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Candida albicans is the most common cause of opportunistic fungal disease in humans. 1) In fact, this fungus is also known as the fourth leading cause of nosocomial infections. 2,3) Over the past few decades, increasing incidence of candidiasis due to this fungus parallels the growing numbers of immmunocompromised people. [2][3][4][5] In immunocompromised people, disseminated candidiasis is sometimes a serious disease which often results in death. 6) In addition to the disseminated disease, C. albicans also causes local infections such as vaginitis and thrush. In case of vulvovaginal candidiasis, C. albicans is the most common etiological agent. 7,8) The treatment for the candidiasis includes antifungal drugs, mainly amphotericin B (Amp B) and the azoles. Furthermore, Amp B has been considered as the drug of choice for the treatment of fungal infections.9,10) However, due to its poor permeability across the membrane, 11) an increased amount of Amp B must be administered to patients in clinical situations, thus often resulting in severe side effects such as renal damage. 12,13) To lessen the severity of the side effects, Amp B is often combined with other antifungal drugs such as the azole, [14][15][16] but data reporting resistance of C. albicans to the azole have been recently increasing. [17][18][19] Thus investigation of reducing the Amp B dose by combining it with a new antifungal product appears to be important.As a part of the investigation, we tested a possibility of combination therapy with an alkaloid berberine that has quaternary nitrogen, polycyclic and planar structure.20) The plants that produce berberine is well protected against microorganisms due to the allelochemical activities of berberine, and thus, it appears likely that the berberine functions as a chemical defence compound. 20) Berberine can be assumed to be quite safe considering that the lethal dose of berberine is known to be approximately 23 mg/kg of body weight in mice.21) According to the previous reports of others berberine is shown to have both antifungal [22][23][24] and antibacterial effects 25,26) under the in vitro condition. We also reported that berberine had antifungal effect on the growth of C. albicans as determined by agar diffusion method.27) Continuing the study of antifungal activity of berberine, we investigated if berberine had a synergic effect with Amp B against disseminated candidiasis caused by C. albicans in animal models. MATERIALS AND METHODSOrganisms and Culture Conditions C. albicans strains CA-1 and A-9, characterized previously, [28][29][30][31] were each grown in glucose-yeast extract-peptone (GYEP) broth at 37°C. Yeast cells collected from the broth cultures were washed with sterile cold Dulbecco's phosphate-buffered saline (DPBS; Sigma, St. Louis, U.S.A.) solution and enumerated by hemocytometer to obtain desired numbers of yeast cells.Mice BALB/c female mice (Charles River Labs, U.S.A.) were used at 6 to 7 weeks of age. The mice were maintained in the animal facility at Dongduk Women's University.Berber...

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Ginsenosides are active compounds isolated from Panax ginseng Meyer. Among these ginsenosides, less polar ginsenosides such as ginsenoside Rg3 and ginsenoside Rh2 have been demonstrated to have tumor inhibitory effects because of their cytotoxicity. In this study, we evaluated the apoptotic effects of ginsenoside Rk1 in SK-MEL-2 human melanoma. Ginsenoside Rk1 isolated from red ginseng is one of the novel ginsenosides that shows strong cytotoxicity compared to ginsenoside Rg3 in dose- and time-dependent manners. The results of DNA fragmentation, 4',6-diamidino-2-phenylindole staining, and flow cytometric analysis are corroborated that ginsenoside Rk1 induced apoptosis in SK-MEL-2 cells. Western blot analysis revealed up-regulation of Fas, FasL, and Bax protein expression and down-regulation of procaspase-8, procaspase-3, mutant p53 and Bcl-2 protein expression. These findings suggest that ginsenoside Rk1 might be a promising compound to induce apoptosis through both extrinsic and intrinsic pathways in SK-MEL-2 cells.

Few researches on the protein stabilization of recombinant human serum albumin (rHSA) have been done. In the present study, we assessed the impact of sugar lyoprotectants on the protein stability of lyophilized rHSA (65 KDa) in the solid state. For the assessment, rHSA was formulated with sucrose and trehalose, respectively, alone or in combination with mannitol, which were lyophilized and stored at 35 degrees C. Degradation and aggregation of the resulting lyophilized formulations was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Induction of amorphous state by the lyophilactants with rHSA was determined by differential scanning calorimetry (DSC). The protein secondary structure of the rHSA in the formulations was analyzed by Fourier transform infrared spectroscopy (FT-IR). Results from SDS-PAGE analysis displayed that mannitol formulation caused aggregation resulting in a few bands that were greater than 65 KDa, whereas sucrose and trehalose formulations revealed no such aggregation. However, the aggregation of the protein decreased when mannitol was combined with sucrose or trehalose. DSC measurement supported the electrophoresis data showing that sucrose and trehalose formed complete amorphous state, but mannitol induced a partial amorphous state. These data indicate during lyophilization the most effective protein protection against aggregation was provided by sucrose and trehalose. The protection lasted during 4 months storage at 35 degrees C. FT-IR analysis displayed that the sucrose formulation inhibited deamidation. In conclusion, our data suggest that sucrose and trehalose as additives seems to be sufficient to protect from lyophilization of rHSA protein and also maintain its stability in the solid state during storage.

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We recently reported that IgM MAb B6.1, specific for β-1, 2-mannotriose on the cell wall of Candida albicans, is therapeutic to disseminated candidiasis due to C. albicans. In the current study, we examined if MAbB6.1 enhances therapeutic effect of fluconazole (FLC) to the disseminated disease. To assess the combination effect, determination by the kidneys-colony forming unit and survival times were used. Results showed that the therapeutic effect of FLC on mice with disseminated candidiasis was dose-dependent, but a FLC dose at 0.8 mg/kg body weight of mice was ineffective. To determine combination effect, mice treated intraperitoneally with a combination of FLC plus MAb B6.1 at 1 h post-infection - a condition of developing partial therapeutic activity - enhanced survival times beyond the effect by only antibody (p < 0.05). The resulting MST (mean survival times) value from the combination-received mice was almost the same as MST value from 3.2 mg FLC dose-given animals (p < 0.05). Another combination of 1.6 mg FLC dose and B6.1 reduced severity of the disseminated disease at almost the same rate as combination efficacy of 0.8 mg FLC dose plus B6.1. This data indicates that B6.1 acts in concert with FLC and that this combination therapy augments protection, which suggests a possibility of reducing FLC dose. The augmentation response was specific because an irrelevant IgM MAb S9 was not effective to the disseminated disease. Thus, our present studies demonstrate that this combination immunotherapy may be a way of solving the problem of limited antifungal drug choices caused by drug-resistant C. albicans.