Utilisation of ferroproteins by Candida albicans during candidastasis by apo-ovotransferrin

한용문 동덕여자대학교 종합약학연구소 2001 동덕약학연구지 Vol.5 No.-

Apotransferrin has a second mechanism for anticandidal activity through binding of Candida albicans

한용문 대한약학회 2014 Archives of Pharmacal Research Vol.37 No.2

It has been reported that transferrin has antibacterialand antifungal activities via iron chelation in theenvironment surrounding the microbes. In the presentstudy, we investigated whether the binding of transferrin toCandida albicans mediates growth inhibition. By usingcultures that contained iron-free (apo)transferrin glycoproteineither in contact with candidal cells or separatedfrom candidal cells by a dialysis membrane, we distinguishedthe growth inhibition by transferrin–cell interactionfrom that of simple iron chelation. Maximal growthinhibition always occurred when the apotransferrin interacteddirectly with the cells. Additionally, there was partialinhibition even when candidal cells were in contact withiron-saturated transferrin. Binding studies with 59Fe3?radiolabeled-transferrin indicated that the apo-protein canbind to the candidal cell surface. The binding sites weresaturable and it was dose dependent. Chemicals (hydrogenperoxide, dithiothreitol, sodium dodecyl sulfate) blockedtransferrin binding to C. albicans, and among the three,hydrogen peroxide (HP) was the most effective for theblocking. When HP-treated yeast cells were added to theculture that was pretreated with apotransferrin, candidalcell growth increased by 5-fold as compared to the growthof HP-untreated candidal cells under apotransferrin-regulation(P\0.05). Combined all data together, it wasconcluded that transferrin has a second mechanism of anticandidalactivity that is mediated by binding to the surfaceof C. albicans yeast cells.

Effects of Sugar Additives on Protein Stability of Recombinant Human Serum Albumin during Lyophilization and Storage

한용문,진병석,Sang-Bong Lee,Yeowon Sohn,Jee-Won Joung,이주희 대한약학회 2007 Archives of Pharmacal Research Vol.30 No.9

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 35oC. 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 35oC. 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.

The identification of surface interaction of apotransferrin with Candida albicans

한용문 대한약학회 2014 Archives of Pharmacal Research Vol.37 No.10

Our recent data indicate that apotransferrin, aniron-chelating protein, has anti-candidal activity by bindingto the Candida albicans surface rather than just simpleiron-chelation. Following that study, in this present study,we investigated the nature of the candidal surface substancethat is responsible for the anticandidal activity byusing 59Fe3?-apotransferrin and biological assay methods. Data resulting from the binding studies showed that theyeast cells had one class of binding sites as analyzed by theScatchard equation, and the binding was specific asdetermined by competitive binding assay with unlabeledand labeled transferrin. All these observations indicate thatthere is a substance(s) that mediates the binding. Thus, amannoprotein-like substance was extracted from C. albicanssurface using hot water-treatment. Radioisotopebinding study revealed that the substance blocked thetransferrin binding. At 25 lg of IHS (inhibitory substance)addition, there was 65 % inhibition of the transferrinbinding to C. albicans (5 9 107 cells/ml) (P\0.05). Theblockage of the transferrin binding disrupted the anticandidalactivity of transferrin, resulting in a full recoveryfrom growth inhibition. These results explain our previousobservation that there is partial growth inhibition when C. albicans interacts directly with iron-saturated transferrin(100 %). Thus, it was concluded that a candidate fortransferrin receptor is involved in the anticandidal activityof transferrin when in direct contact with C. albicans.

Utilization of Ferroproteins by Candida albicans during Candidastasis by Apotransferrin

한용문 대한약학회 2005 Archives of Pharmacal Research Vol.28 No.8

Many reports have stated that some of the pathogenic bacteria can obtain iron from ferroproteins, such as cytochrome C, ferritin, hemin, hemoglobin, and myoglobin. These reports prompted us to determine if an opportunistic pathogenic fungus, Candida albicans, can utilize ferroproteins to circumvent the iron-regulatory effect of transferrin. The following assays were carried out to measure in vitro growth stimulation by the ferroproteins: as an initial step, C. albicans was cultured in iron-free (pretreated with apotransferrin for 24 h) culture medium. Once Candida albicans yeast cell growth reached stasis from iron starvation, individual ferroproteins were added to the culture media. Results showed that hemin, hemoglobin, and myoglobin supported a partial growth recovery. Additional studies with haptoglobin, a serum protein that interacts with the globin moiety of certain ferroproteins, established that C. albicans could obtain iron from the haptoglobin-ferroprotein complexes. These data indicate that the heme part of the ferroproteins is the source of iron. This implies that heme oxygenase, CaHMX1 might be involved in bringing about dissociation of heme-containing protein for iron-acquisition. In addition, anticandidal activity of transferrin takes place not only by the process of iron regulation, but also by direct interaction with the yeast cells

$18{\beta}$-Glycyrrhetinic Acid의 항 감염성관절염효과

한용문,Han, Yong-Moon 대한약학회 2007 약학회지 Vol.51 No.6

A polymorphic fungus, Candida albicans, causes various forms of infections such as disseminated candidiasis and vaginitis. Recent reports indicate that the fungus is a main etiological agent for the arthritis. In search of new sources for treatment of the fungal arthritis, we examined $18{\beta}$-glycyrrhetinic acid ($18{\beta}$-GA) against C. albicans-caused septic arthritis. The compound is isolated from Glycyrrhizae Radix that is known to have various immunomodulating activities and is one of the most popular herbal medicines. For induction of animal model of a septic arthritis, mice were given an emulsion form of C. albicans cell wall mixed with Complete Freund's Adjuvant (CFA) via footpad-injection. To determine prophylactic and therapeutic effects, the component was given to the animals before or after the induction of the arthritis, respectively. Data showed that intraperitoneal administration of $18{\beta}$-GA resulted in reduction of the inflammation, indicating the component had both prophylactic and therapeutic activities. For investigation of mechanism of the $18{\beta}$-GA, inhibitory effects on NO (nitiric oxide) and on T-lymphocyte proliferation were determined. Results demonstrated that $18{\beta}$-GA suppressed NO production from LPS (lipopolysaccharide)-treated macrophages and also inhibited proliferation of Con A (concanavalin A)activated T-cells. Taken together, $18{\beta}$-GA, a pentacyclic triterpene, has anti-arthritic activity against C. albicans-caused septic arthritis, possibly by blocking NO production and T-cell suppression.

18β-Glycyrrhetinic Acid의 면역보조제효능에 의한 항 전신성캔디다증 효과

한용문 대한약학회 2008 약학회지 Vol.52 No.6

The role of antibody in the fungal infections is controversial. However, our previous reports showed a certain epitope in Candida albicans cell wall (CACW) induces protective antibody. A major problem is that the epitope isolation requires tremendous time with high cost. This aspect led us to investigate a simple way inducing protective antibodies against C. albicans. In the present study, we determined if 18β-glycyrrhetinic acid (18β-GA) from Glabrae Radix (a family of Leguminosae) has immunoadjuvant activity. Data displayed that the 18β-GA suppressed proliferations of both T- and Blymphocytes at high concentrations, whereas below 20 μM concentration the compound supported the proliferations. These observations indicate that 18β-GA has immunoregulatory activity. Based on this observation, an immunoadjuvant effect was examined at the low concentration. Results from animal experiments showed that CACW combined with or without 18β- GA produced the anti-C. albicans antiserum in mice. Nevertheless, the CACW combined with 18β-GA formula only protected mice against disseminated candidiasis (P<0.05). These data implicate that 18β-GA has immunoadjuvant activity, which may provoke the CACW antigen to induce protective antibody. Currently, we are investigating possible mechanism of how the 18β-GA provokes such protective immunity against the disseminated disease.

캔디다질염에 효과가 있는 단항체에 대한 호중구의 역할

한용문 대한약학회 2003 약학회지 Vol.47 No.3

As previously reported, an immunoglobulin M (IgM) monoclonal antibody (MAb) B6.1, specific for a cell wall B-l,2-mannotriose, was protective against vaginal infection due to Candida albicans when mice were treated with the antibody. In this study, the role of neutrophil was examined in the protective effect of MAb B6.1 against vaginal infection. To deplete neutrophils, mice were given intravenously rat anti-mouse neutrophile MAb RB6-8C5 prior to intraperitoneal administration of MAb B6.1 to these mice. The mice were examined for antibody in their reproductive tract. By an ELISA, MAb B6.1 was found in the vaginal homogenates, but no antibody was detected in vaginal lavage materials. The neutropenia was induced by a single dose of the anti-neutrophil antibody, but lymphocytes were also partially depleted. The protective effect of MAb B6.1 was decreased when mice pretreated with MAb RB6-8C5 were given the anti-Candida antibody before challenge with C. albicans yeast cells intravaginally. These results show that neutrophils are involved in the MAb B6.1 protection against Candida vaginal infection.

페렴구균 전신감염에 대한 협막. 표면단백질 접합백신의 효과

한용문,이주희,Han , Yong-Moon,Lee , Jue-Hee 대한약학회 2004 약학회지 Vol.48 No.6

ln the present work to determine effect of a Streptococcus pneumoniae conjugate vaccine, S.pneumoniae capsule attached to the surface protein (JY-Pol) was ex amined. This JY-Pol contained approximately 92% and 6% carbohydrate and protein, respectively. Gel electrophoresis revealed the presence of the surface protein in the JY-Pol. By the double immunodiffusion and isotyping ELISA analyses, administration of JY-Pol that was adsorbed to alum adjuvant (JY-Pol/Alum) into mice induced IgM, IgG, and IgA specific for the S.pneumoniae capsule. The ATCC capsular polysaccharide adsorbed to alum (ATCC-Pol/Alum) provoked only IgM in mice. In survival tests, mice that were immunized with the JY-Pol/Alum before intravenous challenge with live S.pneumoniae survived entire period of 46 day-observation, whereas all mice that received ATCC-Pol/Alum or only diluent instead of the vaccination died within 5 and 12 days, respectively. Results from footpad-edema test showed that JY-Pol/Alum formula provoked the cellular immunity as determined by swelling of the mouse footpad. These data indicate that the naturally conjugated JY- Pol enhances resistance of mice against disseminated pneumococcal disease due to S.pneumoniae by both humoral and cellular immune responses.

Comparison of Two Candida Mannan Vaccines: The Role of Complement in Protection against Disseminated Candidiasis

한용문,유기연 대한약학회 2012 Archives of Pharmacal Research Vol.35 No.11

We have previously shown that Candida albicans mannan extract encapsulated in liposomes [Lipo-mann] or conjugated to a protein (bovine serum albumin) [Conju-mann] induces the production of antibody in BALB/c mice with normal complement system that protect against disseminated candidiasis. In this present study, we determined the protective abilities of two formulae in a C5-deficient mouse model of disseminated candidiasis. It is known that the lack of C5 is known to aggravate candidal infection. In experiments, BALB/c or C5-deficient mice-DBA/2J and AKR mice, were immunized with one of the formulae before intravenous challenge with live C. albicans yeast cells and their degrees of survivability were measured. Results showed that Conju-mann was 100% protective in BALB/c mice against disseminated candidiasis, whereas only 60% of Lipo-mann immunized mice survived the entire 50 day observation period (p < 0.05). With the DBA/2J strain, Conju-mann resulted in a partial protection, but Lipo-mann had no protection. The conjugate vaccine enhanced the resistance of AKR mice, which resulted in three survivors of the five Conju-immunized AKR mice until the end of 50 day observation period (p < 0.05). Lipo-mann showed little protection in AKR mice. By agglutination analyses, it was determined that there was the same level of production of polyclonal antisera specific to the mannan regardless of the mouse strains. All data indicate that both formulations require complement in the protection. However, Conju-mann appears to be superior to Lipo-mann because the conjugate vaccine is protective even in the absence of C5. These observations suggest that the conjugate vaccine can be an excellent vaccine formulation against C. alibicans infections.