Life Sci, 1999, 65(15), 1537 - 44 Inhibition of endothelium-dependent relaxation by Candida albicans; Ataoglu H et al.; This study examines the effects of Candida albicans on acethylcholine-induced, endothelium-dependent relaxation of thoracic aorta of rabbits, precontracted by phenylephrine (10(-7) M) . Isolated vessel rings were incubated with C . albicans, Saccharomyces cerevisiae, or their mannans, and endothelium-dependent relaxation was measured by the induction of acethylcholine . Endothelium-dependent relaxation remained unaffected after 3 hours by either C . albicans or S . cerevisiae, or their mannans . After 24 hours, however, incubation with C . albicans had completely abolished relaxation, whereas relaxation was decreased by mannan of C . albicans and continued unaffected by S . cerevisiae . In contrast, no change was registered with a 24 hours incubation of C . Albicans in a sodium nitroprusside-induced, endothelium-independent, vascular smooth muscle relaxation . Microscopical investigation of the morphological structure of vessel walls revealed penetration of C . albicans on the intimal surface after 3 hours incubation and infiltration of the yeast through the vessel wall after 24 hours . No changes in vessel morphology occurred after 3 or 24 hours with S . cerevisiae or the mannan of C . albicans . These results show the ability of C . albicans to inhibit endothelium-dependent, but not endothelium-independent, relaxation of vascular smooth muscle and may have important implications for functional damage to endothelial cells and the regulation of vessel tone and blood flow. Biol Cell, 1999 Sep, 91(7), 525 - 31 Effects of isoproterenol on IL-2 and cAMP production in peripheral T cells from asthmatic and non-asthmatic subjects sensitive to Candida; Aihara M et al.; Immunity to Candida albicans (Candida) is characterized by a Th-1 type pattern of reactivity . Candida is rarely a cause antigen for bronchial asthma . Beta agonists have been found to inhibit secretion of IL-2 from T cells through intracellular cAMP elevation . We examined effects of isoproterenol (ISO) on Candida-stimulated T cells . Peripheral T cells obtained from six Candida-sensitive asthmatics, six Candida-sensitive non-asthmatic subjects, and six normal donors by Ficoll-Hypaque gradient centrifugation and nylon-wool column chromatography were incubated with Candida antigen or concanavalin A (Con A) in the absence or presence of ISO . Secretion of IL-2 and intracellular accumulation of cAMP were assayed by ELISA . Con A induced secretion of IL-2 in each of the three groups . Candida antigen induced IL-2 secretion in the normal and the non-asthmatic subjects, but not in the asthmatics . ISO, which reduced Con A-induced secretion of IL-2 in a dose-dependent manner, had no effect on Candida-induced secretion of IL-2 . Although ISO increased the intracellular concentration of cAMP in untreated and Con A-treated T cells from all donors, cells from the normal and the non-asthmatic subjects, but not from the asthmatics, that were co-incubated with ISO and Candida had lower levels of cAMP than those treated with ISO alone . It is suggested that Candida antigen induces secretion of IL-2 and reduces ISO-inducible accumulation of cAMP in Candida-responsive IL-2 secreting cells, which may make Candida-induced secretion of IL-2 insensitive to ISO. Allerg Immunol (Paris), 1999 Oct, 31(8), 263 - 7 A clinical study of airborne allergens in the United Arab Emirates; Lestringant GG et al.; BACKGROUND: In the past 25 years the United Arab Emirates (UAE) have experienced a socioeconomic boom . The once nomadic Bedouin population of Al Ain, in the emirate of Abu Dhabi, now lives in modern air-conditioned accommodation, and huge desalination plants have allowed afforestation and farming . OBJECTIVE: To evidence responsible airborne allergens in an UAE population . PATIENTS AND METHODS: 263 UAE Nationals who attended Tawam Hospital (Al Ain, UAE) with a respiratory disease suspected of being of allergic origin, were submitted to SPT and RAST . The choice of pollinic allergens was made in accordance with the local flora and market availability . All patients were SPTed with at least the same battery of 15 pollinic and indoor allergens . Most patients were submitted to at least 4 RAST, viz Cynodon dactylon, Salsola kali, Prosopis juliflora and Dermatophagoides pteronyssinus . RESULTS: 71.8% patients were positive for at least one allergen . Pollen accounted for 61.6% of positive patients, with 45.2% positive to chenopodiaceae, 33% to gramineae and 23.5% to P . juliflora . Indoor allergens were positive in 30.4% of patients with 17.9% positive to D . pteronyssinus and D . farinae, 11% to Blatella germanica, 8.3% to Cat fur, 4.9% to Goat hair, 0.7% to Rat hair and Mouse hair and 1.5% to Candida albicans . CONCLUSION: Pollen was the prominent allergen . There is room, however, for further epidemiological studies possibly with new extracts and RAST specifically designed after the species of the Gulf region. Singapore Med J, 1999 Aug, 40(8), 533 - 6 Successful treatment of Candida albicans endocarditis in a child with leukemia--a case report and review of the literature; Ariffin H et al.; Candida species is now being increasingly recognised as an important cause of endocarditis especially in immunocompromised patients . A case of Candida albicans endocarditis in a child with acute lymphoblastic leukemia (ALL) is reported . The child did not have a central venous catheter at any time . Treatment consisted of intravenous amphotericin B and fluconazole for 3 weeks followed by oral fluconazole for 2 weeks . No surgical resection was necessary . We highlight here the importance of echocardiography in the management of prolonged febrile neutropenia and discuss the dilemma of continuing chemotherapy in such patients. Yeast, 1999 Nov, 15(15), 1609 - 18 Transformation of Candida albicans by electroporation; De Backer MD et al.; In contrast to a variety of other yeasts, Candida albicans has proved difficult to transform with high efficiency . Lithium acetate transformation is fast and simple but provides a very low efficiency of DNA transfer (50-100 transformants/microg DNA), while spheroplast transformation, although more efficient ( approximately 300 transformants/microg integrative DNA and 10(3)-10(4) transformants/microg replicative DNA), is complicated and time-consuming . In this study we applied various yeast transformation techniques to C . albicans and selected an electroporation procedure for further optimization . Transformation efficiencies of up to 300 transformants/microg were obtained for an integrative plasmid and up to 4500 transformants/microg for a CARS-carrying plasmid . This reasonably high transformation efficiency, combined with the ease and speed of electroporation in comparison to alternative techniques, make it the preferred method for transformation of C . albicans . Nucleic Acids Res, 1999 Dec 15, 27(24), 4671 - 8 An essential surface motif (WAQKW) of yeast RNA triphosphatase mediates formation of the mRNA capping enzyme complex with RNA guanylyltransferase; Ho CK et al.; Saccharomyces cerevisiae RNA triphosphatase (Cet1p) and RNA guanylyltransferase (Ceg1p) interact in vivo and in vitro to form a bifunctional mRNA capping enzyme complex . Cet1p binding to Ceg1p stimulates the guanylyltransferase activity of Ceg1p . Here we localize the guanylyltransferase-binding and guanylyltransferase-stimulation functions of Cet1p to a 21-amino acid segment from residues 239 to 259 . The guanylyltransferase-binding domain is located on the protein surface, as gauged by protease sensitivity, and is conserved in the Candida albicans RNA triphosphatase CaCet1p . Alanine-cluster mutations of a WAQKW motif within this segment abolish guanylyltransferase-binding in vitro and Cet1p function in vivo, but do not affect the triphosphatase activity of Cet1p . Proteolytic footprinting experiments provide physical evidence that Cet1p interacts with the C-terminal domain of Ceg1p . Trypsin-sensitive sites of Ceg1p that are shielded from proteolysis when Ceg1p is bound to Cet1p are located between nucleotidyl transferase motifs V and VI. J Bacteriol, 1999 Dec, 181(23), 7243 - 7 Roles of three histidine kinase genes in hyphal development and virulence of the pathogenic fungus Candida albicans; Yamada-Okabe T et al.; The pathogenic fungus Candida albicans harbors three histidine kinase genes called CaSLN1, CaNIK1, and CaHK1 . The disruption of any one of these three genes impaired the hyphal formation and attenuated the virulence of C . albicans in a mouse systemic candidiasis model . The effects of the disruption on hyphal formation and virulence were most severe in the cahk1Delta null mutants . Although the double disruption of CaSLN1 and CaNIK1 was impossible, further deletion of CaSLN1 or CaNIK1 in the cahk1Delta null mutants partially restored the serum-induced hypha-forming ability and virulence . When incubated with radiolabelled ATP, the recombinant CaSln1 and CaNik1 proteins, which contained their own kinase and response regulator domains, were autophosphorylated, whereas CaHk1p was not . These results imply that in C . albicans, CaSLN1 and CaNIK1 function upstream of CaHK1 but are in distinct signal transmission pathways. J Bacteriol, 1999 Dec, 181(23), 7235 - 42 Overexpression of a dominant-negative allele of SEC4 inhibits growth and protein secretion in Candida albicans; Mao Y et al.; Candida albicans SEC4 was cloned by complementing the Saccharomyces cerevisiae sec4-8 mutation, and its deduced protein product (Sec4p) was 63% identical to S . cerevisiae Sec4p . One chromosomal SEC4 allele in C . albicans CAI4 was readily disrupted by homologous gene targeting, but efforts to disrupt the second allele yielded no viable null mutants . Although this suggested that C . albicans SEC4 was essential, it provided no information about this gene's functions . Therefore, we constructed a mutant sec4 allele encoding an amino acid substitution (Ser-28-->Asn) analogous to the Ser-17-->Asn substitution in a trans-dominant inhibitor of mammalian Ras protein . GAL1-regulated expression plasmids carrying the mutant sec4 allele (pS28N) had minimal effects in glucose-incubated C . albicans transformants, but six of nine transformants tested grew very slowly in galactose . Incubation of pS28N transformants in galactose also inhibited secretion of aspartyl protease (Sap) and caused 90-nm secretory vesicles to accumulate intracellularly, and plasmid curing restored growth and Sap secretion to wild-type levels . These results imply that C . albicans SEC4 is required for growth and protein secretion and that it functions at a later step in the protein secretion pathway than formation of post-Golgi secretory vesicles . They also demonstrate the feasibility of using inducible dominant-negative alleles to define the functions of essential genes in C . albicans. J Invest Dermatol, 1999 Nov, 113(5), 747 - 51 HIV-Protease inhibitors reduce cell adherence of Candida albicans strains by inhibition of yeast secreted aspartic proteases; Borg-von Zepelin M et al.; Since the introduction of new anti-retroviral agents such as human immunodeficiency virus (HIV) protease inhibitors, oropharyngeal candidiasis is less often observed in acquired immune deficiency syndrome patients . Secretory aspartic proteases of Candida albicans, which have similarities to the HIV aspartic proteases, are pathogenicity factors that have been intensively investigated in recent years . The inhibitory effect of four different HIV aspartic protease inhibitors (ritonavir, saquinavir, indinavir, and nelfinavir), on the activity of different Candida albicans secretory aspartic proteases was demonstrated . These anti-retroviral agents were able to inhibit Candida albicans secretory aspartic proteases 1, 2, and 3 which are involved in Candida adherence . As a consequence of these results we used selected HIV protease inhibitors in an adherence assay of Candida cells to epithelial cells . Ritonavir and saquinavir inhibited adherence of Candida albicans under the chosen experimental conditions similarly to the in vitro results, whereas indinavir had no effect . This inhibition was shown to be concentration dependent . The specificity of these effects with respect to the secretory aspartic proteases was demonstrated by competitive binding experiments using purified recombinant secretory aspartic proteases . On the basis of these studies we conclude that lower rates of oropharyngeal candidiasis in individuals receiving potent anti-retroviral therapy could reflect not only an improvement in the immune system but also direct inhibition of Candida secretory aspartic proteases by HIV protease inhibitors. Cancer, 1999 Nov 15, 86(10), 2133 - 7 Defective activity of monocytes from patients with non-Hodgkin lymphoma . The modulatory effect of granulocyte-macrophage-colony stimulating factor; Burgaleta C et al.; BACKGROUND: Mononuclear phagocytic function is not well defined in non-Hodgkin lymphoma patients although, defective function of those cells has been reported in patients with Hodgkin disease and other solid tumors . The potential application of granulocyte-macrophage-colony stimulating factor (GM-CSF) in the prevention and treatment of infections in those patients is being studied . METHODS: Phagocytosis and microbiocidal activity of monocytes in peripheral blood from 10 newly diagnosed patients and 14 healthy donors were tested cytologically against a strain of Candida albicans, and chemotaxis was evaluated in a Boyden chamber using zymosan-activated serum as a chemotactic agent . Cells were assayed under basal conditions and after incubation with GM-CSF (12 ng/mL) . RESULTS: The phagocytosis and chemotactic activity of monocytes from non-Hodgkin lymphoma patients was lower than results obtained with cells from healthy donors (P < 0.05), and microbiocidal activity against Candida albicanswas similar in both groups . After exposure to GM-CSF, the functional activity of monocytes from control donors was only slightly modified (P > 0.05); by contrast, the percentage of mononuclear phagocytic cells in non-Hodgkin lymphoma (NHL) patients increased from 41 +/- 3% to 53 +/- 3%, the phagocytic index from 0.6 +/- 0.1 to 0.87 +/- 0.1 (P < 0.05), microbiocidal activity against Candida from 54 +/- 5% to 66 +/- 6% (P > 0.05), and chemotaxis from 43 +/- 8 cells per field to 48 +/- 9 cells per field (P > 0.05) . CONCLUSIONS: The results of this study indicate that there is defective phagocytic and chemotactic activity in monocytes from NHL patients at diagnosis . "In vitro" improvement of phagocytic activity was observed after exposure to GM-CSF . Mol Microbiol, 1999 Nov, 34(4), 651 - 62 Filamentous growth of Candida albicans in response to physical environmental cues and its regulation by the unique CZF1 gene; Brown DH Jr et al.; Hyphal growth in the opportunistic fungal pathogen Candida albicans is believed to contribute to the virulence of the organism by promoting penetration of fungal cells into host tissue . In this study, stimulation of hyphal growth by a feature of the physical environment was demonstrated . Specifically, growth of cells embedded within a matrix promoted the formation of hyphae . The CZF1 gene, encoding a putative transcription factor, was shown to be involved in the regulation of hyphal growth under certain conditions, including embedded conditions . Ectopic expression of CZF1 in embedded cells promoted the rapid formation of hyphae . Elimination of CZF1 and CPH1, encoding a homologue of the Saccharomyces cerevisiae Ste12p transcription factor, led to a pronounced defect in filamentous growth of embedded cells . Elimination of CZF1 alone led to a moderate defect in hyphal growth under some conditions, including embedded conditions . Hyphal morphogenesis in response to matrix embedding may occur in the opportunistic pathogen, C . albicans, to promote invasion of fungal cells into host tissue. Infect Immun, 1999 Dec, 67(12), 6652 - 62 Misexpression of the opaque-phase-specific gene PEP1 (SAP1) in the white phase of Candida albicans confers increased virulence in a mouse model of cutaneous infection; Kvaal C et al.; Candida albicans WO-1 switches reversibly and at high frequency between a white and an opaque colony-forming phenotype that includes dramatic changes in cell morphology and physiology . A misexpression strategy has been used to investigate the role of the opaque-phase-specific gene PEP1 (SAP1), which encodes a secreted aspartyl proteinase, in the expression of the unique opaque-phase phenotype and phase-specific virulence in two animal models . The PEP1 (SAP1) open reading frame was inserted downstream of the promoter of the white-phase-specific gene WH11 in the transforming vector pCPW7, and the resulting transformants were demonstrated to misexpress PEP1 (SAP1) in the white phase . Misexpression did not confer any of the unique morphological characteristics of the opaque phase to cells in the white phase and had no effect on the switching process . However, misexpression conferred upon white-phase cells the increased capacity of opaque-phase cells to grow in medium in which protein was the sole nitrogen source . Misexpression of PEP1 (SAP1) had no effect on the virulence of white-phase cells in a systemic mouse model, in which white-phase cells were already more virulent than opaque-phase cells . Misexpression did, however, confer upon white-phase cells the dramatic increase in colonization of skin in a cutaneous mouse model that was exhibited by opaque-phase cells . Misexpression of PEP1 (SAP1) conferred upon white-phase cells two dissociable opaque-phase characteristics: increased adhesion and the capacity to cavitate skin . The addition of pepstatin A to the cutaneous model inhibited the latter, but not the former, suggesting that the latter is effected by released enzyme, while the former is effected by cell-associated enzyme. Infect Immun, 1999 Dec, 67(12), 6637 - 42 Germ tubes and proteinase activity contribute to virulence of Candida albicans in murine peritonitis; Kretschmar M et al.; Peritonitis with Candida albicans is an important complication of bowel perforation and continuous ambulatory peritoneal dialysis . To define potential virulence factors, we investigated 50 strains of C . albicans in a murine peritonitis model . There was considerable variation in their virulence in this model when virulence was measured as release of organ-specific enzymes into the plasma of infected mice . Alanine aminotransferase (ALT) and alpha-amylase (AM) were used as parameters for damage of the liver and pancreas, respectively . The activities of ALT and AM in the plasma correlated with invasion into the organs measured in histologic sections and the median germ tube length induced with serum in vitro . When the activity of proteinases was inhibited in vivo with pepstatin A, there was a significant reduction of ALT and AM activities . This indicates that proteinases contributed to virulence in this model . Using strains of C . albicans with disruption of secreted aspartyl proteinase gene SAP1, SAP2, SAP3, or SAP4 through SAP6 (collectively referred to as SAP4-6), we showed that only a Deltasap4-6 triple mutant induced a significantly reduced activity of ALT in comparison to the reference strain . In contrast to the Deltasap1, Deltasap2, and Deltasap3 mutants, the ALT induced by the Deltasap4-6 mutant could not be further reduced by pepstatin A treatment, which indicates that Sap4-6 may contribute to virulence in this model. Vet Pathol, 1999 Nov, 36(6), 631 - 2 Oxalate-producing pulmonary aspergillosis in an alpaca; Muntz FH; An aging lactating alpaca was presented in sternal recumbency . Although bright and alert, she did not respond to symptomatic treatment and was euthanized 5 weeks after initial presentation . Gross postmortem examination revealed purulent material in the pulmonary airways . Histologic examination of the lungs revealed an extensive pyogranulomatous pneumonia with bronchiectasis . There were abundant fungal hyphae and high numbers of associated oxalate crystals, which were presumed to have been produced by the fungus . Low numbers of yeast cells were also present . Microbiological culture of tissues on horse blood agar and Sabouraud's agar identified the fungus to be Aspergillus niger . There was also moderate growth of Candida albicans . Calcium oxalate crystals in cytologic and histologic preparations can suggest an underlying Aspergillus infection . This is the first reported veterinary case of pneumonia due to Aspergillus niger infection and the associated production of oxalate crystals. Vet Pathol, 1999 Nov, 36(6), 594 - 600 Fatal measles virus infection in Japanese macaques (Macaca fuscata); Choi YK et al.; An outbreak of natural measles virus infection occurred in a group of Japanese macaques (Macaca fuscata) . Over a period of 4 months, 12 of 53 Japanese macaques died following a 2-23-day history of anorexia, diarrhea, and dermatitis . The monkeys were kept in outdoor exhibits but had been moved temporarily into indoor caging and then transferred to new outdoor exhibits . Ten monkeys died while they were in temporary caging, and two monkeys died after they were moved to new outdoor exhibits . The diagnoses were made based on the results of histopathology, immunohistochemistry (IHC), in situ hybridization (ISH), and electron microscopy . Measles virus antigens were detected in the lung, stomach, skin, salivary gland, spleen, and lymph nodes . Tangled, tubular nucleocapsids compatible with paramyxovirus were noted in the lung tissue . As a result of immunosuppression following measles virus infection, various secondary infections including disseminated cytomegalovirus infection, adenoviral and bacterial pneumonia, and Candida albicans-associated gingivitis and esophagitis were noted . The primary infective source or the mode of infection could not be determined in this outbreak, but measles virus may have been transmitted to the monkeys from human visitors while the monkeys were on exhibit. J Clin Microbiol, 1999 Dec, 37(12), 3896 - 900 Epidemiology of oropharyngeal Candida colonization and infection in patients receiving radiation for head and neck cancer; Redding SW et al.; Oral mucosal colonization and infection with Candida are common in patients receiving radiation therapy for head and neck cancer . Infection is marked by oral pain and/or burning and can lead to significant patient morbidity . The purpose of this study was to identify Candida strain diversity in this population by using a chromogenic medium, subculturing, molecular typing, and antifungal susceptibility testing of clinical isolates . These results were then correlated with clinical outcome in patients treated with fluconazole for infection . Specimens from 30 patients receiving radiation therapy for head and neck cancer were cultured weekly for Candida . Patients exhibiting clinical infection were treated with oral fluconazole . All isolates were plated on CHROMagar Candida and RPMI medium, subcultured, and submitted for antifungal susceptibility testing and molecular typing . Infections occurred in 27% of the patients and were predominantly due to Candida albicans (78%) . Candida carriage occurred in 73% of patients and at 51% of patient visits . Yeasts other than C . albicans predominated in carriage, as they were isolated from 59% of patients and at 52% of patient visits . All infections responded clinically, and all isolates were susceptible to fluconazole . Molecular typing showed that most patients had similar strains throughout their radiation treatment . One patient, however, did show the acquisition of a new strain . With this high rate of infection (27%), prophylaxis to prevent infection should be evaluated for these patients. J Clin Microbiol, 1999 Dec, 37(12), 3804 - 8 Identification of Candida dubliniensis based on temperature and utilization of xylose and alpha-methyl-D-glucoside as determined with the API 20C AUX and vitek YBC systems; Gales AC et al.; To have a better understanding of the role of Candida dubliniensis in clinical infections, it is essential that microbiology laboratories can identify this species rapidly and accurately in clinical specimens . C . dubliniensis has been reported to lack the ability to utilize xylose (XYL) and alpha-methyl-D-glucoside (MDG) and to grow poorly or not at all at 45 degrees C, whereas Candida albicans isolates utilize XYL and MDG and usually grow well at 45 degrees C . We tested 66 isolates of C . dubliniensis and 100 isolates of C . albicans with both the API 20C AUX and Vitek YBC systems to evaluate the ability of the XYL and MDG tests contained within each of these systems to distinguish between the two species . The ability to grow at 45 degrees C was also examined . None of the C . dubliniensis isolates grew at 45 degrees C, and 23 of 100 C . albicans isolates (23%) exhibited poor or no growth at 45 degrees C . The XYL and MDG tests contained within the API 20C AUX system were both negative for all 66 C . dubliniensis isolates and were positive for 98 (XYL) and 56 (MDG) of the 100 C . albicans isolates . With the Vitek system, 64 of 66 C . dubliniensis isolates (97.0%) were XYL negative and 63 (95.0%) were MDG negative . Conversely, 96 of 100 C . albicans isolates (96.0%) were XYL positive and 100 (100.0%) were MDG positive with the Vitek system . Clinical microbiology laboratories could use lack of growth at 45 degrees C and a negative XYL test with either the API 20C AUX or Vitek yeast identification system to provide a presumptive identification of C . dubliniensis . A negative MDG test result with either system would also be helpful but may misclassify C . albicans as C . dubliniensis, especially when the API 20C AUX system is used. Yonsei Med J, 1999 Oct, 40(5), 420 - 4 The presumptive identification of Candida albicans with germ tube induced by high temperature; Lee KH et al.; For direct identification of Candida albicans from other Candida species, the chlamydospore formation and the mycelial transition induced by high temperature and by sera were examined in 198 Candida isolates . The germ tubes of C . albicans developed early at 30 min in high temperature-induction, but at 60 min in serum-induction . C . albicans generated germ tubes well at concentrations lower than 2 x 10(7) cells/ml, but the germ tube formation was markedly restrained at concentrations higher than 4 x 10(7) cells/ml . In a serum-free, yeast extract-peptone-dextrose (YEPD) medium, C . albicans grew as a yeast form at 30 degrees C and as a mycelial form at 35-42 degrees C . Mycelial development was maximal at 37 degrees C in serum and at 39 degrees C in YEPD . Germ tubes were formed within 30 min in YEPD at 39 degrees C, but after 60 min in serum at 37 degrees C . Our examination showed that the 39 degrees C-induced germ tube formation tests were very reliable (sensitivity 100%, specificity 100%) at discerning C . albicans from other Candida species . These results suggest that the high temperature-induced germ tube formation testing could be a useful identification method of C . albicans in clinical laboratories. Mol Microbiol, 1999 Nov, 34(4), 792 - 8 The MET3 promoter: a new tool for Candida albicans molecular genetics; Care RS et al.; A central technique used to investigate the role of a Candida albicans gene is to study the phenotype of a cell in which both copies of the gene have been deleted . To date, such investigations can only be undertaken if the gene is not essential . We describe the use of the Candida albicans MET3 promoter to express conditionally an essential gene, so that the consequences of depletion of the gene product may be investigated . The effects of environmental conditions on its expression were investigated, using GFP as a reporter gene . The promoter showed an approximately 85-fold range of expression, according to the presence or absence of either methionine or cysteine in concentrations in excess of 1 mM . In the presence of either amino acid, expression was reduced to levels that were close to background . We used URA3 as a model to demonstrate that the MET3 promoter could control the expression of an essential gene, provided that a mixture of both methionine and cysteine was used to repress the promoter . We describe an expression vector that may be used to express any gene under the control of the MET3 promoter and a vector that may be used to disrupt a gene and simultaneously place an intact copy under the control of the MET3 promoter . During the course of these experiments, we discovered that directed integration into the RP10 locus gives a high frequency of transformation, providing a means to solve a long-standing problem in this field. J Clin Pathol, 1999 May, 52(5), 385 - 7 Pseudomonas aeruginosa pyocyanin and 1-hydroxyphenazine inhibit fungal growth; Kerr JR et al.; AIM: To examine strains of Pseudomonas aeruginosa for specific antifungal factors . METHODS: Two clinical strains of P aeruginosa with strong in vitro inhibition (by cross streak assay) of Candida albicans and Aspergillus fumigatus were examined . Both strains were isolated from sputum--one from a patient with cystic fibrosis and one from a patient with bronchiectasis . Bacterial extracts were fractionated by high performance liquid chromatography and examined by ultraviolet absorbance and mass spectroscopy . Antifungal activity against C albicans and A fumigatus was determined in a well plate assay . RESULTS: Pyocyanin was the major antifungal agent of P aeruginosa; 1-hydroxy-phenazine also possessed activity . Pyocyanin MICs for C albicans and A fumigatus were > 64 micrograms/ml . These phenazines were active against nine other yeast species pathogenic for man . Preliminary experiments also suggested possible inhibition of yeast mycelial transformation in C albicans by pyocyanin . CONCLUSIONS: There may be a role for pyocyanin and 1-hydroxyphenazine in the prevention of pulmonary candidiasis in patients colonised by P aeruginosa. Mol Microbiol, 1999 Nov, 34(3), 451 - 62 Mitogen-activated protein kinase Mkp1 of Pneumocystis carinii complements the slt2Delta defect in the cell integrity pathway of Saccharomyces cerevisiae; Fox D et al.; Signal transduction pathways are important in the adaptive response of microbes to their environment . A Pneumocystis carinii extracellular signal-regulated protein kinase (MAPK) homologue, Mkp1, has been isolated by sequence similarity screening of P . carinii genomic DNA . The Mkp1 of P . carinii shows closest homology to other fungal MAP kinases involved in cell integrity signal transduction cascades, including Slt2p/Mpk1p of Saccharomyces cerevisiae, Mkc1 of Candida albicans and Mps1 of Magnaporthe grisea . Defects of Slt2p in S . cerevisiae result in phenotypes of slow growth, and temperature sensitivity in the absence of an osmostabilizer . Overexpression of mkp1 in a strain with the slt2Delta defect fully restored the normal growth rate, and partially reduced lysis at elevated temperatures . Complementation of the slt2Delta defect by Mkp1 demonstrates that Mkp1 is a functional MAP kinase, and that it may be the MAP kinase component of a similar signal transduction cascade within P . carinii . Furthermore, Mkp1 is activated in vitro upon the exposure of P . carinii to conditions of oxidative stress . The investigation of a MAP kinase signal transduction pathway of P . carinii will result in both a better understanding of the mechanism the organism utilizes to respond to environmental changes, and a system to assay responses to these changes. Clin Exp Dermatol, 1999 Sep, 24(5), 402 - 6 Cell-mediated immunity to Malassezia furfur in patients with seborrhoeic dermatitis and pityriasis versicolor; Bergbrant IM et al.; The lymphocyte transformation response to Malassezia furfur, Candida albicans, phytohaemagglutinin, concanavlin A and tuberculin purified protein derivative of 12 patients with pityriasis versicolor, 15 patients with seborrhoeic dermatitis and matched controls, was studied . Patients with pityriasis versicolor showed a significantly lower response to M . furfur than patients with seborrhoeic dermatitis and controls. Biochim Biophys Acta, 1999 Sep 21, 1421(1), 175 - 82 Effect of hexavalent chromium on eukaryotic plasma membrane studied by EPR spectroscopy; Belagyi J et al.; The effect of Cr(VI) anion on an ergosterol-producing strain of eukaryotic yeast Candida albicans and its mutant with ergosterol-less membrane was studied with EPR spectroscopy . 5- and 14-doxyl stearic acid spin probes were used to label the protoplast membrane after removal of the cell wall . In control experiments, the mutant strain exhibited larger rigidity in the membrane than its parental strain . Addition of Cr(VI), at a minimum inhibitory concentration of 0.6 mM, increased the rotational mobility of the spin labels significantly and decreased the temperature of the structural changes in both strains, in the temperature range between 0 and 30 degrees C . The ergosterol-less mutant, having a membrane composition with increased polyunsaturated fatty acid content, exhibited higher Cr(VI) sensitivity . Treatment of the membrane with Cr(VI) for 10 min already resulted in an increase in membrane fluidity . An EPR signal of Cr(V) was detected which reached maximum amplitude after 120 min of treatment with Cr(VI) . Further chemical reduction of Cr(V) in the absence of extracellular Cr(VI) led to a lack of detectable paramagnetic chromium intermediates within 200 min. Dermatology, 1999, 199(2), 135 - 9 Nummular eczema: An addition of senile xerosis and unique cutaneous reactivities to environmental aeroallergens; Aoyama H et al.; BACKGROUND: The pathogenesis of nummular eczema (NE) is still unknown . It often develops on the lower legs of elderly individuals with xerotic changes during the winter months . Such winter exacerbation is also observed in atopic dermatitis, in which there is a high incidence of cutaneous immune reactivities against environmental aeroallergens . OBJECTIVE: Because of the total lack of information about skin reactivities in NE patients, we performed immunological as well as functional studies in their uninvolved skin . METHOD: Prick tests and chamber scarification patch tests for representative aeroallergens were conducted on the flexor surface of the forearm in 26 NE patients, in 21 age-matched elderly persons without NE and in 43 healthy young controls . RESULTS: We found that the elderly subjects, regardless of their background, showed a significantly higher immediate skin reactivity to Candida albicans than the young controls . In contrast, patch testing revealed that, unlike the age-matched elderly subjects who showed a decrease in incidence of positive patch test reactions, the NE patients retained delayed contact sensitivity at a level comparable to that of the young healthy controls . They showed a significantly higher percentage of positive patch test reactions to Dermatophagoides farinae allergen (46%) and house dust allergen (35%) than the age-matched controls . Moreover, they also showed a significantly higher percentage of delayed hypersensitive reactions to C . albicans allergen (85%) than the age-matched controls (48%) . Noninvasive functional assessment of the stratum corneum (SC) in unaffected skin areas of the lower legs in 8 NE patients demonstrated that, though the water barrier function of the SC was comparable to that of the age-matched controls, they showed a significantly lower hydration state of the SC than the age-matched controls . CONCLUSION: The xerotic skin of elderly individuals facilitates the development of cracking and fissuring of the skin surface in dry and cold winter . Such damage in the SC is sometimes aggravated by inadvertent scratching due to pruritus, allowing skin permeation of various environmental allergens . They may induce eczematous changes in those with preserved adequate delayed hypersensitivity despite their advanced age. J Bacteriol, 1999 Nov, 181(22), 7070 - 9 PHR1 and PHR2 of Candida albicans encode putative glycosidases required for proper cross-linking of beta-1,3- and beta-1,6-glucans; Fonzi WA; PHR1 and PHR2 encode putative glycosylphosphatidylinositol-anchored cell surface proteins of the opportunistic fungal pathogen Candida albicans . These proteins are functionally related, and their expression is modulated in relation to the pH of the ambient environment in vitro and in vivo . Deletion of either gene results in a pH-conditional defect in cell morphology and virulence . Multiple sequence alignments demonstrated a distant relationship between the Phr proteins and beta-galactosidases . Based on this alignment, site-directed mutagenesis of the putative active-site residues of Phr1p and Phr2p was conducted and two conserved glutamate residues were shown to be essential for activity . By taking advantage of the pH-conditional expression of the genes, a temporal analysis of cell wall changes was performed following a shift of the mutants from permissive to nonpermissive pH . The mutations did not grossly affect the amount of polysaccharides in the wall but did alter their distribution . The most immediate alteration to occur was a fivefold increase in the rate of cross-linking between beta-1,6-glycosylated mannoproteins and chitin . This increase was followed shortly thereafter by a decline in beta-1,3-glucan-associated beta-1, 6-glucans and, within several generations, a fivefold increase in the chitin content of the walls . The increased accumulation of chitin-linked glucans was not due to a block in subsequent processing as determined by pulse-chase analysis . Rather, the results suggest that the glucans are diverted to chitin linkage due to the inability of the mutants to establish cross-links between beta-1,6- and beta-1,3-glucans . Based on these and previously published results, it is suggested that the Phr proteins process beta-1,3-glucans and make available acceptor sites for the attachment of beta-1,6-glucans. Oral Surg Oral Med Oral Pathol Oral Radiol Endod, 1999 Nov, 88(5), 573 - 80 Oral Candida dubliniensis as a clinically important species in HIV-seropositive patients in the United States; Meiller TF et al.; OBJECTIVE: Interest in Candida dubliniensis has led to renewed clinical investigations regarding incidence, drug resistance, pathogenesis, and epidemiology of fungal infections in patients with HIV . C dubliniensis phenotypically resembles Candida albicans in many respects, yet it can be identified and differentiated as a unique Candida species by its phenotypic and genetic profiles . The purpose of this study was to prospectively evaluate the prevalence of C dubliniensis in clinical isolates and determine the clinical and demographic characteristics of patients harboring C dubliniensis . STUDY DESIGN: Over a 6-week period, 24 yeast-positive isolates from HIV-positive dental patients were screened for C dubliniensis through use of phenotypic criteria . HIV viral load, CD4 count, and complete oral health evaluations were performed on each patient at the same visit during which the oral fungal surveillance culture was taken . RESULTS: Six isolates from 24 HIV-seropositive and yeast-positive patients were shown to be consistent phenotypically and by electrophoretic karyotyping with the European reference strain of C dubliniensis . Dose-dependent susceptibility to fluconazole was shown in one of the C dubliniensis isolates . Five of the 6 patients demonstrated moderate to high viral loads . General oral health, as evidenced by the presence of advanced periodontal lesions and a high decayed, missing, and filled teeth index (>20), was poor in 3 of the 6 patients with C dubliniensis and 7 of the 18 patients with C albicans . A history of intravenous drug abuse was present in 50% of the C dubliniensis -positive patients, which is representative of the HIV-positive population at the hospital . CONCLUSIONS: In this small sample, C dubliniensis represented 25% of the yeast-positive cultures . The clinical significance of this interesting species in the United States may be related to high viral load, rapid AIDS progression, and/or concomitant oral disease, such as a high caries index or periodontal disease. FEMS Microbiol Lett, 1999 Nov 15, 180(2), 213 - 9 CDR1, a multidrug resistance gene from Candida albicans, contains multiple regulatory domains in its promoter and the distal AP-1 element mediates its induction by miconazole; Puri N et al.; We previously demonstrated that the CDR1 gene, encoding a multidrug transporter in Candida albicans, is differentially upregulated by various drugs and steroids . In order to get an insight into the molecular basis of the induction of this gene we analyzed its promoter region . The transcription start site was mapped to 63 nucleotides upstream of the initiating ATG . Reporter assays revealed the presence of four upstream activating and four upstream repressing sequence domains along the entire promoter . Like the native gene, promoter-luciferase recombinants showed enhanced activity in response to various stresses like drugs, human steroid hormones and heavy metals . Mutational analysis demonstrated that while the proximal promoter (-345/+1) contains all the regulatory domains required for its induction by various other stresses, the miconazole response is mediated via the distal promoter (-857/-1147), harboring an AP-1 site . The involvement of the AP-1 element in mediating the latter effect was evident by an increase in AP-1 binding activity following miconazole treatment. FEMS Microbiol Lett, 1999 Nov 15, 180(2), 171 - 5 Y132H substitution in Candida albicans sterol 14alpha-demethylase confers fluconazole resistance by preventing binding to haem; Kelly SL et al.; Fungal cytochrome P450 sterol 14alpha-demethylase (CYP51) is required for ergosterol biosynthesis and is the target for azole antifungal compounds . The amino acid substitution Y132H in CYP51 from clinical isolates of Candida albicans can cause fluconazole resistance by a novel change in the protein . Fluconazole binding to the mutant protein did not involve normal interaction with haem as shown by inducing a Type I spectral change . This contrasted to the wild-type protein where fluconazole inhibition was reflected in coordination to haem as a sixth ligand and where the typical Type II spectrum was obtained . The Y132H substitution occurred without drastic perturbation of the haem environment or activity allowing resistant mutants to produce ergosterol and retain fitness, an efficient strategy for resistance in nature. AIDS Res Hum Retroviruses, 1999 Nov 1, 15(16), 1413 - 7 Treatment of fluconazole-refractory oropharyngeal candidiasis with itraconazole oral solution in HIV-positive patients; Saag MS et al.; This open-label, multicenter trial evaluated the efficacy and safety of a new oral solution formulation of itraconazole in HIV+/AIDS patients with fluconazole-refractory oropharyngeal candidiasis . Seventy-four HIV+/AIDS patients with mycologically confirmed oropharyngeal candidiasis who failed fluconazole therapy (200 mg/day) were treated with 100 mg of itraconazole oral solution administered twice daily (200 mg/day) for 14 days . Patients who demonstrated an incomplete response to treatment were treated for an additional 14 days (28 days total) . Clinical responders were eligible for participation in a separate 6-month maintenance protocol . If they declined further treatment, responders were monitored for 6 weeks posttreatment . The primary efficacy parameter was clinical response (i.e., no lesions or symptoms) at end of treatment . Fungal cultures were performed at baseline and at the end of treatment . Among the 74 patients who had mycologically confirmed, fluconazole-unresponsive, oropharyngeal candidiasis at baseline, 41 (55%) achieved a clinical response by day 28 . The median time to response was 7 days (range, 7 to 28 days) . Candida albicans was the most common pathogen isolated, either alone (62%) or in combination with another Candida species (31%) . All 22 patients who entered the optional, off-therapy, 6-week follow-up phase relapsed; mean time to relapse was 13 days . Itraconazole oral solution was well-tolerated; adverse events were predominantly gastrointestinal disturbances . This trial demonstrates that itraconazole oral solution is a useful therapy in the treatment of HIV-infected patients with fluconazole-refractory oropharyngeal candidiasis. AIDS Res Hum Retroviruses, 1999 Nov 1, 15(16), 1405 - 12 Diagnosis and treatment of oropharyngeal candidiasis in patients infected with HIV: a critical reassessment; Powderly WG et al.; Oropharyngeal candidiasis is the most common opportunistic infection seen in patients infected with the human immunodeficiency virus (HIV) . As HIV disease progresses and immunosuppression worsens, the incidence and severity of oropharyngeal candidiasis increase . The predominant pathogen in initial and recurrent episodes is Candida albicans, which responds to a variety of topical (nystatin and clotrimazole) and systemic azole antifungal agents (ketoconazole, itraconazole, and fluconazole) . Since the introduction of the oral azoles, increasing evidence indicates that C . albicans strains are developing resistance to azoles, particularly fluconazole, and other Candida strains are emerging that are intrinsically less susceptible to azole therapy . The advent of effective antiretroviral therapies for the treatment of HIV disease has led to a scenario in which antifungal strategies are likely to be highly effective . To minimize the risk of resistance, topical therapies should be considered first-line candidates for treatment of initial or recurrent cases of uncomplicated oropharyngeal candidiasis . Systemic azole therapy should be reserved for cases unresponsive to topical therapies or for more severe oropharyngeal candidiasis with esophageal involvement. Am J Med Sci, 1999 Nov, 318(5), 324 - 9 The pathophysiology of glossal pain in patients with iron deficiency and anemia; Osaki T et al.; BACKGROUND: It is well known that prolonged anemia causes atrophy of tongue papillae, glossal pain, and dysphagia, but it is uncertain whether iron (Fe) deficiency induces glossal pain without any objective manifestation . To resolve this matter, the relationship between Fe deficiency and glossal pain was examined . METHODS: Eighteen patients with Fe deficiency and 7 anemic patients manifesting spontaneous irritation or pain of the tongue without any objective abnormalities participated in this study . To ascertain the cause of glossal pain and the oral pathophysiology in Fe deficiency and anemia, peripheral blood was examined and the glossal pain threshold and salivary flow rates (SFRs) were estimated along with Candida albicans cell culture tests . RESULTS: Compared with patients with Fe deficiency, those with anemia had a longer history of tongue pain . In patients with anemia, painful areas of the tongue were more numerous than in patients with Fe deficiency . Pain thresholds were decreased in the painful portions, and both nonstimulated and stimulated SFRs were suppressed . Each patient was treated with oral Fe; within 2 months, most patients exhibited increased serum ferritin level (P< 0.02, paired t-test), pain threshold (P < 0.05) and salivation (P < 0.05) and glossal pain subsided . CONCLUSIONS: Fe deficiency causes glossal pain and the degree of glossal pain increases as Fe deficiency advances to anemia, manifesting hyposalivation and abnormalities of glossal papillae. Br J Haematol, 1999 Jun, 105(4), 948 - 54 Monocyte dysfunction in patients with multiple myeloma and lymphoplasmacytic disorders is related to serum paraprotein levels; Mainwaring CJ et al.; We have investigated monocyte function in 30 patients with lymphoplasmacytic disorders and in 21 age and sex matched normal controls . Marked abnormalities of all facets of monocyte function were demonstrated in six patients with multiple myeloma (MM) and a single patient with Waldenstrom's macroglobulinaemia (WM) plus significant paraproteinaemia . Serious infection occurred in three of these patients . An inverse relationship between the level of the serum paraprotein and impairment of monocyte phagocytosis plus killing of Candida albicans was observed . Crossover studies suggested that these abnormal findings were constitutive and not reversed by removal of the serum paraprotein . The data suggest that monocyte function is constitutively abnormal in patients with MM and can be further, but reversibly, inhibited by high paraprotein levels . Further research is required to confirm these findings, ascertain whether monocyte function can be normalized using chemotherapy or growth factors, and if so, whether their tumouricidal functions could be harnessed in the treatment of this currently incurable condition. Clin Exp Immunol, 1999 Nov, 118(2), 192 - 6 Differences in cytokine production by peripheral blood mononuclear cells (PBMC) between patients with atopic dermatitis and bronchial asthma; Kimura M et al.; It is widely accepted that type 2 helper T (Th2) lymphocytes play a crucial role in the pathogenesis of atopic dermatitis (AD) as well as bronchial asthma (BA) . We measured the amounts of IL-5 and interferon-gamma (IFN-gamma) produced by PBMC upon stimulation with house dust mite (HDM) or Candida albicans (CA) in 17 children (3-15 years) with AD, and compared these values with those of 16 children with BA . Although IL-5 production by PBMC upon stimulation with HDM in patients with AD was significantly higher than that in 13 non-atopic controls (geometric mean = 23.4 pg/ml versus 5.9 pg/ml, P < 0.05), it was significantly lower than that in patients with BA (177.8 pg/ml, P < 0.001) . The amount of IL-5 produced by PBMC upon stimulation with CA was also significantly lower in patients with AD than in those with BA (7.2 pg/ml versus 100.0 pg/ml, P < 0.001) . The production of IFN-gamma by PBMC stimulated with HDM or CA was also significantly lower in patients with AD than in those with BA (HDM 4 . 3 pg/ml versus 12.6 pg/ml, P < 0.05; CA 6.5 pg/ml versus 60.3 pg/ml, P < 0.001) . Consequently the ratio of IL-5 to IFN-gamma production was high not only in patients with BA but also in those with AD . These findings suggest that there are some differences in the regulation of in vivo cytokine production between patients with AD and those with BA, although a Th2-dominant profile is common to both. Clin Diagn Lab Immunol, 1999 Nov, 6(6), 924 - 9 Use of monoclonal antibody in diagnosis of candidiasis caused by Candida albicans: detection of circulating aspartyl proteinase antigen; Na BK et al.; To develop a serological diagnosis of invasive candidiasis based on detection of circulating secreted aspartyl proteinase (SAP) antigen of Candida albicans, three different enzyme-linked immunosorbent assays (ELISAs) were compared . The first was a standard ELISA to detect anti-SAP antibodies, and the others were an antigen capture ELISA and an inhibition ELISA to detect circulating SAP antigen with monoclonal antibody (MAb) CAP1, which is highly specific for SAP . These tests were applied to 33 serum samples retrospectively selected from 33 patients with mycologically and/or serologically proven invasive candidiasis caused by C . albicans . Serum samples from 12 patients with aspergillosis and serum samples from 13 healthy individuals were also included . The sensitivities and specificities were 69.7 and 76.0% for the standard ELISA and 93.9 and 92.0% for the antigen capture ELISA, respectively . However, these values reached 93.9 and 96.0%, respectively, for the inhibition ELISA . Serum samples from 31 of 33 patients had detectable SAP antigen, with concentrations ranging from 6.3 to 19.0 ng/ml . These results indicate that the inhibition ELISA with MAb CAP1 is effective in detection of circulating SAP antigen and that this assay may be useful for diagnosis and treatment monitoring of invasive candidiasis. Clin Diagn Lab Immunol, 1999 Nov, 6(6), 921 - 3 Successful treatment of fluconazole-resistant oropharyngeal candidiasis by a combination of fluconazole and terbinafine; Ghannoum MA et al.; Increasing incidence of resistance to conventional antifungal therapy has demanded that novel therapies be introduced . Recent in vitro studies have shown that combinations involving azoles and allylamines may be effective in inhibiting fluconazole-resistant fungi . In this report, we describe the case of a 39-year-old woman who presented with white patches on her buccal mucosa, tongue, and palate with a bright erythematous erosive base . A fungal culture revealed Candida albicans . The patient failed to respond to the initially prescribed fluconazole therapy . Failure of therapy can be attributed to a developed resistance to fluconazole from the patient's intermittent use of this antifungal agent at varying dosages for the preceding 2 years due to a diagnosis of onychomycosis . In vitro testing of the culture from the patient showed elevated MICs of fluconazole, itraconzole, and terbinafine (MICs were 32, 0.5, and 64 microg/ml, respectively) . Our goal was to combine therapies of fluconazole and terbinafine in an attempt to clear the fungal infection . Impressively, this combination resulted in the clearing of the clinical symptoms and the patient has successfully been asymptomatic for more than 12 months posttreatment. Clin Diagn Lab Immunol, 1999 Nov, 6(6), 851 - 5 Further characterization of human salivary anticandidal activities in a human immunodeficiency virus-positive cohort by use of microassays; Lin AL et al.; Salivary anticandidal activities play an important role in oral candidal infection . R . P . Santarpia et al . (Oral Microbiol . Immunol . 7:38-43, 1992) developed in vitro anticandidal assays to measure the ability of saliva to inhibit the viability of Candida albicans blastoconidia and the formation of germ tubes by C . albicans . In this report, we describe modifications of these assays for use with small volumes of saliva (50 to 100 microl) . For healthy subjects, there is strong inhibition of blastoconidial viability in stimulated parotid (75%), submandibular-sublingual (74%), and whole (97%) saliva, as well as strong inhibition of germ tube formation (>80%) for all three saliva types . The susceptibility of several Candida isolates to inhibition of viability by saliva collected from healthy subjects is independent of body source of Candida isolation (blood, oral cavity, or vagina) or the susceptibility of the isolate to the antifungal drug fluconazole . Salivary anticandidal activities in human immunodeficiency virus (HIV)-infected patients were significantly lower than those in healthy controls for inhibition of blastoconidial viability (P < 0.05) and germ tube formation (P < 0 . 001) . Stimulated whole-saliva flow rates were also significantly lower (P < 0.05) for HIV-infected patients . These results show that saliva of healthy individuals has anticandidal activity and that this activity is reduced in the saliva of HIV-infected patients . These findings may help explain the greater incidence of oral candidal infections for individuals with AIDS. J Mol Biol, 1999 Nov 12, 293(5), 1039 - 53 The Candida albicans CUG-decoding ser-tRNA has an atypical anticodon stem-loop structure; Perreau VM et al.; In many Candida species, the leucine CUG codon is decoded by a tRNA with two unusual properties: it is a ser-tRNA and, uniquely, has guanosine at position 33 (G33) . Using a combination of enzymatic (V1 RNase, RnI nuclease) and chemical (Pb(2+), imidazole) probing of the native Candida albicans ser-tRNACAG, we demonstrate that the overall tertiary structure of this tRNA resembles that of a ser-tRNA rather than a leu-tRNA, except within the anticodon arm where there is considerable disruption of the anticodon stem . Using non-modified in vitro transcripts of the C . albicans ser-tRNACAG carrying G, C, U or A at position 33, we demonstrate that it is specifically a G residue at this position that induces the atypical anticodon stem structure . Further quantitative evidence for an unusual structure in the anticodon arm of the G33-tRNA is provided by the observed change in kinetics of methylation of the G at position 37, by purified Escherichia coli m(1)G37 methyltransferase . We conclude that the anticodon arm distortion, induced by a guanosine base at position 33 in the anticodon loop of this novel tRNA, results in reduced decoding ability which has facilitated the evolution of this tRNA without extinction of the species encoding it . Mycoses, 1999, 42(7-8), 475 - 8 Tinea pedis and onychomycosis in Danish soldiers before and after service in ex-Yugoslavia; Brocks KM et al.; Seventy-three 1-year-experienced Danish soldiers were examined for tinea pedis as well as onychomycoses before and after a duty period of 6 months in ex-Yugoslavia . The incidence of fungal infections was 16.4% before and 32.3% after their duty period abroad . At first investigation Trichophyton rubrum and T . mentagrophytes were dominant but onychomycosis and tinea pedis were found as well . In contrast, Candida albicans was the predominant pathogen in the second investigation . We explain this by means of the more aggressive nature that yeasts can show when host-parasite relations are disturbed or compromised . Twelve soldiers with positive mycology were offered treatment and the final investigation showed a cure rate of 50% . This result is satisfactory in view of the difficult sanitary conditions. Mycoses, 1999, 42(7-8), 465 - 74 Antifungal action of Hevea brasiliensis latex . Its effect in combination with fluconazole on Candida albicans growth; Giordani R et al.; Latex from Hevea brasiliensis and its subcellular fractions (L-serum and C-serum) were tested for antifungal activity alone or in combination with fluconazole . Candida albicans growth was inhibited with the same efficacy when yeasts were inoculated into culture medium supplemented over the total growth phase with latex as when latex was added during the exponential phase only: the minimum inhibitory concentration (MIC 80%) of H . brasiliensis latex was 123 micrograms protein ml-1 . By means of a non-linear regression analysis of the experimental data, two distinct fixation sites for fluconazole (FCZ) could be determined: one of strong affinity (Kaff = 0.0162 microgram-1 protein ml) and another of low affinity (Kaff = 0.0071 microgram-1 protein ml) . After addition of a mixture of FCZ and latex during the exponential phase, the affinity constant of yeasts for FCZ was calculated: when latex was in a final concentration of 21 micrograms protein ml-1 (Kaff = 1 microgram-1 protein ml) or 42 micrograms protein ml-1 (Kaff = 0.277 microgram-1 protein ml) and without latex (Kaff = 0.0502 microgram-1 protein ml) . In two cases a synergistic effect between latex and FCZ was obtained . The highest efficacy was obtained with a latex concentration of 21 micrograms protein ml-1 . The addition of subcellular fractions of latex, L-serum and C-serum, did not cause an antifungal effect . The indispensable role of rubber particles for raising an antifungal effect is demonstrated . Electron microscopy observations indicated a limited cell wall degradation and a high percentage of coagulated yeasts. Mycoses, 1999, 42(7-8), 453 - 8 Molecular aspects of fluconazole resistance development in Candida albicans; Franz R et al.; Serial Candida albicans isolates from recurrent episodes of oropharyngeal candidosis (OPC) in four AIDS patients which became fluconazole-resistant during therapy were analysed by molecular methods . The CARE-2 fingerprint patterns of the isolates demonstrated that in all four patients fluconazole resistance developed in a previously more susceptible strain . In two cases resistance correlated with enhanced expression of genes encoding multiple drug resistance proteins that mediate active drug efflux . Enhanced mRNA levels of the CDR1/CDR2 genes encoding ABC transporters were observed in fluconazole-resistant isolates from one patient compared with the corresponding susceptible isolates . The fluconazole-resistant isolates from another patient exhibited high mRNA levels of the MDR1 gene encoding a membrane transport protein of the major facilitator superfamily that was not detectably expressed in any of the fluconazole-susceptible isolates . These results demonstrate that in AIDS patients with recurrent OPC the development of fluconazole resistance is usually caused by molecular changes in a previously susceptible C . albicans strain from the same patient. J Immunother, 1999 Sep, 22(5), 431 - 40 A phase I trial of an HLA-A1 restricted MAGE-3 epitope peptide with incomplete Freund's adjuvant in patients with resected high-risk melanoma; Weber JS et al.; Cytolytic and helper T cells recognize small peptide fragments of protein antigens that are intracellularly processed and delivered to the cell surface in conjunction with HLA molecules . In mice, peptide vaccines can protect against lethal virus infections and tumor challenges . To test whether epitope peptides derived from a human tumor antigen can induce immune responses in patients, a vaccine was prepared consisting of an HLA-A1-restricted epitope of the antigen MAGE-3 mixed with a pan-class II epitope peptide PADRE and emulsified with incomplete Freund's adjuvant . Eighteen patients with resected stages III and IV melanoma at high risk for relapse were vaccinated subcutaneously with increasing doses of the MAGE-3 vaccine ranging from 100 to 2,000 micrograms per injection four times, each 4 weeks apart . The purpose of the phase I trial was to assess the toxicity, tolerability, and immune responses to the vaccine . The vaccine was not toxic, with only one case of grade III lethargy, and most patients complaining of grade I or II local pain, swelling, and tenderness at the injection sites . Peripheral blood mononuclear cells (PBMC) were collected from most patients prior to and after vaccination and used for assessment of global levels of immunity prevaccination, and to measure immune responses to the MAGE-3 and PADRE peptides prior to and after vaccination . Significant defects in global immunity shown by anergy to DTH skin testing in 7 of 16 patients and decreased proliferation to PHA (phytohemagglutinin) and CASTA, a Candida albicans protein extract, were observed . Seven of nine patients showed an increased response to PADRE after restimulation in vitro . Five of 14 patients at doses from 100 to 2,000 micrograms demonstrated an immune response to MAGE-3 by cytolysis of MAGE-3-specific target cells . Release of gamma interferon by T cells from 8 patients at the 100, 1,000, or 2,000 micrograms dose was measured after vaccination, and only two of eight patients showed an increase indicating augmented antigen-specific immunity . These data suggest that immune responses can be detected against PADRE and MAGE-3 in vaccinated melanoma patients, albeit with a low frequency of effector cells. Antimicrob Agents Chemother, 1999 Nov, 43(11), 2798 - 800 Overexpression of Erg11p by the regulatable GAL1 promoter confers fluconazole resistance in Saccharomyces cerevisiae; Kontoyiannis DP et al.; The contribution of the dosage of target enzyme P-450 14alpha-demethylase (14alphaDM) to fluconazole resistance in both Candida albicans and Saccharomyces cerevisiae remains unclear . Here, we show that overexpression of Saccharomyces P-450 14alphaDM in S . cerevisiae, under the control of the regulatable promoter GAL1, results in azole resistance. Antimicrob Agents Chemother, 1999 Nov, 43(11), 2753 - 65 The ATP binding cassette transporter gene CgCDR1 from Candida glabrata is involved in the resistance of clinical isolates to azole antifungal agents; Sanglard D et al.; The resistance mechanisms to azole antifungal agents were investigated in this study with two pairs of Candida glabrata clinical isolates recovered from two separate AIDS patients . The two pairs each contained a fluconazole-susceptible isolate and a fluconazole-resistant isolate, the latter with cross-resistance to itraconazole and ketoconazole . Since the accumulation of fluconazole and of another unrelated substance, rhodamine 6G, was reduced in the azole-resistant isolates, enhanced drug efflux was considered as a possible resistance mechanism . The expression of multidrug efflux transporter genes was therefore examined in the azole-susceptible and azole-resistant yeast isolates . For this purpose, C . glabrata genes conferring resistance to azole antifungals were cloned in a Saccharomyces cerevisiae strain in which the ATP binding cassette (ABC) transporter gene PDR5 was deleted . Three different genes were recovered, and among them, only C . glabrata CDR1 (CgCDR1), a gene similar to the Candida albicans ABC transporter CDR genes, was upregulated by a factor of 5 to 8 in the azole-resistant isolates . A correlation between upregulation of this gene and azole resistance was thus established . The deletion of CgCDR1 in an azole-resistant C . glabrata clinical isolate rendered the resulting mutant (DSY1041) susceptible to azole derivatives as the azole-susceptible clinical parent, thus providing genetic evidence that a specific mechanism was involved in the azole resistance of a clinical isolate . When CgCDR1 obtained from an azole-susceptible isolate was reintroduced with the help of a centromeric vector in DSY1041, azole resistance was restored and thus suggested that a trans-acting mutation(s) could be made responsible for the increased expression of this ABC transporter gene in the azole-resistant strain . This study demonstrates for the first time the determinant role of an ABC transporter gene in the acquisition of resistance to azole antifungals by C . glabrata clinical isolates. Antimicrob Agents Chemother, 1999 Nov, 43(11), 2731 - 5 Genetic analysis of azole resistance by transposon mutagenesis in Saccharomyces cerevisiae; Kontoyiannis DP; The increasing resistance of Candida species to fluconazole is cause for concern . To determine the molecular mechanisms involved in resistance to fluconazole, I used a scheme of transposon mutagenesis in Saccharomyces cerevisiae, a genetically tractable yeast that is closely related to Candida albicans . This technique, which permits the generation and analysis of multiple random Tn3::LEU2::lacZ fusions, can be used as a disruption mutagen (N . B . Burns et al., Genes Dev . 8:1087-1105, 1994) . By using the Tn3::LEU2::lacZ library as a disruption mutagen, I found recessive mutations in genes that were previously found to be involved in azole resistance, e.g., PDR5 and CPR1, and in genes previously found to be involved in azole sensitivity, e.g., ERG3 . This approach also enabled me to identify recessive mutations in three genes not previously known to be involved in azole sensitivity . Two of the genes, ADA3 and SPT7, are general transcriptional regulators; the third, YMR034c, is a putative sterol transporter . Finally, by screening the Tn3::LEU2::lacZ library for lacZ fusions induced by a low concentration of fluconazole, I identified genes known to be induced by azoles as well as a variety of other genes not previously known to be induced by the drug . In conclusion, transposon mutagenesis is a promising screening tool for use in identifying novel drug targets and in uncovering the mechanisms involved in the response of S . cerevisiae to antifungal drugs. Clin Otolaryngol, 1999 Sep, 24(5), 398 - 403 Microbial colonization of silicone voice prostheses used in laryngectomized patients; Eerenstein SE et al.; The aim of this study was to identify the microbial colonization of dysfunctioning voice prostheses in laryngectomized patients and determine the influence of patient radiation therapy on prosthesis life span . In a 40-month period, 257 outpatient voice prosthesis replacements were carried out in a laryngectomized group of 31 patients . The voice prostheses were all removed from the tracheo-oesophageal fistula after dysfunctioning of the prosthesis . Of the replaced prostheses 183 were cultured . The microbial cultures showed a predominant colonization with Candida albicans and commensal oral microflora . Radiation therapy induced xerostomia shortened the lifetime of the first inserted prosthesis in particular. Support Care Cancer, 1999 Nov, 7(6), 428 - 31 Nosocomial candidaemias due to species other than Candida albicans in cancer patients . Aetiology, risk factors, and outcome of 45 episodes within 10 years in a single cancer institution; Krcmery V Jr et al.; Forty-five cases of fungaemia due non-albicans Candida spp . (NAC) in a single National Cancer Institution within 10 years were analysed for aetiology, risk factors and outcome . There had been 12 cases of fungaemia that were due to C . krusei, 14 due to C . parapsilosis, 7 due to C . (T.) glabrata, 6 to C . tropicalis, 2 to C . guillermondii, 2 to C . lusitaniae, 1 to C . stellatoidea, and 1 to C . rugosa . Comparison of 45 NAC fungaemia with 75 episodes of C . albicans fungaemia revealed differences only in two risk factors: previous empiric therapy with amphotericin B (16.0 vs 2.2%, P<0.01) appeared more frequently in cases of C . albicans fungaemia, and prior prophylaxis with fluconazole (8.9 vs 0%, P<0.02) was conversely more frequently observed with NAC . The incidence of other risk factors, such as underlying disease, chemotherapy, antibiotic prophylaxis or therapy, treatment with corticosteroids, catheter insertion, mucositis, cytotoxic chemotherapy, and neutropenia, was similar in both groups . There was no difference either in attributable or in overall mortality between NAC and C . albicans fungaemia in our cancer patients. Microbiology, 1999 Oct, 145 ( Pt 10), 2727 - 37 Candida albicans and Yarrowia lipolytica as alternative models for analysing budding patterns and germ tube formation in dimorphic fungi; Herrero AB et al.; The site for bud selection and germ tube emission in two yeasts, Candida albicans and Yarrowia lipolytica, was analysed . Both dimorphic organisms display different patterns of budding, which also differ from those described for Saccharomyces cerevisiae . C . albicans, which is diploid and (until now) lacks a known sexual cycle, buds in an axial budding pattern . During the yeast-hypha transition induced by pH, serum, N-acetylglucosamine (GlcNAc) or temperature, germ tube emergence occurs at approximately 50% in a polar manner, while the other 50% of cells show non-polar germ tube emission . Y . lipolytica, in which most of the natural isolates are haploid and which has a well characterized sexual cycle, buds with a polar budding pattern independently of the degree of ploidy . Germ tube emission during the yeast-hypha transition in both haploid and diploid cells generally occurs at the pole distal from the division site (bipolar) . The addition of hydroxyurea (HU), an inhibitor of DNA synthesis, also produces different effects . In its presence, and therefore in the absence of DNA synthesis, the yeast-hypha transition is completely abolished in Y . lipolytica . By contrast, in C . albicans germ tube emission in the presence of HU is similar to that observed in control cultures for at least 90 min under induction conditions . These results demonstrate that, rather than a single developmental model, several models of development should be invoked to account for the processes involved in the morphological switch in yeasts (the yeast-hypha transition). Microbiology, 1999 Oct, 145 ( Pt 10), 2715 - 25 Multiple amino acid substitutions in lanosterol 14alpha-demethylase contribute to azole resistance in Candida albicans; Favre B et al.; Lanosterol 14alpha-demethylase (14DM) is the target of the azole antifungals, and alteration of the 14DM sequence leading to a decreased affinity of the enzyme for azoles is one of several potential mechanisms for resistance to these drugs in Candida albicans . In order to identify such alterations the authors investigated a collection of 19 C . albicans clinical isolates demonstrating either frank resistance (MICs > or = 32 microg ml(-1)) or dose-dependent resistance (MICs 8-16 microg ml(-1)) to fluconazole . In cell-free extracts from four isolates, including the Darlington strain ATCC 64124, sensitivity of sterol biosynthesis to inhibition by fluconazole was greatly reduced, suggesting that alterations in the activity or affinity of the 14DM could contribute to resistance . Cloning and sequencing of the 14DM gene from these isolates revealed 12 different alterations (two to four per isolate) leading to changes in the deduced amino acid sequence . Five of these mutations have not previously been reported . To demonstrate that these alterations could affect fungal susceptibility to azoles, the 14DM genes from one sensitive and three resistant C . albicans strains were tagged at the carboxyl terminus with a c-myc epitope and expressed in Saccharomyces cerevisiae under control of the endogenous promoter . Transformants receiving 14DM genes from resistant strains had fluconazole MICs up to 32-fold higher than those of transformants receiving 14DM from a sensitive strain, although Western blot analysis indicated that the level of expressed 14DM was similar in all transformants . Amino acid substitutions in the 14DM gene from the Darlington strain also conferred a strong cross-resistance to ketoconazole . In conclusion, multiple genetic alterations in C . albicans 14DM, including several not previously reported, can affect the affinity of the enzyme for azoles and contribute to resistance of clinical isolates. Microbiology, 1999 Oct, 145 ( Pt 10), 2701 - 13 Contribution of mutations in the cytochrome P450 14alpha-demethylase (Erg11p, Cyp51p) to azole resistance in Candida albicans; Marichal P et al.; The cytochrome P450 14alpha-demethylase, encoded by the ERG11 (CYP51) gene, is the primary target for the azole class of antifungals . Changes in the azole affinity of this enzyme caused by amino acid substitutions have been reported as a resistance mechanism . Nine Candida albicans strains were used in this study . The ERG11 base sequence of seven isolates, of which only two were azole-sensitive, were determined . The ERG11 base sequences of the other two strains have been published previously . In these seven isolates, 12 different amino acid substitutions were identified, of which six have not been described previously (A149V, D153E, E165Y, S279F, V452A and G4655) . In addition, 16 silent mutations were found . Two different biochemical assays, subcellular sterol biosynthesis and CO binding to reduced microsomal fractions, were used to evaluate the sensitivity of the cytochromes for fluconazole and itraconazole . Enzyme preparations from four isolates showed reduced itraconazole susceptibility, whereas more pronounced resistance to fluconazole was observed in five isolates . A three-dimensional model of C . albicans Cyp51p was used to position all 29 reported substitutions, 98 in total identified in 53 sequences . These 29 substitutions were not randomly distributed over the sequence but clustered in three regions from amino acids 105 to 165, from 266 to 287 and from 405 to 488, suggesting the existence of hotspot regions . Of the mutations found in the two N-terminal regions only Y132H was demonstrated to be of importance for azole resistance . In the C-terminal region three mutations are associated with resistance, suggesting that the non-characterized substitutions found in this region should be prioritized for further analysis. Microbiology, 1999 Oct, 145 ( Pt 10), 2635 - 46 Microevolutionary changes in Candida albicans identified by the complex Ca3 fingerprinting probe involve insertions and deletions of the full-length repetitive sequence RPS at specific genomic sites; Pujol C et al.; The 11 kb complex DNA fingerprinting probe Ca3 is effective both in cluster analyses of Candida albicans isolates and in identifying microevolutionary changes in the size of hypervariable genomic fragments . A 2.6 kb EcoRI fragment of Ca3, the C fragment, retains the capacity to identify these microevolutionary changes, and when the C fragment is cleaved with SacI, the capacity is retained exclusively by a 1 kb subfragment, C1, which contains a partial RPS repeat element . The microevolutionary changes identified by Ca3, therefore, may involve reorganization of RPS elements dispersed throughout the genome . To test this possibility, hypervariable fragments from several strains of C . albicans were sequenced and compared . The results demonstrate that the microevolutionary changes identified by Ca3 are due to the insertion and deletion of full-length tandem RPS elements at specific genomic sites dispersed throughout the C . albicans genome . The RPS elements at these dispersed sites are bordered by the same upstream and downstream sequences . The frequency of recombination was estimated to be one recombination per 1000 cell divisions by following RPS reorganization in vitro . The results are inconsistent with unequal recombination between homologous or heterologous chromosomes, but consistent with intrachromosomal recombination . Two alternative models of intrachromosomal recombination are proposed: unequal sister-chromatid exchange and slipped misalignment at the replication fork. Nippon Ishinkin Gakkai Zasshi, 1999, 40(4), 209 - 15 Antifungal activities of D0870 against fluconazole-resistant Candida albicans; Kojima M et al.; We compared the in vitro and in vivo antifungal activities of D0870, a new triazole antifungal agent, with those of other antifungal agents against 8 clinical isolates of fluconazole-resistant Candida albicans . Microdilution testing was performed according to National Committee for Clinical Laboratory Standards (NCCLS) document M27-T . Minimal inhibitory concentration of D0870 (<0.004-1.0 micro g/ml) was lower than those of fluconazole (2->64 microEg/ml) and itraconazole (0.031-8.0 microEg/ml) . In systemic infection models with C . albicans in normal and immunosuppressed mice, D0870 at 0 . 3-30 mg/kg/day for 5 days after infection prolonged survival of the animals and showed the highest efficacy among the triazole antifungal agents . At pH 7 and 37C in Sabouraud dextrose broth (SDB), D0870 inhibited the growth of C . albicans and acted cytocidally against one of the middle-resistant strains . In an in vivo study against this strain, D0870 at 10 mg/kg/day for 5 days after infection significantly reduced kidney colony counts (2850+406-997+537 CFU/kidney, P<0.05) on day 7 after infection in comparison with those of the control mice at 24 h after infection . Plasma concentration of D0870 after a single oral administration at 10 mg/kg maintained a sufficient level for interpretation of in vivo antifungal activities . These results suggest that D0870 has strong antifungal activities against clinical isolates of fluconazole-resistant C . albicans in vitro and in vivo, and that these strong activities are at least partially concerned with the fungicidal action. Mycoses, 1999, 42(5-6), 395 - 402 Fungal surveillance cultures during antifungal prophylaxis with itraconazole in neutropenic patients with acute leukaemia; Glasmacher A et al.; Fungal colonization has been associated with an increased rate of invasive fungal infections in neutropenic patients . This study evaluates weekly fungal surveillance cultures from the oropharyngeal and perianal space as well as other suspected sites in 219 courses of myelosuppressive chemotherapy with itraconazole antifungal prophylaxis in 116 neutropenic patients with acute leukaemia . Itraconazole was given from the start of chemotherapy in one of six different dosing regimens . Fungal colonization occurred in 68 (31%) of courses, which was lower than in a historical control group without prophylaxis (53%, P = 0.004) . Twenty-six per cent of these 116 isolates had a growth rate of more than 50 colony forming units (CFU) per culture . Candida glabrata (51%), Candida albicans (18%) and Candida krusei (4%) were the most frequently isolated species . Higher median itraconazole trough concentrations were associated with a lower growth rate in the cultures (< or = 50 CFU/culture versus > 50 CFU/culture): 710 (430-1180) ng ml-1 versus 900 (560-1650) ng ml-1 (P = 0.015) . The use of itraconazole solution--compared with capsules--led to a reduced growth rate (P = 0.035) . In conclusion, compared with historical controls itraconazole antifungal prophylaxis reduces the incidence and the extent of fungal colonization during neutropenia in patients with acute leukaemia. Mycoses, 1999, 42(5-6), 371 - 83 Molecular epidemiology of Candida albicans isolates from AIDS and cancer patients using a novel standardized CARE-2 DNA fingerprinting technique; Lischewski A et al.; A total of 277 Candida isolates from various body sites of 149 AIDS and cancer patients treated in four different university clinics in Wurzburg, Germany were collected over a period of 27 months and phenotypically and genotypically characterized . The fingerprinting patterns of 194 Candida albicans isolates obtained with the moderately repetitive, C . albicans-specific DNA fragment CARE-2 were digitized and retrospectively compared with a highly accurate computer-assisted standardization method . A total of 168 different genotypic patterns (< 100% identity) could be differentiated using this technique . Although comparative analysis of C . albicans subsets revealed a pronounced tendency of C . albicans isolates from HIV patients to form clusters, the mean genetic variability in HIV and cancer patient isolates was virtually identical . Patients with a specific disease condition or in a certain age group were not found to harbour C . albicans isolates displaying a characteristic "signature genotype" . Micro-evolutionary changes were detected by CARE-2 fingerprinting in temporal successive isolates of one patient, but nosocomial transmission of identical isolates between unrelated patients was never seen . Genotyping showed that patient isolates can replace one another; occasionally also species switches were observed . Secreted aspartic protease (SAP) production was not correlated with a specific C . albicans banding pattern; isolates obtained from HIV patients and from an internal control group secreted comparable amounts of SAP . Candida dubliniensis isolates in this study showed an elevated level of SAP production . When used under standardized conditions, CARE-2 fingerprinting is an efficient, reproducible and sensitive technique to characterize C . albicans isolates. FEMS Immunol Med Microbiol, 1999 Nov, 26(2), 175 - 80 Hamycin treatment of candidiasis in normal and diabetic rats; Dhuley JN; Hamycin, a heptaene antifungal antibiotic was compared with amphotericin B in the treatment of established systemic infection with Candida albicans in normal and diabetic rats . In normal rats, orally administered hamycin at 10 mg kg(-1) per day for 7 days reduced Candida colony counts in the kidneys and livers as well as amphotericin B did and was nearly as effective as amphotericin B in a 21-day treatment trial . There was no further reduction in Candida colony counts when normal rats were treated with hamycin at 25 mg kg(-1) twice a day for 7 days . In streptozotocin induced diabetic rats, hamycin at 20 mg kg(-1) per day for either 7 or 21 days compared favourably with amphotericin B in efficacy . Results of the present study suggest that oral hamycin may be useful in the treatment of established disseminated candidiasis in normal as well as diabetic hosts. FEMS Immunol Med Microbiol, 1999 Nov, 26(2), 125 - 30 Experimental murine mycotic mastitis: a sensitive and lenient model for studies of antifungal chemotherapy; Guhad FA et al.; The murine model of mycotic mastitis was used to study the efficacy of amphotericin B (AmB) . Twenty-four BALB/cJ mice at the fifth day of lactation were anesthetized and inoculated through the teat canal (two glands) with 50 microl suspension containing 5.0 x 10(7) cfu ml(-1) Candida albicans blastospores . Mice were randomly divided into two groups: untreated controls and AmB treated . Animals were euthanized 3 and 6 days after infection and treatment (4 mg kg(-1) per day intraperitoneally) . The fungal burden of the mammary gland was determined by quantitative cultures . The number of C . albicans cells recovered from mammary gland homogenates were significantly lower in the AmB treated animals (both 3 and 6 days post-infection) than in the untreated controls (P<0.007 and P<0.003, respectively) . The mammary glands of all untreated control animals showed marked neutrophilic infiltration, severe necrosis, and presence of blastospores, hyphae and pseudohyphae . In contrast, 10 of 12 animals treated with AmB showed only a mild neutrophilic infiltration which was restricted to alveoli and excretory ducts . All extra-mammary organs were free of infection in both groups . The results demonstrate that the murine mycotic mastitis model is suitable for investigations of new antifungal compounds . In addition, this model is more lenient than the systemic candidiasis models. Glycobiology, 1999 Nov, 9(11), 1281 - 6 Differences in the acid-labile component of Candida albicans mannan from hydrophobic and hydrophilic yeast cells; Masuoka J et al.; Cell surface hydrophobicity of the opportunistic fungal pathogen Candida albicans has been linked to the level of cell wall protein glycosylation . Previous work demonstrated that outer chain mannosylation, rather than overall glycosylation, correlated with cell surface hydrophobicity . These studies further suggested that the phosphodiester-linked, acid-labile beta-1,2-mannan was the correlating element . The present work tests this hypothesis and extends the previous results . The composition of bulk mannan from hydrophobic and hydrophilic yeast cells, and the acid-labile mannan from both cell types are compared . Compositional analysis shows that the protein, hexose, and phosphorus content of bulk mannan is similar between the two phenotypes . Electrophoretic separation of acid-released and fluorophore-labeled mannan shows that the acid-labile oligomannosides from hydrophobic cells are longer and potentially in greater abundance than those from hydrophilic cells . These results suggest that regulation of a single step in cell wall protein outer chain mannosylation affects the cell surface ultrastructure and phenotype of C.albicans. Enferm Infecc Microbiol Clin, 1999 Aug-Sep, 17(7), 350 - 3 {Molecular typing and sensitivity of Candida albicans isolates proceeding from critically ill patients}; Ubeda P et al.; BACKGROUND: Nosocomial infection due Candida albicans in immunocompromised patients are recognized as a significant cause of morbidity and mortality . The endogenous forms of infections may require effective strategies for prevention which are different from those for exogenous infections due to transmission of any organism from patient to patient . Typing by PCR of isolates of C . albicans maybe useful for that . METHODS: Twenty-four isolates in blood cultures of 24 critically ill patients were studied . Typing by interrepeat PCR and in vitro antifungal susceptibility tests was performed by a microdilution . RESULTS: Twenty-one different genotypes were obtained . The isolates with same genotype shown different patterns of susceptibility . With one strain a band pattern very different from that obtained with the remaining isolates . CONCLUSIONS: No were relation between strain of same unit . The isolates with same genotype was different critically unit or year of isolation. Infect Immun, 1999 Nov, 67(11), 5820 - 6 Local production of chemokines during experimental vaginal candidiasis; Saavedra M et al.; Recurrent vulvovaginal candidiasis, caused by Candida albicans, is a significant problem in women of childbearing age . Although cell-mediated immunity (CMI) due to T cells and cytokines is the predominant host defense mechanism against C . albicans at mucosal tissue sites, host defense mechanisms against C . albicans at the vaginal mucosa are poorly understood . Based on an estrogen-dependent murine model of vaginal candidiasis, our data suggest that systemic CMI is ineffective against C . albicans vaginal infections . Thus, we have postulated that local immune mechanisms are critical for protection against infection . In the present study, the kinetic production of chemokines normally associated with the chemotaxis of T cells, macrophages (RANTES, MIP-1alpha, MCP-1), and polymorphonuclear neutrophils (MIP-2) was examined following intravaginal inoculation of C . albicans in estrogen-treated or untreated mice . Results showed significant increases in MCP-1 protein and mRNA in vaginal tissue of infected mice as early as 2 and 4 days postinoculation, respectively, that continued through a 21-day observation period, irrespective of estrogen status . No significant changes were observed with RANTES, MIP-1alpha, or MIP-2, although relatively high constitutive levels of RANTES mRNA and MIP-2 protein were observed . Furthermore, intravaginal immunoneutralization of MCP-1 with anti-MCP-1 antibodies resulted in a significant increase in vaginal fungal burden early during infection, suggesting that MCP-1 plays some role in reducing the fungal burden during vaginal infection . However, the lack of changes in leukocyte profiles in vaginal lavage fluids collected from infected versus uninfected mice suggests that MCP-1 functions to control vaginal C . albicans titers in a manner independent of cellular chemotactic activity. Immunobiology, 1999 Sep, 201(1), 133 - 44 Dissimilar attenuation of Candida albicans virulence properties by human immunodeficiency virus type 1 protease inhibitors; Gruber A et al.; The secreted aspartyl proteinase (Sap) of Candida albicans, which is believed to represent an important virulence factor of this opportunistic yeast, and the human immunodeficiency virus type 1 (HIV-1) protease, which is obligatory for the production of infectious virions, both belong to the same family of aspartyl proteinases . We have previously shown that the HIV-1 protease inhibitor Indinavir directly inhibits secretion and proteinase activity of Sap in a dose-dependent manner . Furthermore, at very high concentrations, viability of C . albicans is markedly reduced by Indinavir, indicating that HIV-1 protease inhibitors may possess antifungal activity . We thus proposed that these drugs may add to the resolution of mucosal candidiasis in HIV-1 infected subjects . We have now compared three different HIV-1 protease inhibitors . The rank order of Sap inhibition, already significant at 0.1 mg/ml for all protease inhibitors, was Ritonavir > Indinavir > Saquinavir . However, the cross-reactivity of Ritonavir to pepsin was also more pronounced compared with the other two . Indinavir did not affect Candida viability at concentrations up to 1 mg/ml, in line with our previous study . In contrast, at this concentration Saquinavir was even fungicidal as assessed by three different viability assays (colony formation assay, MTT assay, propidium iodide staining) whereas Ritonavir significantly affected the mitochondrial activity only (MTT assay) . No influence on Candida viability was observed for any of the three at concentrations of 0.1 mg/ml or lower . It remains to be examined whether HIV-1 protease inhibitors or derivatives thereof may be suitable for in vivo therapy of subjects suffering from mucosal candidiasis resistant to current antimycotics. Mycopathologia, 1998-99, 144(3), 147 - 52 Expression of the complement-binding protein (MP60) of Candida albicans in experimental vaginitis; Stringaro A et al.; The expression of the Candida albicans complement-binding C3d protein (MP60) was investigated both in vitro and in vivo by immunogold labelling and electron microscopy . In vivo expression was determined in a rat vaginitis model . Reactivity of in vitro-grown cells to an anti-MP60 rabbit serum was associated with both cytoplasmic and cell wall sites . Immunostaining in the cell wall of both yeast and hyphae was most concentrated in the inner, electron-lucid layer . Immunogold stained preparations of C . albicans from vaginal smears of infected animals also showed intense localization of the MP60 in the inner cell wall, plasma membrane . However, immunogold label was also intense at the cell surface in these samples, mostly in the area of close adherence with the keratinocytes of the vaginal epithelia . These observations indicate that MP60 is expressed both in vitro and in vivo, but to a different degree in the different cell wall layers. Infect Immun, 1999 Nov, 67(11), 6040 - 7 Overexpression of the Candida albicans ALA1 gene in Saccharomyces cerevisiae results in aggregation following attachment of yeast cells to extracellular matrix proteins, adherence properties similar to those of Candida albicans; Gaur NK et al.; Candida albicans maintains a commensal relationship with human hosts, probably by adhering to mucosal tissue in a variety of physiological conditions . We show that adherence due to the C . albicans gene ALA1 when transformed into Saccharomyces cerevisiae, is comprised of two sequential steps . Initially, C . albicans rapidly attaches to extracellular matrix (ECM) protein-coated magnetic beads in small numbers (the attachment phase) . This is followed by a relatively slower step in which cell-to-cell interactions predominate (the aggregation phase) . Neither of these phases is observed in S . cerevisiae . However, expression of the C . albicans ALA1 gene from a low-copy vector causes S . cerevisiae transformants to attach to ECM-coated magnetic beads without appreciable aggregation . Expression of ALA1 from a high-copy vector results in both attachment and aggregation . Moreover, transcriptional fusion of ALA1 with the galactose-inducible promoters GALS, GALL, and GAL1, allowing for low, moderate, and high levels of inducible transcription, respectively, causes attachment and aggregation that correlates with the strength of the GAL promoter . The adherence of C . albicans and S . cerevisiae overexpressing ALA1 to a number of protein ligands occurs over a broad pH range, is resistant to shear forces generated by vortexing, and is unaffected by the presence of sugars, high salt levels, free ligands, or detergents . Adherence is, however, inhibited by agents that disrupt hydrogen bonds . The similarities in the adherence and aggregation properties of C . albicans and S . cerevisiae overexpressing ALA1 suggest a role in adherence and aggregation for ALA1 and ALA1-like genes in C . albicans. J Gastroenterol Hepatol, 1999 Oct, 14(10), 1041 - 4 Case report: spontaneous peritonitis caused by Candida albicans; Yang C et al.; We report a 40-year-old man with decompensated alcoholic liver cirrhosis, who developed spontaneous peritonitis caused by Candida albicans after complete recovery from a recent episode of acute pancreatitis . The patient was successfully treated with amphotericin B . A search of the literature showed that this is the fourth reported case of spontaneous peritonitis caused by Candida albicans. Diagn Microbiol Infect Dis, 1999 Sep, 35(1), 19 - 25 International surveillance of blood stream infections due to Candida species in the European SENTRY Program: species distribution and antifungal susceptibility including the investigational triazole and echinocandin agents . SENTRY Participant Group (Europe); Pfaller MA et al.; The SENTRY Antimicrobial Surveillance Program, an international study of blood stream infections (BSIs), detected 170 episodes of candidemia in 20 European medical centers (13 nations) between January and December, 1997 . Twenty-three percent of the candidal BSI occurred in patients hospitalized in an intensive care unit, 21% in patients in an internal medicine service, 13% in patients in a surgical service, and 9% in patients in an oncology service . Overall, 53% of the BSI were attributable to Candida albicans followed in prevalence by C . parapsilosis (21%), C . glabrata (12%), C . tropicalis (6%), C . famata (2%), C . krusei (1%), and C . inconspicua (1%) . As observed previously in Canada and Latin America, C . parapsilosis and not C . glabrata, was the most common non-albicans species causing yeast BSI in Europe . The proportion of these candidemias attributable to C . albicans varied widely from 0-100% among the 20 European centers . Among the different species of Candida, resistance to fluconazole (MIC, > or = 64 micrograms/mL) and itraconazole (MIC, > or = 1.0 microgram/mL) was observed with C . glabrata and C . krusei and was observed more rarely among other species (e.g., C . inconspicua) . Isolates of C . albicans, C . parapsilosis, C . tropicalis, and C . guilliermondii were all highly susceptible to both fluconazole and itraconazole . Furthermore, the investigational triazoles (BMS-207147, Sch 56592, and voriconazole) and an echinocandin (MK-0991) all demonstrated potent in vitro activity (MIC90s, 0.5, 0.5, 1.0, and 2.0 micrograms/mL, respectively) against these isolates . Continued surveillance at an international level will be important to monitor trends in species distribution and antifungal susceptibility among invasive strains of Candida. Microbiol Immunol, 1999, 43(7), 645 - 51 The expression of the pathogenic yeast Candida albicans catalase gene in response to hydrogen peroxide; Nakagawa Y et al.; The catalase gene of the pathogenic yeast Candida albicans was cloned and its expression was examined . Activity of the catalase was detected when cells which were in the early logarithmic stage were treated with hydrogen peroxide . Additionally, activity was detected without any treatment to cells in the late logarithmic and stationary phases . When cells were cultured in galactose, glycerol, or ethanol, catalase activity was always observed without the hydrogen peroxide treatment, suggesting that glucose represses the induction of catalase expression . To elucidate the molecular mechanism of catalase expression, the putative gene for catalase and its 5' untranscribed region were cloned . Sequences of the gene and its potential regulatory region revealed several motifs, including a GC box-like element and stress-responsive element (STRE), which could be involved in the transcriptional regulation . Northern analysis showed that hydrogen peroxide and sorbitol activated transcription of the catalase . On the other hand, treatment of glucose strictly repressed the expression of the catalase even when co-treated with hydrogen peroxide . The expression of catalase against treatment with hydrogen peroxide took place very quickly and decreased slowly in the experimental condition adopted here . From these results, we assumed that the expression of the catalase in Candida albicans is regulated by various environmental conditions via motifs for transcriptional activation as in other yeast catalases. Rev Rhum Engl Ed, 1999 Jul-Sep, 66(7-9), 434 - 5 Successful lipid-complexed amphotericin B treatment of Candida arthritis in a lymphoma patient; Azaceta G et al.; Fungal arthritis is uncommon but has been increasingly diagnosed over recent years, particularly in patients with immunodeficiency due for instance to hematological malignancies . Candida albicans is the most frequent causative agent, and the knee is the joint most often involved . Amphotericin B is the drug of choice, but is associated with significant toxicity . Recently developed lipid formulations of amphotericin B have been found as effective and less toxic than the conventional formulation . We report a new case of Candida arthritis that occurred after chemotherapy for nonHodgkin's lymphoma and was successfully treated with lipid-complexed amphotericin B. Clin Infect Dis, 1999 Nov, 29(5), 1220 - 5 Molecular epidemiology of the global and temporal diversity of Candida albicans; McCullough MJ et al.; The epidemiology of Candida albicans has changed with the rise in immunocompromised patients and the pressures of antifungal treatment and prophylaxis . We assessed the genotype distribution of recently obtained, globally diverse isolates in comparison with isolates recovered in the United States and United Kingdom before 1985, in order to determine temporal and geographic differences . We used EcoRI digestion of cellular DNA to generate restriction fragment length polymorphisms, dividing the isolates into 4 groups . From 15 diverse geographic areas, 439 isolates obtained over 20 years were divided into 121 genotypes within groups A (289 isolates), B (85), C (56), and D (9) . Differences in genotype distribution existed among the localities (P<.0001) and between isolates obtained before 1990 versus those recovered since then (P=.009) . Comparison of pre-1985 United States/United Kingdom isolates with post-1994 United States isolates revealed a trend toward a changing genotype distribution (P=.057) . Global post-1985 isolates were different in genotype distribution from United States/United Kingdom isolates (P<.0001) . The distribution of isolates from Israel was unique (P<.0001) . These differences could be due in part to the increasing prevalence of group C strains worldwide. Infect Dis Obstet Gynecol, 1999, 7(5), 222 - 6 Germ tube formation changes surface hydrophobicity of Candida cells; Rodrigues AG et al.; Hydrophobic interaction is generally considered to play an important role in the adherence of microorganisms to eukaryotic cells and also to certain inert surfaces . Using a microbe adhesion assay to hydrocarbons (n-hexadecane), 68 strains of Candida albicans and 30 non-albicans strains were studied . Influence of source of isolate, age of the culture, and percentage of germ tube formation on adhesion were studied . C . albicans blastoconidia were found to be hydrophilic; conversely, blastoconidia of non-albicans strains were slightly more hydrophobic . Germ tube formation was associated with a significant rise in cell surface hydrophobicity. J Pediatr Hematol Oncol, 1999 Sep-Oct, 21(5), 431 - 5 Bacillus cereus causing fulminant sepsis and hemolysis in two patients with acute leukemia; Arnaout MK et al.; PURPOSE: Hemolysis is so rarely associated with Bacillus cereus sepsis that only two very well documented cases have been reported . This article reports two unusual cases of Bacillus cereus sepsis with massive intravascular hemolysis in patients who had acute lymphoblastic leukemia (ALL) . PATIENTS AND METHODS: A 20-year-old woman who was 9 weeks pregnant experienced a relapse of ALL . A therapeutic abortion was performed . During week 4 of reinduction the patient had abdominal pain, nausea, and vomiting, with severe neutropenia but no fever . Her condition deteriorated rapidly with cardiovascular collapse, acute massive intravascular hemolysis, and death within hours of the onset of symptoms . Blood cultures were positive for Bacillus cereus . Postmortem histologic examination and cultures revealed Bacillus cereus and Candida albicans in multiple organs . The second patient, a 10-year-old girl, presented with relapsed T-cell ALL . In the second week of reinduction, she had abdominal pain followed by hypotension . Again, no fever was noted . Laboratory studies showed intravascular hemolysis 12 hours after admission . Aggressive support was promptly initiated . Despite disseminated intravascular coagulation; cardiovascular, hepatic, and renal failure; and multiple intracerebral hypodense lesions believed to be infarcts, the patient recovered fully and resumed reinduction therapy . CONCLUSIONS: Bacillus cereus infection can have a fulminant clinical course that may be complicated by massive intravascular hemolysis . This pathogen should be suspected in immunosuppressed patients who experience gastrointestinal symptoms and should not be precluded by the absence of fever, especially if steroids such as dexamethasone are being given . Exchange transfusion may be lifesaving in Bacillus cereus septicemia associated with massive hemolysis. Biochim Biophys Acta, 1999 Sep 3, 1446(3), 443 - 9 Structure of Tetrahymena CCT theta gene and its expression under colchicine treatment; Domingues C et al.; We report here the cloning and the characterization of the Tetrahymena pyriformis chaperonin-containing-TCP1 theta gene (TpCCT theta), an orthologue of the mouse chaperonin gene CCT theta . TpCCT theta gene is interrupted by eight introns, ranging in size between 91 and 419 nucleotides, and encodes a protein consisting of 540 amino acid residues (59.1 kDa), with a putative pI of 5.73 . The amino acid sequence of TpCCT theta reveals 39.4-46.0% identity with the sequences of Candida albicans and mouse CCT theta subunits and 28.0-32.6% identity with the other TpCCT subunits known so far . We have studied the expression of this gene in exponentially growing Tetrahymena cells and in cells treated with colchicine for different times . The steady-state levels of CCT theta mRNA rapidly decrease in the first 30 min of colchicine treatment . Interestingly, treatment for subsequent 60 min gives expression levels higher than those found in exponentially growing cells. J Clin Microbiol, 1999 Nov, 37(11), 3533 - 9 Rapid identification of Candida dubliniensis with commercial yeast identification systems; Pincus DH et al.; Candida dubliniensis is a newly described species that is closely related phylogenetically to Candida albicans and that is commonly associated with oral candidiasis in human immunodeficiency virus-positive patients . Several recent studies have attempted to elucidate phenotypic and genotypic characteristics of use in separating the two species . However, results obtained with simple phenotypic tests were too variable and tests that provided more definitive data were too complex for routine use in the clinical laboratory setting . The objective of this study was to determine if reproducible identification of C . dubliniensis could be obtained with commercial identification kits . The substrate reactivity profiles of 80 C . dubliniensis isolates were obtained by using the API 20C AUX, ID 32 C, RapID Yeast Plus, VITEK YBC, and VITEK 2 ID-YST systems . The percentages of C . dubliniensis isolates capable of assimilating or hydrolyzing each substrate were compared with the percentages from the C . albicans profiles in each kit's database, and the results were expressed as percent C . dubliniensis and percent C . albicans . Any substrate that showed >50% difference in reactivity was considered useful in differentiating the species . In addition, assimilation of methyl-alpha-D-glucoside (MDG), D-trehalose (TRE), and D-xylose (XYL) by the same isolates was investigated by the traditional procedure of Wickerham and Burton (L . J . Wickerham and K . A . Burton, J . Bacteriol . 56:363-371, 1948) . At 48 h (the time recommended by the manufacturer for its new database), we found that the assimilation of four carbohydrates in the API 20C AUX system could be used to distinguish the species, i.e., glycerol (GLY; 88 and 14%), XYL (0 and 88%), MDG (0 and 85%), and TRE (15 and 97%) . Similarly, results with the ID 32 C system at 48 h showed that XYL (0 and 98%), MDG (0 and 98%), lactate (LAT; 0 and 96%), and TRE (30 and 96%) could be used to separate the two species . Phosphatase (PHS; 9 and 76%) and alpha-D-glucosidase (23 and 94%) proved to be the most useful for separation of the species in the RapID Yeast Plus system . While at 24 h the profiles obtained with the VITEK YBC system showed that MDG (10 and 95%), XYL (0 and 95%), and GLY (26 and 80%) could be used to separate the two species, at 48 h only XYL (6 and 95%) could be used to separate the two species . The most useful substrates in the VITEK 2 ID-YST system were TRE (1 and 89%), MDG (1 and 99%), LAT (4 and 98%), and PHS (83 and 1%) . While the latter kit was not yet commercially available at the time of the study, it would appear to be the most valuable for the identification of C . dubliniensis . Although assimilation of MDG, TRE, and XYL proved to be the most useful for species differentiation by the majority of commercial systems, the results with these carbohydrates by the Wickerham and Burton procedure were essentially the same for both species, albeit following protracted incubation . Thus, it is the rapidity of the assimilation achieved with the commercial systems that allows the differentiation of C . dubliniensis from C . albicans. Arch Surg, 1999 Oct, 134(10), 1041 - 8 Six years of surgical wound infection surveillance at a tertiary care center: review of the microbiologic and epidemiological aspects of 20,007 wounds; Weiss CA 3rd et al.; HYPOTHESES: (1) Antibiotic restriction policies result in alteration of microbiologic features of surgical site infections (SSIs) and (2) reported SSI rates are underestimated when postdischarge surveillance is not included in SSI surveillance efforts . DESIGN: Retrospective analysis of prospectively collected SSI surveillance data . PATIENTS AND METHODS: We compared initial microbial isolates from SSIs between (1) January 1, 1993, and December 31, 1995, and (2) January; 1, 1996, and December 31, 1998 . Antibiotic restriction policies were implemented at Fairview-University Medical Center, Minneapolis, Minn, on March 1, 1995 . For the combined periods (January 1, 1993, to December 31, 1998), we determined SSI rates for 20007 operations according to the extent of bacterial contamination at surgery (wound class) . Then, we analyzed SSI rates for 10559 of these operations (selected based on availability of Anesthesia Society of America score and type of procedure) using the surgical wound risk index (wound class, Anesthesia Society of America score, and length of operation) . We categorized SSI rates by 17 procedures for comparison with SSI rates reported by 286 hospitals that contributed data confidentially and voluntarily to the National Nosocomial Infections Surveillance System in 1998 . We compared SSI rates with and without postdischarge surveillance . RESULTS: Coagulase-negative staphylococcus and group D enterococcus were the 2 most frequent isolates before and after antibiotic restriction policies were implemented . Candida albicans isolates decreased from 7.9% (1993-1995) to 6.5% (1996-1998; P=.46) . Methicillin-resistant Staphylococcus aureus (1.8% of isolates) and vancomycin-resistant enterococcus (2.4% of isolates) organisms were first identified between 1996 and 1998 . Our SSI rates were 2.6% for class I wounds, 3.6% for class II wounds, and 10.5% for class III/IV wounds; 53.9% of SSIs were identified after hospital discharge . CONCLUSIONS: Antibiotic restriction policies did not alter the microbial spectrum of SSIs during the observation period . Reporting SSI rates in the absence of postdischarge surveillance dramatically underestimates actual SSI rates, especially in tertiary care hospitals that provide care for large populations of elderly and immunosuppressed patients. J Biol Chem, 1999 Oct 22, 274(43), 30520 - 6 The Candida albicans phospholipomannan is a family of glycolipids presenting phosphoinositolmannosides with long linear chains of beta-1,2-linked mannose residues; Trinel PA et al.; In a series of studies, we have shown that Candida albicans synthesizes a glycolipid, phospholipomannan (PLM), which reacted with antibodies specific for beta-1,2-oligomannosides and was biosynthetically labeled by {(3)H}mannose, {(3)H}palmitic acid, and {(32)P}phosphorus . PLM has also been shown to be released from the C . albicans cell wall and to bind to and stimulate macrophage cells . In this study, we show by thin layer chromatography scanning of metabolically radiolabeled extracts that the C . albicans PLM corresponds to a family of mannose and inositol co-labeled glycolipids . We describe the purification process of the molecule and the release of its glycan fraction through alkaline hydrolysis . Analysis of this glycan fraction by radiolabeling and methylation-methanolysis confirmed the presence of inositol and of 1, 2-linked mannose units . NMR studies evidenced linear chains of beta-1,2-oligomannose as the major PLM components . Mass sp