Effect of YJ-7 (a calcium channel blocker) on blood pressure regulation in spontaneous hypertensive rat model

BACKGROUND: Farnesylacetone compounds that dilate blood vessels by blocking calcium channels in sargassum siliquastrum have been reported. And this study was done to demonstrate the effect of YJ-7, a synthetic material derived from these compounds, on vessel dilation and blood pressure control. METHODS: We used vasoconstricted basilar and carotid artery of rabbits. Changes in blood pressure were measured in vivo at 15, 30, 45, and 60 minutes after intravenous injection of YJ-7 3 microM, EC50 value from in vitro experiment, and nimodipine 10 microM through the tail vein of 20 rats. Spontaneous hypertensive rat (SHR) has its blood pressure higher than 190 mmHg. Measurements of blood pressure were done 6 times and the mean values were used for data analysis. RESULTS: Systolic and diastolic blood pressure before the injection of YJ-7 were 194.2 +/- 6.1 mmHg and 140.2 +/- 6.4 mmHg. Blood pressure were decreased with time, 157.2 +/- 2.6 / 120.8 +/- 4.2 mmHg at 15 minutes, 161.8 +/- 18.3 / 123.2 +/- 13.9 mmHg at 30 minutes, and 156.0 +/- 4.1 / 112.4 +/- 1.7 mmHg at 45 minutes. The blood pressure lowering effect lasted until 45 minutes. However, the blood pressure increased to 182.2 +/- 16.4 / 149.0 +/- 20.4 mmHg at 60 minutes reaching similar levels of before the injection (P < 0.05). CONCLUSIONS: We could see YJ-7 has vasorelaxation effect and would be helpful to control blood pressure with short recovery period than nimodipine.

Different Responses of MSSA and MRSA to Oxacillin of Their Respective MICs

All of the methicillin-resistant Staphylococcus aureus (MRSA) strains exhibit resistance to oxacillin by producing PBP2a encoded by mecA, whereas methicllin-susceptible Staphylococcus aureus (MSSA) strains do not. To investigate phenotypic differences other than oxacillin resistance level in responses to oxacillin between MSSA and MRSA, we compared alterations of viability and ultrastructure of MSSA by oxacillin treatment with those of MRSA. When MSSA and MRSA strains were exposed to oxacillin of their respective MICs, and then were assayed for viability and observed by transmission electron microscope, increase in thickness of cell wall was more prominent in MRSA strains than in MSSA strains, while decrease in number of surviving cells was more evident and change in morphology of growing cross wall was greater in MSSA strains than in MRSA strains. It is assumed that these different responses to oxacillin between MSSA and MRSA strains may be due to activation of some PBP2a unbound to oxacillin. In conclusion, MSSA and MRSA showed different functional and morphological responses to oxacillin, although they were treated with oxacillin of concentrations that respectively inhibit their proliferation.

Relationship between Biofilm Formation Ability and Virulence of Candida albicans

Biofilms are microbial communities that form on a surface and are surrounded by extracellular polymeric substances. Candida biofilms are a cause of infections associated with medical devices. In the present study, an attempt was made to evaluate a significance of biofilm formation ability (BF) in virulence of C. albicans. C. albicans of 98 isolates, 24 commensal strains obtained from the oral cavities of healthy volunteers, 29 from blood culture, 25 from urine culture, and 20 from vaginal candidiasis, were assayed for BF, an ability to adhere to epithelial cells (ADH), cell surface hydrophobicity (CSH), and germ tube forming rate (GT). The relationships of BF with CSH, ADH, and GT were statistically examined. A positive correlation between BF and ADH was obtained, but the correlation (r=0.326) was relatively low. To assess BF as a factor contributing for candidiasis, mice lethality test was performed. The 10 isolates with the highest BF (mean survival rate, 24%) allow to kill mice more than those with the 10 lowest BF (mean survival rate, 47%). In addition, clinical strains isolated from blood culture, urine culture, and vaginal candidiasis showed higher BF than oral commensal strains. These results suggest BF may represent a virulent characteristic of C. albicans.

Protective Effects of Immunization with Proteinase, Phospholipase and Cell Wall Proteins of Candida albicans in Mice Candidosis / 대한의진균학회지

BACKGROUND: The opportunistic fungus Candida albicans is a major pathogen especially to immunocompromised patients. OBJECTIVES: We examined the protective effect of the active and passive immunizations to evaluate the applicability for the treatment of candidosis in Candida-infected mice model. METHODS: Candida cell wall components were obtained by treatment of lyticase, proteinase K, and dithiothreitol. The proteinase was purified from the culture filtrates of C. albicans using a series of chromatographic steps consisting of DEAE-Sepharose FF, Sephacryl S-200 HR and size-exclusion high performance liquid chromatography. The phospholipase was purified from the culture supernatant of C. albicans with DEAE column chromatography, reverse phase column chromatography, revere phase HPLC and size-exclusion HPLC. Antibodies to cell wall protein components, proteinase and phospholipase were produced by immunization into mice of same strain. RESULTS: The mean survival times of active and passive immunized mice groups were longer than those of non-immunized groups. CONCLUSION: These results showed that immunization with proteinase and its antibody were the most effective to prolong survival time in Candida-infected mice.

Effect of Culture Conditions on the Protein Fibril Expression of Candida albicans

Candida albicans is an important human pathogen that causes systemic infections, predominantly among population with weakened immune system. Cell wall structures of C. albicans are important to adhere to host tissue and evade to host immune system. Among cell wall structure, the outermost fibrillar layer of C. albicans is of interest since it may play important roles in antigenicity, phagocytosis, and adherence. The expression of virulent factors could be affected by the growth conditions. The dynamic nature of the cell surface alters the physical properties of the fungal interface with host cells and thereby influences adhesion to the host and recognition by components of the host immune system. In this study, we investigated the effects of culture conditions on cell surface fibril expression of C. albicans by a transmitting electron microscopy and SDS-PAGE. The protein fibril of C. albicans was expressed in the presence of whole serum, however, the fibril expression was decreased in 25% serum and serum containing 1% glucose. Also, germ tube can be induced by serum, RMPI medium, N-acetyl glucosamine, and 39 degrees C culture condition, hence, the fibrillar structure of C. albicans was detected only in serum-induced germ tube. The expression of fibril layer and the major fibril proteins of 66, 47, 30 kDa were reduced as increasing cell concentration of intial inoculum from 2x10(7) cells/ml to 8x10(7) cells/ml. The fibrillar layer of C. albicans was expressed in serum early within 10 min, and the thickness of fibril layer was increased according to the increase of culture time. When the fibrillar proteins were analysed by SDS-PAGE, major protein of 30 kDa was maintained continuously during over night culture although expression of the other proteins were various. These results suggest that expression of serum induced protein fibril is influenced by culture conditions and is not related to hyphal transition of C. albicans.

Potentiality of Anti-idiotypic Antibodies Mimicking GD2 to Induce Cellular Immunity

BACKGROUND: Disialoganglioside GD2 is a tumor-associated antigen that is overexpressed on tumor cells of neuroectodermal origin, such as melanoma, small cell lung carcinoma and neuroblastoma. Immunity against GD2 has anti-tumor activities, but GD2 is poorly immunogenic. Anti-idiotypic antibodies that mimic GD2 may induce more effective immune responses than GD2 antigen itself, because they are protein antigens and are known to be able to break immune tolerance. In our previous study, we produced anti-idiotypic antibodies mimicking GD2 (3A4 and 3H9), which induced humoral immunity. However, cellular immunity is essential to eradicate tumor cells in vivo as well as humoral immunity. In the present study, we investigated whether these anti-idiotypic antibodies 3A4 and 3H9 could induce cellular immunes responses. METHODS: BALB/C mice were immunized with anti-idiotypic antibody 3A4 or 3H9, or normal mouse IgG as a negative control. Lymphoproliferative responses, cytokine production responses, and delayed-type hypersensitivity reactions were measured in mice immunized with the anti-idiotypic antibodies. RESULTS: Both the anti-idiotypic antibody 3A4 and 3H9 induced GD2-specific lymphoproliferative responses and IFN-gamma production of lymph node lymphocytes in BALB/C mice. Only anti-idiotypic antibody 3H9 induced significant GD2-specific delayed-type hypersensitivity in the mice. CONCLUSION: These results show that anti-idiotypic antibodies 3A4 and 3H9 have the potentiality of inducing GD2-specific cellular immune responses that cannot be induced by the native antigen GD2 itself.

Search for Genes Potentially Related to Germ Tube Formation in Candida albicans by Differential-Display Reverse Transcription Polymerase Chain Reaction

Candida albicans exhibits the ability to grow in either a yeast or a mycelia form in response to different environmental factors. The mycelia form, found in infected tissues, is important as a virulence factor in the adherence of the organism to the host epithelium. In vitro, the morphological transition can be induced by environmental shifts in the growing conditions, or by a variety of exogenous factors, including ambient pH, nutritional status and temperature. The differential-display reverse transcription polymerase chain reaction (DDRT-PCR) is a powerful technique for comparing gene expression between cell types, stages of development or differentiation. Hyphae related genes were identified and characterized using a PCR-based differential display. Candida albicans formed a germ tube when cultured in rabbit serum, RPMI 1640 medium or 39degrees C-YPD medium. We gained 21 cDNA bands showing a different expression pattern from that of the uninduced culture. DNA was extracted from the same location of the isolated bands, and PCR was performed under the same conditions, which reamplified the PCR product, showing the specific expression patterns according to the culture conditions. We cloned 18 germ tube-related cDNA clones (inserts average size is 80 - 700 bp) and sequenced them. The nucleotide sequences of the 18 clones were identified through in the present study from GenBank, and were found to have the accession number (AF405213-AF405230). We could not find any nucleotide sequence having a high homology with these clones. This study could form a part of the projects in the search for genes related to the germ tube formation of C. albicans.

Ultrastructure of Fibrillar Layer of Candida albicans in Serum Culture

No abstract available.

The Effect of Rabbit Serum on the Expression of Cell Surface Hydrophobicity in Candida albicans / 대한의진균학회지

No abstract available.

Relative risk of virulence factors in Candida-infected mouse

Candida albicans is one of the most frequently isolated fungal pathogens in human. Recently, the prevalence of candida infection has markedly increased, partially due to the increase of immunocompromised hosts. Proposed virulence factors of the pathogenic Candida are the ability to form hyphae to adhere to epithelial cell surfaces, and to secrete acid proteinases and phospholipases. We measured the relative cell surface hydrophobicity (CSH) and the ability of proteinase production (PROT), phospholipase production (PLase), adherence to host epithelium (ADH), and hyphal transition (Germ). The relative risk of virulence factors was analyzed by lethality test in murine model of hematogeneously disseminated candidal infection. According to Cox's proportional hazard analysis, the statistically significant virulence factors were PROT, ADH, and CSH. PROT was the highest risk factor of them. To evaluate the applicability for the diagnosis and treatment of Candidiasis, we examined the protective effect of the active and passive immunizations with the materials purified from virulence factors and antibodies to them in Candia-infected mice model. The mean survival times of active and passive immunized groups were slightly longer than those of non-immunized groups.

The presumptive identification of Candida albicans with germ tube induced by high temperature

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.

Serum Indeces Protein Fibrils Expression in Candida albicans

The fibrillar coat of Candida albicans is of interest as its significance in antigenicity, antiphagocytosis, and adherence to host tissues. The partial biochemical properties and ultrastructure of fibrillar coat induced by rabbit sera were examined. The induced fibrillar layer was destroyed by treatments of lyticase, proteinase K and dithiothreitol. The total protein concentration of fibrillar cell wall lysate was higher than that of non-fibrillar cell wall lysate, but the total sugar concentration was similar. On SDS-PAGE analysis, the protein profiles between in fibrillar cells and in non-fibrillar cells were shown to be different. In fibrillar cells, the major bands of cell wall lysate were 83, 66, 54, 47, 33, and 26 kDa in dithiothreitol-treated lysate. The proteins of 26 and 19 kDa were predominant in lyticase-treated lysate. Although the fibrillar thickness and protein amount of cell wall lysate were increased in according to the incubation time, the protein profiles did not changed. These results suggest that the proteins of 83, 66, 54, 47, 33, 26, and 19 kDa may be major constituents of fibrillar coat in C. albicans.

Antibacterial Activity of an Antibiotic (K-681) from Streptomyces sp. 681 against Staphylococcus aureus

The antimicrobial agents reduced infectious diseases significantly. However, antibiotic resistance has followed for almost every antimicrobial agent. Especially, Staphylococcus aureus was one of the most notorious for the multidrug resistance. Streptomyces sp. 681 has been selected for antibiotic-producing strain against methicillin-resistant Staphylococcus aureus (MRSA) from 1,000 strains of Actinomycetales which had been isolated from soil. In antimicrobial susceptibility test, all of the test strains were susceptible to vancomycin. However, most strains of Staphylococcus aureus were found to be resistant to methicillin. Ninety eight (75%) strains out of 129 strains showed multiple resistance pattern to more than 5 antimicrobial agents. The MIC values of the purified antibiotic (K-681) were 1-32 ug/ml against Gram-positive bacteria compared to >128 ug/ml against Grarn-negative bacteria or fungi. The MIC was 8 ug/ml for 90% of the 129 clinical isolates of S. aureus. The antibiotic showed no cytotoxicity against P 388, HeLa, and S180 at the concentration of 500 ug/ml.

Detection of Antibody to Candida albicans Proteinase in Sera from C. albicans - Cultured Patients and Healthy Controls

To investigate whether anti-Candida proteinase antibody could be a diagnostic marker, we examined seroreactivity to proteinase in sera from 90 healthy controls and 8 of C. albicans culture-positive patients. Previously we purified proteinases of C. albicans, C. tropicalis, and C. parapsilosis using a series of chromatographic steps consisting of DEAE- Sepharose, Sephacryl S-200, and size-exclusion HPLC. ELISA and Western blot technique were adopted to examine seroreactivity of C. albicans proteinase with sera. On ELISA, the seroreactivities of healthy controls and C. albicans-cultured patients were 0.601 +- 0.014 (mean+SEM), and 0.695 +- 0.079, respectively (P=0.084, t-test). In C. albicans-cultured patients, the positive rate was 62.5% (5/8) and the positive rate of healthy controls was 39% (35/90). On Western blot analysis, C. albicans proteinase molecule was blotted by all sera tested. But the intensity of blotted band was different with the same dilution of sera; the intensity of C. albicans proteinase molecule band blotted by 2 sera of 3 healthy control's sera was distinctively lower than that by C. albicans-cultured patients sera. However, all sera including C. albicans-cultured patient's sera did not blot the proteinase secreted by C. tropicalis and C. parapsilosis. It is necessary to collect sequential sera of patients with candidiasis and to establish a cut-off value for ELISA or serum dilution for Western blot analysis that will give reliable test sensitivity and specificity.

Seroreactivities of proteinases of Candida albicans, C. tropicalis, and C. parapsilosis in sera from various Candida species-infected mice

From the culture filtrates of C. albicans, C. tropicalis and C. parapsilosis, proteinases were purified using a series of chromatographic steps consisting of DEAE-Sepharose, Sephacryl S-200 and size-exclusion HPLC which removed contaminating mannoproteins and extraneous proteins. Anti-Candida proteinase antibodies in sera from mice infected with various Candida species were detected using ELISA for serodiagnosis of candidiasis. Three proteinases were blotted by homologous and heterologous anti-proteinase antisera on Western blot analysis. All sera from six Candida species-infected mice were reactive with proteinases of C. albicans, C. tropicalis, and C. parapsilosis, although C. glabrata, C. guilliermondii, and C. krusei did not secrete proteinase. The seroreactivities of proteinase with sera from mice infected with homologous C. albicans and C. tropicalis were higher than those with sera from heterologous Candida species-infected mice. These results suggest that three proteinases have at least one common epitope, but its application for diagnosis of candidiasis should be considered with limits of specificity.

Effects of Culture Condition on Secretion of Phospholipase from Candida albicans / 대한의진균학회지

BACKGROUND: The dimorphic yeast, Candida albicans, is considered as a dangerous opportunistic pathogen in immunocompromised hosts. Several phospholipases of C. albicans are known to be secreted into the culture medium. Phospholipases have been proposed as a virulence factor in the pathogenesis of Candida infections. OBJECTIVE: In order to investigate enzyme production, we examined culture condition of secreted phospholipase production from C. albicans. METHODS: C. albicans ATCC 10231 was cultivated in various media at 37 degrees C for 3 days. Phospholipase activity was measured by fatty acid soap precipitation in plate containing 0.04% lecithin, 0.1 M citrate buffer, pH 4.2 and 1.5% noble agar. RESULTS: Phospholipase was highly induced when C. albicans was cultivated in broth medium (containing glucose 2%, albumin 0.2% and Fe++ ion 0.01%) and Saboulaud's dextrose agar supplemented with 0.01% sodium deoxycholate. CONCLUSION: Highly induction of secreted phospholipase by albumin from C albicans may be play an important role in tissue invasion in the pathogenesis of C. albicans.

Optimum Culture Conditions for Production of Proteinases secreted by Candida albicans , C. tropicalis , and C. parapsilosis

In the present study, culture conditions to secrete proteinases from C. albicans, C. tropicalis and C. parapsilosis were examined. All three Candida species were found to secrete proteinases from acceleration phase to stationary phase, although the proteinase activities in culture filtrate were maximal during late exponential or early stationary phase. The proteinase activity in the culture filtrate of C. albicans cells grown at 30'C, was much higher than those grown at either 20 or 37'C. In culture of C. tropicalis and C. parapsilosis, the highest activity was found in culture filtrate grown at 37C. C. albicans secreted proteinases well in medium at initial pH 4.0-7.0. The optimal initial pH of medium for proteinase secretion was 7.0 for C. tropicalis and 5.0-6.0 for C. parapsilosis. All three Candida species secreted proteinases to greater amount in aerobic state. The most effective carbon source for proteinase secretion was xylose, glucose, maltose and sucrose for C. albicans, xylose for C. tropicalis and trehalose for C. parapsilosis. The effects of proteins, hydrolyzed proteins, ammonium sulfate as a sole nitrogen source on proteinase secretion were examined. Bovine serum albumin was the most effective nitrogen source of those tested and a little proteinase activity was detected in the culture filtrates when yeast cells were incubated in the medium containing ammonium sulfate. C. parapsilosis secreted proteinases to greater amount than the other Candida species in all nitrogen sources under study, indicating that C. parapsilosis proteinase would not be a inducible but a constitutive enzyme.

Biological Properties of a Polyene Antifungal Substance(AF1) Produced from Streptomyces sp. WCM-9 / 대한의진균학회지

BACKGROUND: Pathogenic fungi infect humans, especially immunocompromised patients, with superficial or deeply invasive pattern. In the past 20 years, fungal infections have been increased dramatically resulted by increment of organ transplantation, cancer, AIDS patients, or use of broad-spectrum antibacterial agents. Fungal infections are now important causes of morbidity and mortality of hospitalized patients. but there is no effective antifungal antibiotics as well as antibacterial antibiotics OBJECTIVE: Effective new antifungal antibiotics are needed for the treatment of mycosis. So in an effort to develop effective antifungal antibiotics, we screened over 600 isolates of Streptomyces sp. from soil. METHODS: Antifungal producing strain was selected using disk diffusion method, An antifungal substance (AF1) was purified with ethyl acetate extraction, silica gel column chromatography and reverse phase HPLC. MICs of AF1 were detected by agar dilition method. RESULTS: The compound showed UV maxima of 307, 321, 340, 359 nm indicating methylpentaene. Minimum inhibitory concentrations of the AF1 were 3.7 microgram/ml against mold, and 3.7 - 7.4 microgram/ml against Candida species. AFI was also active against Crytococcus neoformans, with MIC of 0.9 microgram/ml. The concentration of AF1 for K+ ion release from human red blood cell and hemolysis were 5 microgram/ml. CONCLUSION: The antibiotic purified from culture broth of Streptomyces sp. WCM-9 was a polyene antifungal antibiotic which have broad spectrum antifungal activity.

In vitro antibacterial activity of WCM 302, a substance obtained from culture filtrate of streptomyces sp. 302, against staphylococcus aureus

No abstract available.

Tumor cell growth inhibitory factor in the culture filtrate of the streptomyces 1. in vitro cytotoxicity of tumor cell growth inhibitory factor

No abstract available.