Trypanocidal Activity of Flavokawin B, a Component of Polygonum ferrugineum Wedd.

The trypanocidal potential of the natural chalcone flavokawin B, which was isolated from the hexanic extract of Polygonum ferrugineum Wedd., is reported here. Although flavokawin B is widespread, this is the first report about its trypanocidal properties on both Trypanosoma cruzi (IC50 = 9.5 µM, IC50 = 34.7 µM benznidazol, Y strain) epimastigotes and Trypanosoma brucei (IC50 = 4.8 µM, IC50 = 6.4 µM pentamidine, 29-13 strain) procyclic forms, which was also corroborated on T. brucei strain 427 (IC50 = 6.2 µM). In order to learn more about its properties, unspecific cytotoxicity on Hep G2 cells was investigated as well as the trans-splicing inhibitory potential on T. brucei cells. The results shown here point to flavokawin B as a candidate in the search for new agents. It is also cheaper and less toxic than the available drugs to treat trypanosomiasis with a special focus on sleeping sickness disease.

Detection of invasive infection caused by Fusarium solani and non-Fusarium solani species using a duplex quantitative PCR-based assay in a murine model of fusariosis.

A duplex Real Time PCR (RT-PCR) assay for detecting DNA of members of the genus Fusarium has been developed and validated by using two mouse models of invasive infection. The duplex RT-PCR technique employed two specific molecular beacon probes targeting a highly conserved region of the fungal rDNA gene. This technique showed a detection limit of 10 fg DNA per µl of sample and a specificity of 100%. The sensitivity in a total of 48 samples from a murine model of Fusarium solani infection was 93.9% for lung tissues and 86.7% for serum samples. In comparison, the sensitivity in a total of 45 samples of a F. oxysporum murine model infection was 87% for lung tissues and 42.8% for serum samples. This molecular technique could be a reliable method for the quantification and the evaluation of the disease in animal models and for the clinical diagnosis of fusariosis.

Histoplasmosis and paracoccidioidomycosis in a non-endemic area: a review of cases and diagnosis.

BACKGROUND: Histoplasmosis and paracoccidioidomycosis (PCM) have increased in Spain in recent years, due firstly to the migration from endemic regions and secondly to travelers returning from these regions. In non-endemic areas, diagnosis of both diseases is hampered by the lack of experience, long silent periods, and the resemblance to other diseases such as tuberculosis and sarcoidosis. METHODS: A total of 39 cases of imported histoplasmosis and 6 cases of PCM diagnosed in the Spanish Mycology Reference Laboratory since 2006 were analyzed. Microbiological diagnosis was performed using classical methods and also a specific real-time polymerase chain reaction (RT-PCR) assay for each microorganism. RESULTS: We had 9 cases of probable histoplasmosis in travelers and 30 cases in immigrants, 29 of whom were defined as proven. Paracoccidioidomycosis (PCM) cases were either immigrants or people who had lived for a long period of time in endemic regions, all of whom were classified as proven cases. Cultures showed a good sensitivity in detecting Histoplasma capsulatum in immigrants with proven histoplasmosis (73%); however, growth was very slow. The fungus was never recovered in traveler patients. Paracoccidioides brasiliensis was isolated in a culture only in one case of the proven PCM. Serological methods were not very reliable in immunocompromised patients with histoplasmosis (40%). A PCR-based technique for histoplasmosis detected 55.5% of the cases in travelers (probable cases) and 89% of the cases in immigrants (proven). The PCR method for PCM detected 100% of the cases. CONCLUSIONS: These kinds of mycoses are increasingly frequent in non-endemic areas, and newer and faster techniques should be used to reach an early diagnosis. The RT-PCR techniques developed appear to be sensitive, specific, and fast and could be helpful to detect those mycoses. However, it is also essential that physicians perform differential diagnosis in individuals coming from endemic areas.

Susceptibility profile of clinical isolates of non-Cryptococcus neoformans/non-Cryptococcus gattii Cryptococcus species and literature review.

The in vitro susceptibility profile of 24 clinical isolates of non-Cryptococcus neoformans/non-Cryptococcus gattii Cryptococcus species was analysed. In addition, the susceptibility results of 98 other strains from seven different reports were reviewed. The latter included studies which used antifungal susceptibility testing reference procedures or commercial methods which exhibited high correlation rates with the reference procedures. A total of 122 isolates were analysed (57 Cryptococcus albidus, 39 Cryptococcus laurentii, ten Cryptococcus uniguttulatus, ten Cryptococcus humicola, four Cryptococcus curvatus, and two Cryptococcus luteolus). Amphotericin B was in vitro the most active compound against all species, while flucytosine and candins were inactive. Fluconazole exhibited a limited in vitro activity, particularly against C. albidus, C. uniguttulatus and C. laurentii. Voriconazole, itraconazole and posaconazole were active against most of isolates, but we found significant rates of decreased susceptibility. Identification and susceptibility testing of Cryptococcus spp. should be performed on a routine basis in view of their unpredictable susceptibility profiles.

ATP hydrolysis-driven H(+) translocation is stimulated by sulfate, a strong inhibitor of mitochondrial ATP synthesis.

Sulfate is a partial inhibitor at low and a non-essential activator at high [ATP] of the ATPase activity of F(1). Therefore, a catalytically-competent ternary F(1) x ATP x sulfate complex can be formed. In addition, the ANS fluorescence enhancement driven by ATP hydrolysis in submitochondrial particles is also stimulated by sulfate, clearly showing that the ATP hydrolysis in its presence is coupled to H(+) translocation. However, sulfate is a strong linear inhibitor of the mitochondrial ATP synthesis. The inhibition was competitive (K (i) = 0.46 mM) with respect to Pi and mixed (K (i) = 0.60 and K'(i) = 5.6 mM) towards ADP. Since it is likely that sulfate exerts its effects by binding at the Pi binding subdomain of the catalytic site, we suggest that the catalytic site involved in the H(+) translocation driven by ATP hydrolysis has a more open conformation than the half-closed one (beta(HC)), which is an intermediate in ATP synthesis. Accordingly, ATP hydrolysis is not necessarily the exact reversal of ATP synthesis.

Development and validation of a quantitative PCR assay for diagnosis of scedosporiosis.

Scedosporium apiospermum and Scedosporium prolificans are fungal pathogens that can cause severe human infections, including disseminated mycosis in immunocompromised patients. Two real-time PCR (RT-PCR) assays for the diagnosis of these species were developed and validated for the classification of clinical strains and for the detection of DNA in clinical samples by use of a murine model of invasive infection. A total of 14 clinical strains and 141 samples, including blood, serum, and lung samples from infected CD1 mice, were analyzed. Each RT-PCR methodology used a species-specific molecular beacon probe targeting a highly conserved region of the fungal ribosomal DNA gene. Results showed 100% specificity and a detection limit of 10 fg of DNA for both assays. The sensitivities for the S. prolificans-specific PCR assay were 100% for cultured clinical strains, 95.5% for lung tissues, 85% for serum, and 83.3% for blood. For S. apiospermum, the sensitivities were 100% for clinical strains and 97.2%, 81.8%, and 54.5% for lung tissues, serum, and blood, respectively. Both techniques can be useful for clinical diagnosis, and further studies are warranted.

Inhibition of the mitochondrial ATP synthesis by polygodial, a naturally occurring dialdehyde unsaturated sesquiterpene.

Polygodial is a naturally occurring sesquiterpene dialdehyde that exhibits several pharmacologically interesting activities. Among them, its antifungal properties have been more thoroughly studied. The mitochondrial ATPase has been suggested as one of the possible targets for polygodial action. However, its mechanism of action is not well defined yet. The effect of polygodial on the mitochondrial energy metabolism is described in this paper. Polygodial inhibited ATP synthesis coupled to succinate oxidation in beef-heart submitochondrial particles at concentrations (IC(50)=2.4+/-0.1 microM) which marginally affected electron transport and ATPase activity (IC(50)=97+/-4 microM). A transitory stimulation of the electron transport in intact rat liver mitochondria in state 4 was also obtained at low polygodial concentrations (EC(50)=20+/-4 microM). These results suggest that polygodial uncouples ATP synthesis from electron transport at low concentrations. Similar concentrations of polygodial partially abolished the ANS fluorescence enhancement (IC(50)=2.2+/-0.4 microM) induced by succinate oxidation in submitochondrial particles but did not collapse the DeltapH. We postulate that polygodial uncouples mitochondrial ATP synthesis by affecting the electrical properties of the membrane surface and consequently collapsing the membrane potential (Deltapsi) and/or the localized transmembrane pH difference (DeltapH(S)) without affecting the DeltapH between the two bulk aqueous phases (DeltapH(B)). The relevance of these findings for the understanding of the biochemical basis of the antifungal activity of polygodial and the evaluation of its potentiality as a therapeutic agent are discussed.

In vitro antifungal properties structure-activity relationships and studies on the mode of action of N-phenyl, N-aryl, N-phenylalkyl maleimides and related compounds.

The synthesis, in vitro antifungal evaluation and structure-activity relationship studies of 14 compounds of the N-phenyl-, N-aryl-, N-phenylalkyl- maleimide and 3,4-dichloromaleimide series are reported. The compounds were evaluated against a panel of standardized yeasts and filamentous fungi as well as clinical isolates of Candida albicans. The activities of N-phenylalkyl-3,4-dichloromaleimide derivatives but not those of N-phenylalkyl-maleimide derivatives showed to be dependent on the length of the alkyl chain. N-Phenylpropyl-3,4-dichloromaleimide showed the broadest spectrum of action and lower minimal inhibitory concentrations (MIC) in all of the fungi tested. The nitrogen-carbon distance between the two rings seems to play an important role in the antifungal behavior of these compounds. The most active structure showed inhibited (1,3)beta-D-glucan and chitin synthases, enzymes that catalyze the synthesis of the major fungal cell-wall polymers.

Synthesis, in vitro/in vivo antifungal evaluation and structure-activity relationship study of 3(2H)-pyridazinones.

The synthesis, in vitro/in vivo antifungal evaluation and a structure-activity relationship (SAR) study of 3(2H)-pyridazinones was carried out. The results reported here may be helpful in the structural identification and understanding of the minimum structural requirements for these molecules acting as antifungal agents. In addition, the most active structure in this series was tested for its capacity of inhibiting Saccharomyces cerevisiae beta 1,3-glucan synthase and chitin synthase, enzymes that catalyze the synthesis of the major polymers of the fungal cell wall.

In vitro antifungal activity of new series of homoallylamines and related compounds with inhibitory properties of the synthesis of fungal cell wall polymers.

The synthesis, in vitro antifungal evaluation and SAR studies of 101 compounds of the 4-aryl-, 4-alkyl-, 4-pyridyl or -quinolinyl-4-N-arylamino-1-butenes series and related compounds, are reported here. Active structures showed to inhibit (1,3)-beta-D-glucan and mainly chitin synthases, enzymes that catalyze the synthesis of the major fungal cell wall polymers.

In vitro inhibition of 1,3-beta-glucan synthase by glycolipids from convolvulaceous species.

Sixteen convolvulaceous glycolipids selected from the tricolorin (1 - 7) and orizabin (8 - 16) series, proved to be strong in vitro inhibitors of the enzyme that catalyzes the synthesis of 1,3-beta-D-glucan, a major polymer of fungal cell-walls. Results provide an insight into function of the specific structures of these complex macrocyclic lactones as inhibitors of the 1,3-beta-D-glucan synthase and open the possibility of using these compounds as starting points for the development of antifungal agents that act by inhibiting fungal cell-wall synthesis.