C . albicans) in adult mice . TNF-alpha serum accumulation was inhibited by oxoglaucine in C . albicans infection and adjuvant arthritis . Treatment with oxoglaucine of arthritic mice, followed by inoculation with C . albicans enhanced the host resistance against the pathogen.

J Infect, 2000 Sep, 41(2), 176 - 8
Medical treatment of a pacemaker endocarditis due to Candida albicans and to Candida glabrata; Roger PM et al.; We describe a case of pacemaker infection due to two fungal species: Candida albicans and C . glabrata . Transthoracic echocardiography showed a large vegetation on the intraventricular wires . Because of severe underlying diseases, surgery was believed to be contraindicated . The patient was treated using high dose of fluconazole, resulting in clinical improvement and negative blood cultures . However, 2 months later, the patient underwent a fatal stroke . At autopsy, a large vegetation was found only all along the wires . Postmortem culture of the infected material was positive for both C . albicans and C . glabrata .

J Infect Dis, 2000 Nov, 182(5), 1479 - 85 Epub 2000 Oct 09.
Growth inhibition of Candida by human oral epithelial cells; Steele C et al.; Oropharyngeal candidiasis (OPC) caused by Candida albicans is a significant problem in human immunodeficiency virus (HIV)-infected persons . Recognizing the paucity of information on innate and/or adaptive mucosal host defenses against C . albicans, we recently reported that human and nonhuman primate and mouse vaginal epithelial cells inhibit the growth of C . albicans in vitro . In the present study, oral epithelial cells collected from saliva of healthy volunteers and a purified oral epithelial cell line were found to inhibit blastoconidia and/or hyphal growth of several Candida species . Cell contact was a strict requirement for the epithelial cell anti-Candida activity; neither saliva nor culture supernatants alone inhibited Candida growth, and addition of saliva to the coculture did not modulate the epithelial cell activity . Finally, epithelial cell anti-Candida activity was significantly lower in HIV-infected persons with OPC . Together, these results suggest that oral epithelial cells may play a role in innate resistance against OPC.

Klin Med (Mosk), 2000, 78(8), 47 - 51
{Infectious endocarditis in intravenous drug abusers}; Demin AA et al.; Clinical course of infectious endocarditis (IE) was analysed for 43 intravenous drug abusers . 42 of them had primary IE, one patient--secondary . Acute course and high activity of the disease were registered in 86% of the patients . IE was provoked by Staphylococcus aureus (50%), Staphylococcus epidermidis 920%), Staphylococcus haemolyticus (11%), E . coli (8%), Pseudomonas aeruginosa (2%), Candida albicans (2%), mixed microflora (7%) . Vegetations were detected on the tricuspid, mitral and aortic valves (52, 23 and 19%, respectively), on more than one valve (6%) . Pneumonia, pleuricy, hydrothorax, enlargement of the liver, spleen, nephritis and anemia were found in 76, 44, 9, 100, 75, 70 and 88% of the patients, respectively . Cardiac failure aggravated the disease in half of the patients, lethality was 18% . Thus, IE in intravenous drug abusers is characterized by a primary form, acute active course, prevalent damage to the tricuspid valve, polyorganic involvement, high lethality . IE cure in such patients is feasible only in adequate antibacterial therapy, timely surgical correction and giving up drug abuse.

Sao Paulo Med J, 2000 Sep 7, 118(5), 131 - 8
Epidemiology of bloodstream infections at a cancer center; Velasco E et al.; CONTEXT: Cancer patients are at unusually high risk for developing bloodstream infections (BSI), which are a major cause of in-hospital morbidity and mortality . OBJECTIVE: To describe the epidemiological characteristics and the etiology of BSI in cancer patients . DESIGN: Descriptive study . SETTING: Terciary Oncology Care Center . PARTICIPANTS: During a 24-month period all hospitalized patients with clinically significant BSI were evaluated in relation to several clinical and demographic factors . RESULTS: The study enrolled 435 episodes of BSI (349 patients) . The majority of the episodes occurred among non-neutropenic patients (58.6%) and in those younger than 40 years (58.2%) . There was a higher occurrence of unimicrobial infections (74.9%), nosocomial episodes (68.3%) and of those of undetermined origin (52.8%) . Central venous catheters (CVC) were present in 63.2% of the episodes . Overall, the commonest isolates from blood in patients with hematology diseases and solid tumors were staphylococci (32% and 34.7%, respectively) . There were 70 episodes of fungemia with a predominance of Candida albicans organisms (50.6%) . Fungi were identified in 52.5% of persistent BSI and in 91.4% of patients with CVC . Gram-negative bacilli prompted the CVC removal in 45.5% of the episodes . Oxacillin resistance was detected in 26.3% of Staphylococcus aureus isolates and in 61.8% of coagulase-negative Staphylococcus . Vancomycin-resistant enterococci were not observed . Initial empirical antimicrobial therapy was considered appropriate in 60.5% of the cases . CONCLUSION: The identification of the microbiology profile of BSI and the recognition of possible risk factors in high-risk cancer patients may help in planning and conducting more effective infection control and preventive measures, and may also allow further analytical studies for reducing severe infectious complications in such groups of patients.

Annu Rev Microbiol, 2000, 54, 463 - 98
Recent developments in molecular genetics of Candida albicans; De Backer MD et al.; The frequency of opportunistic infections caused by the fungus Candida albicans is very high and is expected to continue to increase as the number of immunocompromised patients rises . Research initiatives to study the biology of this organism and elucidate its pathogenic determinants have therefore expanded significantly during the last 5-10 years . The past few years have also brought continuous improvement in the techniques to study gene function by gene inactivation and by regulated gene expression and to study gene expression and protein localization by using gene reporter systems . As steadily more genomic sequence information from this human fungal pathogen becomes available, we are entering a new era in antimicrobial research . However, many of the currently available molecular genetics tools are poorly adapted to a genome-wide functional analysis in C . albicans, and further development of these tools is hampered by the asexual and diploid nature of this organism . This review outlines recent advances in the development of molecular tools for functional analysis in C . albicans and summarizes current knowledge about the genomic and genetic variability of this important human fungal pathogen.

Fungal Genet Biol, 2000 Jul, 30(2), 155 - 62
Filamentous growth of Saccharomyces cerevisiae is regulated by manganese; Asleson CM et al.; The Candida albicans INT1 gene is a virulence factor that contributes to both adhesion and filamentous growth of the fungus . Expression of INT1 in the budding yeast Saccharomyces cerevisiae directs both adhesion and filamentous growth . Because Int1p contains two predicted divalent cation-binding motifs, we asked whether divalent cations are important for the role of Int1p in filament formation . In this study, we found that INT1-induced filamentous growth (I-IFG) is sensitive to the divalent cation chelator EDTA and that this EDTA sensitivity can be ameliorated by the addition of Mn(2+), but not Mg(2+) or Ca(2+) ions . The addition of MnCl(2) restored both the proportion of cells forming filaments and the length of filaments formed . Expression of INT1 in S . cerevisiae mutants that reduce the intracellular concentration of Mn(2+) did not affect I-IFG . Interestingly, the Mn(2+) dependence of I-IFG is not dependent upon the presence of the putative divalent cation-binding domains found in INT1 . Rather, we found that polarized growth induced by mutations in CDC12 and CLA4 or by expression of excess SWE1 was also sensitive to EDTA treatment and was restored by the addition of MnCl(2) but not by the addition of CaCl(2) . Thus, our results suggest that in S . cerevisiae polarized growth is dependent upon the presence of Mn(2+) ions .

Fungal Genet Biol, 2000 Jul, 30(2), 127 - 33
Biosynthesis of glycoproteins in Candida albicans: activity of mannosyl and glucosyl transferases; Arroyo-Flores BL et al.; The enzymes dolichol phosphate glucose synthase and dolichol phosphate mannose synthase (DPMS), which catalyze essential steps in glycoprotein biosynthesis, were solubilized and partially characterized in Candida albicans . Sequential incubation of a mixed membrane fraction with increasing concentrations of Nonidet P-40 released a soluble fraction that transferred glucose from UDP-Glc to dolichol phosphate glucose and minor amounts of glucoproteins in the absence of exogenous dolichol phosphate . Studies with the soluble fraction revealed that some properties were different from those previously determined for the membrane-bound enzyme . Accordingly, the soluble enzyme exhibited a twofold higher affinity for UDP-Glc and a sixfold higher affinity over the competitive inhibitor UMP, and the transfer reaction was fourfold more sensitive to inhibition by amphomycin . On the other hand, a previously described protocol for the solubilization of mannosyl transferases that rendered a fraction exhibiting both DPMS and protein mannosyl transferase (PMT) activities operating in a functionally coupled reaction was modified by increasing the concentration of Nonidet P-40 . This resulted in a solubilized preparation enriched with DPMS and nearly free of PMT activity which remained membrane bound . DPMS solubilized in this manner exhibited an absolute dependence on exogenous Dol-P . Uncoupling of these enzyme activities was a fundamental prerequisite for future individual analysis of these transferases .

Pediatrics, 2000 Oct, 106(4), 712 - 8
When to suspect fungal infection in neonates: A clinical comparison of Candida albicans and Candida parapsilosis fungemia with coagulase-negative staphylococcal bacteremia; Benjamin DK Jr et al.; OBJECTIVES: To determine the epidemiology of candidemia in our neonatal intensive care unit; to compare risk factors, clinical presentation, and outcomes for neonates infected with Candida albicans, Candida parapsilosis, and coagulase-negative staphylococcus (CoNS); and to suggest a rational approach to empiric antifungal therapy of neonates at risk for nosocomial infection . DESIGN: Retrospective chart review of all neonatal intensive care unit patients with systemic candidiasis or CoNS infection between January 1, 1995 and July 31, 1998 at Duke University Medical Center . RESULTS: Fifty-one patients were reviewed . Nine of 19 patients infected with C parapsilosis and 5 of 15 patients infected with C albicans died of fungemia . Seventeen neonates had >2 positive cultures for CoNS obtained within 96 hours and 1 died . There was no statistically significant difference in birth weight, gestational age, or age at diagnosis between patient groups; however, candidemic patients had a sevenfold higher mortality rate . Before diagnosis, candidemic patients had greater exposure to systemic steroids, antibiotics, and catecholamine infusions . Of the 51 patients, 32 received third-generation cephalosporins in the 2 weeks before diagnosis and 19 did not . Twenty-nine of the 32 who were treated with third-generation cephalosporins subsequently developed candidemia, while candidemia occurred in only 5 of 19 patients who were not treated with cephalosporins . At the time of diagnosis, candidemic patients were more likely to have required mechanical ventilation and were less likely to be tolerating enteral feeding . Multivariate clustered logistic regression analysis revealed that candidemic patients had more exposure to third-generation cephalosporins . Once the clinician was notified of a positive blood culture for Candida, patients infected with C parapsilosis retained their central catheters longer than patients infected with C albicans . CONCLUSIONS: In this retrospective review, we were able to identify aspects of the clinical presentation and medication history that may be helpful in differentiating between candidemia and CoNS bacteremia . Those key features may be used by clinicians to initiate empiric amphotericin B therapy in premature neonates at risk for nosocomial infections . Prolonged use of third-generation cephalosporins was strongly associated with candidemia . There was no statistically significant difference in the morbidity and mortality between patients infected with C parapsilosis and those infected with C albicans . Observed delays in removal of the central venous catheter may have contributed to finding a mortality rate from C parapsilosis that was higher than was previously reported.

J Clin Microbiol, 2000 Oct, 38(10), 3696 - 704
Differentiation between Candida dubliniensis and Candida albicans by fatty acid methyl ester analysis using gas-liquid chromatography; Peltroche-Llacsahuanga H et al.; Candida dubliniensis is often found in mixed culture with C . albicans, but its recognition is hampered as the color of its colonies in primary culture on CHROMagar Candida varies . Furthermore, definite identification of C . dubliniensis is difficult to achieve, time-consuming, and expensive . Therefore, a method to discriminate between these two closely related yeast species by fatty acid methyl ester (FAME) analysis using gas-liquid chromatography (Sherlock Microbial Identification System {MIS}; MIDI, Inc., Newark, Del.) was developed . Although the chromatograms of these two species revealed no obvious differences when applying FAME analysis, a new library (CADLIB) was successfully created using Sherlock Library Generation Software (MIDI) . The amount and frequency of FAME was analyzed using library training files (n = 10 for each species), preferentially those comprising reference strains . For testing the performance of the CADLIB, clinical isolates genetically assigned to the respective species (C . albicans, n = 32; C . dubliniensis, n = 28) were chromatographically analyzed . For each isolate tested, MIS computed a similarity index (SI) indicating a hierarchy of possible strain fits . When using the newly created library CADLIB, the SIs for C . albicans and C . dubliniensis ranged from 0.11 to 0.96 and 0.53 to 0 . 93 (for all but one), respectively . Only three isolates of C . albicans (9.4%) were misidentified as C . dubliniensis, whereas all isolates of C . dubliniensis were correctly identified . Resulting differentiation accuracy was 90.6% for C . albicans and 100% for C . dubliniensis . Cluster analysis and principal component analysis of the resulting FAME profiles showed two clearly distinguishable clusters matching up with two assigned species for the strains tested . Thus, the created library proved to be well suited to discriminate between these two species.

J Clin Microbiol, 2000 Oct, 38(10), 3595 - 607
Elevated phenotypic switching and drug resistance of Candida albicans from human immunodeficiency virus-positive individuals prior to first thrush episode; Vargas K et al.; Strains of Candida albicans obtained from human immunodeficiency virus (HIV)-positive individuals prior to their first episode of oral thrush were already in a high-frequency mode of switching and were far more resistant to a number of antifungal drugs than commensal isolates from healthy individuals . Switching in these isolates also had profound effects both on susceptibility to antifungal drugs and on the levels of secreted proteinase activity . These results suggest that commensal strains colonizing HIV-positive individuals either undergo phenotypic alterations or are replaced prior to the first episode of oral thrush . They also support the suggestion that high-frequency phenotypic switching functions as a higher-order virulence trait, spontaneously generating in colonizing populations variants with alterations in a variety of specific virulence traits.

Neurosurgery, 2000 Oct, 47(4), 973 - 6; discussion 976-7
Candida albicans cerebral granulomas associated with a nonfunctional cerebrospinal fluid shunt: case report; Soto-Hernandez JL et al.; OBJECTIVE AND IMPORTANCE: We report an unusual case of basal ganglia granulomas caused by Candida albicans that surrounded the proximal segment of a nonfunctional cerebrospinal fluid shunt in a previously healthy patient . CLINICAL PRESENTATION: A 22-year-old woman had undergone ventriculoatrial cerebrospinal fluid shunt placement for posttraumatic hydrocephalus 3 years previously . One year later, a shunt revision was followed by wound dehiscence with local infection at the neck level . She received oral administration of antibiotics for 3 months until the wound closed . Twelve weeks before admission, the patient experienced pulmonary emboli . She received anticoagulants, and the distal segment of the shunt was removed . Five weeks after shunt removal, she presented with headache and left-sided hemiplegia caused by right basal ganglia inflammatory masses . INTERVENTION: A stereotactic brain biopsy was performed, and the shunt remnants were removed . Microscopically, the lesions were acutely and chronically inflamed . C . albicans grew in tissue and in shunt hardware cultures . The patient was treated with 1.1 g of intravenously administered amphotericin B and orally administered ketoconazole; she recovered completely . CONCLUSION: C . albicans brain granulomas occur rarely in immunocompetent patients . Despite the large size of the lesions and severe brain edema, the absence of an underlying disease contributed to complete resolution after shunt removal and antifungal therapy.

Clin Exp Dermatol, 2000 Jul, 25(5), 404 - 5
Persistent annular erythema of infancy associated with intestinal Candida colonization; Stachowitz S et al.; We report a case of persistent annular erythema of infancy in a 4-month-old boy . Physical and laboratory parameters showed no sign of internal disease or specific infection except a massive Candida albicans colonization (> 103 organisms/mm3) of the lower gastrointestinal tract . Oral treatment with amphotericin B for 2 weeks resulted in a complete remission of the skin lesions indicating Candida colonization as a trigger.

Braz J Infect Dis, 2000 Apr, 4(2), 47 - 54
Changing strategies for treatment of systemic mycoses; Graybill JR; There have been a number of changes in strategies in antifungal therapy in the past few years . AIDS related mycoses have decreased, and the increase of fluconazole resistant Candida albicans may be slowing because fewer severely immune depressed patients require constant fluconazole suppression . Candida species continue to be relatively common blood culture isolates . About half of these are C . albicans and half non-albicans species . In recent years, we have moved from the use of amphotericin B to fluconazole for initial treatment of candidemia . We have seen fluconazole resistant isolates emerge, primarily C . glabrata and a few C . krusei, but also C . albicans . It is unclear whether the increasing use of fluconazole in intensive care units will worsen this problem . There appears to be no advantage for the lipid formulations of amphotericin B, though they are useful to reduce or prevent renal toxicity . In the United States and Europe, prevention and treatment of aspergillosis have become increasingly important . There are increasing data suggesting that lipid formulations are more effective for both treatment and prevention of invasive disease in the most vulnerable patients with this infection . Renal toxicity is reduced but not avoided by use of the lipid formulations of amphotericin B . For those patients with less acutely progressing disease, the triazoles may be effective options . It is unclear at present whether itraconazole, voriconazole, or posaconazole will be the most favored drug . One promising new class, now in clinical trials, is the echinocandin group . Other agents, such as the sordarins, the chitin synthase inhibitors, and topoisomerase inhibitors, have promise but are much earlier in development . Unfortunately, we still have >50% treatment failure with acute invasive aspergillosis, and 20%-30% failures with candidemia . Now that we have multiple classes of antifungal drugs available, and others in preclinical trials, it would be advantageous to begin more active exploration of combination therapy with antifungals and with combined immune modulators and antifungals.

Rev Argent Microbiol, 2000 Jul-Sep, 32(3), 123 - 8
Criteria for Candida albicans numerical analysis based on electrophoretic protein patterns; Boriollo MF et al.; Even in normalized gels, some problems with the choice and position of protein bands always have hindered the processing of electrophoretic data . We have developed a way to establish which criterion best fits the necessities in order to maximize the similarity indexes for numerical analysis . Some repetitions of a Candida albicans strain were carried out in eleven different gels . After staining, the bands were scored in numbers within ranges of +/- n values with increases of one quarter steps (+/- 0.25 kDa, +/- 0.5 kDa, +/- 0.75 kDa, +/- 1.0 kDa, +/- 1.25 kDa) that will limit the possibility of variation for the same bands that will appear on the repetitions . Using this criterion, we have determined that values scored within +/- 1.25 kDa could optimize the minimum limiting value of similarity in different dendrograms built.

Biochim Biophys Acta, 2000 Jul 14, 1480(1-2), 117 - 31
Enzymic characteristics of secreted aspartic proteases of Candida albicans; Koelsch G et al.; Candida yeasts are rarely infectious, but frequently cause life-threatening systemic infections in patients immunocompromised by AIDS or by immunosuppressive therapeutics . The secreted aspartic proteases (Saps) are known virulence factors of pernicious Candida species . The most virulent, Candida albicans, possesses at least nine SAP genes, some of which are specifically expressed from cells with morphologies associated with virulence . Only one of these proteases, Sap2, has been previously purified from yeast in sufficient quantities for enzymic studies . The other enzymes are present in low amounts in yeast culture and are difficult to purify . As a consequence, enzyme properties, including the substrate specificities, of all Saps are poorly studied . Therefore, four Saps that are known to be expressed in C . albicans, Sap1, Sap2, Sap3 and Sap6, were produced in Escherichia coli as recombinant zymogens and purified in large quantities . These proenzymes were autoactivated and purified as active proteases . The enzymic properties including the substrate specificities at the P(1) and P(1)' sites were determined using a competitive hydrolysis method employing synthetic substrate mixtures . All four Saps cleave peptide bonds between larger hydrophobic amino acids, but these somewhat broad specificities differ in detail among the four enzymes at both sites . At the P(1) site, Sap1, Sap2 and Sap6 prefer Phe while Sap3 prefers Leu . Positively charged amino acids are also accommodated, especially by Sap2 and Sap3 . The specificities at P(1)' are broader than at P(1) for all four enzymes . Sap6 prefers Ala, whereas other Saps prefer Tyr . Acidic side chains are also accommodated at this site . Analysis of substrates with a hydrophobic amino acid in P(1)' reveals that all the Saps possess a unique preference for Ala at this site . The observed differences of residue preferences among Saps may be utilized for the design of specific substrates and inhibitors.

Biochim Biophys Acta, 2000 Jul 24, 1492(2-3), 369 - 76
Functional cloning and mutational analysis of the human cDNA for phosphoacetylglucosamine mutase: identification of the amino acid residues essential for the catalysis; Mio T et al.; In Saccharomyces cerevisiae, phosphoacetylglucosamine mutase is encoded by an essential gene called AGM1 . The human AGM1 cDNA (HsAGM1) and the Candida albicans AGM1 gene (CaAGM1) were functionally cloned and characterized by using an S . cerevisiae strain in which the endogenous phosphoacetylglucosamine mutase was depleted . When expressed in Escherichia coli as fusion proteins with glutathione S-transferase, both HsAgm1 and CaAgm1 proteins displayed phosphoacetylglucosamine mutase activities, demonstrating that they indeed specify phosphoacetylglucosamine mutase . Sequence comparison of HsAgm1p with several hexose-phosphate mutases yielded three domains that are highly conserved among phosphoacetylglucosamine mutases and phosphoglucomutases of divergent organisms . Mutations of the conserved amino acids found in these domains, which were designated region I, II, and III, respectively, demonstrated that alanine substitutions for Ser(64) and His(65) in region I, and for Asp(276), Asp(278), and Arg(281) in region II of HsAgm1p severely diminished the enzyme activity and the ability to rescue the S . cerevisiae agm1Delta null mutant . Conservative mutations of His(65) and Asp(276) restored detectable activities, whereas those of Ser(64), Asp(278), and Arg(281) did not . These results indicate that Ser(64), Asp(278), and Arg(281) of HsAgm1p are residues essential for the catalysis . Because Ser(64) corresponds to the phosphorylating serine in the E . coli phosphoglucosamine mutase, it is likely that the activation of HsAgm1p also requires phosphorylation on Ser(64) . Furthermore, alanine substitution for Arg(496) in region III significantly increased the K(m) value for N-acetylglucosamine-6-phosphate, demonstrating that Arg(496) serves as a binding site for N-acetylglucosamine-6-phosphate.

FEMS Microbiol Lett, 2000 Oct 1, 191(1), 151 - 5
Phagocytosis, oxidative burst, and killing of Candida dubliniensis and Candida albicans by human neutrophils; Peltroche-Llacsahuanga H et al.; Candida dubliniensis is a phylogenetically closely related species to Candida albicans . So far virtually nothing is known about the virulence factors of C . dubliniensis . Cell surface hydrophobicity (CSH) plays a critical role in adhesion of microorganisms to phagocytic cells; hydrophobic cells of C . albicans have been reported to be less sensitive to phagocytic killing than hydrophilic cells . C . dubliniensis displays CSH at 37 degrees C in contrast to C . albicans . To elucidate this issue, we determined levels of phagocytosis, oxidative burst and killing by human neutrophils of C . dubliniensis (n=10) compared to C . albicans (n=10) both cultured at 37 degrees C . Obtained test results revealed no statistically significant differences between these two yeast species for the level of phagocytosis (77.3 vs . 76.2% after 60 min), evoked oxidative burst (64.5 vs . 67.3% after 30 min) and killing (72.7 vs . 73.1% after 240 min) . Therefore, human neutrophils can be considered to be equally efficient against these two yeast species.

J Bacteriol, 2000 Oct, 182(20), 5730 - 6
A single-transformation gene function test in diploid Candida albicans; Enloe B et al.; The fungal pathogen Candida albicans is naturally diploid, and current gene disruption strategies require two successive transformations . We describe here a genetic construct (UAU1) for which two copies may be selected . Insertion of UAU1 into one genomic site, after a single transformation, allows selection for segregants with two copies of the insertion . Major classes of segregants are those carrying homozygous insertion mutations and allelic triplications, which have two insertion alleles and a wild-type allele . Thus nonessential and essential genes may be distinguished rapidly through PCR tests for homozygosis and triplication . We find that homozygous mutations may be isolated at three nonessential loci (ADE2, RIM20, and YGR189), while only allelic triplications were found at two essential loci (SNF1 and CDC28) . We have unexpectedly isolated homozygous mutants with mutations at CDC25; they are viable but defective in filamentation on serum-containing medium . The UAU1 cassette is thus useful to assess rapidly the essentiality of C . albicans genes.

Eur J Med Res, 2000 Sep 18, 5(9), 369 - 74
In vitro effects of interleukin-10, prednisolone, and GM-CSF on the non-specific immune function of human polymorphonuclear leucocytes and monocytes; Ziege SU et al.; A wide range of immune-modulating effects make IL-10 a potential therapeutic option in the treatment of numerous diseases pathophysiological based on a dysregulation of cytokine production . The background of this study was to investigate, whether the beneficial effects of a therapeutic immunosuppression with IL-10 may be countered by an increased risk for infections due to impaired effector cell functions of unspecific immunity . We demonstrated the in vitro effects of IL-10 on phagocytosis (P), intracellular killing (K), and chemotactic activity (C) by human neutrophils (PMN) and monocytes (MON) using Candida albicans as test strain and compared the results to the effects of prednisolone and GM-CSF . IL-10 reduced significantly the intracellular killing rate of PMN compared to untreated phagocytes (60 +/- 16% versus 68 +/- 13%, mean +/- SD, p = 0.0002) . High dose IL-10 (100 ng/ml) had a stimulating effect on the percentage of phagocytizing MON (70.2 +/- 12.7% vs . 66.9 +/- 14.2%, p = 0.0436), without impairing intracellular killing . Prednisolone reduced significantly the Candida uptake by MON (57 +/- 18.1% vs . 66 . 9 +/- 14.2%, p = 0.0019) . In contrast to prednisolone, neither MON nor PMN chemotaxis was suppressed by IL-10 . In conclusion, IL-10 had only marginal immunosuppressive effects on the unspecific immunity compared to prednisolone.

Biochim Biophys Acta, 2000 Jul 26, 1475(3), 265 - 72
The VIG9 gene products from the human pathogenic fungi Candida albicans and Candida glabrata encode GDP-mannose pyrophosphorylase; Ohta A et al.; We have identified two genomic DNA fragments from the human pathogenic fungi, Candida albicans (CaVIG9) and Candida glabrata (CgVIG9) that encode GDP-mannose pyrophosphorylase, a key enzyme for protein glycosylation . The VIG9 homologues of CaVIG9 and CgVIG9 complement an identified protein glycosylation-defective mutation, vig9, of Saccharomyces cerevisiae . The nucleotide sequences of the ORFs, which are 83 and 90% identical to that of the ScVIG9 protein, respectively, showed a predicted gene product homologous to S . cerevisiae GDP-mannose pyrophosphorylase . We examined the enzyme activity of a glutathione S-transferase fusion of each VIG9 gene to synthesize GDP mannose in the cell extracts of a heterologous Escherichia coli expression system . We also developed a method for detecting the enzyme activity using a non-radioactive substrate that would be applicable to high throughput screening.

J Microbiol Methods, 2000 Sep, 42(1), 17 - 27
FT-IR microspectroscopy for microbiological studies; Orsini F et al.; In this article we present an infrared microspectroscopic investigation on Candida albicans microcolonies, taken as a model system for studies on other microorganisms . Excellent Fourier transform infrared (FT-IR) absorption spectra from 4000 to 850 cm(-1) have been collected in only 20 s from sampling areas of 100x100 microm(2) in microcolonies, which had been transferred from the agar plate onto zinc selenide (ZnSe) windows . When different regions within a single microcolony were investigated, absorption spectra with important differences in the carbohydrate absorption (from 1200 to 850 cm(-1)) were detected for the cells in the center and in the periphery of the colony . Results obtained on microcolonies grown on solid agar with increasing dextrose concentrations indicated that the observed spectral heterogeneity was related to differences in dextrose uptake, which was lower for the old cells in the center of the colony than for the metabolically active cells at the periphery . Although it is otherwise difficult to quantitatively evaluate the dextrose uptake in a microcolony, FT-IR absorption microspectroscopy offers a new and rapid method for the analysis of this process . The possibility of studying highly absorbing colonies by attenuated total reflection (ATR) by means of an ATR microscope germanium objective is also presented here for the first time . An evaluation of the contact area sampled by this technique is reported with a discussion of the spatial resolution, the quality and the potential of the ATR measurements.

Arch Intern Med, 2000 Sep 25, 160(17), 2659 - 64
Candida krusei fungemia . An escalating serious infection in immunocompromised patients; Abbas J et al.; BACKGROUND: Candida krusei is inherently resistant to fluconazole and is emerging as a frequent cause of fungemia in patients with hematologic malignant neoplasms . OBJECTIVE: To determine the risk and prognostic factors associated with C krusei fungemia in comparison with Candida albicans fungemia in patients with cancer . METHODS: Retrospective study of 57 cases of C krusei fungemia occurring at the M . D . Anderson Cancer Center, Houston, Tex, from 1989 to 1996 . The C krusei cases were compared with 57 cases of C albicans fungemia with respect to demographics, underlying cancer, Acute Physiology and Chronic Health Evaluation II score, immunosuppression status, chemotherapy, and the use of central venous catheters, as well as fluconazole prophylaxis . RESULTS: At our institution, C krusei accounted for 5% of fungemias during 1989 through 1992 and for 10% during 1993 through 1996 . Patients with C krusei fungemia more often had leukemia than patients with C albicans (77% vs 11%; P =.02), whereas catheter-related infections were more common among patients with C albicans fungemia (42% vs 0%; P<.001) . Patients with C krusei fungemia had a lower response rate (51% vs 69%; P =.05), largely because they more frequently were neutropenic and had disseminated infection . Mortality related to fungemia was 49% in the cases with C krusei vs 28% in C albicans . Multiple logistic regression analysis showed that persistent neutropenia (P =.02) and septic shock (P =.002) were predictors of poor prognosis . CONCLUSION: In neutropenic patients, C krusei fungemia is associated with high mortality . It should be suspected in patients with leukemia who are receiving fluconazole prophylaxis and should be treated aggressively with an amphotericin B regimen.

Folia Microbiol (Praha), 1999, 44(5), 523 - 6
Inhibition of germ tube formation, filamentation and ergosterol biosynthesis in Candida albicans treated with 6-amino-2-n-pentylthiobenzothiazole; Fabry S et al.; Three representative Candida albicans strains were selected out of 26 clinical isolates and strains from culture collections on the basis of their high level of conversion to germ tubes and mycelial form at mycelium-promoting culture conditions, and on their different sensitivity to 6-amino-2-n-pentylthiobenzothiazole (APB) . When these strains were treated with APB at mycelium-promoting culture conditions, a concentration-dependent decrease in the proportion of germ tubes and hyphae was observed, while the proportion of the yeast from increased . When non-saponifiable lipids were extracted from these cultures and analyzed, a concentration-dependent decrease in ergosterol and an increase in 4-methylated sterols was observed . However, the sensitivity of sterol biosynthesis did not directly relate to the sensitivity of the morphological conversion, and was exhibited at higher concentrations of APB . On the basis of these results it is suggested that the inhibition of germ tube formation and filamentation is not a consequence of inhibition of ergosterol biosynthesis in APB-treated C . albicans.

J Agric Food Chem, 2000 Sep, 48(9), 3785 - 8
Bioactive compounds and 1,3-Di{(cis)-9-octadecenoyl}-2-{(cis,cis)-9, 12-octadecadienoyl}glycerol from Apium graveolens L . seeds; Momin RA et al.; Bioassay-directed isolation and purification of the hexane extract of Apium graveolens L . seeds led to the characterization of three compounds: beta-selinene (1), 3-n-butyl-4,5-dihydrophthalide (2) and 5-allyl-2-methoxyphenol (3) . The structures of these compounds were established by using (1)H and (13)C NMR spectral methods . Compounds, 1-3 demonstrated 100% mortality on fourth-instar Aedes aegyptii larvae at 50, 25, and 200 microg mL(-)(1), respectively, in 24 h . Also, 2 inhibited the growth of Candida albicans and Candida kruseii at 100 microg mL(-)(1) . It inhibited both topoisomerase-I and -II enzyme activities at 100 microg mL(-)(1) . Compound 2 displayed 100% mortality at 12.5 and 50 microg mL(-)(1), respectively, when tested on nematodes, Panagrellus redivivus and Caenorhabditis elegans . The triglyceride, 1,3-di{(cis)-9-octadecenoyl}-2-{(cis,cis)-9, 12-octadecadienoyl}glycerol (4) and 3 were isolated for the first time from A . graveolens seeds, although 4 was not biologically active.

Infect Immun, 2000 Oct, 68(10), 5953 - 9
Candida albicans RIM101 pH response pathway is required for host-pathogen interactions; Davis D et al.; The ability of Candida albicans to respond to diverse environments is critical for its success as a pathogen . The RIM101 pathway controls gene expression and the yeast-to-hyphal transition in C . albicans in response to changes in environmental pH in vitro . In this study, we found that the RIM101 pathway is necessary in vivo for pathogenesis . First, we show that rim101(-)/rim101(-) and rim8(-)/rim8(-) mutants have a significant reduction in virulence using the mouse model of hematogenously disseminated systemic candidiasis . Second, these mutants show a marked reduction in kidney pathology . Third, the rim101(-)/rim101(-) and rim8(-)/rim8(-) mutants show defects in the ability to damage endothelial cells in situ . Finally, we show that an activated allele of RIM101, RIM101-405, is a suppressor of the rim8(-) mutation in vivo as it rescues the virulence, histological, and endothelial damage defects of the rim8(-)/rim8(-) mutant . These results demonstrate that the RIM101 pathway is required for C . albicans virulence in vivo and that the function of Rim8p in pathogenesis is to activate Rim101p.

Infect Immun, 2000 Oct, 68(10), 5771 - 7
Cellular and cytokine correlates of mucosal protection in murine model of oral candidiasis; Elahi S et al.; Host protection against Candida albicans infection in a model of oral candidiasis involving infection-prone {DBA/2 (H-2(d))} and less infection-prone {BALB/c (H-2(d))} mouse strains was analyzed in terms of antibody and cellular responses, and in terms of cytokine patterns from regional lymph node cells . There was a selective expansion of gamma/delta(+) T-cell receptor cells, which correlated with the patterns of colonization in both mouse strains, with higher numbers of gamma/delta T cells detected in BALB/c mice . Antigen-induced T-cell proliferation was significantly higher in BALB/c mice than in DBA/2 mice . Higher levels of serum immunoglobulin G (IgG) and salivary IgA antibodies were detected in BALB/c mice than in DBA/2 mice, but only after the infection was cleared . The cervical lymph node cells from infected mice were assessed for interleukin-4 (IL-4), IL-12, and gamma interferon (IFN-gamma) mRNA gene expression by reverse transcription-PCR and protein production in the culture supernatants following restimulation in vitro . In BALB/c mice, an early increase in levels of IL-4, IFN-gamma, and IL-12 correlated with rapid elimination of C . albicans . In DBA/2 mice, where resolution of infection was delayed, IL-4 message expression was delayed and the IL-4 secretion level was lower . Neutralization of IL-4 by multiple injections of an anti-IL-4 monoclonal antibody in BALB/c mice resulted in increased carriage rate and delayed clearance of the yeasts . Collectively, the data suggest that the T-cell response to C . albicans in the regional lymph nodes which correlates best with rapid oral clearance of C . albicans is a balanced Th0 cytokine response involving early secretion of both IFN-gamma and IL-4.

Infect Immun, 2000 Oct, 68(10), 5628 - 34
Defective induction of interleukin-12 in human monocytes by germ-tube forms of Candida albicans; Chiani P et al.; Yeast (Y) to germ-tube (GT) transition of Candida albicans is considered a putative virulence trait . On the other hand, interleukin-12 (IL-12) is a key promoter of T-helper type 1 protective immunity against this human opportunistic pathogen . We studied IL-12 production by human monocytes cocultured in vitro with Y or GT forms of C . albicans . Following stimulation by Y cells, monocytes produced appreciable levels of IL-12, which, upon addition of gamma interferon (IFN-gamma), compared to those achievable by lipopolysaccharide (100 ng/ml) stimulation (140 +/- 65 and 185 +/- 80 pg/ml, respectively {mean +/- standard deviation in four independent experiments}) . In contrast, IL-12 production by GT cell-stimulated monocytes was much lower or absent (<5 pg/ml) and could not be brought to the level induced by Y cells by the addition of IFN-gamma (30 +/- 10 pg/ml in the four independent experiments above) . Besides being observed as actual cytokine production, this lower response was also observed as specific IL-12 p40 mRNA transcript and was not associated with hyperproduction of the IL-12-competing cytokine IL-10 . Phagocytosis and killing experiments in the presence of cytochalasin D showed that IL-12 production by Y cell-stimulated monocytes was phagocytosis dependent and that GT cells of C . albicans were not phagocytized by the human monocytes . Importantly, however, Y and GT cells were equally killed by the monocytes . Thus, the virulence trait attributed to the Y-GT transition of C . albicans might also be related to the lack of induction by GT cells of a protective anticandidal immunity through defective IL-12 production.

Yeast, 2000 Sep 30, 16(13), 1205 - 15
MET15 as a visual selection marker for Candida albicans; Viaene J et al.; To develop better molecular genetic tools for the diploid yeast Candida albicans, the suitability of the MET15 gene as a visual selection marker was studied . Both MET15 alleles of C . albicans CAI-4 were isolated by functional complementation of a Saccharomyces cerevisiae strain lacking the MET15 gene . Growth of this complemented strain on Pb(2+)-containing medium was associated with a colour shift of brown into white colonies . The MET15 alleles of C . albicans were located on chromosome 4 by pulsed-field gel electrophoresis and Southern blotting . A met15-deficient strain of C . albicans CAI-4 was generated using the ura blaster technique . This strain showed a brown colony colour on Pb(2+)-containing medium, which corresponded with the colony colour of a S . cerevisiae strain lacking the MET15 gene . Unexpectedly, the met15-deficient strain of C . albicans still grew on methionine-depleted medium . However, this growth was severely delayed . In addition, complementation of this strain with an integrative or replicative plasmid containing either of the MET15 alleles resulted in the formation of white transformants on Pb(2+)-containing medium . These transformants grew very well on methionine-depleted medium . Colony sectoring was obtained with the replicative plasmid and not with the integrative one . This study demonstrates that the MET15 gene of C . albicans is suitable as a visual marker and therefore can be used to identify transformants and study plasmid stability . GenBank Accession Nos for MET15 nucleotide sequences are AF188273, AF188274 and AF188275 .

Antimicrob Agents Chemother, 2000 Oct, 44(10), 2887 - 90
In vitro pharmacodynamic characteristics of nystatin including time-kill and postantifungal effect; Gunderson SM et al.; Four Candida albicans isolates and six non-albicans Candida isolates were evaluated by time-kill methods to characterize the relationship between nystatin concentrations, the rate and extent of fungicidal activity, and the postantifungal effect (PAFE) . Against Candida species, nystatin exhibits concentration-dependent fungicidal activity and a pronounced PAFE.

Antimicrob Agents Chemother, 2000 Oct, 44(10), 2873 - 5
Undecylenic acid inhibits morphogenesis of Candida albicans; McLain N et al.; Resilient liners are frequently used to treat denture stomatitis, a condition often associated with Candida albicans infections . Of 10 liners tested, 2 were found to inhibit the switch from the yeast form to hyphae and a third was found to stimulate this switch . The inhibitor was determined to be undecylenic acid.

Antimicrob Agents Chemother, 2000 Oct, 44(10), 2853 - 4
Anti-Candida activity of a new platinum derivative; Watanabe T et al.; A new platinum derivative of the form H{Pt(IV)(Hdigly)Cl(2)(OH)(2)} (Hdigly==glycylglycine) damaged the Candida albicans cell membrane and inhibited the growth of the cells . The cytotoxic activity of H{Pt(IV)(Hdigly)Cl(2)(OH)(2)} on mammalian cells was 10-fold lower than that of cis-diammine-dichloroplatinum (cisplatin) . Substitution of platinum for peptides is effective for enhancement of antifungal activity and reduction of the toxicity to mammalian cells.

Antimicrob Agents Chemother, 2000 Oct, 44(10), 2693 - 700
Upregulation of ERG genes in Candida species by azoles and other sterol biosynthesis inhibitors; Henry KW et al.; Infections due to Candida albicans are usually treated with azole antifungals such as fluconazole, but treatment failure is not uncommon especially in immunocompromised individuals . Relatedly, in vitro studies demonstrate that azoles are nonfungicidal, with continued growth at strain-dependent rates even at high azole concentrations . We hypothesized that upregulation of ERG11, which encodes the azole target enzyme lanosterol demethylase, contributes to this azole tolerance in Candida species . RNA analysis revealed that ERG11 expression in C . albicans is maximal during logarithmic-phase growth and decreases as the cells approach stationary phase . Incubation with fluconazole, however, resulted in a two- to fivefold increase in ERG11 RNA levels within 2 to 3 h, and this increase was followed by resumption of culture growth . ERG11 upregulation also occurred following treatment with other azoles (itraconazole, ketoconazole, clotrimazole, and miconazole) and was not dependent on the specific medium or pH . Within 1 h of drug removal ERG11 upregulation was reversed . Azole-dependent upregulation was not limited to ERG11: five of five ERG genes tested whose products function upstream and downstream of lanosterol demethylase in the sterol biosynthetic pathway were also upregulated . Similarly, ERG11 upregulation occurred following treatment of C . albicans cultures with terbinafine and fenpropimorph, which target other enzymes in the pathway . These data suggest a common mechanism for global ERG upregulation, e.g., in response to ergosterol depletion . Finally, azole-dependent ERG11 upregulation was demonstrated in three additional Candida species (C . tropicalis, C . glabrata, and C . krusei), indicating a conserved response to sterol biosynthesis inhibitors in opportunistic yeasts.

Antimicrob Agents Chemother, 2000 Oct, 44(10), 2653 - 8
CD4(+)-T-Cell-mediated resistance to systemic murine candidiasis induced by a membrane fraction of Candida albicans; Mizutani S et al.; We induced resistance to systemic Candida albicans infection through CD4(+)-cell-mediated immunity in mice by immunization with subcutaneous injections of live C . albicans cells emulsified in incomplete Freund adjuvant . Using the resistant mice, we tested subcellular fractions of C . albicans cells for antigenicity . The fractions were derived from digested surface cell walls, insoluble membranes, or soluble and insoluble cytoplasmic materials, which were prepared by treatment with cell wall-digesting enzymes followed by lysis of the consequent protoplasts . Interestingly, the live-cell-immunized mice showed strong cell-mediated immune responses to the membrane fraction (C . albicans membrane antigen {CMA}) . In addition, immunization with CMA induced resistance to systemic candidiasis, which disappeared upon administration of anti-CD4 monoclonal antibody . Infusion of splenocytes from the CMA-immunized mice conferred resistance on SCID mice, whereas infusion of CD4(+)-T-cell-depleted splenocytes was unable to induce resistance, indicating the importance of CD4(+) lymphocytes for resistance . These results suggest a potential for the membrane fraction to act as an antigen conferring resistance to systemic candidiasis in place of live cells and also as a source for the isolation of a new antigen.

Microb Drug Resist, 2000 Summer, 6(2), 155 - 61
Cross-Resistance of clinical isolates of Candida albicans and Candida glabrata to over-the-counter azoles used in the treatment of vaginitis; Cross EW et al.; Antifungal drug resistance in Candida spp . continues to increase in response to the widespread application of triazole therapeutics among immunosuppressed patients . Azole-based over-the-counter (OTC) antifungal agents used to treat vaginitis have the potential to exacerbate this problem by contributing to the selection of highly resistant strains of Candida in otherwise healthy women . In this study, we show that fluconazole-resistant (MIC > 64 microg/mL) blood stream isolates of Candida albicans and Candida glabrata obtained from cancer patients were cross-resistant to the root drugs miconazole, clotrimazole, and tioconazole (found in several over-the-counter products), but remained susceptible to butoconazole . We also provide evidence that spontaneous mutants of Candida glabrata selected for resistance to clotrimazole were cross-resistant to other azolebased drugs, including fluconazole . Our findings demonstrate cross-resistance of Candida strains to fluconazole and OTC azole antifungals, and support the notion that OTC drugs can promote azole resistance in Candida spp.

Acta Ophthalmol Scand, 2000 Aug, 78(4), 448 - 50
Efficacy of fluconazole and liposome entrapped fluconazole for C . albicans induced experimental mycotic endophthalmitis in rabbit eyes; Gupta SK et al.; PURPOSE: To study the efficacy of intravitreally injected plain and liposome encapsulated fluconazole in the doses of 100 and 200 microg in Candidal endophthalmitis in rabbit eyes . METHOD: Endophthalmitis was induced by injecting Candida albicans (1000 CFU/0.1 ml) . Seventy two hours after inoculation, plain and liposome encapsulated fluconazole (REVs) were injected . At day 16 sterility was studied using vitreous culture . RESULTS: In the 100 & 200 microg plain fluconazole group, vitreous sterility was seen in 62.5% and 75%, respectively . In the liposome entrapped fluconazole a culture sterility of 25% and 50% for 100 microg and 200 microg, respectively, was seen . CONCLUSION: Plain fluconazole in the dose of 100 and 200 microg was equally effective against Candidal endophthalmitis in rabbits, but a failure of 25-37.5% of the eyes to respond, discourages one from using fluconazole as a sole therapy . Liposome entrapped fluconazole was found to be inferior to plain fluconazole in this model.

Vutr Boles, 1999, 31(4), 45 - 7
{An alternative to conventional antibiotic therapy in respiratory infections--Bioparox Spray}; Chalukov I; As the main target of influenza viral aggression, the respiratory tract is subject to easier bacterial infection superimposition . The researchers from Les Laboratoires Servier--France, managed to isolate a substance--fusafungine--from the microspore of the fungus Fusarium lateritium, which demonstrates unique anti-inflammatory and antibiotic action, and is the active ingredient of Bioparox Spray, an inhalant . The principal indications of Bioparox Spray for treatment of respiratory tract infections fall within the range from the sinuses to the finest alveolar duct, namely: rhinitis, sinusitis, tonsillitis, pharyngitis, laryngitis, tracheitis and bronchitis . In terms of technology Bioparox is unique due to the fact that 90% of the aerosol particles are less than one micron large, while generally the particles needed for penetration through the alveolar duct should be less than three microns . Due to such micronization, after inhalation Bioparox Spray reaches from the sinuses to the finest bronchial branches . Bioparox Spray possesses sound and broad antibiotic spectrum of action on the most common causative agents of respiratory infections, and more over, it acts upon Candida albicans, unlike the remaining broad-spectrum antibiotics . Bioparox Spray also has an independent anti-inflammatory effect by blocking the inflammation mediators: Bioparox Spray inhibits the synthesis of free radicals and the action of IL1 and TNF as pro-inflammatory factors, and it potentiates the action of IL2 and interferon-gamma which are anti-inflammatory factors . By its dual antibiotic and anti-inflammatory action Bioparox Spray is an excellent alternative to the conventional antibiotic therapy.

Org Lett, 2000 Sep 21, 2(19), 2939 - 42
Synthesis of a beta1,2-mannopyranosyl tetrasaccharide found in the phosphomannan antigen of Candida albicans; Nitz M et al.; The synthesis of a portion of the challenging beta1,2-mannosyl polymer found in the cell walls of Candida albicans was undertaken to develop a conjugate vaccine against C . albicans and to facilitate NMR conformational studies of this unique polysaccharide . The novel approach to the synthesis of tetrasaccharide 1 employed the modified ulosyl bromide 11 as the glycosyl donor which provided high diastereoselectivity . A participating solvent as well as p-chlorobenzyl protection facilitated the new approach.

Planta Med, 2000 Aug, 66(6), 541 - 4
New antimicrobial mono- and sesquiterpenes from Soroseris hookeriana subsp . erysimoides; Meng JC et al.; A new monoterpene and a new guaianolide were isolated from the aerial parts of the Tibetan medicinal plant Soroseris hookeriana subsp . erysimoides (Asteraceae), in addition to (1R,4R,5R)-5-hydroxybornan-2-one 5-O-beta-D-glucopyranoside, beta-sitosterol, daucosterol, diosmetin, isoluteolin, p-methoxybenzoic acid, isovanillic acid, two phenylmethanol derivatives (vanilioloside and phenylmethanol glucopyranoside), and five guaianolides {3 beta,8 beta-dihyroxyguaia-4(15),10(14),11(13)-triene-12,6 alpha-olide, dentalactone, 10 alpha-hydroxy-8-deoxy-10,14-dihydrodeacylcinaropicrin, glucozaluzanin C and 8-epideacylcinaropicrin glucoside} . By a combination of spectroscopic methods (IR, EI-MS, 1H- and 13C-NMR, and DEPT), the structure of the new guaianolide was established as 3 beta,8 beta-dihydroxy-11 alpha H-guaia-4(15),10(14)-diene-12,6 alpha-olide, and that of the new monoterpene as (1R,4R,5R)-5-benzoyloxybornan-2-one . The antimicrobial activity of all isolates except the two sterols were measured using Escherichia coli, Bacillus subtilis, Staphylococcus aureus, Candida albicans, Aspergillus niger, and Trichophyton rubrum as test microorganisms . The new guaianolide was shown to be equally active (MIC: 50 micrograms/ml) against E . coli, B . subtilis and A . niger . The new monoterpene inhibited exclusively the growth of B . subtilis with MIC at 25 micrograms/ml . p-Methoxybenzoic acid and isovanillic acid were inhibitory against A . niger (MIC: 25 micrograms/ml), the latter being also active against B . subtilis with MIC at 25 micrograms/ml . The flavonoids diosmetin and isoluteolin almost equally inhibited the growth of B . subtilis (MIC: 25 micrograms/ml) and the human pathgenic fungus T . rubrum (MIC: 50 micrograms/ml).

Farmaco, 2000 May, 55(5), 397 - 405
Synthesis and microbiological activity of some novel 5-benzamido- and 5-phenylacetamido-substituted 2-phenylbenzoxazole derivatives; Sener EA et al.; The synthesis and microbiological activity of a new series of 5-benzamido- and 5-phenylacetamidosubstituted-2-phenylbenzoxazole derivatives (1-26) were described . The in vitro microbiological activity of the compounds was determined against Gram-positive, Gram-negative bacteria and the yeast Candida albicans in comparison with standard drugs . Microbiological results indicated that the synthesized compounds possessed a broad spectrum of activity against the tested microorganisms . The compounds 1, 21, 25 showed higher activity than tetracycline and streptomycin against Pseudomonas aeruginosa.

FEMS Microbiol Lett, 2000 Sep 1, 190(1), 35 - 8
Production of pyruvate by Candida albicans: proposed role in virulence; Saeed FA; Proposed herein is a mechanism for virulence by Candida albicans based upon this organism's ability to produce high levels of pyruvate, potentially resulting in localized tissue ketosis and undermining the normal defensive function of neutrophil myeloperoxidase . Neutrophils, a key component of our innate defense against microbial infections, seem to play a particularly important role protecting us against fungal agents such as C . albicans . In this regard, it is myeloperoxidase which is central to many of the antimicrobial properties of neutrophils . We have previously shown that metabolic ketones inactivate myeloperoxidase and impair phagocytosis . Thus, production of pyruvate by C . albicans may indeed be a significant virulence factor.

J Antimicrob Chemother, 2000 Sep, 46(3), 479 - 83
In vitro activity of fluconazole and voriconazole against isolates of Candida albicans from patients with haematological malignancies; Girmenia C et al.; We evaluated the activity of fluconazole and voriconazole against 83 Candida albicans isolates from patients with haematological malignancies, comparing the NCCLS microdilution method (M27-A) with a modified method with RPMI-2% glucose and MIC endpoint at 50% inhibition . Both drugs were highly active regardless of the year, the site of isolation of the strain and the test method employed . In several strains isolated during the last few years, trailing growth leading to difficulty in interpretation of the endpoint of the test has been observed for both drugs by the NCCLS method, but not by the modified method . In our experience, azole resistance of C . albicans is still not a clinical problem, however, the emerging phenomenon of the 'trailing effect' by the NCCLS method, even though resolvable by technical modifications, seems at least to indicate a reduction in the inhibitory activity of the azoles.

J Infect Dis, 2000 Oct, 182(4), 1272 - 5 Epub 2000 Sep 05.
Zinc-reversible antimicrobial activity of recombinant calprotectin (migration inhibitory factor-related proteins 8 and 14); Sohnle PG et al.; Recombinant calprotectin, consisting of 2 individual peptide chains also called migration inhibitory factor-related protein (MRP)-8 and MRP14, was tested for antimicrobial activity in a Candida albicans growth inhibition assay . Both chains contain HEXXH zinc-binding sites and might be expected to manifest zinc-reversible, antimicrobial activity similar to that of native calprotectin . When tested alone, neither MRP8 nor MRP14 showed activity in the Candida growth assay . A synthetic 20-amino acid peptide containing the HEXXH sequence of MRP14, along with a nearby HHH sequence, was also inactive in this assay . However, equimolar concentrations of MRP8 and MRP14 demonstrated a potent growth inhibitory effect that was reversible by 30 microM zinc . Truncated MRP14 (missing the C-terminal GHHHKPGLGEGTP tail) used in combination with MRP8 demonstrated zinc-reversible activity that was somewhat less than that with complete MRP14 . These results suggest that intact calprotectin, consisting of a heterodimer of MRP8 and MRP14, is necessary to form a zinc-binding site capable of inhibiting microbial growth.

Genetics, 2000 Sep, 156(1), 31 - 44
Identification and characterization of TUP1-regulated genes in Candida albicans; Braun BR et al.; TUP1 encodes a transcriptional repressor that negatively controls filamentous growth in Candida albicans . Using subtractive hybridization, we identified six genes, termed repressed by TUP1 (RBT), whose expression is regulated by TUP1 . One of the genes (HWP1) has previously been characterized, and a seventh TUP1-repressed gene (WAP1) was recovered due to its high similarity to RBT5 . These genes all encode secreted or cell surface proteins, and four out of the seven (HWP1, RBT1, RBT5, and WAP1) encode putatively GPI-modified cell wall proteins . The remaining three, RBT2, RBT4, and RBT7, encode, respectively, an apparent ferric reductase, a plant pathogenesis-related protein (PR-1), and a putative secreted RNase T2 . The expression of RBT1, RBT4, RBT5, HWP1, and WAP1 was induced in wild-type cells during the switch from the yeast form to filamentous growth, indicating the importance of TUP1 in regulating this process and implicating the RBTs in hyphal-specific functions . We produced knockout strains in C . albicans for RBT1, RBT2, RBT4, RBT5, and WAP1 and detected no phenotypes on several laboratory media . However, two animal models for C . albicans infection, a rabbit cornea model and a mouse systemic infection model, revealed that rbt1Delta and rbt4Delta strains had significantly reduced virulence . TUP1 appears, therefore, to regulate many genes in C . albicans, a significant fraction of which are induced during filamentous growth, and some of which participate in pathogenesis.

Med Mycol, 2000 Aug, 38(4), 301 - 8
D-cecropin B: proteolytic resistance, lethality for pathogenic fungi and binding properties; De Lucca AJ et al.; L-Cecropin B (LCB) is a potent fungicidal peptide that is subject to proteolytic degradation by extracellular enzymes produced by Aspergillus flavus . We hypothesized that D-cecropin B (DCB), containing all D-amino acids, should resist proteolysis while retaining its fungicidal and target specificities . DCB was synthesized by solid phase methods using Fmoc chemistry . In vitro, at pH 6 x 0, DCB was lethal against the germinating conidia of A . flavus (LD90, 25 microM) and A . fumigatus (LD98, 25 microM) and for nongerminating and germinating conidia of Fusarium moniliforme (LD98, 1 x 25 microM) and F . oxysporum (LD95, 2 x 5 microM) at concentrations similar to those previously reported for LCB . It was lethal for Candida albicans with an LD98 at 12 x 5, microM . DCB was not active for the nongerminating conidia of A . fumigatus or A . flavus . Papain, trypsin, pepsin A and Staphylococcus aureus V8 protease degraded LCB but not DCB . Binding assays and circular dichroism showed DCB and LCB bound to cholesterol, ergosterol, beta-1,3-glucan, mannan and chitin . Data show that DCB retains the potent fungicidal properties of the L-form while being resistant to proteolytic enzymes that degrade the latter peptide . This study demonstrates that D-enantiomerization of cecropin B yields a novel fungicidal peptide, which resists proteolytic degradation and is lethal for pathogenic fungi.

Support Care Cancer, 2000 Sep, 8(5), 427 - 30
Prospective study on fungemia in children with cancer: analysis of 35 cases and comparison with 130 fungemias in adults; Krupova Y et al.; The aim of this prospective study on fungemia in children with cancer compared with adults with cancer appearing during the last 10 years in a pediatric hospital and in national cancer institutions was to investigate risk factors, etiology, therapy, complications and outcome . Univariate analysis showed significant differences in 35 children with cancer and fungemia in comparison with 130 cases of fungemias in adults with cancer . It was found that (1) therapy with corticosteroids (40 vs 18.5%, P<0.03), (2) breakthrough fungemia during ketoconazole prophylaxis (20 vs 7.7%, P<0.025), and (3) meningitis as a complication of fungemia (11.4 vs 0.8%, P< 0.001) occurred more frequently in the pediatric subgroup with fungemia . Candida albicans was more common as the causative agent of fungemia among adults (58.5 vs 37.1, P<0.02) than in children . However, mortality was similar in children with cancer and in adults with cancer and fungemia (31.4 vs 23.1%, NS) . Comparison of risk factors revealed no differences between adults and children with cancer and fungemia except in etiology, breakthrough fungemia during prophylaxis with ketoconazole, prior therapy with corticosteroids and meningitis as a complication . The outcome was also similar in pediatric and adult cancer patients with fungal bloodstream infection.

Microbiology, 2000 Sep, 146 ( Pt 9), 2105 - 12
Effect of salivary secretory IgA on the adhesion of Candida albicans to polystyrene; San Millan R et al.; Attachment of Candida albicans to plastic materials of dental prostheses or to salivary macromolecules adsorbed on their surface is believed to be a critical event in the development of denture stomatitis . In an earlier study, it was shown that adhesion of C . albicans to polystyrene, a model system to study the adhesion of C . albicans to plastic materials, can be partially inhibited with an mAb directed against cell wall polysaccharides of C . albicans . In the present study, the role of whole saliva in the adhesion of C . albicans to polystyrene has been investigated, and three mAbs directed against epitopes of cell wall mannoproteins have been used to mimic the inhibitory effect observed with salivary secretory IgA (sIgA) on the adhesion of C . albicans to polystyrene . In the absence of whole saliva, adherence of C . albicans 3153 increased with germination . However, the presence of whole saliva enhanced the adhesion to polystyrene of C . albicans 3153 yeast cells but decreased the adhesion of germinated cells . The enhancement of adhesion of yeast cells to polystyrene mediated by saliva was confirmed with an agerminative mutant of C . albicans 3153 . The inhibition of the adhesion of C . albicans 3153 germ tubes to polystyrene was due to the salivary sIgA since sIgA-depleted saliva enhanced the adhesion of C . albicans 3153 to polystyrene . The inhibitory effect mediated by sIgA was not related to the inhibition of germination but to the blockage of adhesins expressed on the cell wall surface of the germ tubes . The three mAbs studied reduced the adhesion of C . albicans 3153 to polystyrene at levels equivalent to those for purified sIgA . The highest reduction in the adhesion was obtained with the IgA mAb N3B . The best results were obtained when the three mAbs were combined . The results suggest that whole saliva plays a different role in the adhesion of C . albicans to polystyrene depending on the morphological phase of C . albicans . These results may give new insights into the conflicting role of saliva in the adhesion of C . albicans to plastic materials of dental prostheses.

Microbiology, 2000 Sep, 146 ( Pt 9), 2097 - 104
Allele-specific gene targeting in Candida albicans results from heterology between alleles; Yesland K et al.; The opportunistic fungal pathogen Candida albicans is asexual and diploid . Thus, introduction of recessive mutations requires targeted gene replacement of two alleles to effect expression of a recessive phenotype . This is often performed by recycling of a URA3 marker gene that is flanked by direct repeats of hisG . After targeting to a locus, recombination between the repeats excises URA3 leaving a single copy of hisG in the disrupted allele . The remaining functional allele is targeted in a second transformation with the same URA3 marked construct . Replacement can be highly biased toward one allele . At the PHR1 locus, there was an approximately 50-fold preference for replacement of the disrupted versus the functional allele in a heterozygous mutant . This preference was reduced six- to eightfold when the transforming DNA lacked the hisG repeats . Nonetheless, there remained a sixfold preference for targeting a particular allele of PHR1 and this was evident even in transformations of the parental strain containing two wild-type alleles of PHR1 . Both wild-type alleles were cloned and nucleotide sequence comparison revealed 24 heterologies over a 2 kb region . Using restriction site polymorphisms to distinguish alleles, it was observed that transformation with the cloned DNA of allele PHR1-1 preferentially targeted allele 1 of the genome . Transformations with PHR1-2 exhibited the reciprocal specificity . In both these instances, heterology was present in the flanking regions of the transforming DNA . When the transforming DNA was chosen from a region 100% identical in both alleles, alleles 1 and 2 were targeted with equal frequency . It is concluded that sequence heterology between alleles results in an inherent allele specificity in targeted recombination events.

J Hosp Infect, 2000 Aug, 45(4), 288 - 92
Fungal surveillance of an open haematology ward; Richardson MD et al.; Air sampling and surveillance cultures for fungi were performed in a Scottish general haematology ward over a five-month period in 1997 . The mean total fungal count from the air sampling appeared to be correlated with the number of patients colonized by Aspergillus . The most commonly isolated species were Aspergillus versicolor, A . fumigatus and A . niger . Rooms with portable air filtration units had significantly lower total fungal counts than the others . Swabs were taken from 70 patients (mean age 62 years); 114 of the 563 cultures (20.2%) were positive . The most commonly isolated species were A . fumigatus, Candida albicans, C . glabrata and C . parapsilosis . Samples taken from the tongue and perineum showed colonization more often than those taken from the nostrils . Almost half the patients (48.6%) were colonized on, or within seven days of, admission; 11.4% became colonized whilst on the unit . One patient developed fatal aspergillosis . We conclude that colonization or high air-borne spore concentrations are not necessarily predictive of fungal infection but may prompt early treatment or more aggressive prophylaxis of potentially fatal invasive infections.

J Appl Microbiol, 2000 Aug, 89(2), 215 - 24
The lactoperoxidase system: the influence of iodide and the chemical and antimicrobial stability over the period of about 18 months; Bosch EH et al.; The lactoperoxidase (LP) system is a natural antimicrobial system, the use of which has been suggested as a preservative in foods and pharmaceuticals . The effect of adding iodide to the LP system, the chemical stability and the change in antimicrobial effectiveness during storage was studied . Addition of iodide with thiocyanate increased the fungicidal and bactericidal effect against Candida albicans, Escherichia coli and Staphylococcus aureus . Pseudomonas aeruginosa showed the same inhibition in the LP system with iodide and without iodide . Storage of the LP system in completely filled airtight containers for 18 months caused a 35% loss of the initial thiocyanate concentration . The antimicrobial activity of this LP system was strong enough to kill inocula of 106 cfu ml-1 of the four test organisms within 2 h of contact time . During storage of the air-containing LP system, the concentration of thiocyanate was reduced below detection limit within 7 d, the concentrations of hypothiocyanite and hypoiodite within 350 d . After 516 d the antimicrobial activity of air-containing LP system was strong enough to kill inocula of 106 cfu ml-1 Ps . aeruginosa within 2 h, Staph . aureus within 4 h and Candida albicans and E . coli within 1 week of contact time.

Ear Nose Throat J, 2000 Aug, 79(8), 606 - 9
Otomycosis: a clinicomycologic study; Kaur R et al.; Otomycosis is a common fungal infection of the ear that is seen in the tropical and subtropical regions of the world . We performed mycologic analyses on debris and scraping samples from the external ear canals of 95 patients who had been clinically diagnosed with otomycosis . Seventy-one samples (74.7%) were positive for fungal growth; two of these samples contained two fungi, bringing the total number of isolates to 73 . The most common pathogens were Aspergillus fumigatus (41.1% of all isolates), A niger (36.9%), and Candida albicans (8.2%).

Infect Dis Obstet Gynecol, 2000, 8(3-4), 124 - 37
Antifungal activity of local anesthetics against Candida species; Pina-Vaz C et al.; OBJECTIVE: To evaluate the activity of benzydamine, lidocaine, and bupivacaine, three drugs with local anesthetic activity, against Candida albicans and non-albicans strains and to clarify their mechanism of activity . METHODS: The minimal inhibitory concentration (MIC) was determined for 20 Candida strains (18 clinical isolates and two American Type Culture Collection strains) . The fungistatic activity was studied with the fluorescent probe FUN-1 and observation under epifluorescence microscopy and flow cytometry . The fungicidal activity of the three drugs was assayed by viability counts . Membrane alterations induced in the yeast cells were evaluated by staining with propidium iodide, by quantitation of intracellular K+ leakage and by transmission electron microscopy of intact yeast cells and prepared spheroplasts . RESULTS: The MIC ranged from 12.5-50.0 microg/mL, 5.0-40.0 mg/mL, and 2.5-10.0 mg/mL for benzydamine, lidocaine, and bupivacaine, respectively . The inhibitory activity of these concentrations could be detected with the fluorescent probe FUN-1 after incubation for 60 minutes . A very fast fungicidal activity was shown by 0.2, 50, and 30 mg/mL of benzydamine, lidocaine, and bupivacaine, respectively . CONCLUSIONS: At lower concentrations, the tested drugs have a fungistatic activity, due to yeast metabolic impairment, while at higher concentrations they are fungicidal, due to direct damage to the cytoplasmic membrane.

FEMS Immunol Med Microbiol, 2000 Sep, 29(1), 69 - 76
Measurement of blood clearance time by Limulus G test of Candida-water soluble polysaccharide fraction, CAWS, in mice; Kurihara K et al.; The Limulus G test, responsive to beta-1,3-D-glucan, is a well-established method for the detection of invasive fungal infection . We have recently found that Candida albicans released a water-soluble polysaccharide fraction (CAWS) into synthetic medium (Uchiyama et al., FEMS Immunol . Med . Microbiol . 24 (1999) 411-420) . CAWS was composed of a mannoprotein-beta-glucan complex and activated Limulus factor G, and thus would be similar to the Limulus active substance in patient's blood . In a preliminary investigation, we have found that CAWS is lethal when administered intravenously in a murine system . In this study, we examined the toxicity and then the fate of CAWS in mice . The lethal toxicity was strain-dependent and strain DBA/2 was the most resistant . The toxicity was, at least in part, reduced by salbutamol sulfate and prednisolone treatment in the sensitive strains . On intravenous administration, the half clearance time (t1/2) was approximately 40 min in mice (DBA/2) . On intraperitoneal administration, CAWS appeared in the blood with a peak concentration at 1 h . In order to establish a treatment plan, it is important to demonstrate the onset and the termination of deep-seated mycosis . The Limulus G test is suitable for the above purpose; however, it is necessary to fully understand the fate of beta-1,3-D-glucan in patients' blood.

J Med Microbiol, 2000 Sep, 49(9), 831 - 40
Antifungal activity of ibuprofen alone and in combination with fluconazole against Candida species; Pina-Vaz C et al.; Ibuprofen, a non-steroidal anti-inflammatory drug, exhibited antimicrobial activity against Candida albicans and non-albicans strains . At 10 mg/ml, ibuprofen showed a rapid cidal activity against exponential growth phase C . albicans, accompanied by rapid and extensive leakage of intracellular K+, permeation to propidium iodide, lysis of spheroplasts and severe membrane ultrastructural alterations . These results indicate that the killing of Candida cells is due to direct damage to the cytoplasmic membrane . At 5 mg/ml, ibuprofen inhibited growth; however, it did not kill the yeasts and did not directly affect the cytoplasmic membrane . Evaluation of yeast metabolic vitality with the fluorescent probe FUN-1 showed that growth inhibition induced by the fungistatic drug concentration was due to metabolic alterations . The combination of ibuprofen with fluconazole resulted in synergic activity with eight of the 12 Candida strains studied, including four of the five fluconazole-resistant strains . The MICs of fluconazole for the fluconazole-resistant strains decreased 2-128-fold when the drug was associated with ibuprofen . When in combination with fluconazole, MICs for ibuprofen decreased by up to 64-fold for all the 12 strains studied . These results point to the practicability of using ibuprofen, alone or in combination with azoles, in the treatment of candidosis, particularly when applied topically, taking advantage of the drug's antifungal and anti-inflammatory properties.

Acta Biochim Pol, 2000, 47(1), 133 - 40
Comparative studies on cell stimulatory, permeabilizing and toxic effects induced in sensitive and multidrug resistant fungal strains by amphotericin B (AMB) and N-methyl-N-D-fructosyl amphotericin B methyl ester (MFAME); Szlinder-Richert J et al.; N-Methyl-N-D-fructosyl-amphotericin B methyl ester (MFAME) is a new derivative of amphotericin B, which is characterised by low toxicity to mammalian cells and good solubility in water of its salts . The antifungal activity and effects of MFAME towards Candida albicans and Saccharomyces cerevisiae multidrug resistant MDR(+) and sensitive MDR(-) strains was compared with those of parent compound . The results obtained indicate that MDR(+) S . cerevisiae was sensitive to MFAME as well as to AMB . MFAME exhibited the same effects on fungal cells studied as parent antibiotic . The two antibiotics, depending on the dose applied induced cell stimulation, K+ efflux, and/or had a toxic effect.

Stomatologiia (Mosk), 2000, 79(4), 22 - 7
{The antagonistic activity of the eubiotic Maxilin towards wound infection and its effect on the antibiotic resistance of microorganisms}; Pichkhadze GM et al.; Antagonistic activity of lactic acid bacteria of strain N 2585 of "Maxilin" probiotic in respect to the strains S . aureus, P . aeruginosa, E . coli, Candida albicans and their effect on the level of sustainability of the antibiotic resistant strains S . aureus, E . coli, P . aeruginosa, which are extracted from the patients with open fractures of the mandible under various course of wound process, have been studied . The studies showed that lactic acid bacteria have suppressed growth of strains Candida most actively, followed by P . aeruginosa, S . aureus and E . coli . The use of "Maxilin" probiotic allowed reducing the epidemiological significance of pathogenetic strains already in 3 to 4 days . The sensibility studies showed high activity of the lactic acid bacteria in reducing antibiotic resistance of the strains investigated . It was revealed that "Maxilin" probiotic affects Ps . aeruginosa effectively, somewhat less St . aureus and E . coli . The extracted strains demonstrated sensibility to all investigated antibiotics already in 3 to 4 days.

Eur J Med Chem, 2000 Jul-Aug, 35(7-8), 761 - 71
Synthesis, characterization and pharmacological properties of some 4-arylhydrazono-2-pyrazoline-5-one derivatives obtained from heterocyclic amines; Guniz Kucukguzel S et al.; The synthesis of a new series of 4-arylhydrazono-2-pyrazoline-5-ones 7-24 and 22a is described . Structures of the synthesized compounds were confirmed using UV, IR, 1H-NMR, 13C-NMR and EI-mass spectral data . These compounds were tested in vitro against one Gram-positive and two Gram-negative bacterial strains, two mycobacterial strains and a fungus, Candida albicans . Compound 22 was found to be more active against Staphylococcus aureus than the other compounds at a concentration of 15.6 microg/mL . Some related compounds were evaluated for anticonvulsant activity . Compound 11 showed 40% protection against pentylenetetrazole-induced seizures in albino Swiss mice . In vitro antituberculosis activity of 4-arylhydrazono-2-pyrazoline-5-ones 7-12, 14-24, 22a and coupling products 6a-f were tested on Mycobacterium tuberculosis H37Rv . Of these compounds, only 24, which exhibited > 90% inhibition in the primary screen at 12.5 microg/mL against this strain was re-examined for determination of its actual MIC . However, level II assay revealed that the MIC value was not less than 12.5 microg/mL . The same compound was also tested against Mycobacterium avium, which was observed not to be susceptible to 24.

Pediatr Infect Dis J, 2000 Aug, 19(8), 729 - 34
Esophageal candidiasis in pediatric acquired immunodeficiency syndrome: clinical manifestations and risk factors; Chiou CC et al.; BACKGROUND: Little is known about the epidemiology and clinical features of esophageal candidiasis (EC) in pediatric AIDS . We therefore investigated the clinical presentation and risk factors of EC in a large prospectively monitored population of HIV-infected children at the National Cancer Institute . PATIENTS AND METHODS: We reviewed the records of all HIV-infected children (N = 448) followed between 1987 and 1995 for a history of esophageal candidiasis to characterize the epidemiology, clinical features, therapeutic interventions and outcome of esophageal candidiasis . To understand further the risk factors for EC in pediatric AIDS, we then performed a matched case-control analysis of 25 patients for whom control cases were available . RESULTS: There were 51 episodes of EC documented in 36 patients with 23 male and 13 female patients (0.2 to 17 years; median CD4, count 11/microl), representing a frequency of EC of 8.0% . Concurrent oropharyngeal candidiasis (OPC) was the most common clinical presentation of EC (94%); other signs and symptoms included odynophagia (80%), retrosternal pain (57%), fever (29%), nausea/vomiting (24%), drooling (12%), dehydration (12%), hoarseness (6%) and upper gastrointestinal bleeding (6%) . The causative organism documented in 36 episodes (18 from OPC, 17 from endoscopic biopsy and 1 from autopsy) was Candida albicans in all cases . Patients received treatment for EC with amphotericin B (63%), fluconazole (29%), ketoconazole (4%) or itraconazole (1%) . A clinical response was documented in all 45 evaluable episodes . In 6 other cases, EC was a final event without contributing to the cause of death . By a conditional logistic regression model for matched data, the best predictor of EC was the presence of prior OPC (P<0.0001), followed by CD4 count and CD4 percentage (P = 0.0002) and use of antibacterial antibiotics (P = 0.0013) . The risks associated with low CD4 count were independent of that of prior OPC . CONCLUSION: EC in pediatric AIDS is a debilitating infection, which develops in the setting of prior OPC, low CD4 counts and previous antibiotics.

Int J Med Microbiol, 2000 Jul, 290(3), 231 - 8
Molecular responses to changes in the environmental pH are conserved between the fungal pathogens Candida dubliniensis and Candida albicans; Heinz WJ et al.; In this work we cloned CdPHR1 and CdPHR2 from the human fungal pathogen Candida dubliniensis . The two genes are homologues to the pH-regulated genes PHR1 and PHR2 from Candida albicans . The pH-dependent pattern of expression of CdPHR1 and CdPHR2 was conserved in C . dubliniensis . CdPHR1 could be shown to be functionally equivalent to PHR1 . The pH-regulated mode of expression was maintained when CdPHR1 was integrated in C . albicans . This indicates a fundamentally similar mode of expressional regulation in the two species . CdPHR1 was furthermore capable of reversing the aberrant phenotype of a Saccharomyces cerevisiae GAS1 deletion mutant . In this species, however, expression of CdPHR1 was no longer under control of the external pH . Expression of CdPHR1 was not detected when it was introduced into Aspergillus nidulans . In conclusion, C . dubliniensis and C . albicans respond to changes in the environmental pH with a change in cell shape and differential gene expression.

Biochemistry, 2000 Aug 29, 39(34), 10532 - 41
Stabilization of a novel enzyme.substrate intermediate in the Y206F mutant of Candida albicans EBP1: evidence for acid catalysis; Buckman J et al.; EBP1-catalyzed reduction of alpha,beta-unsaturated ketones and aldehydes is proposed to proceed via transfer of hydride from the flavin to the beta-position of the olefinic bond, concomitant with or followed by uptake of a proton at the alpha-position . Structural analysis suggests that this proton is donated from Tyr206, and, hence, a protein was constructed in which it was replaced by phenylalanine . The mutation results in a slightly less stable protein than the wild type that nevertheless retains the fundamental flavin and phenol binding properties of EBP1 characterized previously . The pH profile for binding of phenol was characterized over the pH range 6.5-9.5 and was found to be simpler than that for the wild-type enzyme . Most importantly, a pK(a) of 8.7 that is perturbed to 9.4 upon binding of phenol to the wild-type enzyme is missing in the mutant, allowing assignment of this pK(a) to the Y206 hydroxyl group . Additionally, the pK(a) of phenol is further lowered from its value of 10.0 in solution to approximately 6.4 in the active site of the mutant, as compared to 7.1 in the wild type . Together, these perturbations lead to an increase of approximately 35-fold in the binding affinity of the mutant for phenol at high pH relative to the affinity of the wild-type enzyme . As expected, the mutation has little effect on the reductive half-reaction, in which a hydride equivalent is transferred from NADPH to the flavin . In contrast, the reduction of trans-2-hexenal by the reduced enzyme is significantly affected . The results indicate formation of a previously unobserved charge-transfer (CT) complex following formation of the Michaelis complex between substrate and reduced enzyme and preceding reduction of the substrate, which occurs at a greatly reduced rate (>/=440-fold) relative to wild type . Thus, while the oxidative half-reaction with wild-type enzyme is limited by the rate of formation of the CT complex, it is the chemical step that is rate-limiting in the reaction with EBP1:Y206F, consistent with the role of this residue as a general acid.

Biochemistry, 2000 Aug 29, 39(34), 10521 - 31
Transient kinetics and intermediates formed during the electron transfer reaction catalyzed by Candida albicans estrogen binding protein; Buckman J et al.; The transient kinetics of the reaction of the estrogen binding protein (EBP1) from Candida albicans in which hydride is transferred from NADPH to trans-2-hexenal (HXL) in two half-reactions were analyzed using UV-visible spectrophotometric and fluorometric stopped-flow techniques . The simplest model of the first half-reaction involves four steps including very rapid, tight binding (K(d) </= 50 nM) characterized by loss of NADPH fluorescence, subsequent rapid formation of a charge-transfer complex between NADPH and oxidized enzyme-bound flavin mononucleotide (FMN) cofactor, followed by rate-limiting reduction of the FMN, and finally dissociation of the oxidized pyridine nucleotide . The UV-visible absorbance behavior of this half-reaction is described by two apparent phases, with k(f)(obs) = 355 +/- 6 and k(s)(obs) = 3.30 +/- 0.03 s(-)(1), for the fast and slow phases, respectively . The reaction has also been evaluated in terms of the full, multi-equilibrium reaction scheme, and microscopic rate constants that lead to the observed behavior have been determined through convergent experimental techniques and computer simulation . Significant intrinsic kinetic isotope effects were noted on both the bond cleavage step and the preceding formation of the charge-transfer complex . The enzyme is reoxidized by transfer of hydride from the FMN to HXL in the second half-reaction that appears to consist of substrate binding to form a Michaelis-type complex and the subsequent chemical step . Characterization of the reaction in this simple manner allows determination of an apparent K(d) = 100 +/- 9 microM for the reduced enzyme.HXL complex.

Fungal Genet Biol, 2000 Jun, 30(1), 1 - 15
Fungal transporters involved in efflux of natural toxic compounds and fungicides; Del Sorbo G et al.; Survival of microorganisms in natural environments is favored by the capacity to produce compounds toxic to competing organisms and the ability to resist the effects of such toxic compounds . Both factors contribute to a competitive advantage of organisms in ecosystems . All organisms have evolved active transport mechanisms by which endogenous and exogenous toxicants can be secreted . Two major classes of transporter proteins are the ATP-binding cassette (ABC) and the major facilitator superfamily (MFS) transporters . Members of both classes can have broad and overlapping substrate specificities for natural toxic compounds and can be regarded as a "first-line defense barrier" in survival mechanisms . In plant pathogens, these transporters can play an essential role in protection against plant defense compounds during pathogenesis . Also, some transporters actively secrete host-specific and non-host-specific toxins . Remarkably, ABC and MFS transporters can also play a major role in fungicide sensitivity and resistance . Their role in multidrug resistance of Aspergillus nidulans, Candida albicans, and Saccharomyces cerevisiae to azoles and other fungitoxic compounds is well established . Knowledge of ABC and MFS transporters opens possibilities of developing novel strategies for controlling plant diseases, either by modulation of transporter activity or by transgenic expression of transporter genes in plants.

Med Pregl, 2000 Jan-Feb, 53(1-2), 85 - 8
{Mycotic disease of the mucous membranes of the head and neck}; Mitrovic S et al.; INTRODUCTION: Candidiasis is usually a superficial infection of the moist areas of the body and is generally caused by Candida albicans . Visceral infections occur in diabetes, lymphomas and leukemias, malnutrition, avitaminosis and they are associated with antibiotic, corticosteroid and immunosuppressive therapy . Candida albicans was isolated from middle ear inflammation . The diagnosis is made on the basis of microscopic appearance of colonies and characteristic smell . Candidiasis is successfully treated with nystatin, imidazol derivatives (fluconazole, ketoconazole and intraconazole), amphotericin B, 5-fluorocystosine and 1% iodine solution . CASE DESCRIPTION: This is a case report of a 46-year-old patient with a persistent nasal, sinus and ear infection of unknown origin . The patient first received antibiotic and steroid therapy and trepanation of the right maxillary sinus was performed . As the patient's condition aggravated with increase of temperature and bad laboratory findings, he was hospitalized . Radiography revealed a pathological process in both maxillary sinuses and both mastoids, so mastoidectomy and left maxillary sinus trepanation were performed . Histopathological examination of the right mastoid revealed a mould infection . The immunologic status pointed to hypogammaglobulinemia IgG . The following diseases were excluded: systemic diseases, blood diseases, Reiter's syndrome, AIDS, Hepatitis B, other viral diseases, toxoplasmosis, trichinellosis, borreliosis, typhus, paratyphus and exanthematous typhus . The diagnosis of candidiasis caused by Candida crusei and Candida kefyr was made on the basis of macroscopic and microscopic findings and biochemical identification . Ketoconazole was introduced (400 mg/per day) as well as high doses of vitamins and povidone-iodine locally . After a period of remission the patient died due to myocarditis, sepsis, acute kidney failure associated with severe mucosal necrosis of the mouth, esophagus and throat . Differential diagnosis in fever of unknown origin must include the possibility of mycotic infection, whereas the therapy of mycotic diseases must include two antimycotics at the same time . DISCUSSION AND CONCLUSION: Candida albicans is often found in the oral cavity and skin as well as in intestines of 18% of healthy subjects . It is unknown why it causes clinical illness . Antibiotic therapy of bacterial infections enables candida colonization especially in immunosuppressed patients . In our patient two types were found: Candida krusei and Candida kefyr . It is of special importance to perform differential diagnosis in cases with fever of unknown origin in order to include the possibility of mycotic infections, whereas treatment of systemic fungal infections requires a team of physicians.

Yeast, 2000 Sep 15, 16(12), 1121 - 9
Evaluation of the CaMAL2 promoter for regulated expression of genes in Candida albicans; Backen AC et al.; An expression vector (CIp10-MAL2p) for use in Candida albicans has been constructed in which a gene of interest can be placed under the control of the CaMAL2 maltase promoter and stably integrated at the CaRP10 locus . Using this vector to express the Candida URA3 gene from the CaMAL2 promoter, we have demonstrated tight regulation of CaURA3 expression by carbon source . Thus under conditions when the CaMAL2 promoter is not induced, expression of Candida URA3 was unable either to complement a C . albicans ura3 mutation or to confer sensitivity to 5-fluoroorotic acid, a compound which is highly toxic to URA3 strains . Since Candida albicans is an obligate diploid organism, analysis of gene function requires manipulation of both copies of any gene of interest . Our expression vector provides a strategy by which the remaining copy of a gene of interest can be placed under CaMAL2 promoter control in a strain where the first copy has been deleted, permitting analysis of gene function by manipulation of carbon source . CIp10-MAL2p should therefore provide a useful means for functional analysis of genes in C . albicans . We have used this strategy with C . albicans DPB2 to demonstrate that the gene is essential and that loss of function leads cells to adopt a hypha-like morphology as they cease proliferation .

Antimicrob Agents Chemother, 2000 Sep, 44(9), 2373 - 81
Potent synergism of the combination of fluconazole and cyclosporine in Candida albicans; Marchetti O et al.; Several types of drugs currently used in clinical practice were screened in vitro for their potentiation of the antifungal effect of the fungistatic agent fluconazole (FLC) on Candida albicans . These drugs included inhibitors of multidrug efflux transporters, antimicrobial agents, antifungal agents, and membrane-active compounds with no antimicrobial activity, such as antiarrhythmic agents, proton pump inhibitors, and platelet aggregation inhibitors . Among the drugs tested in an agar disk diffusion assay, cyclosporine (Cy), which had no intrinsic antifungal activity, showed a potent antifungal effect in combination with FLC . In a checkerboard microtiter plate format, however, it was observed that the MIC of FLC, as classically defined by the NCCLS recommendations, was unchanged when FLC and Cy were combined . Nevertheless, if a different reading endpoint corresponding to the minimal fungicidal concentration needed to decrease viable counts by at least 3 logs in comparison to the growth control was chosen, the combination was synergistic (fractional inhibitory concentration index of <1) . This endpoint fitted to the definition of MIC-0 (optically clear wells) and reflected the absence of the trailing effect, which is the result of a residual growth at FLC concentrations greater than the MIC . The MIC-0 values of FLC and Cy tested alone in C . albicans were >32 and >10 microg/ml, respectively, and decreased to 0.5 and 0.625 microg/ml when the two drugs were combined . The combination of 0.625 microg of Cy per ml with supra-MICs of FLC resulted in a potent antifungal effect in time-kill curve experiments . This effect was fungicidal or fungistatic, depending on the C . albicans strain used . Since the Cy concentration effective in vitro is achievable in vivo, the combination of this agent with FLC represents an attractive perspective for the development of new management strategies for candidiasis.

Antimicrob Agents Chemother, 2000 Sep, 44(9), 2356 - 60
Efficacy of oral cochleate-amphotericin B in a mouse model of systemic candidiasis; Santangelo R et al.; Amphotericin B (AMB) remains the principal therapeutic choice for deep mycoses . However, its application is limited by toxicity and a route of administration requiring slow intravenous injection . An oral formulation of this drug is desirable to treat acute infections and provide prophylactic therapy for high-risk patients . Cochleates are a novel lipid-based delivery system that have the potential for oral administration of hydrophobic drugs . They are stable phospholipid-cation crystalline structures consisting of a spiral lipid bilayer sheet with no internal aqueous space . Cochleates containing AMB (CAMB) inhibit the growth of Candida albicans, and the in vivo therapeutic efficacy of CAMB administered orally was evaluated in a mouse model of systemic candidiasis . The results indicate that 100% of the mice treated at all CAMB doses, including a low dosage of 0.5 mg/kg of body weight/day, survived the experimental period (16 days) . In contrast, 100% mortality was observed with untreated mice by day 12 . The fungal tissue burden in kidneys and lungs was assessed in parallel, and a dose-dependent reduction in C . albicans from the kidneys was observed, with a maximum 3.5-log reduction in total cell counts at 2.5 mg/kg/day . However, complete clearance of the organism from the lungs, resulting in more than a 4-log reduction, was observed at the same dose . These results were comparable to a deoxycholate AMB formulation administered intraperitoneally at 2 mg/kg/day (P < 0.05) . Overall, these data demonstrate that cochleates are an effective oral delivery system for AMB in a model of systemic candidiasis.

Antimicrob Agents Chemother, 2000 Sep, 44(9), 2327 - 32
Single-dose AmBisome (Liposomal amphotericin B) as prophylaxis for murine systemic candidiasis and histoplasmosis; Garcia A et al.; AmBisome is a liposomal formulation of amphotericin B that has broad-spectrum antifungal activity and greatly reduced toxicity compared to the parent drug . In this study, amphotericin B deoxycholate (Fungizone) (1 mg/kg) and AmBisome (1 to 20 mg/kg) were tested as single-dose prophylactic agents in both immunocompetent and immunosuppressed C57BL/6 mice challenged with either Candida albicans or Histoplasma capsulatum . Prophylactic efficacy was based on survival and fungal burden in the target organ (kidneys or spleen) . At 9 to 10 days after histoplasma challenge, 80 to 90% of both immunocompetent and immunosuppressed mice in the control and Fungizone groups had died . All AmBisome-treated mice survived, although in the AmBisome groups given 1 mg/kg, the mice became moribund by day 10 to 12 . No spleen CFU were detected in the histoplasma-challenged mice given 10 or 20 mg of AmBisome per kg . By 23 to 24 days after histoplasma challenge, fungal growth and/or death had occurred in all immunosuppressed mice except for four mice receiving 20 mg of AmBisome per kg . There were still no detectable fungi in the spleens of immunocompetent mice given 10 or 20 mg of AmBisome per kg . In the C . albicans experiment at 7 days postchallenge, all animals in both untreated and treated groups were alive with culture-positive kidneys . The kidney fungal burdens in AmBisome groups given 5 to 20 mg/kg were at least 1 log unit lower than those in the Fungizone group and significantly lower than those in the untreated control group (P < 0.05) . There was a trend toward decreasing fungal growth in the kidneys as the dose of AmBisome was increased . In conclusion, these results show that a single high dose of AmBisome (5 to 20 mg/kg) had prophylactic efficacy in immunocompetent and immunosuppressed murine H . capsulatum and C . albicans models.

Antimicrob Agents Chemother, 2000 Sep, 44(9), 2310 - 8
Efficacy of the echinocandin caspofungin against disseminated aspergillosis and candidiasis in cyclophosphamide-induced immunosuppressed mice; Abruzzo GK et al.; The in vivo efficacy of the echinocandin antifungal caspofungin acetate (caspofungin; MK-0991) was evaluated in models of disseminated aspergillosis and candidiasis in mice with cyclophosphamide (CY)-induced immunosuppression . Caspofungin is a 1, 3-beta-D-glucan synthesis inhibitor efficacious against a number of clinically relevant fungi including Aspergillus and Candida species . Models of CY-induced transient or chronic leukopenia were used with once daily administration of therapy initiated 24 h after microbial challenge . Caspofungin was effective in treating disseminated aspergillosis in mice that were transiently leukopenic (significant prolongation of survival at doses of > or =0.125 mg/kg of body weight and a 50% protective dose {PD(50)} of 0.245 mg/kg/day at 28 days after challenge) or chronically leukopenic (50 to 100% survival at doses of > or =0.5 mg/kg and PD(50)s ranging from 0.173 to 0.400 mg/kg/day) . Caspofungin was effective in the treatment and sterilization of Candida infections in mice with transient leukopenia with a 99% effective dose based on reduction in log(10) CFU of Candida albicans/gram of kidneys of 0.119 mg/kg and 80 to 100% of the caspofungin-treated mice having sterile kidneys at caspofungin doses from 0.25 to 2.0 mg/kg . In Candida-infected mice with chronic leukopenia, caspofungin was effective at all dose levels tested (0.25 to 1.0 mg/kg), with the log(10) CFU of C . albicans/gram of kidneys of caspofungin-treated mice being significantly lower (>99% reduction) than that of sham-treated mice from day 4 to day 28 after challenge . Also, 70 to 100% of the caspofungin-treated, chronic leukopenic mice had sterile kidneys at caspofungin doses of 0.5 to 1.0 mg/kg from day 8 to 28 after challenge . Sterilization of Candida infections by caspofungin in the absence of host leukocytes provides compelling in vivo evidence for fungicidal activity against C . albicans . Further human clinical trials with caspofungin against serious fungal infections are in progress.

Antimicrob Agents Chemother, 2000 Sep, 44(9), 2296 - 303
Transcriptional analyses of antifungal drug resistance in Candida albicans; Lyons CN et al.; Oral infections with the pathogenic yeast Candida albicans are one of the most frequent and earliest opportunistic infections in human immunodeficiency virus-infected patients . The widespread use of azole antifungal drugs has led to the development of drug-resistant isolates . Several molecular mechanisms that contribute to drug resistance have been identified, including increased mRNA levels for two types of efflux pump genes: the ATP binding cassette transporter CDRs (CDR1 and CDR2) and the major facilitator MDR1 . Using Northern blot analyses, the expression patterns of these genes have been determined during logarithmic and stationary phases of cell growth and during growth in different carbon sources in a set of matched susceptible and fluconazole-resistant isolates that have been characterized previously . MDR1, CDR1, and CDR2 are expressed early during logarithmic growth, CDR4 is expressed late during logarithmic growth, and CDR1 is preferentially expressed in stationary-phase cells . There is a small decrease in expression of these genes when the cells are grown in carbon sources other than glucose . While increased mRNA levels of efflux pump genes are commonly associated with azole resistance, the causes of increased mRNA levels have not yet been resolved . Southern blot analysis demonstrates that the increased mRNA levels in these isolates are not the result of gene amplification . Nuclear run-on assays show that MDR1 and CDR mRNAs are transcriptionally overexpressed in the resistant isolate, suggesting that the antifungal drug resistance in this series is associated with the promoter and trans-acting factors of the CDR1, CDR2, and MDR1 genes.

J Infect Dis, 2000 Sep, 182(3), 955 - 9 Epub 2000 Aug 17.
In vivo virulence of Candida albicans isolates causing mucosal infections in people infected with the human immunodeficiency virus; Taylor BN et al.; Mucosal candidiasis is common in human immunodeficiency virus (HIV) infection . Susceptibility to such infections may be attributed to reduced host defense mechanisms and/or virulence of the organism . In the present study, we compared the virulence of mucosal Candida albicans isolates from HIV-infected people, with and without fluconazole-refractory infection, in established murine models of systemic and vaginal candidiasis . Compared with the mortality rate ( approximately 70%) after intravenous challenge with 2 virulent reference isolates, challenge with most clinical isolates (66%-77%) resulted in prolonged survival . In contrast, fungal burden induced by intravaginal challenge of nearly all (97%) isolates was similar to that of the virulent controls . There were no differences in in vitro growth rates for any of the isolates, and there was no association between reduced mortality and clinical failure to fluconazole, in vitro antifungal susceptibility, site of infection, or other host factors . These results suggest that virulence of C . albicans is tissue specific and is not a factor in the development of fluconazole-refractory infections in advanced HIV disease.

J Infect Dis, 2000 Sep, 182(3), 917 - 22 Epub 2000 Aug 17.
Role of pulmonary surfactant protein D in innate defense against Candida albicans; van Rozendaal BA et al.; Pulmonary surfactant protein D (SP-D), which is a member of the collectin family, is implicated in pulmonary defense against pathogens . To determine whether SP-D is involved in first-line immunity against Candida albicans, an important respiratory fungus, the interaction of SP-D with C . albicans was studied . SP-D was found to bind C . albicans, resulting in agglutination of the microorganisms . Binding was calcium dependent and was inhibited by competing sugars maltose or mannose . Incubation of C . albicans with SP-D resulted in profound fungal growth inhibition and decreased hyphal outgrowth . Furthermore, it was found that SP-D inhibited phagocytosis of C . albicans by alveolar macrophages . These data suggest that the lung collectin SP-D has an important role in first-line defense against C . albicans in the lung, by agglutinating C . albicans and limiting their growth, without the need for macrophage activation.

Electrophoresis, 2000 Jul, 21(13), 2651 - 9
Cross-species identification of novel Candida albicans immunogenic proteins by combination of two-dimensional polyacrylamide gel electrophoresis and mass spectrometry; Pardo M et al.; We have previously reported the usefulness of two-dimensional gel electrophoresis followed by Western blotting with sera from patients with systemic candidiasis in the detection of the major Candida albicans antigens (Pitarch et al., Electrophoresis 1999, 20, 1001-1010) . The identification of these antigens would be useful for the characterization of good markers for the disease, and for the development of efficient diagnostic strategies . In this work we have used nanoelectrospray tandem mass spectrometry to obtain amino acid sequence information from the immunogenic proteins previously detected . We report here the cross-species identification of these antigens by matching of tandem mass spectrometry data to Saccharomyces cerevisiae proteins . Using this approach, we unambiguously identified the four C . albicans immunogenic proteins analyzed, namely aconitase, pyruvate kinase, phosphoglycerate mutase and methionine synthase . Furthermore, we report for the first time that aconitase, methionine synthase and phosphoglycerate mutase have antigenic properties in C . albicans.

Mycoses, 2000, 43(5), 185 - 9
Adherence in tissues of immunocompromised mice of a non-mycelium producing strain of Candida albicans; Vespa MN; A non-mycelial strain of wild-type Candida albicans strain 3153 A was produced by repeated subculturing on Sabouraud glucose agar, and maintained on yeast extract-peptone-glucose medium resulting in hydrophobic cells at 26 degrees C and hydrophilic cells at 37 degrees C . The behaviour of cells of this strain was studied in male BALB/c mice, immunocompromised by treatment with cyclophosphamide and cortisone acetate . An ex-vivo assay of cell adherence to tissue sections of liver, spleen, kidney and lymph-nodes was used . The adherence of yeast cells at 26 and 37 degrees C was predominantly produced by hydrophobic cells and was significantly greater in spleen and liver of immunosuppressed mice compared with the organs of control animals . Adhesion was observed in the white and red pulp as well as in the marginal zone of the spleen.

Mycoses, 2000, 43(5), 173 - 5
Antibodies to antigens of Histoplasma, Blastomyces and Candida in HIV patients and carriers in Nigeria; Muotoe-Okafor FA et al.; Serum samples from 60 subjects with confirmed HIV-1 infection including 28 AIDS patients and 32 carriers were examined by immunodiffusion for precipitating antibodies to antigens of Histoplasma, Blastomyces and Candida . Seven of the subjects, four patients and three carriers, showed antibodies to histoplasmin prepared from mycelial cultural filtrate of Histoplasma capsulatum var . capsulatum and H . capsulatum var . duboisii although without any clinical signs of classical or African histoplasmosis . Another eight subjects comprising five patients and three carriers demonstrated antibodies to yeast cell antigen of Candida albicans; three of the patients had oral lesions clinically suggestive of Candida infection . None of the serum samples revealed antibodies to "A" antigen (yeast cell antigen) of Blastomyces dermatitidis.

Infect Immun, 2000 Sep, 68(9), 5126 - 31
Interleukin 18 restores defective Th1 immunity to Candida albicans in caspase 1-deficient mice; Mencacci A et al.; Caspase 1, formerly designated interleukin 1beta (IL-1beta)-converting enzyme, processes pro-IL-1beta and pro-IL-18 to yield active cytokines that play a pivotal role in inflammation and cell activation . We show here the effect of caspase 1 deficiency on the inflammatory and adaptive immune responses to the fungus Candida albicans . Caspase 1 deficiency did not affect susceptibility to primary systemic infection with the fungus, as revealed by survival and fungal growth . However, Th1-mediated resistance to reinfection was greatly impaired in caspase 1-deficient mice, and this correlated with low-level production of IL-12 and gamma interferon . Early in infection, production of these cytokines and that of tumor necrosis factor alpha, IL-6, and, interestingly, IL-1beta occurred normally in caspase 1-deficient mice, while that of IL-18 was severely impaired . Exogenous administration of IL-18, more than IL-12, restored the Th1-mediated resistance to the infection . We conclude that, while caspase 1 is not indispensable for release of mature IL-1beta in candidiasis, the caspase 1-dependent production of IL-18 may represent an important and novel pathway for the expression of sustained Th1 reactivity to the fungus.

J Leukoc Biol, 2000 Aug, 68(2), 175 - 9
Innate and adaptive immunity in Candida albicans infections and saprophytism; Romani L; Underlying acquired immunity to the fungus Candida albicans is usually present in adult immunocompetent individuals and is presumed to prevent mucosal colonization progressing to symptomatic infection . Exploration of immunological events leading to Candida resistance or susceptibility has indicated the central role of the innate and adaptive immune systems, the relative contribution of which may vary depending on the site of the primary infection . Nevertheless, acquired resistance to infection results from the development of Th1 responses . Cytokines produced by Thl cells activate phagocytic cells to a candidacidal state . In contrast, cytokines produced by Th2 cells inhibit Th1 development and deactivate phagocytic effector cells . Because reciprocal influences have been recognized between innate and adaptive Th immunity, it appears that an integrated immune response determines the life-long commensalism of the fungus at the mucosal level, as well as the transition from mucosal saprophyte to pathogen.

Yakugaku Zasshi, 2000 Aug, 120(8), 715 - 9
{Prophylactic efficacy of a basidiomycetes preparation AHCC against lethal opportunistic infections in mice}; Ishibashi H et al.; The prophylactic effects of a Basidiomycetes preparation, AHCC, against experimental opportunistic infections were investigated in leukopenic mice . In cyclophosphamide-induced leukopenic mice, oral or intraperitoneal administration of the AHCC at doses of 1000 or 50 mg/kg/day, respectively, for 4 consecutive days prior to Candida albicans infection significantly prolonged the survival periods of the infected mice, and decreased the viable counts of C . albicans cells recovered from their kidneys . Similarly, the oral treatment with AHCC protected mice from lethal infection with Pseudomonas aeruginosa and intraperitoneal one also protected mice from infection with methicillin-resistant Staphylococcus aureus (MRSA) . These results suggest a potential usefulness of the AHCC as a prophylactic agent for the management of patients with opportunistic infections.

Ann Vasc Surg, 2000 Jul, 14(4), 370 - 5
Binding kinetics of triclosan (Irgasan) to alloplastic vascular grafts: an in vitro study; Hernandez-Richter T et al.; The aim of this study was to investigate the binding kinetics of triclosan (Irgasan) to alloplastic vascular grafts and to examine its antimicrobial activity against various microbial pathogens in vitro . Vascular grafts made by Intergard (Intervascular), Fluoropassiv (Vascutek), and Gore-tex (Gore) were examined . Grafts were incubated in 10 g/L triclosan (Irgasan), dried, sterilized, and incubated in RPMI medium . One-centimeter segments of the grafts were resected under sterile conditions at intervals of minutes, then hours, followed by days and up to 4 weeks . Samples were stored frozen at -20 degrees C for the measurement of triclosan bound to the vascular graft by high-performance liquid chromatography (HPLC) . The binding kinetics under perfusion conditions were determined for Intergard grafts, which were perfused with 50 mL of nutrient medium for 24 hr . Samples were taken at various time intervals for the measurement of triclosan . The antimicrobial activity of triclosan against Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans as well as Enterococcus faecium was determined . Triclosan effectively binds to vascular graft without the use of intermediate binding substances . It stayed on the graft for the duration of 4 weeks . Under both static and perfusion conditions, the binding kinetics are similar . Triclosan binds most effectively to Intergard grafts, less so to Fluoropassiv grafts, and not at all to Gore-tex material . Antimicrobial activity of triclosan is very effective against S . aureus and E . faecium but not against P . aeruginosa.

Eur J Immunol, 2000 Jul, 30(7), 1894 - 901
In vivo reduction of telomere length in human antigen-reactive memory T cells; Burns JB et al.; There is a reduction in the average telomere lengths of CD4+ "memory" T cells, defined by the CD45RO+ phenotype, compared to CD54RA+ "naive" T cells . However, other studies suggest that telomerase activity often is sufficient to maintain the telomere length of certain B and T cell populations following immune activation in vivo . Thus it is uncertain whether genuine memory CD4+ T cells, defined by an immune response to specific recall antigens, would display telomeres of reduced length, or whether telomere size would be maintained . Therefore, we examined the telomere lengths of T cells responding to two common recall antigens, tetanus toxoid and Candida albicans . Telomere terminal restriction fragment length was assessed by Southern blots or by flow cytometry following in situ hybridization with telomere-specific peptide nucleic acid probes . For the five subjects tested, the Candida- or tetanus-reactive memory T cell populations demonstrated a significant reduction of telomere length even when compared to the phenotypically defined memory CD45RO+ T cell populations isolated from peripheral blood mononuclear cells . This finding suggests that telomerase activity does not fully compensate for the effects of in vivo activation and proliferation of some antigen-specific CD4+ T cell populations . This may contribute to immune senescence.

J Ethnopharmacol, 2000 Aug, 71(3), 377 - 82
Anti-microbial activity of a new vaginal contraceptive NIM-76 from neem oil (Azadirachta indica); SaiRam M et al.; Efficacy of NIM-76, a spermicidal fraction from neem oil, was investigated for its antimicrobial action against certain bacteria, fungi and Polio virus as compared to whole neem oil . The NIM-76 preparation showed stronger anti-microbial activity than the whole neem oil . It inhibited growth of various pathogens tested including Escherichia coli and Kleibsiella pneumoniae which were not affected by the whole neem oil . NIM-76 also exhibited antifungal activity against Candida albicans and antiviral activity against Polio virus replication in vero cell lines . It also protected mice from systemic candidiasis as revealed by enhanced % survival and reduced colony forming units of C . albicans in various tissues . This shows that NIM-76 has a potent broad spectrum anti-microbial activity.

J Ethnopharmacol, 2000 Aug, 71(3), 479 - 82
Effect of Inula viscosa extract on chitin synthesis in dermatophytes and Candida albicans; Maoz M et al.; An antimycotic effect of an extract from Inula viscosa leaves was demonstrated affecting chitin synthesis in dermatophytes and Candida albicans . The antimycotic effect was compared to the effect caused by miconazole nitrate--an antifungal drug . The inhibition effect on chitin synthesis was not correlated to the extent of growth inhibition caused by the antifungal agents: both miconazole nitrate and the I . viscosa extract inhibited the growth of dermatophytes and C . albicans . Miconazole nitrate did not affect chitin synthesis--except for M . canis--whereas I . viscosa extract caused a significant decline in chitin content.

J Bacteriol, 2000 Sep, 182(17), 4899 - 905
Role of a Candida albicans P1-type ATPase in resistance to copper and silver ion toxicity; Riggle PJ et al.; Copper ion homeostasis is complicated in that copper is an essential element needed for a variety of cellular processes but is toxic at excess levels . To identify Candida albicans genes that are involved in resistance to copper ion toxicity, a library containing inserts of C . albicans genomic DNA was used to complement the copper sensitivity phenotype of a Saccharomyces cerevisiae cup1Delta strain that is unable to produce Cup1p, a metallothionein (MT) responsible for high-level copper ion resistance . A P1-type ATPase (CPx type) that is closely related to the human Menkes and Wilson disease proteins was cloned . The gene encoding this pump was termed CRD1 (for copper resistance determinant) . A gene encoding a 76-amino-acid MT similar to higher eukaryotic MTs in structure was also cloned, and the gene was termed CRD2 . Transcription of the CRD1 gene was found to increase upon growth with increasing copper levels, while the CRD2 mRNA was expressed at a constant level . Strains with the CRD1 gene disrupted were extremely sensitive to exogenous copper and failed to grow in medium containing 100 microM CuSO(4) . These crd1 strains also exhibited increased sensitivity to silver and cadmium, indicating that Crd1p is somewhat promiscuous with respect to metal ion transport . Although strains with the CRD2 gene disrupted showed reduced growth rate with increasing copper concentration, the crd2 mutants eventually attained wild-type levels of growth, demonstrating that CRD2 is less important for resistance to copper ion toxicity . Crd1p is the first example of a eukaryotic copper pump that provides the primary source of cellular copper resistance, and its ability to confer silver resistance may enhance the prevalence of C . albicans as a nosocomial pathogen.

New Microbiol, 2000 Jul, 23(3), 329 - 37
Candida albicans strain differentiation in complete denture wearers; Abu-Elteen KH; Strain differentiation of 66 clinical isolates of Candida albicans obtained from healthy dentate and complete denture wearers was performed . Resistogram method based on differences in the resistance of C . albicans isolates to sodium selenite, boric acid, cetrimide, sodium periodate and silver nitrate was used for strain differentiation . Of the 32 potential strains that can be distinguished, 14 different resistogram strains of C . albicans were found among the 66 isolates tested . Strain-C--was the most predominant (24.3% of total isolates), while strain A-CDE was the least predominant (1.5%) . The results showed no particular association of certain strains with Candida infections in complete denture wearers . Sensitivity to antifungal agents showed that isolates from different strains were most sensitive to amphotericin B and nystatin and least sensitive to miconazole.

Nippon Ishinkin Gakkai Zasshi, 2000, 41(3), 157 - 60
{Learning from fungus allergy in atopic dermatitis patients}; Terui T et al.; It has been recognized that there are considerable variations in their skin reactivity to environmental allergens as well as in immunoreactivities, even in AD patients with similar signs and symptoms . Some AD patients have high serum IgE antibody levels, while others show low levels . There are also differences in the kinds of triggering factors that are related to the development and maintenance of AD, e.g., allergic or non-allergic . Even among AD patients with high titers of serum IgE antibodies, the kinds and number of allergens involved in the exacerbation of AD are different and can change with time . The types of the underlying allergic reactions vary as well, i.e., some show immediate reactions, while others show delayed type hypersensitivity responses to environmental allergens . Thus, even AD patients diagnosed by the established criteria may have remarkably different backgrounds . When we looked over our published data, we noticed that there were differences in levels of IgE RAST and skin reactions between AD with atopic respiratory diseases (ARD) and pure AD without ARD . Levels of IgE RAST against airborne allergens, which come into the body mainly through the respiratory tract, were higher in AD with ARD, while those against allergens such as Candida albicans and Malassezia furfur, which can colonize on the skin, were higher in pure AD . In addition to these Th2-mediated immunological abnormalities, Th1-mediated DTH reaction and lymphocyte proliferation indices against airborne allergens were remarkably low in AD with ARD, whereas those against Candida albicans and Malassezia furfur were relatively preserved, although they were lower than those found in normal subjects . We understand from these findings that routes of allergen entry are important for the outcome of the resultant allergic reactions . This point of view is important answering questions such as how AD develops and how it can be prevented from the insults of each allergen.

Nippon Ishinkin Gakkai Zasshi, 2000, 41(3), 149 - 55
{The role of fungal allergy in bronchial asthma}; Akiyama K; Fungus is known to be one of the causative allergens inducing bronchial asthma as are housedustmites, pollen and pet dander . Outdoor airborne fungi such as Cladosporium, Alternaria, Penicillium and Aspergillus are important inducing IgE antibody formation . In addition to these common fungi, the indoor fungi Aspergillus restrictus, Neurospora and Eurotium are important allergenic fungi which have recently been identified . The yeast Candida albicans, is a common commensal of the human oral and vaginal mucosae and gastrointestinal tract and part of the normal flora, is known as one of the main allergens causing bronchial asthma . We examined the allergenicity of mannan (Mn) as a cell-wall constituent and acid protease (CAAP) as a secreted enzyme of C . albicans . We previously reported cases of atopic asthma caused by CAAP and stressed the role of CAAP as an important allergen in mucosal allergy to C . albicans 9) . The levels of the antibodies to these antigens in the sera of asthmatic patients who showed positive immediate intradermal response to crude C . albicans (n=86) were measured . Anti-Mn IgE and IgG antibody levels were measured by liquid-phase assay (AlaSTAT) . Anti-CAAP and anti-crude C . albicans IgE and IgG antibody levels were measured by RAST and AlaSTAT . Anti-Mn A and anti-Mn B IgE antibody titers were strongly correlated (r=0.87), while anti-Mn A and anti-CAAP IgE titers were not correlated . However, all of the anti-Mn A IgE positive sera and all of the anti-CAAP IgE positive sera were positive for IgE to crude-C . albicans . This indicates that both Mn and CAAP are C . albicans-related allergens . Titers of IgG antibodies to Mn A and crude C . albicans were highly correlated (r=0 . 90) . Results of inhibition assays performed using other fungal antigens as inhibitors showed that Mn is a cross reactive allergen among different fungi and that CAAP is a C . albicans specific allergen causing human mucosal allergic reaction.

Immunopharmacology, 2000 Jul 20, 48(2), 145 - 56
Differential effects of buprenorphine and morphine on immune and neuroendocrine functions following acute administration in the rat mesencephalon periaqueductal gray; Gomez-Flores R et al.; The effects of the mu-opioid receptor agonists buprenorphine and morphine on immune and neuroendocrine functions through acute action in the rat mesencephalon periaqueductal gray (PAG) were evaluated . Buprenorphine is an analgesic recently approved for the treatment of drug dependency . In this study, it was shown that injection of an equianalgesic dose of buprenorphine (related to morphine) into the ventral-caudal PAG did not alter splenic NK cell, T cell, and macrophage functions, whereas morphine significantly (p<0.001) suppressed splenic NK cell cytotoxic activity (14-50% reduction), splenic and thymic T cell proliferation to concanavalin A (Con A, 43-76% reduction), antiTCR (T cell receptor) (85% reduction) and IL-2 (36-48% reduction), and macrophage functions including nitric oxide (36-41% reduction) and TNF-alpha production (26%), and phagocytosis of Candida albicans (39%) . In addition, buprenorphine was associated with significant (p<0.0001) reductions in adrenocorticotropic hormone (ACTH) and corticosterone (CSO) plasma levels, without altering norepinephrine (NE) and serotonin splenic dialysate levels . In contrast, morphine significantly (p<0.0001) increased glucocorticoid and catecholamine levels in plasma and spleen dialysates, respectively . These results indicated that buprenorphine did not activate either the hypothalamic-pituitary-adrenal (HPA) axis with glucocorticoid release, or the sympathetic nerve (SNS) activity with bioamine production, and was not associated with immunosuppression . The lack of effects of buprenorphine on neuroendocrine systems may be related to its partial agonist properties, the absence of effects on immune system function, and may be associated with the reduction in craving observed in addictive disorders.

Vojnosanit Pregl, 2000 Mar-Apr, 57(2), 187 - 90
{Analysis of the results of mycological examination of skin samples from 1997 to 1998}; Bulajic et al.; Mycological findings of 2,447 skin specimens were analyzed . The aim of the study was to estimate the methodology for mycological examination, frequency of particular fungal species isolation and localization on the skin . Fungi were found in 591 specimens: 53.64% microscopically and isolated in cultures, 31.47% only microscopically and 14.89% only isolated in cultures . Total number of isolated strains was 405 (356 strains grew on Sabouraud agar with antibiotics and 272 on mycobiotic agar) . The most frequently isolated species were: Trichophyton mentagrophytes (27.9%), Trichophyton rubrum (20%) and Candida albicans (9.14%) . The most frequent localizations on skin where fungi had been found were foot (41.47%), toe-nail (22.06%) and finger-nail (10.88%) . Sabouraud agar is more reliable for isolation of dermatophytes and mycobiotic agar for Candida species . Usage of both media for primoisolation provides better and faster identification of fungi causing dermatomycoses . Comparing our with other studies, the patterns of fungal appearance in our population had changed and the number of nail specimens, where fungi were found had increased.

Braz J Infect Dis, 2000 Jun, 4(3), 113 - 8
Epidemiology and treatment of hematogenous candidiasis: a Brazilian perspective; Colombo AL; Hematogenous candidemia is an increasingly frequent problem among patients who are immunosuppressed, receiving parenteral nutrition and/or antibiotics, or who have invasive medical devices such as indwelling catheters . In Brazil, Candida albicans was responsible for 53/145 (37%) of candidemia in 6 different tertiary care hospitals . The most common non-albicans species were C . parasilosis (25%), C . tropicalis (24%), C . rugosa (5%) and C . glabrata (4%) . The main risk factors for infection were antibiotic use and the presence of a central venous catheter . The main risk factors for mortality were patient age (older patients at risk) and not removing the catheter . Because of the great number of non-albicans species and varied degrees of antifungal drug sensitivity, laboratory identification and sensitivity testing is very important . All patients with documented candidemia should be treated . Drugs to be used are amphotericin B and/or fluconazole . Fluconazole resistance is not yet a problem in Brazil, perhaps because it is rarely used as prophylaxis due to its high cost . Intravenous catheters should be removed immediately if the patient has a short term catheter, or if the patient is clinically unstable due to the infection and has a long term catheter in place.

Braz J Infect Dis, 2000 Jun, 4(3), 108 - 12
Prevention of systemic mycoses in patients who are not neutropenic: should we do it? Can we do it?
Graybill JR.
It has been well documented that serious fungal infections may cause death in 5% to 10% of patients in certain high risk groups, such as those undergoing lung, pancreas, or liver transplantation . Patients in intensive care units, such as those with underlying severe disease, multi-organ fungal infection, those with catheters, those on broad spectrum antibacterial agents, and those in renal failure are also at risk and may be candidates for antifungal prophylaxis . However, recommendations regarding the use of antifungal drugs for prophylaxis in non-neutropenic patients are unclear . Several clinical trials in transplant recipients have supported the use of fluconazole for prophylaxis, particularly in liver transplantation, though the data are too few to permit generalized conclusions for all organ transplant recipients . There is also a trial in which antifungal prophylaxis has been successful after gut perforation . However, there are also reports in which high doses of fluconazole have not reduced fungal infection . The appropriate circumstances for prophylaxis are still undergoing definition . It is the author's opinion that effective prophylaxis will become more problematic in the future . In a year or two, once the drug becomes generic, the price of fluconazole will fall dramatically . A sharp increase in use is likely to occur, and is likely to be followed by increasing fluconazole resistance in both Candida albicans and non-albicans colonization and infections . The situation is similar to the consequences of widespread fluconazole use in AIDS patients . The best methods to delay resistance include strict handwashing, careful control of antibacterials, restricting fluconazole use to those situations where it has been most clearly shown to be beneficial, and carefully monitoring patients in intensive care units.

J Antimicrob Chemother, 2000 Aug, 46(2), 291 - 5
Development of simultaneous resistance to fluconazole in Candida albicans and Candida dubliniensis in a patient with AIDS; Ruhnke M et al.; In this report, we describe a patient with recurrent episodes of oral candidosis who finally suffered from fluconazole-refractory oral and oesophageal candidosis . The patient was monitored for 4 years until his death from AIDS . During the observation period, persistent colonization with both Candida albicans and Candida dubliniensis was observed . From the appearance of the first episode of oral candidosis, the patient was treated with fluconazole for 18 months . The infection became unresponsive to fluconazole 400 mg/day . In vitro susceptibility testing revealed the development of resistance to fluconazole in C . albicans and C . dubliniensis . Molecular typing confirmed the persistence of the same C . albicans and C . dubliniensis strains which developed resistance after up to 3 years of asymptomatic colonization . This observation demonstrates that Candida spp . other than C . albicans may develop resistance to fluconazole in a patient who is repeatedly exposed to the drug.

J Antimicrob Chemother, 2000 Aug, 46(2), 199 - 203
Efflux-mediated resistance to fluconazole could be modulated by sterol homeostasis in Saccharomyces cerevisiae; Kontoyiannis DP; Saccharomyces cerevisiae has long been used as a model organism in the study of the ergosterol pathway and its inhibitors . The Pdr5 protein (Pdr5p), an ATP binding cassette transporter, plays an important role in active efflux of azole antifungals and therefore in azole sensitivity and resistance in S . cerevisiae . We have identified the Fluconazole Dominant Resistance-1 (FDR-1) mutant, which has a single dominant mutation conferring high-level resistance to fluconazole . FDR-1 has been found to be an activated allele of the Pleiotropic Drug Resistance-1 (PDR-1) gene (termed PDR1-100) and to upregulate PDR5 transcription . Resistance of PDR1-100 to fluconazole decreased in the background of mutations known to affect sterol homeostasis . Hence, the resistance to fluconazole of PDR1-100 was paradoxically decreased in an erg3 PDR1-100 double mutant . The erg3 mutants are resistant to azoles and accumulate 14-methyl-fecosterol instead of ergosterol in the presence of azoles . These results reinforce the emerging evidence in both S . cerevisiae and Candida albicans that sterols could serve as substrates for Pdr5p for transport across membranes.

J Antimicrob Chemother, 2000 Aug, 46(2), 191 - 7
Modulation of fluconazole sensitivity by the interaction of mitochondria and erg3p in Saccharomyces cerevisiae; Kontoyiannis DP; We studied the effects of fluconazole, an ergosterol-depleting agent, in Saccharomyces cerevisiae, a genetically tractable fungus closely related to Candida albicans . The wild-type Saccharomyces strain was sensitive to fluconazole, but the isogenic cytoplasmic petite mutant (rho-) was resistant . The mechanism of resistance of rho- mutants appeared to involve uncoupling of oxidative phosphorylation . However, the petite strain with a mutation in cent5, 6 desaturase (erg3 rho-) was sensitive to fluconazole, in contrast to its erg3 rho+ counterpart . It is known that erg3 mutants are azole resistant through the accumulation of 14-methyl-fecosterol, a less toxic ergosterol intermediate . These results indicate that mitochondria function as important physiological partners with Erg3p in the accumulation of toxic sterol intermediates in the presence of azoles.

Microbiology, 2000 Aug, 146 ( Pt 8), 1881 - 9
Survival in experimental Candida albicans infections depends on inoculum growth conditions as well as animal host; Odds FC et al.; Evidence is presented that the growth medium used to prepare a CANDIDA: albicans challenge inoculum is a significant factor determining the ability of a fungus strain to gain an initial invasive hold immediately after injection into an animal host, and thus determining gross strain lethality . Three C . albicans strains, one known to be attenuated in virulence, were grown in two broth media and injected intravenously at different doses into female NMRI mice and male albino guinea pigs . For each fungus strain and challenge dose, survival was longer from inocula grown in a diluted, buffered peptone-based broth than from inocula grown in Sabouraud glucose broth . When animals were challenged intravenously with yeast doses adjusted to give the same mean survival time regardless of strain or growth medium, the progression of fungus tissue burdens (c . f.u . g(-1)) in kidneys, lungs, liver, spleen and brain samples was broadly similar for all three C . albicans strains but differed between the two animal hosts . The morphological form of C . albicans recovered from infected tissues differed at the level of both the fungus strain and the host tissue . Use of survival-standardized inocula provides a means of distinguishing differences in progression of experimental disseminated Candida infections that are related to the infecting strain from those related to the animal host.

Mol Microbiol, 2000 Jun, 36(6), 1250 - 64
Morphogenesis in Aspergillus nidulans requires Dopey (DopA), a member of a novel family of leucine zipper-like proteins conserved from yeast to humans; Pascon RC et al.; DopA is the founding member of a novel protein family required for correct cell morphology and spatiotemporal organization of multicellular structures in the filamentous fungus Aspergillus nidulans . DopA homologues from Saccharomyces cerevisiae (Dop1), Candida albicans, Caenorhabditis elegans, Rattus norvegicus and Homo sapiens have been identified from genome sequencing projects . S . cerevisiae DOP1 is essential for viability and, like DopA, affects cellular morphogenesis . dopA encodes a large protein (207 kDa) containing several putative domains, including three leucine zipper-like domains . Strains with either the temperature-sensitive dopA1(ts) allele, which alters one of the leucine zippers, or the null deltadopA allele, had abnormal morphology of the vegetative hyphae, delayed and asynchronous initiation of asexual development, aberrant morphogenesis of the conidiophore and an early block in the sexual cycle . The expression patterns of key transcriptional regulators of the asexual and sexual cycle (brlA, abaA and steA) are altered in a deltadopA background, suggesting that DopA functions upstream in the developmental pathway . Double mutant analysis showed that dopA interacts genetically with constitutively active and inactive forms of A . nidulans Aras to modulate hyphal morphogenesis and asexual development.

FEMS Microbiol Lett, 2000 Aug 15, 189(2), 225 - 32
Cellular actin is affected by interaction with Candida albicans; Tsarfaty I et al.; Attachment of Candida albicans, an important opportunistic pathogen, to host tissues is an initial step in the development of the infection . The events occurring in the fungal and in the host cells after interaction are poorly understood . In this study we concentrated on the events occurring in the mammalian cells after the interaction with Candida, with emphasis on the cytoskeleton actin . Human cell line cells (HEp2) were exposed to C . albicans or C . albicans-secreted material (culture filtrate) (actin-rearranging Candida-secreted factor, arcsf) . The HEp2 cells were examined for cellular changes using confocal laser microscopy (CLSM), transmission and scanning electron microscopy (TEM and SEM) . The CLSM studies, using fluorescein isothiocyanate-labeled C . albicans and rhodamine phalloidin actin staining, revealed yeasts adhering to the HEp2 cells or internalized into the cells, with actin surrounding the fungi . Furthermore, actin rearrangement from filamentous network to actin aggregates was noticed . Interaction between the HEp2 cells and C . albicans could be demonstrated also by SEM and TEM after a 2-4-h exposure of the cells to the fungus . Yeasts and hyphae were found attaching to the surface and within the cells . CLSM studies revealed that exposure of HEp2 cells to arcsf was also followed by cellular actin rearrangement, reduced membrane ruffling and decreased cellular motility . The effect was dose- and time-dependent . All these data indicate that the interaction of Candida with HEp2 cells involves signaling events and affects the cellular actin.

J Biol Chem, 2000 Oct 20, 275(42), 32901 - 5
Identification of a novel inhibitor specific to the fungal chitin synthase . Inhibition of chitin synthase 1 arrests the cell growth, but inhibition of chitin synthase 1 and 2 is lethal in the pathogenic fungus Candida albicans; Sudoh M et al.; As in Saccharomyces cerevisiae, the pathogenic fungus Candida albicans harbors three chitin synthases called CaChs1p, CaChs2p, and CaChs3p, which are structurally and functionally analogous to the S . cerevisiae ScChs2p, ScChs1p, and ScChs3p, respectively . In S . cerevisiae, ScCHS1, ScCHS2, and ScCHS3 are all non-essential genes; only the simultaneous disruption of ScCHS2 and ScCHS3 is lethal . The fact that a null mutation of the CaCHS1 is impossible, however, implies that CaCHS1 is required for the viability of C . albicans . To gain more insight into the physiological importance of CaCHS1, we identified and characterized a novel inhibitor that was highly specific to CaChs1p . RO-09-3143 inhibited CaChs1p with a K(i) value of 0.55 nm in a manner that was non-competitive to the substrate UDP-N-acetylglucosamine . RO-09-3143 also hampered the growth of the C . albicans cells with an MIC(50) value of 0.27 microm . In the presence of RO-09-3143, the C . albicans cells failed to form septa and displayed an aberrant morphology, confirming the involvement of the C . albicans Chs1p in septum formation . Although the effect of RO-09-3143 on the wild-type C . albicans was fungistatic, it caused cell death in the cachs2Delta null mutants but not in the cachs3Delta null mutants . Thus, it appears that in C . albicans, inhibition of CaChs1p causes cell growth arrest, but simultaneous inhibition of CaChs1p and CaChs2p is lethal.

J Nat Prod, 2000 Jul, 63(7), 911 - 4
Thalicosides A1-A3, minor cycloartane bisdesmosides from Thalictrum minus; Gromova AS et al.; Three new cycloartane bisdesmosides, two of which are based on a new genin, were isolated from the above-ground parts of Thalictrum minus . Thalicosides A1-A3 (1-3) were characterized as 3-O-beta-D-galactopyranosyl-29-O-beta-D-glucopyranosyl-3beta,16beta++ +, 29-trihydroxy-22(S),25-epoxycycloartane (1); 3-O-alpha-L-arabinopyranosyl-29-O-beta-D-glucopyranosyl-3beta,1 6beta, 29,22(S)-tetrahydroxycycloart-24-ene (2); and 3-O-alpha-L-arabinopyranosyl-29-O-beta-D-glucopyranosyl-3beta,1 6beta, 29-trihydroxy-22(S),25-epoxycycloartane (3), respectively . The structural assignments of these new compounds were based on interpretation of spectroscopic data . Thalicoside A2 showed in vitro inhibition of the fungus Candida albicans and also activity against Staphylococcus aureus.

Yeast, 2000 Aug, 16(11), 1045 - 51
Identification of a Candida albicans homologue of the PHO85 gene, a negative regulator of the PHO system in Saccharomyces cerevisiae; Miyakawa Y; In a screen for the protein kinase genes of the human pathogenic yeast Candida albicans, a putative homologue (CaPHO85) of PHO85, a negative regulator of the PHO system of Saccharomyces cerevisiae, which is one of the cyclin-dependent protein kinases (CDKs), was isolated . An open reading frame (ORF) of this gene was identified encoding a predicted protein of 326 amino acids with a calculated molecular weight of 37.6 kDa . The amino acid sequence is highly homologous to S . cerevisiae Pho85 (62% identity) and its Aspergillus nidulans homologue (70% identity), but less homologous to Cdc28 (50% identity) of S . cerevisiae and to its C . albicans homologue CaCdc28 (49% identity), both of which are also CDK . The coding region for the C . albicans gene was interrupted by an intron of 81 nucleotides near the sequence encoding the N-terminal region, similarly to the case of the S . cerevisiae PHO85 gene . Alignment of CaPho85 with various yeast CDKs revealed that most of the domains for ATP-binding and protein kinase activity are conserved among fungal species . Southern blot analysis indicated that CaPHO85 is most likely present as a single copy gene . This gene complemented the pho85 mutation of S . cerevisiae by transformation.

Hum Exp Toxicol, 2000 Apr, 19(4), 230 - 43
Preclinical development of keliximab, a Primatized anti-CD4 monoclonal antibody, in human CD4 transgenic mice: characterization of the model and safety studies; Bugelski PJ et al.; The preclinical safety assessment of biopharmaceuticals necessitates that studies be conducted in species in which the products are pharmacologically active . Monoclonal antibodies are a promising class of biopharmaceuticals for many disease indications; however, by design, these agents tend to have limited species cross-reactivity and tend to only be active in primates . Keliximab is a human-cynomolgus monkey chimeric (Primatized) monoclonal antibody with specificity for human and chimpanzee CD4 . In order to conduct a comprehensive preclinical safety assessment of this antibody to support chronic treatment of rheumatoid arthritis in patients, a human CD4 transgenic mouse was used for chronic and reproductive toxicity studies and for genotoxic studies . In addition, immunotoxicity studies were conducted in these mice with Candida albicans, Pneumocystis carinii and B16 melanoma cells to assess the effects of keliximab on host resistance to infection and immunosurveillance to neoplasia . The results of these studies found keliximab to be well tolerated with the only effects observed being related to its pharmacologic activity on CD4+ T lymphocytes . The use of transgenic mice expressing human proteins provides a useful alternative to studies in chimpanzees with biopharmaceutical agents having limited species cross-reactivity.

J Clin Microbiol, 2000 Aug, 38(8), 3016 - 21
Simple and rapid detection of Candida albicans DNA in serum by PCR for diagnosis of invasive candidiasis; Wahyuningsih R et al.; A rapid and sensitive PCR assay for the detection of Candida albicans DNA in serum was established . DNA from human serum samples was purified using the QIAamp blood kit, which proved to be a fast and simple method for isolating minute amounts of Candida DNA from clinical specimens for diagnosis of invasive candidiasis . Universal primer sequences used in the PCR assay are derived from the internal transcribed spacer rRNA gene of fungi, whereas the biotinylated hybridization probe used in a DNA enzyme immunoassay (DEIA) binds specifically to C . albicans DNA . The sensitivity of this PCR-DEIA method is very high; the detection limit for genomic Candida DNA is one C . albicans genome per assay . Blood from uninfected and infected persons, ranging from healthy volunteers, patients with mucocutaneous infections, and patients at risk to develop a systemic Candida infection to patients with an established systemic candidiasis, was analyzed for the presence of C . albicans to diagnose fungal infection . Candida DNA could not be detected in sera of 16 culture-negative controls and from 11 nonsystemic candidal infections by PCR or DEIA . Blood cultures from patients at risk were all negative for Candida, whereas all blood cultures from systemic candidiasis patients were positive . However, Candida DNA could be detected by PCR and DEIA in the serum from three out of nine patients who were at risk for a systemic infection and in the serum of all seven patients who had already developed an invasive Candida infection . PCR is more sensitive than blood culture, since some of the patients at risk for invasive yeast infection, whose blood cultures were all negative for Candida, tested positive in the PCR amplification . These results indicate the potential value of PCR for detecting C . albicans in serum samples and for identifying patients at risk for invasive candidiasis.

J Clin Microbiol, 2000 Aug, 38(8), 2902 - 8
Rapid detection and identification of Candida, Aspergillus, and Fusarium species in ocular samples using nested PCR; Jaeger EE et al.; A protocol for the rapid detection of fungal DNA in ocular samples, derived from three species, Candida albicans, Aspergillus fumigatus, and Fusarium solani, has been developed . Two novel panfungal primers complementary to 18S rRNA sequences present in all three species were designed . Panfungal PCR was followed by three nested PCRs utilizing species-specific primers . PCR sensitivity ranged from 50 to 100 fg of free DNA and between one and two C . albicans organisms . In addition, we also developed a rapid and reliable DNA extraction protocol . This protocol minimized DNA loss during extraction, whilst removing compounds from vitreous and aqueous fluids that have previously been shown to have inhibitory effects on PCR . Preliminary results obtained after testing the protocol on three patient samples support culture results and medical history . However, one patient was PCR positive but culture negative, suggesting that the sensitivity of this protocol may exceed that of traditional culture techniques . This system, therefore, constitutes an additional protocol that may significantly aid patient management in cases where fungal endophthalmitis is suspected.

J Clin Microbiol, 2000 Aug, 38(8), 2893 - 6
Estimation of minimum sterol 14alpha-demethylation-inhibitory concentration of azoles in Candida yeasts using acetate-mediated growth inhibition: potential utility in susceptibility testing; Shimokawa O et al.; We have recently shown that 14alpha-demethylation-deficient cells of Candida albicans are subject to growth arrest by 0.24 M acetate in a yeast extract-peptone-glucose medium and that the minimum concentration of an azole antifungal agent required for total inhibition of sterol 14alpha-demethylation (MDIC for minimum demethylation-inhibitory concentration) is practically identical to its MIC determined in the acetate-supplemented medium (O . Shimokawa and H . Nakayama, Antimicrob . Agents Chemother . 43:100-105, 1999) . In the present study we estimated the MDICs of three different azoles (fluconazole, ketoconazole, and itraconazole) for strains of various Candida species using this method and compared them with the MICs determined in the corresponding acetate-free medium . The results demonstrated that the test strains were divided into two classes . One class of strains was characterized by tolerance to 14alpha-demethylation deficiency (MIC > MDIC) and consisted of strains of C . albicans, C . guilliermondii, C . kefyr, and C . tropicalis . The other class was intolerant to 14alpha-demethylation deficiency (MIC approximately MDIC) and comprised strains of C . glabrata, C . krusei, and C . parapsilosis . We also showed that replacement of the yeast extract-peptone-glucose medium with RPMI 1640 medium did not affect the results substantially . Furthermore, the 80% inhibitory concentration (IC(80)) in RPMI 1640 medium, recommended as a substitute for the conventional MIC in susceptibility testing, was found to be close to the MDIC.

J Clin Microbiol, 2000 Aug, 38(8), 2862 - 9
Relative abundance of oligosaccharides in Candida species as determined by fluorophore-assisted carbohydrate electrophoresis; Goins TL et al.; Fluorophore-assisted carbohydrate electrophoresis (FACE) is a straightforward, sensitive method for determining the presence and relative abundance of individual oligomannosyl residues in Candida mannoprotein, the major antigenic determinant located on the outer surface of the yeast cell wall . The single terminal aldehydes of oligomannosyl residues released by hydrolysis were tagged with the charged fluorophore 8-aminonaphthalene-1,3,6-trisulfonate (ANTS) and separated with high resolution on the basis of size by polyacrylamide gel electrophoresis . ANTS fluorescence labeling was not biased by oligomannoside length; therefore, band fluorescence intensity was directly related to the relative abundance of individual oligomannoside moieties in heterogeneous samples . FACE analysis revealed the major oligomannosides released by acid hydrolysis and beta-elimination of Fehling-precipitated mannan from Candida albicans, which were the same as those previously reported in studies based on mass and nuclear magnetic spectroscopic analysis . FACE was also amenable to the analysis of samples obtained by direct hydrolysis of whole yeast cells . Whole-cell acid hydrolysis and whole-cell beta-elimination of two isolates each of C . albicans, C . glabrata, C . krusei, C . lusitaniae, C . parapsilosis, C . rugosa, C . stellatoidea, and C . tropicalis resulted in oligomannoside gel banding patterns that were species and strain specific for the 16 isolates surveyed . Whereas some bands were specific for an individual isolate or species, other bands were shared by two or three species in various groupings . Differences in the mannoprotein composition of C . albicans A9 and four spontaneous cell surface mutants were also detected . Mannan "fingerprints," or banding pattern profiles, derived from the electrophoretic mobilities of individual bands relative to the migration of acid-hydrolyzed dextran (relative migration index) yielded profiles characteristic of individual isolates not revealed by standard assimilation and biochemical profiles . FACE represents an accessible, sensitive, and quantitative analytical tool enabling the characterization of yeast mannan complexity.

J Perinatol, 2000 Jul-Aug, 20(5), 335 - 7
Candida chorioamnionitis after serial therapeutic amniocenteses: a possible association; Rode ME et al.; BACKGROUND: Reduction amniocentesis is used in cases of severe polyhydramnios to decrease maternal discomfort and the risk of preterm labor . In a MEDLINE search (1966 to present, English language, keywords: amniocentesis, chorioamnionitis), no report of Candida chorioamnionitis after serial reduction amniocentesis exists . CASE: A 29-year-old primigravida with a history of four therapeutic amniocenteses for idiopathic polyhydramnios developed preterm labor at 30 and 5/7 weeks' gestation, rupture of membranes, and Candida albicans chorioamnionitis . Despite aggressive therapy with amphotericin B, the neonate succumbed to overwhelming systemic candidiasis . CONCLUSION: Serial amniocentesis may place patients at elevated risk for Candida chorioamnionitis and subsequent preterm delivery . Clinicians should consider early diagnostic amniocentesis in patients in preterm labor with a history of prior amniocentesis, and the routine Gram stain and culture of amniotic fluid.

Protein Expr Purif, 2000 Aug, 19(3), 343 - 9
Purification to homogeneity of Candida albicans glucosamine-6-phosphate synthase overexpressed in Escherichia coli; Sachadyn P et al.; The Candida albicans GFA1 gene encoding glucosamine-6-phosphate synthase, an enzyme of cell wall biosynthesis pathway in fungi and bacteria, recently an object of interest as a target for the chemotherapy of systemic mycoses, was PCR amplified and cloned to an Escherichia coli expression vector pET23b . The activity of the enzyme in the lysates from the overproducing E . coli strain was approximately 50-100 times higher than in the lysates from the control E . coli strain . This abundant overproduction allows to purify milligram amounts of the enzyme to homogeneity .

Planta Med, 2000 Jun, 66(5), 435 - 8
In vitro synergism between nyasol, an active compound isolated from Anemarrhena asphodeloides, and azole agents against Candida albicans; Iida Y et al.; The antifungal activity of nyasol (NYS) alone or with various antifungal agents was measured in vitro against Candida albicans, Aspergillus fumigatus, and Trichophyton mentagrophytes . NYS is a compound recently purified from a medicinal plant, Anemarrhena asphodeloides . Among 12 agents, miconazole (MCZ), ketoconazole (KCZ), clotrimazole (CTZ), and cerulenin showed marked synergistic effects against C . albicans . The fractional inhibition concentration (FIC) indices against 4 strains of C . albicans were 0.067-0.31 for MCZ plus NYS, 0.078-0.31 for KCZ plus NYS, and 0.098-0.13 for CTZ plus NYS . These values indicate the possibility of using NYS as an adjuvant to azole agents in the chemotherapy of candidiasis.

Obstet Gynecol, 2000 Aug, 96(2), 301 - 3
Fungal culture findings in cyclic vulvitis; Handa VL et al.; OBJECTIVE: To estimate the prevalence of fungal infection in cyclic vulvitis . METHODS: We retrospectively reviewed 40 cases of cyclic vulvitis . We examined the historic characteristics, physical findings, and laboratory results in this population, including the results of potassium hydroxide preparations of vaginal secretions and fungal cultures . RESULTS: The median age was 32 years and the mean duration of symptoms was 3.8 years . Thirty women (75%) reported prior antifungal therapies . Fungal cultures were positive in 24 of 39 (61.5%) . Candida albicans was the species isolated in 13 of 24 cases (54%) . Potassium hydroxide wet mounts contained evidence of fungal infection in 15 of 37 cases (40.5%) . The sensitivity of the potassium hydroxide preparation was only 61% . Potassium hydroxide preparations were more sensitive when the species isolated was C albicans . CONCLUSIONS: Many women with cyclic vulvitis have positive vaginal fungal cultures . Potassium hydroxide preparations of vaginal secretions are not sufficiently sensitive to exclude fungal infection in this setting, possibly because of the relatively high incidence of fungal species other than C albicans . Fungal culture should be considered in the evaluation of women with recurrent episodes of vulvar discomfort, even when potassium hydroxide wet mounts do not contain fungal elements.

Mycoses, 2000, 43(3-4), 119 - 23
Phenotypic variation and antifungal susceptibility patterns of Candida albicans strains isolated from neutropenic patients; Kiraz N et al.; The aim of this study was to investigate the relationship between phenotypes of Candida albicans strains isolated from clinical specimens and the susceptibility of the strains to three antifungal agents, fluconazole, amphotericin B and flucytosine . Oropharyngeal, gastrointestinal and urogenital tract specimens were collected from 122 neutropenic patients who had received no previous prophylactic treatment . Each of 122 C . albicans strains recovered was found to express one of the six phenotypes: smooth, fuzzy, irregular, star, ring and stipple . The mean minimum inhibitory concentrations (MICs) of fluconazole was consistently higher for C . albicans strains expressing the stipple phenotype . The mean MICs for the six phenotypes of C . albicans strains ranged between 1.22 and 7.94 micrograms ml-1 for fluconazole, 0.99 and 2.55 micrograms ml-1 for amphotericin B and 1.23 and 1.83 micrograms ml-1 for flucytosine . The antifungal susceptibility of the stipple phenotype requires attention, especially in patients who are clinically unresponsive to fluconazole chemotherapy or in cases of life-threatening C . albicans infections of immunocompromised hosts . Long-term use of fluconazole may explain the outcome of the resistant stipple phenotype.

Mycoses, 2000, 43(3-4), 109 - 17
Multicentric genetic study of Candida albicans isolates from non-neutropenic patients using MLEE typing: population structure and mode of reproduction; Arnavielhe S et al.; A mycological survey was conducted on non-neutropenic patients in three distinct intensive care units in two hospitals in Marseille (France) from November 1993 to November 1995 . Candida albicans positive cultures from 62 patients were included in this study . Every first isolate of each patient was typed by multilocus enzyme electrophoresis (MLEE) . The enzyme profiles obtained from 15 polymorphic loci were then compared . This analysis demonstrated a strong population differentiation of C . albicans infective strains within and between the different care units and confirmed the probable preponderant clonal mode of reproduction of this yeast.

Enferm Infecc Microbiol Clin, 2000 Mar, 18(3), 120 - 4
{Identification of yeast and sensitivity in vitro against different antifungal agents}; Muriel MA et al.; INTRODUCTION: The increasing incidence of Candida yeasts infections and its hospital and community repercussion (vaginal thrush), as well as the will to acquire the knowledge of the new antifungal that were launched to the therapeutic store, have motivated us to identify that type of yeasts from different sources, as well as to study their behaviour against the antifungal, using commercial procedures with easy clinical application . MATERIALS AND METHODS: An amount of 317 Candida yeasts were identified through commercial procedures (CHROMagar Candida and Auxacolor): 108 vaginals, 138 from ICU newborn children and 71 from ICU adults, while the antifungal drug susceptibilities was done to 199 of the isolated ones using another commercial procedure (Fungitest) . RESULTS AND CONCLUSIONS: Candida albicans is identified as the most frequent in both hospital and community samples (78.7 and 45.93%, respectively), followed by Candida glabrata (19.44 and 28.23%, respectively) . The sensitivity to amphotericin B and to 5-flucytosine was very high in every studied group, while sensitivity to imidazole derivatives depends on the samples source (lower sensitivity in the ICU newborn isolated ones) and the species (C . glabrata is less sensitive than C . albicans).

Clin Microbiol Rev, 2000 Jul, 13(3), 408 - 27
Applications of differential-display reverse transcription-PCR to molecular pathogenesis and medical mycology; Sturtevant J; The host-fungus interaction is characterized by changes in gene expression in both host and pathogen . Differential-display reverse transcription PCR (DDRT-PCR) is a PCR-based method that allows extensive analysis of gene expression among several cell populations . Several limitations and drawbacks to this procedure have now been addressed, including the large number of false-positive results and the difficulty in confirming differential expression . Modifications that simplify the reaction time, allow the use of minute quantities of RNA, or address unusual species- or gene-specific sequences have been reported . DDRT-PCR has been used to address biological questions in mammalian systems, including cell differentiation, cell activation, cell stress, and identification of drug targets . In microbial pathogenesis and plant pathogenesis, DDRT-PCR has allowed the identification of virulence factors, genes involved in cell death, and signaling genes . In Candida albicans, DDRT-PCR studies identified TIF-2, which may play a role in the upregulation of phospholipases, and the stress-related genes, CIP1 and CIP2 . In Histoplasma capsulatum and C . albicans, genes involved in the host-pathogen interaction, including a member of the 100-kDa family in Histoplasma and an ALS and 14-3-3 gene in Candida, were potentially identified by DDRT-PCR . Although very few reports have been published in medical mycology, studies in mammalian, nonfungal microbial, and plant pathogen systems are easily applied to basic questions in fungal pathogenesis and antifungal therapeutics.

Rev Esp Quimioter, 2000 Mar, 13(1), 60 - 3
{Susceptibility to fluconazole and itraconazole in isolates of Candida spp . from HIV-positive and HIV-negative patients}; Bernal S et al.; We studied the possible differences in the pattern of susceptibility to fluconazole and itraconazole in 393 isolates of Candida spp . from the oral cavity of HIV-positive patients and 102 isolates from HIV-negative patients with candidemia or candiduria . We used the broth microdilution method according to the NCCLS guidelines . We observed a decrease in the susceptibility to fluconazole in the group of HIV-positive patients in comparison to those who were HIV negative, especially in Candida albicans (MIC(90) 32 mg/l vs . 1 mg/l and Candida glabrata (MIC(90) 64 mg/l vs . 16 mg/l) . Furthermore, we did not find any resistant strains in the HIV-negative group . For itraconazole, the MIC(90) was two dilutions greater in the HIV-positive patients, except for C . albicans, which had a much higher MIC(90) (4 mg/l vs . 0.12 mg/.) . Therefore, the decrease in the susceptibility of Candida spp . in the HIV-positive patients must be taken into account when establishing a specific antifungal therapy.

Biochim Biophys Acta, 2000 Jul 19, 1486(2-3), 299 - 311
Arachidonic acid stimulates cell growth and forms a novel oxygenated metabolite in Candida albicans; Deva R et al.; Infection of human tissues by Candida albicans has been reported to cause the release of arachidonic acid (AA), eicosanoids and other proinflammatory mediators from host cells . Therefore, we investigated the interaction of this pathogen with AA . AA stimulated cell growth at micromolar concentrations when used as a sole carbon source . Moreover, it selectively inhibited the antimycin A-resistant alternative oxidase . {1-(14)C}AA was completely metabolised by C . albicans . Only one-seventh of the radioactivity metabolised was found in CO(2), whereas two-thirds occurred in carbohydrates suggesting a predominant role of the glyoxalate shunt of citrate cycle . About 1% of radioactivity was found in polar lipids including eicosanoids . A novel AA metabolite, which revealed immunoreactivity with an antibody against 3(R)-hydroxy-oxylipins, was identified as 3, 18-dihydroxy-5,8,11,14-eicosatetraenoic acid . Using immunofluorescence microscopy, endogenous 3(R)-hydroxy-oxylipins were found in hyphae but not in yeast cells . Such compounds have recently been shown to be connected with the sexual stage of the life cycle of Dipodascopsis uninucleata . Together, we propose that infection-mediated release of AA from host cells may modulate cell growth, morphogenesis and invasiveness of C . albicans by several modes . A better understanding of its role is thus promising for novel approaches towards the treatment of human mycoses.

J Cardiovasc Surg (Torino), 2000 Apr, 41(2), 317 - 9
Implantation of a composite bifurcated cryopreserved aorto-iliac-femoral homograft in a patient with Candida albicans endocarditis; Abad C et al.; A 35 year old woman, cocaine addict, suffered Candida albicans aortic valve endocarditis complicated with embolisation of infected vegetations in the distal abdominal aorta . She underwent successful staged aortic valve replacement followed by transaortic and transfemoral thrombectomy . One month later an arteriogram disclosed partial occlusion of the left iliac artery, bilateral aneurysmal degeneration of both iliac arteries and right iliac artery-right iliac vein fistula . She was operated again, performing re-laparotomy and re-exploration . A composite bifurcated cryopreserved homograft was implanted end-to-side between the infrarenal abdominal aorta, right external iliac artery and left common femoral artery . The right iliac artery-iliac vein fistula was obliterated with suture . The patient had an uneventful recovery but a relapsing arterio-venous fistula was diagnosed by arteriography . Three months later she underwent percutaneous transluminal closure of the reopened fistula . At present, 17 months after the implantation of the homograft, the patient is symptom-free, on antifungal agents and with arteriographic and cinical evidence of a well-functioning arterial homograft.

Clin Immunol, 2000 Aug, 96(2), 162 - 7
Deficiency of human complement factor I associated with lowered factor H; Naked GM et al.; Deficiencies of factor I and/or factor H result in an increased consumption of C3 and higher susceptibility to recurrent infections . Here we describe a case of human factor I deficiency and lowered factor H levels . C3 concentration was 50% lower than normal, the classical pathway-dependent hemolytic activity was reduced to almost 30% of normal, and alternative pathway-dependent activity was completely absent . The killing by peripheral leukocytes of Candida albicans treated with deficient serum and the production of complement-dependent chemotactic factors were reduced in the proband's serum when compared with normal serum . Finally, we observed that C3 antigen present in the proband's serum has a different electrophoretic mobility than native C3 (most likely C3b), confirming the deregulation of complement activation due to the lack of regulatory proteins factors I and H . The impaired complement system described in this case, the first of its kind described in a Chile, explains the higher susceptibility to infections found in the proband .

Infect Immun, 2000 Aug, 68(8), 4391 - 8
beta-1,2-linked oligomannosides from Candida albicans bind to a 32-kilodalton macrophage membrane protein homologous to the mammalian lectin galectin-3; Fradin C et al.; beta-1,2-linked oligomannoside residues are present, associated with mannan and a glycolipid, the phospholipomannan, at the Candida albicans cell wall surface . beta-1,2-linked oligomannoside residues act as adhesins for macrophages and stimulate these cells to undergo cytokine production . To characterize the macrophage receptor involved in the recognition of C . albicans beta-1,2-oligomannoside we used the J774 mouse cell line, which is devoid of the receptor specific for alpha-linked mannose residues . A series of experiments based on affinity binding on either C . albicans yeast cells or beta-1,2-oligomannoside-conjugated bovine serum albumin (BSA) and subsequent disclosure with biotinylated conjugated BSA repeatedly led to the detection of a 32-kDa macrophage protein . An antiserum specific for this 32-kDa protein inhibited C . albicans binding to macrophages and was used to immunoprecipitate the molecule . Two high-pressure liquid chromatography-purified peptides from the 32-kDa tryptic digest showed complete homology to galectin-3 (previously designated Mac-2 antigen), an endogenous lectin with pleiotropic functions which is expressed in a wide variety of cell types with which C . albicans interacts as a saprophyte or a parasite.

Int Arch Allergy Immunol, 2000 Jul, 122(3), 195 - 9
IFN-gamma plays a dominant role in upregulation of Candida-specific IgE synthesis in patients with atopic dermatitis; Kimura M et al.; BACKGROUND: Although Candida albicans (CA) is known to induce Th1 clones that suppress IgE synthesis, serum IgE antibody against CA is often increased in atopic patients . This study aims to elucidate the mechanism of IgE synthesis against CA in atopic patients . METHODS: We measured the production of IL-4 and IFN-gamma by peripheral blood mononuclear cells (PBMCs) from atopic patients upon stimulation with CA and examined the correlation with the level of serum IgE antibody against CA . Results: The level of serum CA-specific IgE antibody (CA-IgE) was significantly higher in patients with atopic dermatitis (AD) than in patients with bronchial asthma (BA) (geometric mean = 3.6 vs . 0.27 U(A)/ml, p < 0.02) (U(A) = unit allergen), while there was no difference in the level of house dust mite-specific IgE antibody between them (67.6 vs . 87.1 U(A)/ml) . Although IL-4 production by PBMCs upon stimulation with CA in patients with AD was not significantly different from that in patients with BA (mean = 359.1 vs . 515.3 fg/ml), IFN-gamma production was significantly lower in the former than in the latter group (8.1 vs . 56.2 pg/ml, p < 0.001) . Consequently, the ratio of IL-4/IFN-gamma production was apparently higher in patients with AD than in those with BA, which corresponds to the difference between them in the level of serum CA-IgE . A significant negative correlation was seen in patients with AD between IFN-gamma production by CA-stimulated PBMCs and the level of serum CA-IgE (p < 0.05) . CONCLUSIONS: IgE synthesis against CA in atopic patients may be precipitated not by enhancing IL-4 production, but by reducing IFN-gamma secretion .

Biochim Biophys Acta, 2000 Jul 14, 1480(1-2), 132 - 44
Expression and characterization of protein geranylgeranyltransferase type I from the pathogenic yeast Candida albicans and identification of yeast selective enzyme inhibitors; Smalera I et al.; Protein geranylgeranyltransferase type I (GGTase I) is a heterodimeric zinc metalloenzyme catalyzing protein geranylgeranylation at cysteine residues present in C-terminal signature sequences referred to as CaaX (X=Leu) motifs . We have studied GGTase I as a potential antifungal target and recently reported its purification and cloning from the yeast Candida albicans (Ca GGTase I), an important human pathogen . Here, we report the high yield bacterial expression of Ca GGTase I by coexpression of maltose binding protein fusion proteins of both the alpha (Ram2p) and beta (Cdc43p) subunits . The cleaved and purified recombinant Ca GGTase I was demonstrated to be functional and structurally intact as judged by the presence of one equivalent of a tightly bound zinc atom and the near stoichiometric formation, isolation and catalytic turnover of a geranylgeranyl pyrophosphate-GGTase I complex . Kinetic analysis was performed with a native substrate protein, Candida Cdc42p, which exhibited significant pH dependent substrate inhibition, a feature not observed with other Ca GGTase I substrates . Prenyl acceptor substrate specificity was studied with a series of peptides in which both the CaaX motif, and the sequence preceding it, were varied . The prenyl acceptor K(M)s were found to vary nearly 100-fold, with biotinyl-TRERKKKKKCVIL, modeled after a presumably geranylgeranylated Candida protein, Crl1p (Rho4p), being the optimal substrate . A screen for inhibitors of Ca GGTase I identified compounds showing selectivity for the Candida versus human GGTase I . The most potent and selective compound, L-689230, had an IC(50) of 20 nM and >12,500-fold selectivity for Ca GGTase I . The lack of significant anti-Candida activity for any of these inhibitors is consistent with the recent finding that GGTase I is not required for C . albicans viability {R . Kelly et al., J . Bacteriol . 182 (2000) 704-713}.

Antimicrob Agents Chemother, 2000 Aug, 44(8), 2081 - 5
Quantitation of Candida albicans ergosterol content improves the correlation between in vitro antifungal susceptibility test results and in vivo outcome after fluconazole treatment in a murine model of invasive candidiasis; Arthington-Skaggs BA et al.; MIC end point determination for the most commonly prescribed azole antifungal drug, fluconazole, can be complicated by "trailing" growth of the organism during susceptibility testing by the National Committee for Clinical Laboratory Standards approved M27-A broth macrodilution method and its modified broth microdilution format . To address this problem, we previously developed the sterol quantitation method (SQM) for in vitro determination of fluconazole susceptibility, which measures cellular ergosterol content rather than growth inhibition after exposure to fluconazole . To determine if SQM MICs of fluconazole correlated better with in vivo outcome than M27-A MICs, we used a murine model of invasive candidiasis and analyzed the capacity of fluconazole to treat infections caused by C . albicans isolates which were trailers (M27-A MICs at 24 and 48 h, </=1.0 and >/=64 microg/ml, respectively; SQM MIC, </=1.0 microg/ml), as well as those which were fluconazole sensitive (M27-A and SQM MIC, </=1.0 microg/ml) and fluconazole resistant (M27-A MIC, >/=64 microg/ml; SQM MIC, 54 microg/ml) . Compared with the untreated controls, fluconazole therapy increased the survival of mice infected with a sensitive isolate and both trailing isolates but did not increase the survival of mice infected with a resistant isolate . These results indicate that the SQM is more predictive of in vivo outcome than the M27-A method for isolates that give unclear MIC end points due to trailing growth in fluconazole.

J Comput Aided Mol Des, 2000 Jul, 14(5), 495 - 506
Selectivity analysis of 5-(arylthio)-2,4-diaminoquinazolines as inhibitors of Candida albicans dihydrofolate reductase by molecular dynamics simulations; Gokhale VM et al.; A series of 5-(arylthio)-2,4-diaminoquinazolines are known as selective inhibitors of dihydrofolate reductase (DHFR) from Candida albicans . We have performed docking and molecular dynamics simulations of these inhibitors with C . albicans and human DHFR to understand the basis for selectivity of these agents . Study was performed on a selected set of 10 compounds with variation in structure and activity . Molecular dynamics simulations were performed at 300 K for 45 ps with equilibration for 10 ps . Trajectory data was analyzed on the basis of hydrogen bond interactions, energy of binding and conformational energy difference . The results indicate that hydrogen bonds formed between the compound and the active site residues are responsible for inhibition and higher potency . The selectivity index, i.e the ratio of I50 against human DHFR to I50 against fungal DHFR, is mainly determined by the conformation adapted by the compounds within the active site of two enzymes . Since the human DHFR active site is rigid, the compound is trapped in a higher energy conformation . This energy difference between the two conformations deltaE mainly governs the selectivity against fungal DHFR . The information generated from this analysis of potency and selectivity should be useful for further work in the area of antifungal research.

Oral Microbiol Immunol, 1999 Dec, 14(6), 364 - 70
Candida albicans triggers interleukin-6 and interleukin-8 responses by oral fibroblasts in vitro; Dongari-Bagtzoglou A et al.; Oral candidiasis is the most frequent opportunistic infection associated with an immunocompromised host . Production of proinflammatory cytokines, such as interleukin-6 (IL-6) and IL-8, by host cells in response to Candida albicans can be expected to have a major impact in the activation of immune effector cells against the invading microorganism . Using a human cell--C . albicans coculture model system, we determined that this microorganism can trigger secretion of these potent chemoattractant and proinflammatory cytokines by oral mucosal fibroblasts . This response varied depending on the infecting strain and required fungal viability, germination of yeast into hyphae and mannose-mediated direct contact between the host cell and Candida . The secretion of proinflammatory cytokines by oral mucosal fibroblasts in response to C . albicans suggests that these cells have the potential to enhance the host defense against this organism in vivo . This may have important implications in controlling fungal overgrowth in the oral cavity.

Science, 2000 Jul 14, 289(5477), 310 - 3
Induction of mating in Candida albicans by construction of MTLa and MTLalpha strains; Magee BB et al.; Although the diploid fungus Candida albicans, a human pathogen, has been thought to have no sexual cycle, it normally possesses mating-type-like orthologs (MTL) of both of the Saccharomyces cerevisiae mating-type genes (MAT) a and alpha . When strains containing only MTLa or MTLalpha were constructed by the loss of one homolog of chromosome 5, the site of the MTL loci, MTLa and MTLalpha strains mated, but like mating types did not . Evidence for mating included formation of stable prototrophs from strains with complementing auxotrophic markers; these contained both MTL alleles and molecular markers from both parents and were tetraploid in DNA content and mononucleate.

Science, 2000 Jul 14, 289(5477), 307 - 10
Evidence for mating of the "asexual" yeast Candida albicans in a mammalian host; Hull CM et al.; Since its classification nearly 80 years ago, the human pathogen Candida albicans has been designated as an asexual yeast . In this report, we describe the construction of C . albicans strains that were subtly altered at the mating-type-like (MTL) locus, a cluster of genes that resembles the mating-type loci of other fungi . These derivatives were capable of mating after inoculation into a mammalian host . C . albicans is a diploid organism, but most of the mating products isolated from a mouse host were tetrasomic for the two chromosomes that could be rigorously monitored and, overall, exhibited substantially higher than 2n DNA content . These observations demonstrated that C . albicans can recombine sexually.

Gac Med Mex, 2000 May-Jun, 136(3), 193 - 9
{Prevalence and sensitivity of Candida albicans in cultures obtained at an oncologic hospital}; Pliego-Castaneda A et al.; BACKGROUND: Prevalence Candida infections have increased and oncologic patients have risk factors for contacting them . They are associated with a long hospital stay and high mortality rate . Candida resistance to antifungal drugs has been reported . OBJECTIVES: To determine the prevalence of Candida grown in cultures from oncologic patients . To identify isolated species and to determine C albicans sensitivity to antifungal drugs . MATERIAL AND METHODS: During one year, different species of Candida spp . were isolated . They were inoculated in API-C-20 at 48 h . and cultivated in RPMI-1640 . RESULTS: Cultures of 5,820 patients were collected, 66.68% from outpatients and 33% from hospitalized patients . Candida spp grew in 394 Candida albicans obtained from 81 cultures was the most frequent species (46.3%) . One hundred were susceptible to Amphotericin B, 63% to Fluconazol, 59% to Ketoconazol and 31% to Itraconazol . Candidemia accounted for 24.6% of bloodstream infections . CONCLUSIONS: C . albicans had significant resistance to imidazoles and 100% were susceptible to Amphotericin B . Identification of species and susceptibility of Candida infections a useful tools because of the high proportion of Candida non-albicans species (53.7%) . Candidemia accounted for 5.076% of the infections.

Med Mycol, 2000 Jun, 38(3), 213 - 9
Treatment of orogastrointestinal candidosis in SCID mice with fluconazole alone or in combination with recombinant granulocyte colony-stimulating factor or interferon-gamma; Clemons KV et al.; Mucosal candidosis is common in acquired immune deficiency syndrome (AIDS) patients, where there is extensive mucosal involvement, but rarely dissemination . To mimic this disease, SCID mice were inoculated orally with Candida albicans, which could be recovered from standardized tissue samples of the esophagus, stomach, small intestine and caecum of all mice . Treatment with fluconazole at 5 or 10 mg kg(-1) per day were equivalent to each other and efficacious in reducing the fungal burden from all four tissues compared with no treatment or lower doses of fluconazole (P < 0.01-0.001) . Fluconazole at 5 or 10 mg kg(-1) reduced fungal burden in the stomach by about 200 or 580-fold, respectively, and by approximately 25-fold in the other tissues, with 80 or 100% of mice cleared of esophageal infection, and 40 or 80% cleared of infection in the small intestine, respectively; the same doses cleared < or =20% of stomach infection and none of caecal infection . Treatment with recombinant human granulocyte colony-stimulating factor (G-CSF) up to 500 microg kg(-1) per day or 10(5) U of murine interferon-gamma (IFN-gamma) alone was ineffective, nor were combinations with a suboptimal dose fluconazole synergistic . Overall, fluconazole had dose-responsive efficacy, whereas neither G-CSF nor IFN-gamma alone or in combination with fluconazole improved efficacy . These studies demonstrate the utility of this model for examining antifungal efficacy in a situation that mimics clinical disease in AIDS patients.

FEMS Immunol Med Microbiol, 2000 Aug, 28(4), 283 - 9
Enhanced resistance to experimental systemic candidiasis in tilorone-treated mice; Ortega E et al.; Candida albicans is an increasingly important opportunistic fungal pathogen in immunocompromised patients . Natural killer (NK) cells constitute an important immune effector mechanism and are involved in the response to different pathological disorders . We wished to determine if this immune mechanism is involved in the specific response to C . albicans . Tilorone hydrochloride and related compounds have been described to display antiviral and antitumoral activity, as well as to enhance NK cell activity . In this study, we show the antimicrobial activity of different tilorone analogues and the enhanced resistance of tilorone-treated mice in experimental systemic candidiasis . We also present data suggesting that there is a correlation between NK cell activation and the resistance to experimental systemic candidiasis . Thus, it seems that the immunosurveillance of metastatic spread and the infection by C . albicans share some immune effector mechanisms, in particular activation of NK cells.

Biochem Biophys Res Commun, 2000 Jul 14, 273(3), 1025 - 32
Adenine-induced selective apoptosis toward HIV chronically infected cells in vitro; Hirasawa K et al.; A novel strategy for anti-HIV therapy is the clearance of the residual infected cells from the body . Here, we show that 6-aminopurine, adenine, induced selective apoptosis toward HIV-1 producing chronically infected MOLT4 cells (MOLT4/HIV) without augmentation of virus production, whereas the growth of uninfected MOLT4 was stimulated . This selective apoptosis did not occur with other adenine nucleotides or with other bases . The purine ring and the amino residue of adenine were responsible for the apoptosis induction and selectivity, respectively . In addition, adenine slightly but consistently reduced viable cell numbers and the production of virus in a fraction of HIV-1 chronically infected human peripheral blood mononuclear cells (PBMCs/HIV) at day 7 . On the other hand, blastogenic response of normal PBMCs to PHA, PWM and Candida albicans were potentiated in the presence of adenine . These results indicated that the effect of adenine may be attributable to activation-induced selective apoptosis toward virus-infected cells .

Biochem Biophys Res Commun, 2000 Jul 14, 273(3), 799 - 804
Sterol 14-demethylase P450 (CYP51) provides a breakthrough for the discussion on the evolution of cytochrome P450 gene superfamily; Yoshida Y et al.; Biodiversity is the most characteristic feature of cytochrome P450 . Finding of CYP51 distributing widely in biological kingdoms provided breakthroughs for the discussion on the evolution and diversification of P450 . Molecular phylogenetic analysis demonstrated that CYP51 appeared in the prokaryotic era and distributed into most kingdoms concomitant with phylogenetic divergence . This is the first evolutionary evidence indicating the prokaryotic origin of P450 . Modification of substrate specificity of eukaryotic CYP51s occurred independently to adapt to the different sterol precursors existing in each kingdom . Formation of CYP51 variants through the mutation of active site and the selection of the advantageous ones from them were demonstrated by the emergence of azole-resistant CYP51s in Candida albicans under the environments rich in azole antifungal agents . These findings illustrate the most probable core process of P450 diversification consisting of modification of active site and selection of the resulting variants through interaction with endogenous and exogenous chemicals .

J Med Chem, 2000 Jun 29, 43(13), 2493 - 505
A three-dimensional model of lanosterol 14alpha-demethylase of Candida albicans and its interaction with azole antifungals; Ji H et al.; The three-dimensional structure of lanosterol 14alpha-demethylase (P450(14DM), CYP51) of Candida albicans was modeled on the basis of crystallographic coordinates of four prokaryotic P450s: P450BM3, P450cam, P450terp, and P450eryF . The P450(14DM) sequence was aligned to those of known proteins using a knowledge-based alignment method . The main chain coordinates of the core regions were transferred directly from the corresponding coordinates of P450BM3 . The side chain conformations of the core regions were determined by the conformations of the equivalent residues with the highest homologous scores in four crystal structures . The model was then refined using molecular mechanics and molecular dynamics . The reliability of the resulting model was assessed by Ramachandran plots, Profile-3D, hydropathy plot analysis, and by analyzing the consistency of the model with the experimental data . The structurally and functionally important residues such as the heme binding residues, the residues interacting with redox-partner protein and/or involved in electron transfer, the residues lining substrate access channel, and the substrate binding residues were identified from the model . These residues are candidates for further site-directed mutagenesis and site-specific antipeptide antibody binding experiments . The active analogue approach was employed to search the pharmacophoric conformations for 14 azole antifungals . The resulting bioactive conformations were docked into the active site of lanosterol 14alpha-demethylase of Candida albicans . All 14 azole antifungals are shown to have a similar docking mode in the active site . The halogenated phenyl group of azole inhibitors is deep in the same hydrophobic binding cleft as the 17-alkyl chain of substrate . The pi-pi stacking interaction might exist between halogenated phenyl ring of inhibitors and the aromatic ring of residue Y132 . The long side chains of some inhibitors such as itraconazole and ketoconazole surpass the active site and interact with the residues in the substrate access channel . To compare with mammalian enzymes, structurally selective residues of the active site of fungal lanosterol 14alpha-demethylase are distributed in the C terminus of F helix, beta6-1 sheet and beta6-2 sheet.

J Dent Res, 2000 Jun, 79(6), 1439 - 42
Influence of environmental pH on the reactivity of Candida albicans with salivary IgA; Bikandi J et al.; Salivary secretory IgA reacts with a group of heat-shock mannoproteins preferentially expressed on Candida albicans yeast cells and germ tubes grown at 37 degrees C . Since other environmental factors can also modulate the expression of those antigens, we have investigated the influence of the pH of the culture medium on the expression of the antigens reacting with human salivary IgA by C . albicans . By indirect immunofluorescence, yeast cells grown in Sabouraud glucose broth at 37 degrees C showed a statistically significant increase in reactivity with salivary IgA (p < 0.0001) when compared with cells grown at 25 degrees C at the 4 pH values studied (3.3, 5.9, 7.5, and 9.5), the highest reactivity and the major heat-shock effect being observed at pH 5.9 . The decrease in reactivity with salivary IgA observed in C . albicans cells grown at pH values of 3.3 and 9.5 was confirmed by Western blotting . Salivary IgA reacted with polydispersed materials from the cell walls of molecular masses > 55 kDa, which were more expressed at neutral pH than at acidic or alkaline pH values . A similar reactivity was observed when the antigenic extracts were stained with an antiserum directed against oligosaccharides present in antigen 6 of C . albicans serotype A . The differences in reactivity presented by salivary IgA may be related to a decrease in the expression of polysaccharides present on the surfaces of the yeast cells of C . albicans grown at acidic or alkaline pH values . The low reactivity of salivary IgA with C . albicans cells grown at acidic pH values may help to explain the association between acidic saliva and the carriage of Candida in the oral cavity, as well as with oral candidiasis.

Bioorg Med Chem Lett, 2000 Jul 3, 10(13), 1459 - 62
Synthesis and structure-activity relationships of novel fungal chitin synthase inhibitors; Masubuchi K et al.; A novel Candida albicans chitin synthase 1 (CaChs1) inhibitor, RO-41-0986 (1) was discovered by random screening . Systematic modification led to the identification of a highly potent CaChs1 inhibitor, RO-09-3024 (2), having strong antifungal activity against Candida spp . in vitro.

Scand J Immunol, 2000 Jul, 52(1), 13 - 20
Interleukin-10 produced by the innate immune system masks in vitro evidence of acquired T-cell immunity to E . coli; Hessle C et al.; Bacteria trigger stimulation of antigen-specific, as well as innate, immune responses . Cytokines and other products of cells belonging to the innate immune system may interfere with the detection of acquired immunity to whole bacteria in vitro . Proliferation and cytokine production by human blood mononuclear cells in response to a whole UV-killed Escherichia coli was compared with the response to commonly used antigen preparations: purified protein derivative (PPD), Candida albicans and influenza proteins . E . coli induced a weaker proliferative response with later onset than did the other antigens, and production of interferon (IFN)-gamma comparable with that in response to C . albicans and influenza vaccine, but lower than that induced by PPD . Both proliferation and IFN-gamma production were abolished by removal of CD4 cells or blocking of HLA-D, but not CD1 antigen-presenting molecules . Purified lipopolysaccharide (LPS) induced neither proliferation nor IFN-gamma production . None of the antigen preparations stimulated interleukin (IL)-4 production but, in contrast to the other antigens, whole E . coli, as well as purified O6 LPS, induced large quantities of IL-10 . IL-10 production was independent of CD4 cells or HLA-D molecules . Blocking of IL-10 by neutralizing antibodies increased both E . coli-induced proliferation and IFN-gamma production markedly . Conversely, the addition of whole E . coli or LPS to cultures stimulated with other antigens (C . albicans or Staphylococcus aureus) down-regulated proliferative and IFN-gamma responses, an effect which was at least partly IL-10 dependent . The results indicate a substantial T-cell memory to commensal E . coli, but suggest that the evidence of such memory, e.g . proliferation and IFN-gamma production, is effectively prevented by IL-10 and perhaps other factors produced by monocytes in response to bacteria . Thus, the innate immune responses must be taken into account when acquired immune responses to microbes are measured.

Lett Appl Microbiol, 2000 Jul, 31(1), 52 - 6
Effect of ionic strength on the inactivation of micro-organisms by microwave irradiation; Watanabe K et al.; Microwave irradiation at 2450 MHz inactivated the cells of Escherichia coli, Staphylococcus aureus and Candida albicans suspended in a phosphate buffer . The rate of cell inactivation was proportional to that of the increase in temperature accompanied by microwave irradiation . The inactivation rates of E . coli and C . albicans were affected by addition of NaCl and KCl, but not by sucrose . The maximal inactivation effect was exerted at concentrations of 0.5-1.0 mol l-1, and the end-point temperature was the highest at the same salt concentrations . Correlation of both the electroconductivity and di-electric loss of ionic solutions with the heating by microwave irradiation was discussed.

Photodermatol Photoimmunol Photomed, 2000 Jun, 16(3), 121 - 4
Increased phototoxicity of hydrochlorothiazide by photodegradation; Han KD et al.; The photodegradation products of hydrochlorothiazide produced by ultraviolet (UV) radiation were investigated for their phototoxicity utilizing the photohemolysis and Candida albicans tests . Hydrochlorothiazide was irradiated for 30, 60, 90 and 120 min with a 250 W xenon arc lamp using a WG295 cut-off filter . Irradiation of hydrochlorothiazide resulted in the gradual decrease of all three absorption bands (225, 270 and 320 nm), the blue shift of the 225 nm band, and the appearance of a new band around 290 nm . Since previous results demonstrated that photosubstitution of chloride could occur, the main product of this photolysis most likely is ethoxyhydrochlorothiazide . The photohemolysis test revealed a significant increase in photohemolysis observed in the photodegradation products produced after 60, 90 and 120 min of UV irradiation . This increase in hemolysis value directly correlated with the UV-irradiation time . However, there was no significant phototoxic killing of yeast in the Candida albicans test . This suggests photodegradation products of hydrochlorothiazide may play an important role in phototoxicity by acting on the cell membrane, but not on DNA . Considering the high in vitro phototoxicity observed in bendroflumethiazide and the data presented here, substitution of chloride seems to be responsible for the increased phototoxicity of hydrochlorothiazide.

Theriogenology, 2000 May, 53(8), 1591 - 608
Peripheral and intrauterine neutrophil function in the cow: the influence of endogenous and exogenous sex steroid hormones; Subandrio AL et al.; It has been accepted for many years that the susceptibility of the genital tract to infection is reduced during the follicular phase compared with the luteal phase of the estrous cycle . Since the role of intrauterine neutrophils is paramount in the elimination of bacteria, it can be hypothesized that these differences in resistance to infection could be mediated by differences in uterine-derived neutrophil function . In order to test this hypothesis two groups of cows were used in this study . Group 1 cows (n=5) were studied at estrus, diestrus, after ovariectomy, after exogenous estradiol and after progesterone treatment, at which time they underwent intrauterine infusion with 1% oyster glycogen (OG) and a bacterial-free filtrate (BFF) of Actinomyces genes (BFF), the latter having been recovered from a clinical case of endometritis; neutrophils were harvested by flushing from the lumen 15 to 18 h later . A peripheral blood sample was collected at the time of flushing for the assay of estradiol and progesterone for a WBC and differential count and for the harvesting of neutrophils using a Percoll single-stage discontinuous gradient . After the recovery of the cells they were re-suspended in HBSS . Group 2 (n=4) were infused with BFF during during all reproductive states as Group 1, but with OG only after ovariectomy and after treatment with progesterone and estradiol . Neutrophil chemotaxis was assessed by measuring their migration using a modified Boyden chamber and Zymogen-activated serum as a chemoattractant . Phagocytic activity was measured by determining the number of Candida albicans ingested by each neutrophil after incubation . The percentage of kill was determined using a radiometric assay in which C . albicans was labeled with L-(5-3H) Proline . Peripheral WBC concentration was not influenced by the reproductive state of the cow; however, the mean neutrophil concentration was significantly different between the reproductive states (P<0.001) and between individual cows (P<0.001) . In Group 1, there was little difference in the function of the peripheral and uterine neutrophils, and while there were differences in all 3 aspects of neutrophil function from both sources between reproductive states and individual cows, of which some were statistically significant, there was no consistent pattern . In Group 2, neutrophils recovered after the infusion of BFF had poorer function compared with those recovered after the infusion of OG . There was no consistent influence of the reproductive state or individual animal . The hypothesis that the influence of the reproductive state of the cow on the resistance of the uterus to infection is mediated by the inherent differences in either peripheral or intrauterine neutrophil function was not supported by this study.

J Infect Dis, 2000 Jul, 182(1), 274 - 82 Epub 2000 Jul 06.
Comparative efficacy and distribution of lipid formulations of amphotericin B in experimental Candida albicans infection of the central nervous system; Groll AH et al.; The central nervous system (CNS) distribution and antifungal efficacy of all 4 approved formulations of amphotericin B (AmB) were investigated in a rabbit model of hematogenous Candida albicans meningoencephalitis . Treatment with AmB deoxycholate (1 mg/kg/day) or liposomal AmB (5 mg/kg/day) yielded the highest peak plasma concentration (C(max)), area under concentration versus time curve from zero to 24 h (AUC(0-24)), and time during dosing level tau Ttau>minimum inhibitory complex (MIC) values and led to complete eradication of C . albicans from brain tissue (P<.05 vs . untreated controls) . By comparison, AmB colloidal dispersion and AmB lipid complex (5 mg/kg/day each) were only partially effective (not significant vs . untreated controls) . There was a strong correlation of C(max), AUC(0-24), C(max)/MIC, AUC(0-24)/MIC, and Ttau>MIC with clearance of C . albicans from brain tissue (P</=.0002) . Although pharmacodynamic parameters derived from the MIC of free AmB were highly predictive of antifungal efficacy, parameters derived from MICs of individual formulations were not predictive . AmB deoxycholate and liposomal AmB had the greatest antifungal efficacy . This activity was concentration and time dependent.

J Infect Dis, 2000 Jul, 182(1), 266 - 73 Epub 2000 Jun 30.
Increased resistance to systemic candidiasis in athymic or interleukin-10-depleted mice; Tavares D et al.; Candida albicans produces a virulence-associated immunomodulatory protein (p43), which activates lymphocytes polyclonally, suppresses specific antibody responses to Candida antigens, and potentiates systemic growth of C . albicans . In this study, athymic, unlike euthymic, C57BL/6 mice were resistant to systemic candidiasis and produced lower serum levels of interleukin (IL)-4 and IL-10 . Pretreatment with p43 stimulated the production of both cytokines . Depletion of IL-10, but not of IL-4, in euthymic animals reestablished resistance and abrogated p43-dependent suppression of the specific antibody response and facilitated C . albicans growth . In agreement with these results, both immunosuppression and p43-mediated facilitation of the fungus growth were abolished in IL-10 knockout mice . These observations demonstrate the relevance of IL-10 in facilitating systemic candidiasis and suggest a critical role for the immunosuppressive virulence factor p43 in the process.

Rev Fac Cien Med Univ Nac Cordoba, 1999, 56(2), 9 - 20
{Basic aspects of neuroendocrinoimmunology}; Correa SG et al.; The purpose of this article is to discuss basic aspects of the interplay between the neuroendocrine and the immune systems . Two pathways link the brain and the immune system: the autonomic nervous system and the neuroendocrine outflow via the pituitary . Most of the influence of the brain on immune events is exerted through the hypothalamic-pituitary-adrenal (HPA) axis . Moreover, certain neurotransmitters, neuropeptides, and neurohormones affect immune function both in vivo and in vitro . Receptors for these molecules are present on immune cells . This cell-to-cell communication is bi-directional, since impulses from the immune system can affect many functions of the central nervous system . Cytokines released during the activation of the immune system, in turn, can alter the function of the HPA axis . In this context, we also describe our main findings working with a model of Candida albicans infection in rats exposed to chronic varied stress.

J Lab Clin Med, 2000 Jul, 136(1), 66 - 73
Regulation of Candida albicans growth and adhesion by saliva; Ueta E et al.; To examine the local regulation of oral Candida albicans growth, we examined non-stimulated and stimulated salivary flow rates (SFRs) and the C . albicans growth and adhesion inhibitory activities of saliva in 60 patients with oral candidiasis (divided into two groups: 25 patients with oral candidiasis only (group OC) and 35 patients with oral candidiasis and systemic diseases (group CS)) and 30 healthy control subjects . Both non-stimulated and stimulated SFRs in patients, especially in group CS; were decreased in comparison with those in the healthy control subjects . The levels of secretory immunoglobulin A (sIgA) in group OC and group CS and the lactoferrin level in group CS were decreased as compared with those in control individuals, although there were no differences in transferrin and total secretory component (SC) levels between the three groups . The secretion amounts (microg/min) of these proteins were statistically significantly decreased in the patients, especially in group CS . Saliva from the patients showed a lesser inhibitory effect on C . albicans growth and adhesion to HeLa cells than did saliva from the control subjects . In addition, polymorphonuclear leukocytes (PMNs) in patients' saliva generated smaller amounts of superoxide than did those in control subjects' saliva, and phagocytic and C . albicans killing activities were suppressed in the patients . These results indicate that the decreases in SFR, secretion of antimicrobial proteins in saliva, and salivary PMN activity are risk factors for oral candidiasis associated with aging and systemic diseases.

Cancer Gene Ther, 2000 Jun, 7(6), 845 - 52
Breast cancer-specific expression of the Candida albicans cytosine deaminase gene using a transcriptional targeting approach; Anderson LM et al.; We constructed a series of adenoviral (Ad) vectors that express the Candida albicans cytosine deaminase (CD) suicide gene under the transcriptional control of either the human alpha-lactalbumin (ALA) or ovine beta-lactoglobulin (BLG) promoter (Ad.ALA.CD and Ad.BLG.CD, respectively) . The Ad.ALA.CD and the Ad.BLG.CD vectors converted the prodrug 5-fluorocytosine (5-FC) to the toxic nucleotide analog 5-fluorouracil in a breast cancer cell-specific manner, with a conversion rate of 40% and 52% in T47D cells and 50% and 41% in MCF7 cells, respectively . No significant conversion (< or =3%) was observed in an immortalized nontumorigenic breast epithelial cell line (MCF10A) and a human osteosarcoma cell line (U2OS) . Adenovirus vector-based prodrug conversion of the 5-FC in T47D and MCF7 in the presence of 1 mg/mL of 5-FC led to cytotoxicity that resulted in a nearly complete cell death (> or =90%) after 5 days, whereas MCF10A and U2OS cells remained resistant (< or =10%) . Nude mice harboring T47D-derived breast tumors that were injected intratumorally (i.t.) with therapeutic adenovirus vectors at a dose of 2 x 10(8) plaque-forming units and treated systemically with 5-FC at a concentration of 500 mg/kg/day showed a marked reduction in tumor mass within 30 days when compared with animals that received vector alone . Animal survival was significantly prolonged after 72 days in mice treated with therapeutic vectors in conjunction with prodrug when compared with control animals . These preclinical data are sufficiently promising to warrant further studies of this transcriptional targeting approach to breast cancer treatment.

Microbiology, 2000 Jul, 146 ( Pt 7), 1753 - 8
Reduced virulence of Candida albicans mutants lacking the GNA1 gene encoding glucosamine-6-phosphate acetyltransferase; Mio T et al.; The yeast GNA1 gene encodes glucosamine-6-phosphate acetyltransferase which catalyses the reaction of glucosamine 6-phosphate with acetyl-CoA to form N-acetylglucosamine 6-phosphate, a fundamental precursor in UDP-N-acetylglucosamine biosynthesis . Candida albicans mutants lacking GNA1 were viable in the presence of N-acetylglucosamine . To confirm the physiological importance of C . albicans GNA1, the virulence of a C . albicans gna1Delta null mutant was examined in a mouse model of candidiasis . When injected intravenously into mice, the virulence of the C . albicans gna1Delta null mutant was significantly attenuated . The reduced virulence appeared to be the result of rapid clearance from host tissue . These data suggest that C . albicans GNA1 is required for survival of the fungus in host animals, probably because an insufficient level of N-acetylglucosamine is available from the host tissues.

Rev Esp Quimioter, 2000 Mar, 13(1), 60 - 63
{Susceptibility to fluconazole and itraconazole in isolates of Candida spp . from HIV-positive and HIV-negative patients}; Bernal S et al.; We studied the possible differences in the pattern of susceptibility to fluconazole and itraconazole in 393 isolates of Candida spp . from the oral cavity of HIV-positive patients and 102 isolates from HIV-negative patients with candidemia or candiduria . We used the broth microdilution method according to the NCCLS guidelines . We observed a decrease in the susceptibility to fluconazole in the group of HIV-positive patients in comparison to those who were HIV negative, especially in Candida albicans (MIC(90) 32 mg/l vs . 1 mg/l and Candida glabrata (MIC(90) 64 mg/l vs . 16 mg/l) . Furthermore, we did not find any resistant strains in the HIV-negative group . For itraconazole, the MIC(90) was two dilutions greater in the HIV-positive patients, except for C . albicans, which had a much higher MIC(90) (4 mg/l vs . 0.12 mg/.) . Therefore, the decrease in the susceptibility of Candida spp . in the HIV-positive patients must be taken into account when establishing a specific antifungal therapy.

Philos Trans R Soc Lond B Biol Sci, 2000 May 29, 355(1397), 695 - 704
Evolution of microbial pathogens; Morschhauser J et al.; Various genetic mechanisms including point mutations, genetic rearrangements and lateral gene transfer processes contribute to the evolution of microbes . Long-term processes leading to the development of new species or subspecies are termed macroevolution, and short-term developments, which occur during days or weeks, are considered as microevolution . Both processes, macro- and microevolution need horizontal gene transfer, which is particularly important for the development of pathogenic microorganisms . Plasmids, bacteriophages and so-called pathogenicity islands (PAIs) play a crucial role in the evolution of pathogens . During microevolution, genome variability of pathogenic microbes leads to new phenotypes, which play an important role in the acute development of an infectious disease . Infections due to Staphylococcus epidermidis, Candida albicans and Escherichia coli will be described with special emphasis on processes of microevolution . In contrast, the development of PAIs is a process involved in macroevolution . PAIs are especially important in processes leading to new pathotypes or even species . In this review, particular attention will be given to the fact that the evolution of pathogenic microbes can be considered as a specific example for microbial evolution in general.

Retina, 2000, 20(3), 262 - 8
Fungal endophthalmitis after a single intravenous administration of presumably contaminated dextrose infusion fluid; Gupta A et al.; PURPOSE: To report fungal endophthalmitis in nonimmunocompromised patients, each of whom received a single intravenous administration of presumably contaminated dextrose infusion fluid for minor ailments in rural settings . METHODS: This noncomparative case series included 12 nonimmunocompromised patients (12 eyes) with culture-positive fungal endophthalmitis . All eyes underwent initial vitreous tap with injection of intravitreal antibiotics . Eleven eyes required pars plana vitrectomy and oral fluconazole or itraconazole for 4 to 6 weeks . One patient with panophthalmitis was treated with intravenous amphotericin B . To support the hypothesis that contaminated intravenous fluid was the possible risk factor, samples from 72 sealed bottles of 5% dextrose were subjected to fungal culture . RESULTS: Patients presented 1 to 11 weeks (mean, 4.6 weeks) after the intravenous infusion . All eyes had a positive smear and cultures for fungi . Aspergillus specimen was isolated in nine eyes, Candida in two eyes, and Mucor in one eye . Final visual acuity was 20/80 or better in 8 (66.6%) eyes . Eleven of the 72 samples from dextrose bottles were culture-positive for fungi: six for Aspergillus fumigatus, three for Aspergillus niger, and two for Candida albicans . CONCLUSION: A presumed contaminated intravenous infusion administered in a rural setting was found as a new risk factor for development of endogenous fungal endophthalmitis . These patients were successfully treated with pars plana vitrectomy and oral fluconazole and itraconazole therapy.

Mycopathologia, 1999, 147(1), 29 - 32
Onychomycosis in Malaysia; Ng KP et al.; The common etiological agents of onychomycosis are dermatophytes, molds and yeasts . A mycological nail investigation of onychomycosis using direct microscopy and culture was conducted by the Mycology Unit, Department of Medical Microbiology, University of Malaya from March 1996 to November 1998 . The study involved 878 nail clippings or subungal scrapings from subjects with onychomycosis . On direct microscopy examination, 50% of the specimens were negative for fungal elements . On culture, 373 specimens had no growth; bacteria were isolated from 15 nail specimens . Among the 490 specimens with positive fungal cultures, 177 (36.1%) were dermatophytes, 173 (35.5%) were molds and 130 (26.5%) were Candida . There were 2% (10/490) mixed infections of molds, yeasts and dermatophytes . Trichophyton rubrum (115/177) and Trichophyton mentagrophytes (59/177) were the main dermatophytes isolated . The molds isolated were predominantly Aspergillus niger (61/173), Aspergillus nidulans (30/173), Hendersonula toruloidea (26/173) and Fusarium species (16/173) . 96.9% of the Candida species identified were Candida albicans.

Mycopathologia, 1999, 147(1), 13 - 20
Isolation and identification of a 92-kDa stress induced protein from Candida albicans; Burt ET et al.; It was previously shown that the presence of estrogen enhances survival of Candida albicans under heat and oxidative stresses . A 92-kDa protein is inducible by heat shock and estrogen in C . albicans . Previous studies have described this protein as hsp90 because of its molecular size and heat inducibility as seen on electrophoretic gels and Western blots . In this study, ion exchange, hydroxyapatite and size exclusion chromatography were used to isolate a 92-kDa-protein band . The N-terminal sequence of isolated protein blotted onto a PVDF membrane was determined to be V-Q-S-?-V-L-G-F-P-R . This sequence is homologous to the N-terminal sequence of the MET6 gene product, cobalamin-independent methionine synthase, from Saccharomyces cerevisiae . The results of this study suggest that a cobalamin-independent methionine synthase homolog is inducible by heat and estrogen in C . albicans . This study also suggests that Candida hsp90 is more likely to exist as an 82-kDa protein as predicted by a previously described cDNA and not as a 92-kDa protein as reported in the literature.

Med Arh, 2000, 54(1), 9 - 11
{Cytomegalovirus disease in immunocompromised patients}; Zvizdic S et al.; Human Cytomegalovirus are a ubiquitous herpesvirus establishing virus-infections which are usually asymptomatic in immunocompetent individuals . In patients with Acquired Immunodeficiency Syndrome (AIDS) and immunocompromised hosts, the virus causes primary, latent or chronic persistent infection . Primary CMV infection is very severe in immunocompromised patients as well as among healthy population . Among patients with AIDS Cytomegalovirus (CMV) is usually isolated from patients specimen in association with other pathogens (Pneumocystis carinii, Candida albicans) . The prevalence of serious CMV (chorioretinitis, gastrointestinal disease, interstitial pneumonia and central nervous system disease), in AIDS population are respectable.

Yeast, 2000 Jul, 16(10), 959 - 66
Functional cloning of the adenylate cyclase gene of Candida albicans in Saccharomyces cerevisiae within a genomic fragment containing five other genes, including homologues of CHS6 and SAP185; Mallet L et al.; We have cloned a genomic fragment of Candida albicans by complementation of a Saccharomyces cerevisiae cyr1 mutant . This fragment contains the two-thirds C-terminal part of the adenylate cyclase CaCYR1 . The complete gene has been sequenced from PCR fragments amplified from genomic DNA, and contains an ORF of 1690 amino acids closely related to other fungal adenylate cyclases . Adjacent to the adenylate cyclase gene, we have sequenced six other putative genes . CaCHS6, CaYNL191 and CaYJL098 are named on the basis of their close similarity with S . cerevisiae genes . ORFs CaYJL097a and CaYJL097b represent two repeated homologues of the S . cerevisiae YJL097w, which probably arose from an ancient duplication . The last one is a hypothetical ORF, CaYKR049, which presents only a very weak similarity with YKR049 . The S . cerevisiae homologues of three of these genes are co-localized on chromosome X but with a different order and orientation .

Yeast, 2000 Jul, 16(10), 933 - 44
A phosphatidylinositol 3-kinase of Candida albicans: molecular cloning and characterization; Eck R et al.; A phosphatidylinositol (PI) 3-kinase gene (CaVPS34) of the human pathogenic yeast Candida albicans was cloned by a PCR-based homology approach . The open reading frame encodes a 1020 amino acid protein with a calculated molecular weight of 118 kDa and a relative isoelectric point of 6.9 . It shares 47% sequence identity with Saccharomyces cerevisiae Vps34p . Southern pattern indicated that CaVPS34 is probably present as a single copy gene per haploid genome in C . albicans . We localized the CaVPS34 gene on chromosome 1 . Under all conditions tested a major CaVPS34 transcript of approximately 3 . 5 kb could be detected . CaVPS34 mRNA levels increased during exponential growth up to 12-fold followed by a decline upon entry into stationary phase . The size of a 6xHis tag-CaVps34p fusion protein purified from Escherichia coli is in agreement with the calculated molecular mass of CaVps34p . It exhibits in vitro PI 3-kinase activity and produces only phosphatidylinositol 3-phosphate . The CaVPS34 gene under the control of its own promoter were not able to complement the temperature-sensitive growth of S . cerevisiae vps34 . However, overexpression of CaVPS34 was sufficient to rescue the temperature-sensitive vps34 phenotype, suggesting a functional conservation in C . albicans .

Biotechniques, 2000 Jun, 28(6), 1112 - 6
PCR- and ligation-mediated synthesis of split-marker cassettes with long flanking homology regions for gene disruption in Candida albicans; de Hoogt R et al.; Because Candida albicans is a diploid organism, two consecutive steps of gene disruption are required to generate a gene knock-out . The same marker (URA3) is often used for disruption of both copies of the gene . This is possible because, after the first round of disruption, homologous recombination between direct repeats flanking the URA3 marker and the subsequent counterselection allow for the efficient recovery of Ura- revertants . Unfortunately, the URA-blaster disruption cassette cannot be used in a PCR-based disruption approach . The hisG repeats flanking the URA3 gene in the disruption cassette anneal to one another during PCR and thereby prevent amplification of the complete cassette . We explored the use of transformation based on split-marker recombination to circumvent this problem . To avoid any cloning steps and to retain the advantage of long flanking regions for disruption, we combined this with a PCR- and ligation-mediated approach for generating marker cassettes . We used this approach to disrupt the C . albicans FAL1 (ATP-dependent RNA helicase) gene . Long 5' and 3' FAL1-specific regions were amplified by PCR and individually ligated to a URA-blaster cassette . The resulting ligation reactions were used separately as templates to generate two FAL1 disruption cassettes with overlapping URA3 marker regions . Simultaneous transformation with both overlapping disruption cassettes yielded efficient disruption of one FAL1 allele.

Arch Dermatol Res, 2000 May, 292(5), 260 - 4
Generation of reactive oxygen species by Candida albicans in relation to morphogenesis; Schroter C et al.; Candida albicans is able to generate significant amounts of reactive oxygen species (ROS) . In this study, ROS generation by yeast and hyphal forms of the strain 3153 A was analyzed to determine whether ROS generation could be a major factor in the invasive behavior of germinative cells . Furthermore, the virulent strain CA6 and its avirulent and agerminative mutant VIR3 were compared . ROS were measured by lucigenin-enhanced chemiluminescence and a cytochrome c assay . During the blastoconidial phase of all strains moderate amounts of ROS were found at cell concentrations > 1 x 10(5)/ml . However, ROS generation appeared to be specifically inhibited at cell concentrations > 1 x 10(8)/ml, and this was found in both assays . As shown in comparative experiments, the medium used for measurement markedly affected the total amount of ROS . Hyphae of strain 3153 A generated a significantly higher amount of ROS than yeast cells and cells with germ tubes (P < 0.001) . The strain CA6 showed significantly higher ROS generation than the VIR3 strain for both blastoconidiae and after 30 min of induction of hypha formation (P < 0.05) . In conclusion, hypha formation, usually acknowledged as a major factor in Candida pathogenicity, was associated with markedly increased ROS formation . ROS generation was not closely linked to the ability to form hyphae, but was highest in germinative cells.

Am Fam Physician, 2000 Jun 1, 61(11), 3306 - 12, 3317
Treatment of recurrent vulvovaginal candidiasis; Ringdahl EN; Vulvovaginal candidiasis is considered recurrent when at least four specific episodes occur in one year or at least three episodes unrelated to antibiotic therapy occur within one year . Although greater than 50 percent of women more than 25 years of age develop vulvovaginal candidiasis at some time, fewer than 5 percent of these women experience recurrences . Clinical evaluation of recurrent episodes is essential . Patients who self-diagnose may miss other causes or concurrent infections . Known etiologies of recurrent vulvovaginal candidiasis include treatment-resistant Candida species other than Candida albicans, frequent antibiotic therapy, contraceptive use, compromise of the immune system, sexual activity and hyperglycemia . If microscopic examination of vaginal secretions in a potassium hydroxide preparation is negative but clinical suspicion is high, fungal cultures should be obtained . After the acute episode has been treated, subsequent prophylaxis (maintenance therapy) is important . Because many patients experience recurrences once prophylaxis is discontinued, long-term therapy may be warranted . Patients are more likely to comply when antifungal therapy is administered orally, but oral treatment carries a greater potential for systemic toxicity and drug interactions.

Mycoses, 1999, 42 Suppl 2, 77 - 82
Antimicrobial peptides as potential new antifungals; Muller FM et al.; Ribosomally synthesized natural antimicrobial peptides (AP) and their synthetic derivatives are small, cationic, amphipathic molecules of 12-50 amino acids with unusually broad activity spectra . These peptides kill microorganisms by a common mechanism, which involves binding to the lipid bilayer of biological membranes, forming pores, and ultimately followed by cell lysis . Several AP from mammals, amphibians, insects, plants and their synthetic derivatives demonstrate promising in vitro activity against various pathogenic fungi including azole-resistant Candida albicans strains . In addition to their antimicrobial activity, some AP such as lactoferrin, interact with a variety of host cells and can increase the activity of natural killer and lymphokine activated killer cells . Pretreatment of polymorphonuclear neutrophil leukocytes (PMN) or monocytes with these AP also may upregulate superoxide release . AP as potential new antifungal agents offer some advantages, such as rapid killing of pathogenic fungi and the difficulty to raise mutants resistant to these peptides . AP are limited by their nonselective toxicity, stability, immunogenicity and their costs of production . Potential clinical applications of AP in the future have to be further explored in preclinical and clinical studies to assess their impact as a new class of antifungals.

Mycoses, 1999, 42 Suppl 2, 69 - 72
Standardized molecular typing; Muller FM et al.; Molecular typing methods are useful tools in molecular mycology . The results of these biotyping procedures may help to identify pathogenic strains in order to detect sources of nosocomial infection and for the investigation of epidemiological relationships . With respect to the facultative pathogen, Candida albicans, various methods such as pulse-field gel electrophoresis (PFGE), restriction fragment length polymorphism (RFLP), DNA fingerprinting methods and hybridization with repetitive DNA elements have been described as useful tools in molecular epidemiology . The previously described hybridization method with the Candida albicans specific CARE-2 probe and subsequent rehybridization with a molecular size marker is a standardized reproducible typing method for comparison of results obtained in different laboratories . In a larger epidemiological study conducted at the University Hospital of Wurzburg analysing clinical C . albicans isolates, we were able to describe relationships between sequential patient isolates . These findings demonstrate that standardized molecular typing methods are a powerful tool in molecular mycology studies.

Mycoses, 1999, 42 Suppl 2, 61 - 7
Caco-2 monolayer as a model for transepithelial migration of the fungal pathogen Candida albicans; Weide MR et al.; A model for transepithelial migration of human fungal pathogens was established, in which Candida albicans was shown to migrate across a monolayer of Caco-2 intestinal cells in a two-chamber system . Electron microscopy revealed typical stages of epithelial penetration by C . albicans including phagocytosis at the apical side, intra- and intercellular migration and exit on the basolateral side of the monolayer . Hyphal growth forms appeared particularly involved in penetration of the Caco-2 monolayer . The model was examined using defined C . albicans mutants defective in hyphal development (efg1/efg1) or growth (ura3/ura3) . Transmigration of the efg1/efg1 mutant strain was reduced, while transmigration of the ura3/ura3 strain was blocked completely in the absence of uridine . Because these results parallel virulence characteristics of the mutants the Caco-2 monolayer system appears a useful model for the study of fungal-human host cell interactions.

Mycoses, 1999, 42 Suppl 2, 45 - 8
Cytokine modulation of specific and nonspecific immunity to Candida albicans; Romani L; Recent observations on reciprocal regulation of humoral and cell-mediated immunity to Candida albicans are presented and their significance in the exploration of the host-fungus-relationship is discussed.

Mycoses, 1999, 42 Suppl 2, 19 - 24
Parameters for determination of Candida albicans virulence in murine peritonitis; Kretschmar M et al.; Using intraperitoneal (i.p.) infection of mice with Candida albicans we determined which parameters might be useful for characterization of virulence in this model . Upon i.p . infection of mice with two reference strains striking differences in lethality were detected . These differences in virulence corresponded with invasion of the liver and pancreas by the virulent strain and with a lack of invasion by the avirulent strain . The virulent strain was able to release high amounts of the enzymes alanine aminotransferase (ALT) and alpha-amylase (AM) from liver and pancreas into the blood plasma . Most likely, these enzymes were released by penetration of hyphae into the cytoplasm which was shown with electron microscopy . When invasion slowed down, there was also a drop in the activities of ALT and AM measured in the blood of infected mice . As both strains disseminated to the heart, kidneys, and lungs, dissemination into these organs was no reliable parameter for virulence in this model . However, only the virulent strain was able to reach the brain and to germinate in the kidneys and brain . In contrast to invasion and enzyme activities, the fungal load in the peritoneal cavity and in the neighbouring organs appeared not to be related with virulence . This may be concluded from the fact that there were no differences in the absolute colony forming units (cfu) and the length of persistence of both strains when similar inocula were used . We conclude that the ability of a given strain of C . albicans to invade neighbouring organs, to reach the brain upon dissemination and germination in the brain and kidneys may be used for measurement of virulence in this model when virulence is defined as lethality.

Planta Med, 2000 May, 66(4), 356 - 8
Local anaesthetic, antibacterial and antifungal properties of sesquiterpenes from myrrh; Dolara P et al.; We extracted, purified and characterized 8 sesquiterpene fractions from Commyphora molmol . In particular, we focused our attention on a mixture of furanodiene-6-one and methoxyfuranoguaia-9-ene-8-one, which showed antibacterial and antifungal activity against standard pathogenic strains of Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans, with minimum inhibitory concentrations ranging from 0.18 to 2.8 micrograms/ml . These compounds also had local anaesthetic activity, blocking the inward sodium current of excitable mammalian membranes.

Planta Med, 2000 May, 66(4), 391 - 3
Terpenoids and flavonoids from Artemisia species; Tang HQ et al.; A phytochemical reinvestigation of the aerial parts of Artemisia sieversiana gave a new guaianolide and two known flavones (chrysosplenetin and 5-hydroxy-3',4',6,7-tetramethoxyflavone) . Antifungal fractions derived from the chloroform extract of A . annua afforded two cadinane derivatives (arteannuin B and artemisinin), oleanolic acid, beta-sitosterol, stigmasterol, and the four flavones artemetin, bonanzin, eupalitin and chrysosplenetin . Their structures were elucidated by spectral methods . All isolates from the two species were tested in vitro for antifungal activity . Arteannuin B, a main sesquiterpenoid in A . annua, showed antifungal activity against one human (Candida albicans, MIC: 100 micrograms/ml) and four plant pathogenic fungi (Gaeumannomyces graminis var . tritici, Rhizoctonia cerealis, Gerlachia nivalis and Verticillium dahliae, MICs: 150, 100, 150 and 100 micrograms/ml, respectively) whereas others showed no antifungal activity . The MIC value of ketoconazole to C . albicans was 1.0 microgram/ml, and those of triadimefon to G . graminis var . tritici and R . cerealis 150 and 100 micrograms/ml.

J Mass Spectrom, 2000 Jun, 35(6), 672 - 82
Novel procedure for the identification of proteins by mass fingerprinting combining two-dimensional electrophoresis with fluorescent SYPRO red staining; Valdes I et al.; The fluorescent sensitive SYPRO Red dye was successfully employed to stain proteins in two-dimensional gels for protein identification by peptide mass fingerprinting . Proteins which are not chemically modified during the SYPRO Red staining process are well digested enzymatically in the gel and hence the resulting peptides can be efficiently eluted and analysed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) . A SYPRO Red two-dimensional gel of a complex protein extract from Candida albicans was analysed by MALDI-TOF MS . The validity of SYPRO Red staining was demonstrated by identifying, via peptide mass fingerprinting, 10 different C . albicans proteins from a total of 31 selected protein spots . The peptide mass signal intensity, the number of matched peptides and the percentage of coverage of protein sequences from SYPRO Red-stained proteins were similar to or greater than those obtained in parallel with the modified silver protein gel staining . This work demonstrates that fluorescent SYPRO Red staining is compatible with the identification of proteins separated on polyacrylamide gel and that it can be used as an alternative to silver staining . As far as we know, this is the first report in which C . albicans proteins separated using 2-D gels have been identified by peptide mass fingerprinting . The improved technique described here should be very useful for carrying out proteomic studies .

Yeast, 2000 Jun 30, 16(9), 847 - 55
The ALS6 and ALS7 genes of Candida albicans; Hoyer LL et al.; ALS genes of Candida albicans encode a family of cell-surface glycoproteins that are composed of an N-terminal domain, a central domain of a tandemly repeated motif, and a relatively variable C-terminal domain . Although several ALS genes have been characterized, more ALS-like sequences are present in the C . albicans genome . Two short DNA sequences with similarity to the 5' domains of known ALS genes were detected among data from the C . albicans genome sequencing project . Probes developed from unique regions of these sequences were used to screen a genomic library from which two full-length genes, designated ALS6 and ALS7, were cloned . ALS6 and ALS7 encode features similar to other genes in the ALS family and map to chromosome 3, a chromosome previously not known to encode ALS sequences . ALS6 and ALS7 are present in all C . albicans strains examined . Additional analysis suggested that some C . albicans strains have another ALS gene with a 5' domain similar to that of ALS6 . Characterization of ALS7 revealed a novel tandemly repeated sequence within the C-terminal domain . Unlike other ALS family tandem repeats, the newly characterized ALS7 repeat does not appear to define additional genes in the ALS family . However, our data and information from the C . albicans genome sequencing project suggest that there are additional ALS genes remaining to be characterized .

Yakugaku Zasshi, 2000 Jun, 120(6), 587 - 9
{The anti-Candida activity of shikon}; Sasaki K et al.; Antifungal activity of Shikon, roots of Lithospermum erythrorhizon and Arnebia euchroma was investigated in vitro . The extracts containing the pigments of Ko-shikon or Nan-shikon showed the antifungal activities against Candida albicans . Acetylshikonin, one of these pigments, inhibited the fungal growth at MIC 15.6 micrograms/ml (RPMI24 h) or 3.9 micrograms/ml (YNB24 h).

Chemotherapy, 2000 Jul-Aug, 46(4), 245 - 52
Influence of temperature and concentration on the postantifungal effect and the effects of sub-MIC concentrations of four antifungal agents on previously treated Candida species; Garcia MT et al.; BACKGROUND: The objective of this study was to investigate the impact of different temperatures (22, 35 and 37 degrees C) on the postantifungal effect (PAFE) and the effect of sub-MIC concentrations (1/4 x MIC) on Candida albicans and Candida glabrata in PAFE stage (PAFSE) . METHODS: This stage was induced by a 1.5-hour pretreatment with different doses (1 x, 4 x and 8 x MIC) of four antifungal agents that are fundamental to modern candidiasis therapy . RESULTS: The temperature, as well as the dose of the antifungal agent that was applied during the pretreatment, determined the duration of the two studied effects . An increase in the temperature and/or the dose prolonged the duration of the PAFE and PAFSE in both species, independent of the applied antifungal agent . Amphotericin B and 5-fluorocytosine always induced significant PAFEs (0.5-4.8 h and 0.5-3.0 h, respectively), which were increased (0.7-3.4 h and 0.5-3 . 2 h, respectively) by posterior exposure to 1/4 x MIC of the respective antifungal agent . In the case of ketoconazole and fluconazole, temperature and concentration were especially important . Although neither antimycotics was able to induce a significant PAFE, posterior exposure to 1/4 x MIC of each of the two azoles led in both yeast species to a significant PAFSE of up to 0.8 h (if the concentrations and/or the temperatures were high enough) . CONCLUSION: Factors such as temperature and concentration could be important when choosing an antifungal agent .

Chemotherapy, 2000 Jul-Aug, 46(4), 235 - 44
Comparative in vitro antifungal activity of amphotericin B lipid complex, amphotericin B and fluconazole; Carrillo-Munoz AJ et al.; Amphotericin B (AMB) is considered the gold standard in the treatment of serious systemic mycoses in spite of its nephrotoxicity and adverse effects . Association with lipids enables larger doses of AMB to be given with a longer t((1/2)) and C(max), without the toxic effects at lower concentrations . Liposome-encapsulated AMB shows a lower affinity for mammalian cells and improves V(d), thus decreasing toxicity . Amphotericin B lipid complex (ABLC) is an AMB formulation associated with a biodegradable phospholipid matrix (5% molar) from which the drug is released by cell phospholipases . ABLC is recommended for serious mycoses refractory to conventional antifungal therapy or when AMB is contraindicated . We compared the in vitro antifungal activity of ABLC, AMB and fluconazole (FLZ) against 328 strains of clinically significant opportunistic fungi using a microdilution method (NCCLS, M-27A) . 64.9% of the yeasts were inhibited by MIC of ABLC </= AMB resulting in a similar or slightly superior efficacy compared to AMB when tested against Candida albicans, C . glabrata, C . guilliermondii, C . parapsilosis and C . tropicalis . Effectiveness against C . krusei was lower for ABLC (5.99 microg/ml for ABLC, 1.58 microg/ml for AMB) . However, for Aspergillus fumigatus, the activities of AMB and ABLC were 1.62 and 2.46 microg/ml, respectively; A . niger 0.72 microg/ml, 0.76 microg/ml (ABLC and AMB, respectively); A . clavatus, A . candidus, A . tenuissima, A . corymbifera and Exophiala jeanselmei, Scedosporium spp . and Miceliophtora spp . showed a low susceptibility to both AMB formulations . ABLC is a useful alternative to AMB or FLZ for the treatment of severe fungal infections, due to the broad spectrum of antifungal actions observed in this study .

Infect Immun, 2000 Jul, 68(7), 3840 - 7
Measurement of T-cell-derived antigen binding molecules and immunoglobulin G specific to Candida albicans mannan in sera of patients with recurrent vulvovaginal candidiasis; Little CH et al.; Immunoglobulin G (IgG) and T-cell-derived antigen binding molecules (TABM) specific to whole Candida extract and to Candida-derived mannans prepared by both the cetryltrimethylammonium bromide (CTAB) and alkaline degradation (PEAT) methods were measured in the sera of women with vulvovaginal candidiasis and controls . In the patients there were significantly higher levels of IgG to both CTAB and PEAT mannans and of TABM to CTAB mannan . TABM specific to CTAB mannan was purified from the serum of a patient with a high titer of this TABM . The purified TABM bound specifically to CTAB mannan and to other yeast and mold extracts . This TABM preparation was associated with transforming growth factor beta2 (TGF-beta2), and on specific binding to mannan there was a marked increase in the level of detectable TGF-beta2 . This increase in TGF-beta2 level was critically dependent on the relative concentrations of the purified TABM and mannan, being smallest when either was in excess . The TABM specific to CTAB mannan was also shown to inhibit Candida-stimulated gamma interferon production . The results suggest that CTAB mannan-specific TABM may increase susceptibility to vulvovaginal candidiasis in association with a shift in the immune response to the Th2 type.

Int J Food Microbiol, 2000 May 25, 56(1), 3 - 12
Antimicrobial effects of Finnish plant extracts containing flavonoids and other phenolic compounds; Rauha JP et al.; Plant phenolics, especially dietary flavonoids, are currently of growing interest owing to their supposed functional properties in promoting human health . Antimicrobial screening of 13 phenolic substances and 29 extracts prepared from Finnish plant materials against selected microbes was conducted in this study . The tests were carried out using diffusion methods with four to nine microbial species (Aspergillus niger, Bacillus subtilis, Candida albicans, Escherichia coli, Micrococcus luteus, Pseudomonas aeruginosa, Saccharomyces cerevisiae, Staphylococcus aureus and Staphylococcus epidermidis) . Flavone, quercetin and naringenin were effective in inhibiting the growth of the organisms . The most active plant extracts were purple loosestrife (Lythrum salicaria L.) against Candida albicans, meadowsweet (Filipendula ulmaria (L.) Maxim.), willow herb (Epilobium angustifolium L.), cloudberry (Rubus chamaemorus L.) and raspberry (Rubus idaeus L.) against bacteria, and white birch (Betula pubescens Ehrh.), pine (Pinus sylvestris L.) and potato (Solanum tuberosum . L.) against gram-positive Staphylococcus aureus.

Int J Antimicrob Agents, 2000 Jun, 15(1), 43 - 8
Prevention by L-alpha-phosphatidylcholine of antifungal activity in vitro of liposome-encapsulated imidazoles determined by using time-killing curves; De Logu A et al.; The antifungal activity of the imidazole derivatives miconazole and ketoconazole was reduced when they were entrapped in liposomal structures and significant differences were detected between small unilamellar vesicles (SUV) and multilamellar vesicles (MLV) . To understand which component of liposomes interfered with the antifungal activity of miconazole and ketoconazole, we examined the influence of pure egg and soy L-alpha-phosphatidylcholine and cholesterol on activity against Candida albicans ATCC E10231 by time killing curves . Association of phospholipids-cholesterol-imidazole leads to an inhibitory effect on the antifungal activity comparable to that shown when miconazole or ketoconazole were entrapped in SUV liposomes or when miconazole and ketoconazole were incubated in the presence of L-alpha-phosphatidylcholine . The antifungal activity determined in the presence of cholesterol was comparable to that observed with the free drugs . Inhibition of the antifungal activity of miconazole and ketoconazole by phospholipids is dependent on the phospholipid concentration but is independent of the source of phospholipids (egg or soy) . Cholesterol had no influence on the antifungal activity of the imidazoles, unlike the effect on other antifungal drugs, such as amphotericin B.

Gene, 2000 May 30, 250(1-2), 159 - 69
Characterization of CaGSP1, the Candida albicans RAN/GSP1 homologue; Clement M et al.; Gsp1p is a small nuclear-located GTP binding protein from the yeast Saccharomyces cerevisiae . It is highly conserved among eucaryotic cells and is involved in numerous cellular processes, including nucleocytoplasmic trafficking of macromolecules . To learn more about the GSP1 structure/function, we have characterized its Candida albicans homologue . CaGsp1p is 214 amino acids long and displays 91% identity to the ScGsp1p . There is functional complementation in S . cerevisiae, and its mRNA is constitutively expressed in the diploid C . albicans grown under various physiological conditions . Disruption of both alleles was not possible, suggesting that it could be an essential gene, but heterozygous mutants exhibited genomic instability.

Blood, 2000 Jun 15, 95(12), 3725 - 33
"Emergency" granulopoiesis in G-CSF-deficient mice in response to Candida albicans infection; Basu S et al.; Granulocyte colony-stimulating factor (G-CSF) is a glycoprotein believed to play an important role in regulating granulopoiesis both at steady state and during an "emergency" situation . Generation of G-CSF and G-CSF receptor-deficient mice by gene targeting has demonstrated unequivocally the importance of G-CSF in the regulation of baseline granulopoiesis . This study attempted to define the physiologic role of G-CSF during an emergency situation by challenging a cohort of wild-type and G-CSF-deficient mice with Candida albicans . Interestingly, after infection, G-CSF-deficient mice developed an absolute neutrophilia that was observed both in blood and bone marrow . In addition, 3 days after Candida infection increased numbers of granulocyte-macrophage (GM) and macrophage (M) progenitors were observed in the bone marrow of G-CSF-deficient mice . Of the cytokines surveyed, interleukin (IL)-6 levels in serum were elevated; interestingly, levels of IL-6 were higher and more sustained in G-CSF-deficient mice infected with C albicans than similarly infected wild-type mice . Despite the higher levels of serum IL-6, this cytokine is dispensable for the observed neutrophilia because candida-infected IL-6-deficient mice, or mice simultaneously deficient in G-CSF and IL-6, developed neutrophilia . Similarly, mice lacking both G-CSF and GM-CSF developed absolute neutrophilia and had elevated numbers of GM and M progenitors in the bone marrow; thus, G-CSF and GM-CSF are dispensable for promoting the emergency response to candidal infection . (Blood . 2000;95:3725-3733)

Ann Dermatol Venereol, 2000 Apr, 127(4), 405 - 7
{Major eosinophilia in a premature infant induced by topical ketoconazole}; Michel JL et al.; INTRODUCTION: Ketoconazole is a member of a newer group of imidazole antifungals . Except for treatment of oral candidiasis, there is no reported experience of use in neonates . Thus, its use in neonatalogy must be considered experimental . Herein we report a major eosinophilia in a very low birth weight neonate induced by transcutaneous resorption of topical ketoconazole . CASE REPORT: A 26 week premature was intubated and ventilated for a hyaline membrane disease . At more than 2 weeks, he developed diaper dermatitis with Candida albicans . Treatment consisted of local application of ketoconazole . After 6 days of application, WBC showed a major eosinophilia {20,000/microL} . Discontinuation of the drug was followed by a prompt normalization of the eosinophil count . The challenge test by topical ketoconazole reproduced eosinophilia, implicating this drug, which, to our knowledge, has not been described previously to cause eosinophilia in very low weight neonates . DISCUSSION: Peripheral eosinophilia is an uncommon finding present in a relatively limited number of conditions in children . A minor eosinophilia often seen in premature neonates may in part be due to intubation . This case illustrates the potential danger of percutaneous application of drugs in newborn and infants as it has previously been pointed out way various chemicals . Any deterioration of the corneal layer such as observed in many dermatosis prompts an increase in the cutaneous permeability . Immaturity of the premature skin may have aiso played a role . Dermatologists should be aware of potential toxic topical ointments before prescribing them to very low weight babies.

Cytokine, 2000 Jun, 12(6), 666 - 70
A single injection of polyethylene-glycol granulocyte colony-stimulating factor strongly prolongs survival of mice with systemic candidiasis; van Spriel AB et al.; Systemic candidiasis is a life-threatening disease occurring in immunocompromized patients . Granulocyte colony-stimulating factor (G-CSF) reduces mortality in experimental invasive candidiasis . Covalent conjugation of polyethylene-glycol (peg) to proteins increases their stability and in vivo bioactivity . In this study, the effect of a single subcutaneous injection of peg-G-CSF on lethal candidiasis was assessed . This was performed in acute and chronic candidiasis models in non-neutropenic FVB/N mice . Peg-G-CSF rapidly increased circulating polymorphonuclear leukocyte (PMNL) numbers in mice, sustaining high for >4 days . Candida albicans outgrowth from kidneys of infected mice was strongly reduced after peg-G-CSF treatment (5.76 log cfu/g kidney vs 7.66 control), with absence of hyphal outgrowth and enhanced PMNL influx . Moreover, peg-G-CSF increased survival of C . albicans -infected mice, whereas efficacy of uncoupled G-CSF was obtained only after repeated treatment . These data document a potent in vivo biological effect of peg-G-CSF, resulting in strongly enhanced resistance against systemic candidiasis .

Australas Radiol, 2000 May, 44(2), 206 - 7
Candida jejunitis: a rare cause of intestinal pneumatosis in the immunocompromised patient; Tie ML et al.; A case of fatal necrotizing jejunitis caused by Candida albicans is described in a patient with acute myeloid leukaemia undergoing chemotherapy . The diagnosis was made at autopsy . Computed tomography findings were of small bowel dilatation with pneumatosis.

Mol Cell Biol, 2000 Jul, 20(13), 4635 - 47
Dominant active alleles of RIM101 (PRR2) bypass the pH restriction on filamentation of Candida albicans; El Barkani A et al.; Morphological development of the fungal pathogen Candida albicans is profoundly affected by ambient pH . Acidic pH restricts growth to the yeast form, whereas neutral pH permits development of the filamentous form . Superimposed on the pH restriction is a temperature requirement of approximately 37 degrees C for filamentation . The role of pH in development was investigated by selecting revertants of phr2Delta mutants that had gained the ability to grow at acid pH . The extragenic suppressors in two independent revertants were identified as nonsense mutations in the pH response regulator RIM101 (PRR2) that resulted in a carboxy-terminal truncation of the open reading frame . These dominant active alleles conferred the ability to filament at acidic pH, to express PHR1, an alkaline-expressed gene, at acidic pH, and to repress the acid-expressed gene PHR2 . It was also observed that both the wild-type and mutant alleles could act as multicopy suppressors of the temperature restriction on filamentation, allowing extensive filamentation at 29 degrees C . The ability of the activated alleles to promote filamentation was dependent upon the developmental regulator EFG1 . The results suggest that RIM101 is responsible for the pH dependence of hyphal development.

Shock, 2000 Jun, 13(6), 453 - 8
The Candida albicans INT1 gene facilitates cecal colonization in endotoxin-treated mice; Bendel CM et al.; Increased intestinal colonization with Candida albicans is believed to be a major predisposing factor to systemic candidiasis . Previous evidence has implicated the C . albicans INT1 gene in hyphal development, epithelial adherence, and mouse virulence . The effect of INT1 on mouse cecal colonization was measured using a parent strain (CAF2, INT1/INT1), an int1 deletion homozygote (CAG3, int1/int1), and a heterozygous reintegrant (CAG5, int1/int1 + INT1) . Forty-eight hours after oral inoculation of 10(7) C . albicans into normal mice, only low numbers of each strain were recovered from the cecal flora . In mice pretreated with oral bacitracin/streptomycin, cecal colonization of each C . albicans strain was increased compared to the corresponding strain inoculated into untreated mice, with the CAF2 parent strain greater (P < 0.01) than the two mutant strains, and with the heterozygous and homozygous mutants not different from each other . In mice pretreated with parenteral lipopolysaccharide (LPS), in addition to oral antibiotics, numbers of cecal CAF2, CAG5, and CAG3 were increased (P < 0.01) compared to the corresponding strain inoculated into mice treated with antibiotics alone . In LPS-treated mice, numbers of cecal C . albicans CAF2 (INT1/INT1) were greater (P < 0.05) than C . albicans CAG3 (int1/int1) . Thus, parenteral LPS had an additive effect on C . albicans cecal colonization in antibiotic-treated mice, and the presence of two functional copies of the INT1 gene appeared to facilitate colonization in both antibiotic-treated mice and in mice treated with antibiotics plus parenteral endotoxin.

Int J Hematol, 2000 Apr, 71(3), 273 - 7
Changes in microbial flora in neutropenic patients with hematological disorders after the Hanshin-Awaji earthquake; Takatsuka H et al.; The earthquake that struck the Hanshin-Awaji district on January 17, 1995, was one of the most destructive to occur in Japan . We analyzed the changes in microorganisms isolated from patients with hematological disorders in relation to this earthquake . We reviewed a total of 4137 microbial cultures obtained over 1-year periods before and after the earthquake . There were 123 neutropenic patients admitted during the study period (54 before the earthquake and 69 afterwards) . No significant differences in clinical characteristics and underlying diseases were found between the 2 groups . Polynomial analysis revealed a significant increase in the isolation of Candida albicans, Candida glabrata, and Pseudomonas aeruginosa from sputum and stool cultures after the earthquake . These microorganisms increased markedly in patients from severe earthquake-damaged areas . In neutropenic patients with hematological disorders, microbial isolates changed significantly after the Hanshin-Awaji earthquake . These changes may have been related to physical and emotional stress, as well as to the deterioration of environmental factors such as food, air, and water.

Pediatr Dent, 2000 May-Jun, 22(3), 234 - 8
Identification of Candida dubliniensis in a study of HIV-seropositive pediatric dental patients; Brown DM et al.; PURPOSE: The combination of an immature immune system and suppressed cellular immunity in children with HIV infections provides optimal conditions for rapid disease progression . As a result, pediatric AIDS has become a major epidemiological challenge . Oral fungal colonization remains one of the most common opportunistic infections observed in both adult and pediatric HIV infected patients . Although Candida albicans is the most frequently isolated opportunistic fungal species, a recently characterized Candida species, C . dubliniensis, has gained considerable attention due to its almost exclusive association with HIV-seropositive individuals . The purpose of this study was to prospectively screen for the presence of C . dubliniensis among pediatric HIV+ patients . METHODS: Oral samples taken from twenty-seven children were cultured for the presence of yeast . All positive yeast isolates obtained were screened for the presence of C . dubliniensis by use of tests for germ tube and chlamydospore production, detection of inability to grow at 45 degrees C, by colony color on CHROMagar Candida medium, coaggregation with Fusobacterium nucleatum ATCC 49256 and by the results of sugar assimilation testing with the API 20C AUX yeast identification system . RESULTS: Among the 27 patients tested, 3 patients were found to harbor C . dubliniensis, one of which also grew C . glabrata; 12 patients were colonized with C . albicans, while the remaining 12 patients were negative for yeast . Identification of the three C . dubliniensis isolates was genetically confirmed by electrophoretic karyotyping . All three C . dubliniensis isolates were found to be susceptible to fluconazole (MIC < or = 0.25 ug/ml) . CONCLUSIONS: These results confirm the presence of this novel species in a dental pediatric HIV seropositive population and support the need for further investigation into the prevalence and pathogenesis of C . dubliniensis.

Mol Microbiol, 2000 Jun, 36(5), 1156 - 66
Glycosylation deficiency phenotypes resulting from depletion of GDP-mannose pyrophosphorylase in two yeast species; Warit S et al.; The genes encoding GDP-mannose pyrophosphorylase from Saccharomyces cerevisiae (SRB1/PSA1) and Candida albicans (CaSRB1) were expressed under the control of the tightly regulated promoters of MET3 and CaMET3 respectively . Northern analysis showed that the addition of methionine effectively blocks the transcription of pMET3-SRB1/PSA1 and pCaMET3CaSRB1 expression cassettes, which had been integrated into the genomes of appropriate mutants . Methionine-mediated repression of CaSRB1 caused loss of viability in C . albicans, demonstrating that, as in S . cerevisiae, the gene is essential for growth . Depletion of GDP-mannose pyrophosphorylase had a highly pleiotropic effect in the two yeasts . The major phenotypes observed were lysis, failure of cell separation and/or cytokinesis, impaired bud growth and bud's site selection, clumping and flocculation, as well as increased sensitivity to a wide range of antifungal drugs and cell wall inhibitors, and impaired hyphal switching ability . These phenotypes resulted from defects in glycosylation, as demonstrated by reduced affinity for Alcian blue and sensitivity to hygromycin B . Our results provide new information about the roles of protein glycosylation in yeast and, in particular, the steps that require GDP-mannose in the fungal pathogen C . albicans.

Mol Microbiol, 2000 May, 36(4), 856 - 65
Targeted gene disruption in Candida albicans wild-type strains: the role of the MDR1 gene in fluconazole resistance of clinical Candida albicans isolates; Wirsching S et al.; Resistance of the pathogenic yeast Candida albicans to the antifungal agent fluconazole is often caused by active drug efflux out of the cells . In clinical C . albicans strains, fluconazole resistance frequently correlates with constitutive activation of the MDR1 gene, encoding a membrane transport protein of the major facilitator superfamily that is not expressed detectably in fluconazole-susceptible isolates . However, the molecular changes causing MDR1 activation have not yet been elucidated, and direct proof for MDR1 expression being the cause of drug resistance in clinical C . albicans strains is lacking as a result of difficulties in the genetic manipulation of C . albicans wild-type strains . We have developed a new strategy for sequential gene disruption in C . albicans wild-type strains that is based on the repeated use of a dominant selection marker conferring resistance against mycophenolic acid upon transformants and its subsequent excision from the genome by FLP-mediated, site-specific recombination (MPAR-flipping) . This mutagenesis strategy was used to generate homozygous mdr1/mdr1 mutants from two fluconazole-resistant clinical C . albicans isolates in which drug resistance correlated with stable, constitutive MDR1 activation . In both cases, disruption of the MDR1 gene resulted in enhanced susceptibility of the mutants against fluconazole, providing the first direct genetic proof that MDR1 mediates fluconazole resistance in clinical C . albicans strains . The new gene disruption strategy allows the generation of specific knock-out mutations in any C . albicans wild-type strain and therefore opens completely novel approaches for studying this most important human pathogenic fungus at the molecular level.

Mol Microbiol, 2000 May, 36(3), 618 - 29
Analysis of the oxidative stress regulation of the Candida albicans transcription factor, Cap1p; Zhang X et al.; CAP1 encodes a basic region-leucine zipper (bZip) transcriptional regulatory protein that is required for oxidative stress tolerance in Candida albicans . Cap1p is a homologue of a Saccharomyces cerevisiae bZip transcription factor designated Yap1p that is both required for oxidative stress tolerance and localized to the nucleus in response to the presence of oxidants . Oxidant-regulated localization of Yap1p to the nucleus requires the presence of a carboxy-terminal cysteine residue (C629) that is conserved in Cap1p as C477 . To examine the role of this conserved cysteine residue, C477 was replaced with an alanine residue . This mutant protein, C477A Cap1p, was analysed for its behaviour both in S . cerevisiae and C . albicans . Wild type and C477A Cap1p were able to complement the oxidant hypersensitivity of a Deltayap1 S . cerevisiae strain . Whereas a Yap1p-responsive lacZ fusion gene was oxidant inducible in the presence of YAP1, the C . albicans Cap1p derivatives were not oxidant responsive in S . cerevisiae . Introduction of wild type and C477A Cap1p-expressing plasmids into C . albicans produced differential resistance to oxidants . Glutathione reductase activity was found to be inducible by oxidants in the presence of Cap1p but was constitutively elevated in the presence of C477A Cap1p . Western blot assays indicate Cap1p is post-translationally regulated by oxidants . Green fluorescent protein fusions to CAP1 showed that this protein is localized to the nucleus only in the presence of oxidants while C477A Cap1p is constitutively nuclear localized . Directly analogous to S . cerevisiae Yap1p, regulated nuclear localization of C . albicans Cap1p is crucial for its normal function.

J Infect, 2000 Mar, 40(2), 191 - 2
Candida albicans meningitis: clinical case; Aleixo MJ et al.; Candida spp . meningitis is still a rare clinical situation, although it is becoming more frequent . Literature references to it and therapeutic options are scarce . We present a case of a young male, HIV-positive drug addict, with Candida albicans meningitis which was treated with oral fluconazole, having a good outcome.

Chemistry, 2000 Apr 14, 6(8), 1355 - 60
Oxepinamides A-C and fumiquinazolines H--I: bioactive metabolites from a marine isolate of a fungus of the genus Acremonium; Belofsky GN et al.; Three new oxepin-containing natural products (1-3) and two new fumiquinazoline metabolites (4-5) have been isolated from organic extracts of the culture broth and mycelia of an Acremonium sp., a fungus obtained from the surface of the Caribbean tunicate Ecteinascidia turbinata . The structures of the five compounds were determined through extensive analysis of 1D- and 2D-NMR data, and mass spectrometry . Compound 1 exhibited good anti-inflammatory activity in a topical RTX-induced mouse ear edema assay . Compounds 4 and 5 exhibited weak antifungal activity toward Candida albicans in a broth microdilution assay.

Am J Infect Control, 2000 Jun, 28(3), 251 - 7
Microbicidal activity of MDI-P against Candida albicans, Staphylococcus aureus, Pseudomonas aeruginosa, and Legionella pneumophila; Baltch AL et al.; BACKGROUND: MDI-P (Medical Discoveries, Inc-Pharmaceutical, Layton, Utah) is a clear, colorless liquid generated by electrolysis of preservative-free and endotoxin-free, nonpyrogenic, sterile, injection saline (0.9% NaCl, wt/vol) . It contains numerous highly reactive chlorine and oxygen species, including HOCl(-1,) OCl-(1), Cl(-1), Cl(2), O(2-)(1), and O(3) . This report presents data on the in vitro microbicidal activity of MDI-P against 4 clinically relevant microbial pathogens that are often difficult to eradicate . METHODS: MDI-P was generated from injection saline by using a patented electrolysis instrument . It was then tested for microbicidal activity at concentrations ranging from 0.01% to 50% against Staphylococcus aureus, Pseudomonas aeruginosa, Legionella pneumophila, and Candida albicans (10(5) to 10(9) colony-forming units/mL) . The effect of serum (50% and 90%) and pH on MDI-P activity were also tested . The morphologic effects of MDI-P on microbial cells were studied by light microscopy of cells stained by Gram's method and by transmission electron microscopy . Morbidity, mortality, and the effect of MDI-P on tissues were studied by using a mouse model . RESULTS: The microbicidal activity of MDI-P occurred within the first minute of exposure for all the organisms tested . When 50% MDI-P was tested against cell titers of 10(5) or 10(7) colony-forming units/mL, all test organisms were killed within 1 minute; at lower MDI-P concentrations, C albicans was the most sensitive organism, and L pneumophila was the most resistant . Even with beginning cell titers of 10(9) colony-forming units/mL, killing by 50% MDI-P was >99.9% for all test strains . Furthermore, at the same beginning cell titer, killing of C albicans by MDI-P diluted to 50% with normal human serum rather than injection saline was only slightly reduced . No acute morbidity, mortality, or tissue damage was detected in mice that were intravenously given 17 mL/kg of undiluted MDI-P . CONCLUSIONS: MDI-P is a very fast-acting, broad-spectrum microbicidal material . The lack of evidence for acute morbidity, mortality, or tissue injury, ease of preparation, and low cost suggest that it may be useful for various sterilization and disinfection applications.

Phytomedicine, 2000 Apr, 7(2), 123 - 7
Anti-inflammatory, antioxidant and antimicrobial activity of Ophthacare brand, an herbal eye drops; Mitra SK et al.; In the present study, the herbal preparation of Ophthacare brand eye drops was investigated for its anti-inflammatory, antioxidant and antimicrobial activity, using in vivo and in vitro experimental models . Ophthacare brand eye drops exhibited significant anti-inflammatory activity in turpentine liniment-induced ocular inflammation in rabbits . The preparation dose-dependently inhibited ferric chloride-induced lipid peroxidation in vitro and also showed significant antibacterial activity against Escherichia coli and Staphylococcus aureus and antifungal activity against Candida albicans . All these findings suggest that Ophthacare brand eye drops can be used in the treatment of various ophthalmic disorders.

Eur Respir J, 1999 Jan, 13(1), 180 - 6
Pre-emptive therapy with azoles in lung transplant patients . Geneva Lung Transplantation Group; Hamacher J et al.; Pulmonary fungal infection is diagnosed in up to 15-25% of lung transplant recipients and frequently bears a fatal outcome . This prospective uncontrolled study addresses the efficacy and safety of pre-emptive azole therapy against fungal infection in these patients . Fluconazole or itraconazole have been systematically used according to reported fungus sensitivity after the discovery of fungi in lower respiratory tract samples . Patients were treated until the bronchial suture was normal and the cultures of the following bronchoscopy remained negative . Fungi were found post-transplantation in the lower respiratory tract specimens of 26 out of 31 (84%) patients, predominantly Candida albicans (20 patients) and Aspergillus fumigatus (16 patients) . Mycelia characteristic of Candida spp . or Aspergillus spp . were found in necrotic tissue at the bronchial suture of nine patients . The mean duration of the 38 treatments was 3.6+/-2.6 months (range, 0.5-12 months) . After a median follow-up of 16 (range, 0-48) months, two cases of extended ulcerative and pseudo membranous Aspergillus fumigatus bronchitis were observed and healed under itraconazole treatment . In conclusion, pre-emptive azole therapy may be effective and well-tolerated in lung transplant patients where fungi are found in the airways or pleura.

J Clin Microbiol, 2000 Jun, 38(6), 2423 - 6
Retrospective identification and characterization of Candida dubliniensis isolates among Candida albicans clinical laboratory isolates from human immunodeficiency virus (HIV)-infected and non-HIV-infected individuals; Jabra-Rizk MA et al.; Fungal opportunistic infections, and in particular those caused by the various Candida species, have gained considerable significance as a cause of morbidity and, often, mortality . The newly described species Candida dubliniensis phenotypically resembles Candida albicans so closely that it is easily misidentified as such . The present study was designed to determine the frequency at which this new species is not recognized in the clinical laboratory, to determine the patient populations with which C . dubliniensis is associated, to determine colonization versus infection frequency, and to assess fluconazole resistance . Over a 2-year period, 1,251 isolates that were initially identified as C . albicans by a hospital clinical laboratory were reevaluated for C . dubliniensis by inability to grow at 45 degrees C, colony color on CHROMagar Candida medium, coaggregation assay with Fusobacterium nucleatum, and sugar assimilation profiles (API 20C AUX yeast identification system) . A total of 15 (1.2%) isolates from 12 patients were identified as C . dubliniensis . Ten of the patients were found to be immunocompromised (these included patients with human immunodeficiency virus infection or AIDS, cancer patients receiving chemotherapy, and patients awaiting transplantation) . Thirteen isolates were highly susceptible to fluconazole (MIC, <0.5 microgram/ml) . Three isolates from one patient, genotypically confirmed as the same strain, showed variable susceptibility to fluconazole . The first isolate was susceptible, whereas the other two isolates were dose-dependent susceptible (MIC, 16.0 microgram/ml) . These data confirm the close association of C . dubliniensis with immunocompromised states and that increased fluconazole MICs may develop in vivo . This study emphasizes the importance of screening germ-tube-positive yeasts for the inability to grow at 45 degrees C followed by confirmatory tests in order to properly identify this species.

J Clin Microbiol, 2000 Jun, 38(6), 2369 - 73
Correlation between in vitro and in vivo antifungal activities in experimental fluconazole-resistant oropharyngeal and esophageal candidiasis; Walsh TJ et al.; Oropharyngeal and esophageal candidiasis (OPEC) is a frequent opportunistic mycosis in immunocompromised patients . Azole-resistant OPEC is a refractory form of this infection occurring particularly in human immunodeficiency virus (HIV)-infected patients . The procedures developed by the Antifungal Subcommittee of the National Committee for Clinical Laboratory Standards (NCCLS) are an important advance in standardization of in vitro antifungal susceptibility methodology . In order to further understand the relationship between NCCLS methodology and antifungal therapeutic response, we studied the potential correlation between in vitro susceptibility to fluconazole and in vivo response in a rabbit model of fluconazole-resistant OPEC . MICs of fluconazole were determined by NCCLS methods . Three fluconazole-susceptible (FS) (MIC, </=0.125 microgram/ml) and three fluconazole-resistant (FR) (MIC, >/=64 microgram/ml) isolates of Candida albicans from prospectively monitored HIV-infected children with OPEC were studied . FR isolates were recovered from children with severe OPEC refractory to fluconazole, and FS isolates were recovered from those with mucosal candidiasis responsive to fluconazole . Fluconazole at 2 mg/kg of body weight/day was administered to infected animals for 7 days . The concentrations of fluconazole in plasma were maintained above the MICs for FS isolates throughout the dosing interval . Fluconazole concentrations in the esophagus were greater than or equal to those in plasma . Rabbits infected with FS isolates and treated with fluconazole had significant reductions in oral mucosal quantitative cultures (P < 0.001) and tissue burden of C . albicans in tongue, soft palate, and esophagus (P < 0.001) . In comparison, rabbits infected with FR isolates were unresponsive to fluconazole and had no reduction in oral mucosal quantitative cultures or tissue burden of C . albicans versus untreated controls . We conclude that there is a strong correlation between in vitro fluconazole susceptibility by NCCLS methods and in vivo response to fluconazole therapy of OPEC due to C . albicans.

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