The onset of leukaemia caused by type C retroviruses (MLV) in mice is accelerated by the emergence of recombinant polytropic or mink cell focus-forming (MCF) viruses. Susceptibility to infection by polytropic/MCF and also by closely related xenotropic MLV has been mapped to Rmc1 on mouse chromosome 1 (refs 5-7). To identify this gene, we introduced an expression cDNA library prepared from mouse NIH3T3 fibroblasts into nonpermissive hamster cells and screened these cells for acquired susceptibility to MCF viruses encoding beta-galactosidase and G418 resistance. From hamster cell clones identified in the screen, we recovered a mouse cDNA that maps to Rmc1 and confers MCF MLV infection when expressed in nonpermissive cell lines. It encodes a membrane protein related to Syg1p (suppressor of yeast G alpha deletion; ref. 8). The receptor-binding domain of the MCF MLV envelope protein binds specifically to Xenopus laevis oocytes that express mouse Syg1, suggesting it functions as a receptor that mediates virus entry. We also obtained the cDNA encoding human SYG1. When expressed in hamster cells, it establishes infectivity by MCF MLV as well as xenotropic MLV, which do not infect laboratory mice.

Non-albicans Candida species had higher rates of resistance to fluconazole than did C. albicans (44 of 395 [11.2%] vs 9 of 237 [3.8%]; P = .002). The increasing rate of fluconazole resistance in C. tropicalis (15%) is important because C. tropicalis is one of the most commonly isolated non-albicans Candida species.

Overexpression of CDR1, an efflux pump, is one of the major mechanisms contributing to drug resistance in Candida albicans. CDR1 p-lacZ was constructed and transformed into a Saccharomyces cerevisiae strain so that the lacZ gene could be used as the reporter to monitor the activity of the CDR1 promoter. Overexpression of CaNDT80, the C. albicans homolog of S. cerevisiae NDT80, increases the ␤-galactosidase activity of the CDR1 p-lacZ construct in S. cerevisiae. Furthermore, mutations in CaNDT80 abolish the induction of CDR1 expression by antifungal agents in C. albicans. Consistently, the Candt80/Candt80 mutant is also more susceptible to antifungal drugs than the wild-type strain. Thus, the gene for CaNdt80 may be the first gene among the regulatory factors involved in drug resistance in C. albicans whose function has been identified.The prevalence of fungal infections has increased significantly in the past few decades. Among the organisms causing these infections, Candida albicans is the most frequently isolated fungal pathogen in humans and has caused morbidity in seriously debilitated and immunocompromised hosts (6). Coincident with the increased use of antifungal drugs, the incidences of drug resistance have also increased (24,33,36). The limited variety of antifungal agents and emerging drug resistance highlight the need to identify potential targets and elucidate the molecular mechanisms involved in drug resistance for the development of new effective antifungal agents.Overexpression of efflux pumps, either major facilitators or ATP binding cassette (ABC) transporters, has been shown to be one of the major mechanisms of drug resistance in clinical isolates (9,18,19). The CDR1 gene, which encodes an ABC efflux pump, is identified by complementation of the pdr5 mutant, which is hypersensitive to cycloheximide, chloramphenicol, and azole drugs, in Saccharomyces cerevisiae (25). Mutations in CDR1 in C. albicans resulted in increased susceptibilities to azole drugs (29), which is consistent with the observation that overexpression of CDR1 contributes to the drug resistance of clinical isolates of C. albicans (17, 36). The AP-1 site and the drug-responsive element of the CDR1 promoter have been reported to be the cis-regulatory elements (5, 26). Furthermore, the existence of trans-regulatory factors of CDR1 has also been suggested (26). However, the molecular mechanism and the gene network regulating the expression of CDR1 and drug resistance are poorly understood.In this study, as in previous studies (15, 16), we have successfully used S. cerevisiae as a model to study C. albicans, despite the differences between these two organisms. We have identified one predicted transcription factor, CaNdt80, the C. albicans homolog of S. cerevisiae Ndt80, which is a meiosisspecific transcription factor in S. cerevisiae (2, 3) and which is involved in drug resistance through the regulation of CDR1 in C. albicans. MATERIALS AND METHODSStrains and media. The S. cerevisiae strains, C. albicans strains, and plasmids used in t...

Background: Invasive fungal infections, such as candidemia, caused by Candida species have been increasing. Candidemia is not only associated with a high mortality (30% to 40%) but also extends the length of hospital stay and increases the costs of medical care. Sepsis caused by Candida species is clinically indistinguishable from bacterial infections. Although, the clinical presentations of the patients with candidemia caused by Candida albicans and non-albicans Candida species (NAC) are indistinguishable, the susceptibilities to antifungal agents of these species are different. In this study, we attempted to identify the risk factors for candidemia caused by C. albicans and NAC in the hope that this may guide initial empiric therapy.

Limited structural information of drug targets, cellular toxicity possessed by lead compounds, and large amounts of potential leads are the major issues facing the design-oriented approach of discovering new leads. In an attempt to tackle these issues, we have developed a process of virtual screening based on the observation that conformational rearrangements of the dengue virus envelope protein are essential for the mediation of viral entry into host cells via membrane fusion. Screening was based solely on the structural information of the Dengue virus envelope protein and was focused on a target site that is presumably important for the conformational rearrangements necessary for viral entry. To circumvent the issue of lead compound toxicity, we performed screening based on molecular docking using structural databases of medical compounds. To enhance the identification of hits, we further categorized and selected candidates according to their novel structural characteristics. Finally, the selected candidates were subjected to a biological validation assay to assess inhibition of Dengue virus propagation in mammalian host cells using a plaque formation assay. Among the 10 compounds examined, rolitetracycline and doxycycline significantly inhibited plaque formation, demonstrating their inhibitory effect on dengue virus propagation. Both compounds were tetracycline derivatives with IC50s estimated to be 67.1 µM and 55.6 µM, respectively. Their docked conformations displayed common hydrophobic interactions with critical residues that affected membrane fusion during viral entry. These interactions will therefore position the tetracyclic ring moieties of both inhibitors to bind firmly to the target and, subsequently, disrupt conformational rearrangement and block viral entry. This process can be applied to other drug targets in which conformational rearrangement is critical to function.

To understand the Candida colonization of human immunodeficiency virus (HIV)-infected outpatients inTaiwan, we have conducted a prospective cohort study of Candida colonization and its risk factors at the National Taiwan University Hospital from 1999 to 2002. More than 50% of the patients were colonized with Candida species, and 12% developed symptomatic candidiasis. Patients colonized with fluconazole-resistant strains of Candida species had a higher prevalence of candidiasis than those colonized with susceptible strains. Our analysis found that antibiotic treatment and lower CD4؉ counts (<200 cells/mm 3 ) increased the rate of oropharyngeal candidiasis in HIV-infected patients, while antiretroviral therapy protected patients from the development of candidiasis.Mucosal candidiasis, including oropharyngeal, esophageal, and vaginal candidiasis, is common among human immunodeficiency virus (HIV)-infected patients (4, 11). In particular, oropharyngeal candidiasis occurs in up to 90% of patients during the course of HIV infection (17). Progressive cellmediated immunodeficiency, with CD4 ϩ lymphocyte counts less than 200 cells/mm 3 , is a risk factor for colonization with Candida species and the development of candidiasis (3). The widespread use of azole antifungal agents for the treatment of mucosal candidiasis results in colonization with less susceptible organisms and the development of resistance (4, 15). Thus, oropharyngeal candidiasis due to drug-resistant fungi is an emerging problem for patients infected with HIV (18).The overall prevalence of known HIV infection in Taiwan remains relatively low (0.01%) (9). As in most other industrialized countries, the majority of HIV-infected patients in Taiwan receive care in the outpatient setting. Therefore, to better understand the epidemiology of Candida species carriage among HIV-infected outpatients in Taiwan, we undertook a study to determine the prevalence of oropharyngeal colonization. Our objectives were to assess the colonization status and the risk factors for colonization and the development of candidiasis in HIV-infected outpatients in Taiwan. The susceptibilities of those Candida isolates to antifungal drugs were also determined. MATERIALS AND METHODSStudy population and data collection. HIV-infected patients were monitored regularly in the outpatient infectious diseases clinic of National Taiwan University Hospital, a major referral hospital for the management of HIV-related complications. The patients were enrolled after they provided informed verbal consent. This was a prospective study performed by the use of three surveys, conducted from May to June 1999, May to September 2001, and January to April 2002. A standardized data collection form was used to retrieve demographic information, the most recent CD4 ϩ lymphocyte count, and the highly active antiretroviral therapy (HAART) prescribed. In addition, clinical information for the previous 3 months was obtained and included information on whether the patient had a history of oral or esophageal candidiasi...

Infections caused by treatment-resistant non-albicans Candida species, such as C. tropicalis, has increased, which is an emerging challenge in the management of fungal infections. Genetically related diploid sequence type (DST) strains of C. tropicalis exhibiting reduced susceptibility to fluconazole circulated widely in Taiwan. To identify the potential source of these wildly distributed DST strains, we investigated the possibility of the presence in soil of such C. tropicalis strains by pulsed field gel electrophoresis (PFGE) and DST typing methods. A total of 56 C. tropicalis isolates were recovered from 26 out of 477 soil samples. Among the 18 isolates with reduced susceptibility to fluconazole, 9 belonged to DST149 and 3 belonged to DST140. Both DSTs have been recovered from our previous studies on clinical isolates from the Taiwan Surveillance of Antimicrobial Resistance of Yeasts (TSARY) program. Furthermore, these isolates were more resistant to agricultural azoles. We have found genetically related C. tropicalis exhibiting reduced susceptibility to fluconazole from the human hosts and environmental samples. Therefore, to prevent patients from acquiring C. tropicalis with reduced susceptibility to azoles, prudent use of azoles in both clinical and agricultural settings is advocated.

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