LPS Sensing Mechanism of Human Astrocytes: Evidence of Functional TLR4 Expression and Requirement of Soluble CD14

Among a myriad of pathogen-associated molecular pattern-sensing receptors, toll-like receptors (TLRs) are the principal core sensors of the host. Despite intensive studies for the expression of TLRs and their roles in the central nervous system, controversies remain regarding the expression and the function of TLR4 in human astrocytes. In order to clarify this issue, we attempted to verify functional expression of TLR4 in human astrocytes. Using Reverse transcription-polymerase chain reaction (RT-PCR), we confirmed that the human astrocytes express the TLR4 constitutively. To determine the function of TLR4, astrocytes were treated with TLR4 ligand or lipopolysaccharide (LPS), and then inflammatory cytokines expressions were checked using RT-PCR and enzyme-linked immunosorbent assay. Nuclear factor (NF)-κB activation was checked using electrophoretic mobility shift assay. Treatment of astrocytes with LPS increased tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-8 expression and induced NF-κB activation. Neutralizing anti-TLR4 antibody blocked the effect of LPS on cytokine production and NF-κB activation in astrocytes. The effect of LPS on cytokine production and NF-κB activation was shown in the presence of serum but not in the absence of serum. Therefore, we investigated the sensing mechanism of LPS in human astrocytes. Human astrocytes were treated with LPS following neutralizing anti-CD14 antibody treatment in the presence of serum. Neutralizing anti-CD14 antibody treatment abolished the effect of LPS on cytokine expression and NF-κB activation. Additionally, supplement of recombinant CD14 in serum-free media induced LPS effect on cytokine production and NF-κB activation. In these results, we showed that human astrocytes constitutively express functional TLR4 and require soluble CD14 to recognize LPS.

Hemorrhagic Rupture of Arachnoid Cyst into the Intradural Space

A 7-year-old boy, diagnosed with an arachnoid cyst and subdural effusion on initial MRI, was admitted with left limb weakness and no history of head trauma. A subsequent follow-up MRI showed different stages of hematoma within multilayered enhancing membranes and in the arachnoid cyst, which was separated by the cerebrospinal fluid cleft. Craniotomy and fenestration of the cyst wall and hematoma removal were performed. The patient was diagnosed as a having a hemorrhagic rupture of an arachnoid cyst into the intradural space, probably via some one-way valve-like defect, based on the MRI and surgical findings. The MRI features and possible mechanism of this rare disease are discussed within the literature review.

Proteus vulgaris and Proteus mirabilis Decrease Candida albicans Biofilm Formation by Suppressing Morphological Transition to Its Hyphal Form

PURPOSE: Candida albicans (C. albicans) and Proteus species are causative agents in a variety of opportunistic nosocomial infections, and their ability to form biofilms is known to be a virulence factor. In this study, the influence of co-cultivation with Proteus vulgaris (P. vulgaris) and Proteus mirabilis (P. mirabilis) on C. albicans biofilm formation and its underlying mechanisms were examined. MATERIALS AND METHODS: XTT reduction assays were adopted to measure biofilm formation, and viable colony counts were performed to quantify yeast growth. Real-time reverse transcriptase polymerase chain reaction was used to evaluate the expression of yeast-specific genes (rhd1 and rbe1), filament formation inhibiting genes (tup1 and nrg1), and hyphae-related genes (als3, ece1, hwp1, and sap5). RESULTS: Candida biofilm formation was markedly inhibited by treatment with either living or heat-killed P. vulgaris and P. mirabilis. Proteus-cultured supernatant also inhibited Candida biofilm formation. Likewise, treatment with live P. vulgaris or P. mirabilis or with Proteus-cultured supernatant decreased expression of hyphae-related C. albicans genes, while the expression of yeast-specific genes and the filament formation inhibiting genes of C. albicans were increased. Heat-killed P. vulgaris and P. mirabilis treatment, however, did not affect the expression of C. albicans morphology-related genes. CONCLUSION: These results suggest that secretory products from P. vulgaris and P. mirabilis regulate the expression of genes related to morphologic changes in C. albicans such that transition from the yeast form to the hyphal form can be inhibited.

Influence of Bacterial Presence on Biofilm Formation of Candida albicans

PURPOSE: Candida albicans is an opportunistic pathogen that is commonly found in human microflora. Biofilm formation (BF) is known as a major virulence factor of C. albicans. The aim of this study was to examine the influence of bacterial presence on biofilm formation of C. albicans. MATERIALS AND METHODS: The BF of Candida was investigated when it was co-cultured with C. albicans (C. albicans 53, a yeast with a low BF ability, and C. albicans 163, a yeast with high BF ability) and bacteria. BF was assessed with XTT reduction assay. A scanning electron microscope was used to determine the structure of the biofilm, and real-time reverse transcriptase polymerase chain reaction was used to amplify and quantify hyphae-associated genes. RESULTS: Co-culturing with two different types of bacteria increased the BF value. Co-culturing with C. albicans 53 and 163 also increased the BF value compared to the value that was obtained when the C. albicans was cultured individually. However, co-culturing with bacteria decreased the BF value of C. albicans, and the BF of C. albicans 163 was markedly inhibited. The expression of adherence and morphology transition related genes were significantly inhibited by co-culturing with live bacteria. CONCLUSION: Bacteria have a negative effect on the formation of biofilm by C. albicans. This mechanism is the result of the suppression of genes associated with the hyphae transition of C. albicans, and bacteria particles physically affected the biofilm architecture and biofilm formation.

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.

The Expression Pattern of Toll-like Receptor (TLR) and Cytokine Production to TLR Agonists in Human Retinal Pigment Epithelial Cells

Retinal pigment epithelium (RPE) constituting the outer blood-retina barrier plays an important role in ocular defense mechanism. Many studies reported that RPE participates in ongoing immune responses in the retina. However, the exact mechanism is still uncertain. Toll-like receptors (TLRs) participate in the recognition of pathogen-associated molecular patterns (PAMP), such as LPS, zymosan, lipoprotein, and dsRNA. The expression and function of TLRs in human RPE have not been established. In this study, we investigated TLRs expression in human fetal RPE and their recognition of PAMP to determine how human RPE participates in ocular defense mechanism against microbial component. RT-PCR and real time PCR revealed that TLR1 through 5 were constitutively expressed in human fetal RPE, and their expressions were slightly increased by LPS. We determined the TNF-alpha, IL-6, and IL-8 expression in human fetal RPE after treatment with LPS, zymosan, petidoglycan, or poly I:C. RT-PCR demonstrated that LPS and poly I:C treatment increased the production of TNF-alpha, IL-6, and IL-8 in human fetal RPE. LPS showed more potent effects on TNF-alpha and IL-8 production. Peptidoglycan and zymosan did not induce the production of TNF-alpha. CD14, the co-receptor of LPS was weakly expressed and functioned in recognizing LPS in human fetal RPE. These results suggest that human RPE may participate in ocular defense mechanism against microbial component through toll-like receptors.

The Expression of TLR3 and Cytokines Induced by Poly I:C in Human Retinal Pigment Epithelial Cells

In this study, we examined the expression of Toll-like receptor3 (TLR3) by human retinal pigment epithelial cells (RPE) and determined whether exposure to the TLR3 agonist polyinosinic-polycytidylic acid (poly I:C) would induced the expression of cytokines in these cells. RT-PCR revealed that TLR3 was constitutively expressed in human RPE, and its expression was increased by treatment with poly I:C. After treatment with poly I:C, we determined the expression levels of pro-inflammatory cytokines in human RPE using RT-PCR and ELISA. We demonstrated that poly I:C treatment increased the production of TNF-alpha, IL-6, and IL-8 in human RPE. Upon exposure to poly I:C, human RPE initiated antiviral response resulting in the induction of IFN-beta mRNA expression and 2',5'-oligoadenylate synthetase mRNA expression. These results suggest that human RPE may participate in ocular defense mechanism against viral infection through TLR3.

Genetic Analysis of Epstein-Barr Virus Latent Membrane Protein 1 and Immunohistochemical Expression of Transforming Growth Factor (TGF)-beta1, TGF-betaRII, p21, p16, E2F1, Thymidylate Synthase, and NF-kappaB in Epstein-Barr Virus Encoded RNA-positive Gast

BACKGROUND :Although clinicopathologic differences have been described between Epstein-Barr virus (EBV)-positive and negative gastric adenocarcinomas, the pathogenetic basis for these differences remains unclear. In this study, efforts were made to confirm that expression of EBV-latent membrane protein (LMP1) and immunohistochemical characteristics of EBVpositive gastric adenocarcinomas. METHODS: We investigated genomic deletion, and RNA & protein expression of the EBV-LMP1, as well as immunohistochemical protein expression of transforming growth factor (TGF)-beta1, TGF-bata RII, p21, p16, E2F1, thymidylate synthase, and NF-kappaB in relation to EBV positive gastric adenocarcinoma. RESULTS: A total of 38 Epstein-Barr Virus Encoded RNA-positive and 80 negative gastric carcinomas were examined. A 30 bp DNA deletion in the EBV-LMP1 gene, initiating at codon 342, was detected in 94.4% of EBVpositive cases. By RT-PCR and western blotting, EBV-LMP1 mRNA and protein expressions were absent in all cases, re-gardless of DNA deletion. No significant differences in TGF-bata1, TGF-betaRII, p21, NF-kappaB, E2F1, or thymidylate synthase expression were identified. However, the decreased expression of p16 was found in 84.2% of EBV-positive carcinomas, relative to only 57.5% of EBV-negative tumors (p=0.024). CONCLUSION: EBV-LMP1 DNA deletion, mRNA and protein losses are highly prevalent in EBV-positive gastric adenocarcinoma among Korean patients, along with decreased p16 expression.

The Present Situation of Infection Control Professionals, Organization, and Activities in Korean Acute Care General Hospitals / 병원감염관리

BACKGROUND: It has been more than 15 years since infection control was first introduced in Korea, but there is little information available on the status of infection control program in the country. METHODS: Included in the study were 139 acute care hospitals with more than 300 inpatient beds. A questionnaire, modified from US SENIC (Study on the Efficacy of Nosocomial Infection Control) and Canadian RICH (Resources for Infection Control in Canadian Acute Care Hospitals) survey, was mailed to the hospitals in the winter of 2003. RESULTS: Ninety-eight (70.5%) of 139 hospitals responded. There was an average of 1.2 (SD, 0.7) Infection Control Practitioners (lCPs) in each hospital and 95.7% were nurses and only 56.5% of the ICPs worked as full-time. The 71.4% of the hospitals had a position for Infection Control Doctor. All hospitals had an Infection Control Committee, which met an average of 3.7 (SD, 1.7) times a year. The 85.7% of the hospitals performed surveillance, but only 31.6% were monitoring surgical site infections. Review of microbiology data was the most common method for case-finding. More than 90% of the hospitals had infection control policies and guidelines, but an adherence to the policies and guidelines was not monitored regularly. CONCLUSION: This study reports the first comparable profile of infection control program of general acute care hospitals in Korea. Although the foundation for infection control program appears to have been established, there is the need for a further increase in the number of ICPs, the standardization of the surveillance method, and the promotion of adherence to the infection control guidelines.

Therapeutic radionuclides / 핵의학분자영상

Since the development of sophisticated molecular carriers such as octereotides for peptide receptor targeting and monoclonal antibodies against various antigens associated with specific tumor types, radionuclide therapy (RNT) employing open sources of therapeutic agents is promising modality for treatment of tumors. Furthermore, the emerging of new therapeutic regimes and new approaches for tumor treatment using radionuclide are anticipated in near future. In targeted radiotherapy using peptides and other receptor based carrier molecules, the use of radionuclide with high specific activity in formulating the radiopharmaceutical is essential in order to deliver sufficient number of radionuclides to the target site without saturating the target. In order to develop effective radiopharmaceuticals for therapeutic applications, it is crucial to carefully consider the choice of appropriate radionuclides as well as the carrier moiety with suitable pharmacokinetic properties that could result in good in vivo localization and desired excretion. Up to date, only a limited number of radionuclides have been applied in radiopharmaceutical development due to the constraints in compliance with their physical half-life, decay characteristics, cost and availability in therapeutic applications. In this review article, we intend to provide with the improved understanding of the factors of importance of appropriate radionuclide for therapy with respect to their physical properties and therapeutic applications.

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.

Dual Expression of Two Transgenes Introduced by Lentiviral Vectors

Lentiviruses can infect mitotic and non-dividing cells owing to the karyophilic properties of their pre-integrating complex, which allow its active import through the nucleopore. Thus lentiviral vectors derived from human immunodeficiency virus type 1 can mediate an efficient transfer integration, and stable expression of transgenes into proliferating and stationary cells both in vivo and in vitro. By adopting the internal ribosome entry site of encephalomyocarditis virus for bicistronic expression or two promoters of EF-1alpha and SV40 for separate expression of two genes of interest, we developed two lentiviral vectors that express two genes. On FACS analysis, RT-PCR, and immunofluorescence assay, it was shown that the target cells expressed two genes of interest at different levels as the transducing vectors designed for. This vector system is useful especially for a stable, dual-gene expression and two transgene deliveries to non-dividing cells.

Efficacy of Influenza Vaccination among Chronic Ill Patients: Retrospective Case Control Study / 감염과화학요법

BACKGROUND: Influenza is one of the preventable respiratory disease by annual vaccination. Elderly people and patients with chronic medical disease are the primary target for influenza vaccination according to ACIP (Advisory Committee on Immunization). This study was done to determine whether influenza vaccination affects hospitalization due to influenza like illness or it's related complications among chronic ill patients. MATERIALS AND METHODS: Retrospective, case-control study was conducted for 5 months (Nov. 1999-Mar. 2000). Physicians from two-university hospitals in Korea were instructed to collect clinical data, sera from subjects presenting with a flu-like illness or flu-related exacerbation in patients with chronic underlying diseases. We compared the vaccination history of case patients with control. Logistic regression analysis was used to calculate the odd ratio and efficacy of influenza vaccination. RESULTS: Hospitalization due to ILI (influenza like illness) or it's related complications were observed more frequently in patients with chronic lung disease (25.3%) and chronic cardiac disease (31.3%) compared to others. Influenza vaccination resulted in fewer hospitalization with ILI or ILI-related acute exacerbation of underlying disease in chronic ill patients (adjusted odd ratio, 0.328 [CI, 0.14 to 0.73]) during influenza seasons. CONCLUSION: For patients with chronic disease, influenza vaccination is associated with substantial health benefit including fewer hospitalization. Health care providers should take advantage of all opportunities to immunize these high-risk patients.

Factors Influencing Decision Regarding Influenza Vaccination: A Survey of Healthcare Workers in One Hospital / 감염과화학요법

BACKGROUND: In spite of yearly recalls, influenza immunization rate among healthcare workers (HCWs) remained low in Korea University Guro Hospital. This study was conducted to identify the causes of non-compliance against influenza immunization and to analyze the barrier factors for the immunization. MATERIALS & METHODS: Questionnaires were distributed in March of 2000 at Korea University Guro Hospital. We evaluated factors associated with acceptance of influenza vaccination and opinions regarding influenza prevention (knowledge about influenza vaccination efficacy, que to action in vaccinee, perceptible benefit, barrier to vaccination). RESULTS: 309 completed questionnaires from HCWs were returned. Mean age and mean duration of work in hospital were higher and longer among vaccinee than non-vaccinee. Even though the necessity of influenza vaccination among HCWs were accepted well in vaccinee compared to non- vaccinee, the accurate reasons for vaccination were not quite understood among HCWs regardless of compliance. Vaccine campaign (30.8%) and advise from doctors (24.7%) were important for the promotion of vaccination among vaccinee. However, major reason for non-compliance among nursing staff and was mis-confidence that their bodies' self defense mechanisms would ward off infection (33.5%) and 'too busy to get injection' for doctors (65%). CONCLUSION: We conclude that regular education about perceptible benefits and wrong concerns about influenza vaccination among HCW's and easy accessibility to vaccination were important determinants to improve the influenza vaccination. On-site availability of a vaccination-nurse also proved to be important.

Efficacy of Influenza Vaccination among Chronic Ill Patients: Retrospective Case Control Study / 감염과화학요법

BACKGROUND: Influenza is one of the preventable respiratory disease by annual vaccination. Elderly people and patients with chronic medical disease are the primary target for influenza vaccination according to ACIP (Advisory Committee on Immunization). This study was done to determine whether influenza vaccination affects hospitalization due to influenza like illness or it's related complications among chronic ill patients. MATERIALS AND METHODS: Retrospective, case-control study was conducted for 5 months (Nov. 1999-Mar. 2000). Physicians from two-university hospitals in Korea were instructed to collect clinical data, sera from subjects presenting with a flu-like illness or flu-related exacerbation in patients with chronic underlying diseases. We compared the vaccination history of case patients with control. Logistic regression analysis was used to calculate the odd ratio and efficacy of influenza vaccination. RESULTS: Hospitalization due to ILI (influenza like illness) or it's related complications were observed more frequently in patients with chronic lung disease (25.3%) and chronic cardiac disease (31.3%) compared to others. Influenza vaccination resulted in fewer hospitalization with ILI or ILI-related acute exacerbation of underlying disease in chronic ill patients (adjusted odd ratio, 0.328 [CI, 0.14 to 0.73]) during influenza seasons. CONCLUSION: For patients with chronic disease, influenza vaccination is associated with substantial health benefit including fewer hospitalization. Health care providers should take advantage of all opportunities to immunize these high-risk patients.

Factors Influencing Decision Regarding Influenza Vaccination: A Survey of Healthcare Workers in One Hospital / 감염과화학요법

BACKGROUND: In spite of yearly recalls, influenza immunization rate among healthcare workers (HCWs) remained low in Korea University Guro Hospital. This study was conducted to identify the causes of non-compliance against influenza immunization and to analyze the barrier factors for the immunization. MATERIALS & METHODS: Questionnaires were distributed in March of 2000 at Korea University Guro Hospital. We evaluated factors associated with acceptance of influenza vaccination and opinions regarding influenza prevention (knowledge about influenza vaccination efficacy, que to action in vaccinee, perceptible benefit, barrier to vaccination). RESULTS: 309 completed questionnaires from HCWs were returned. Mean age and mean duration of work in hospital were higher and longer among vaccinee than non-vaccinee. Even though the necessity of influenza vaccination among HCWs were accepted well in vaccinee compared to non- vaccinee, the accurate reasons for vaccination were not quite understood among HCWs regardless of compliance. Vaccine campaign (30.8%) and advise from doctors (24.7%) were important for the promotion of vaccination among vaccinee. However, major reason for non-compliance among nursing staff and was mis-confidence that their bodies' self defense mechanisms would ward off infection (33.5%) and 'too busy to get injection' for doctors (65%). CONCLUSION: We conclude that regular education about perceptible benefits and wrong concerns about influenza vaccination among HCW's and easy accessibility to vaccination were important determinants to improve the influenza vaccination. On-site availability of a vaccination-nurse also proved to be important.

Characterization of the Two Genes Differentially Expressed During Development in 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 that they 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 were isolated at intervals of 5 days for 50 days. Using 24 primer combinations, we identified a set of 18 candidate cDNAs deriving from the excised DDRT-PCR bands. DNA sequencing revealed 16 genes that have been described already. We found that RTP, TG, hTM-alpha, SPARC, TRIP7, and RPL7 genes were expressed increasingly, while HMGCR, RPL27a, NACA, NPM, and TARBP2 genes were expressed decreasingly, according to their culture stages. We also found two unidentified genes, A3 and C8, which were expressed differently in culture stages; the former was expressed decreasingly and the latter increasingly. These two genes were found in the same amount in genomic DNA from various human cells such as astrocytes, astrocytoma, trophoblasts and lymphocytes. The A3 gene was found only in human genomic DNA, but not in rat (ATr5), mouse (RAW264.7), or monkey (Vero) cells, whereas the C8 gene was found in human genomic DNA and monkey cells, but not in rat or mouse cells. We analysed these two genes for identification. There was > 92% nucleotide sequence identity between the A3 gene (3, 626 bp) and the Homo sapiens general transcription factor 3 (GTF3), and > 96% nucleotide sequence identity between the C8 gene (2, 401 bp) and the transmembrane receptor Unc5h2. These findings suggest that these two genes may participate in some functional roles within the cells.

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.