Differential Expression, Shedding, Cytokine Regulation and Function of TNFR1 and TNFR2 in Human Fetal Astrocytes

Tumor necrosis factor (TNF) -alpha induces pleiotropic cellular effects through a 55kDa, type 1 receptor (TNFR1) and a 75kDa type 2 receptor (TNFR2). Moreover, it participates in the pathogenesis of several CNS diseases, including demyelinating diseases. TNF- receptors are differentially expressed and are regulated in many cell types. However, data regarding the TNF-alpha receptor expression and regulation in human astrocytes is limited to date. We investigated TNF-alpha receptor expression, its regulation by cytokines, and its functional role in primary cultured human fetal astrocytes, which are the most abundant cellular population in the central nervous system and are known to be immunologically active. In this study, astrocytes were found to constitutively and predominantly transcribe, translate and shed TNFR1 rather than TNFR2, but TNFR2 expression was increased by adding TNF-alpha, IL-1, and IFN-gamma, but not by adding LPS. To determine the functional roles of TNFR1 and TNFR2 on TNF induction, we investigated NF-kappaB activation and TNF-alpha induction after neutralizing TNFR1 and TNFR2 by an antibody treatment. We found that NF-kappaB activation and TNF-alpha induction are blocked by TNFR1 neutralizing antibody treatments.

Purification and Characterization of Novel Genes of Human Fetal Astrocytes

Astrocytes are ubiquitous in the brain and have multiple functions. It is becoming clear that they play an important role in monitoring the neuromicroenvironment, information processing, and signaling in the central nervous system (CNS) in normal conditions and respond to CNS injuries. During the development of the CNS, astrocytes play a key role as a substrate for neuronal migration and axonal growth. To identify genes that could participate in astrocyte maturation, we used the differential display reverse transcription-PCR (DDRT-PCR) method. Human fetal astrocytes were cultured and total RNAs are isolated at intervals of 5 days for 50 days. Using 24 primer combinations, we have identified a set of 18 candidate cDNAs deriving from excised DDRT-PCR bands. DNA sequencing revealed 16 genes that have been described already (HMGCR, thyroid receptor interactor gene, NPM, transglutaminase mRNA, and SPARC etc.). We have also found two novel genes (A3 and C8), which were expressed differently in culture stages. A3 expressed decreasingly and C8 expressed increasingly in accordance with to culture stages. We have analysed these two genes. A3 (3,626 bp) showed 93% homology with the Homo sapiens general transcription factor 3 (GTF3) and C8 (2,401 bp) had 97% homology with the transmembrane receptor Unc5H2. Temporal expression of these two genes in this study suggests that the proteins of these genes may have different roles in maturation of the human fetal astrocytes.

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.

Effects of Th1 type cytokines on secretion of vascular endothelial growth factor by human first trimester trophoblast cell line / 대한산부인과학회잡지

Embryo implantation and development are critically dependent upon the regulation of angiogenesis and adequate immunologic acceptance. These local angiogenesis and vascular permeability are regulated by the interaction between fetal trophoblast, uterine decidua, and endothelial cells through the key mediator, vascular endothelial growth factor (VEGF). PROBLEM: The mechanism through which VEGF regulation occurs at the feto-maternal interface is poorly understood. The Th1 type cytokines are known to be harmful to the successful maintenance of early pregnancy at the feto-maternal interface. OBJECTIVE: To clarify whether the Th1 type cytokines could be involved in the regulation of VEGF secretion at the feto-maternal interface. Method of Study : we investigated the effects of Th1 type cytokines on VEGF secretion in human first trimester trophoblast cell-line by using reverse transcription polymerase chain reaction(RT-PCR)and enzyme-linked immunosorbent assay (ELISA). RESULTS: The trophoblast cells expressed VEGF constitutively and the main isoforms were VEGF121 and VEGF165. When cultured in the presence of IFN-gamma or IL-2, VEGF secretion was most significantly increased by IFN-gamma treatment but not affected by IL-2 treatment. The level of intracellular VEGF was also increased by IFN-gamma treatment. CONCLUSION: These results suggest that IFN-gamma, despite of harmful Th1 type cytokine to the maint enance of early pregnancy, may regulate the production of VEGF in early gestational trophoblasts.

Regulation of TNF - alpha Gene Expression in Human Fetal Astrocytes

Tumor necrosis factor-n (TNF - alpha) involved in the pathogenesis of multiple sclerosis and contribute to the degeneration of oligodendrocytes as well as neurons. TNF - alpha is produced by miocroglia and astrocytes, which also produce hormones and cytokines that influence its biological activity. Astrocytes, the major glial cells in the CNS, are capable of producing TNF - alpha at both the mRNA and protein levels in response to interleukine-1 (IL-1) or TNF - alpha. Two immunosuppressive cytokines, transforming growth factor - beta (TGF - beta) and IL-10, have been shown to influence glial cell function. TGF - beta can modulate the activity of glial cells by inhibiting interferon-gamma (IFN - gamma) induced expression of class II major histocompatibility complex (MHC) molecules on astrocytes and microglia. To explore the role of astrocytes in the production of TNF - alpha, astrocytes were pretreated with IL-10 or TGF - beta and then stimulated with IL-1p to determine their effects on TNF - alpha production. The secretion of TNF - alpha by human fetal astrocytes was markedly inhibited by TGF - beta at a low concentration. In contrast IL-10 had no effect on TNF - alpha mRNA level. These results show that TGF - beta may regulate the expression of TNF - alpha in activated human fetal astrocytes.

Ultrastructure of Fibrillar Layer of Candida albicans in Serum Culture

No abstract available.

Establishment of a Culture Method and Characterization for Human Fetal Astrocytes

No abstract available.

Characterization of Gene Expression Pattern in Human Astrocytes using DDRT - PCR Method

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

No abstract available.

Vascular Endothelial Growth Factor Expression in Human Trohoblast Cell Line

No Abstract Available.

Vascular Endothelial Growth Factor Expression in Human Trohoblast Cell Line

No Abstract Available.

Identification of Differentially Expressed Genes in Human Fetal Astrocytes

No Abstract Available.

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.

The p16INK4A Expression in Stomach Cancer , Colon Cancer and Hepatoma Cell Lines / 대한암학회지

PURPOSE: The p16(INK4A) gene encodes a specific inhibitor of cell cycle progression. In recent years, genetic deletion and altered expression of p16(INK4A) gene were frequently showed in many human cancers. So, the p16(INK4A) gene is considered as tumor suppressor gene. However, there has been a few data for the p16(INK4A) in gastric cancer, colon cancer, and hepatoma.So.we investigated the genetic deldtion and altered expression of p16(INK4A) in gastric cancer, colon cancer and hepatoma cell lines. MATERIALS AND METHODS: The homozygous deletion of p16(INK4A) was examined by using PCR and the protein expression of p16(INK4A) by using Western blotting in cancer cell lines established from Korean patients: stomach cancer, colon cancer and hepatoma cell lines. RESULTS: Homozygous deletion of p16(INK4A) was detected only 1 stomach cancer cell line out of 13 cell lines examined. The p16(INK4A) was detected in 3 of 13 cancer cell line. These results showed the low frequency of p16(INK4A) homozygous deletion and high frequency of p16(INK4A) expression alteration in stomach cancer, colon cancer and hepatoma cell lines. CONCLUSION: In this study, it may be suggested that the altered pl6(INK4A) expression as well as p16(INK4A) gene deletion play important role in oncogenesis. Further studies to determine the mechanism of p16(INK4A) gene inactivation are expected.

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.

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.

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.