Functional characterization of Clonorchis sinensis choline transporter.

Clonorchis sinensis is commonly found in East Asian countries. Clonorchiasis is prevalent in these countries and can lead to various clinical symptoms. In this study, we used overlap extension polymerase chain reaction (PCR) and the Xenopus laevis oocyte expression system to isolate a cDNA encoding the choline transporter of C. sinensis (CsChT). We subsequently characterized recombinant CsChT. Expression of CsChT in X. laevis oocytes enabled efficient transport of radiolabeled choline, with no detectable uptake of arginine, α-ketoglutarate, p-aminohippurate, taurocholate, and estrone sulfate. Influx and efflux experiments showed that CsChT-mediated choline uptake was time- and sodium-dependent, with no exchange properties. Concentration-dependent analyses of revealed saturable kinetics consistent with the Michaelis-Menten equation, while nonlinear regression analyses revealed a Km value of 8.3 µM and a Vmax of 61.0 pmol/oocyte/h. These findings contribute to widen our understanding of CsChT transport properties and the cascade of choline metabolisms within C. sinensis.

Functional characterization of Plasmodium vivax hexose transporter 1.

Plasmodium vivax is the most widely distributed human malaria parasite. The eradication of vivax malaria remains challenging due to transmission of drug-resistant parasite and dormant liver form. Consequently, anti-malarial drugs with novel mechanisms of action are urgently demanded. Glucose uptake blocking strategy is suggested as a novel mode of action that leads to selective starvation in various species of malaria parasites. The role of hexose transporter 1 in Plasmodium species is glucose uptake, and its blocking strategies proved to successfully induce selective starvation. However, there is limited information on the glucose uptake properties via P. vivax hexose transporter 1 (PvHT1). Thus, we focused on the PvHT1 to precisely identify its properties of glucose uptake. The PvHT1 North Korean strain (PvHT1NK) expressed Xenopus laevis oocytes mediating the transport of [3H] deoxy-D-glucose (ddGlu) in an expression and incubation time-dependent manner without sodium dependency. Moreover, the PvHT1NK showed no exchange mode of glucose in efflux experiments and concentration-dependent results showed saturable kinetics following the Michaelis-Menten equation. Non-linear regression analysis revealed a Km value of 294.1 µM and a Vmax value of 1,060 pmol/oocyte/hr, and inhibition experiments showed a strong inhibitory effect by glucose, mannose, and ddGlu. Additionally, weak inhibition was observed with fructose and galactose. Comparison of amino acid sequence and tertiary structure between P. falciparum and P. vivax HT1 revealed a completely conserved residue in glucose binding pocket. This result supported that the glucose uptake properties are similar to P. falciparum, and PfHT1 inhibitor (compound 3361) works in P. vivax. These findings provide properties of glucose uptake via PvHT1NK for carbohydrate metabolism and support the approaches to vivax malaria drug development strategy targeting the PvHT1 for starving of the parasite.

Geographical distribution and genetic diversity of Plasmodium vivax reticulocyte binding protein 1a correlates with patient antigenicity.

Plasmodium vivax is the most widespread cause of human malaria. Recent reports of drug resistant vivax malaria and the challenge of eradicating the dormant liver forms increase the importance of vaccine development against this relapsing disease. P. vivax reticulocyte binding protein 1a (PvRBP1a) is a potential vaccine candidate, which is involved in red cell tropism, a crucial step in the merozoite invasion of host reticulocytes. As part of the initial evaluation of the PvRBP1a vaccine candidate, we investigated its genetic diversity and antigenicity using geographically diverse clinical isolates. We analysed pvrbp1a genetic polymorphisms using 202 vivax clinical isolates from six countries. Pvrbp1a was separated into six regions based on specific domain features, sequence conserved/polymorphic regions, and the reticulocyte binding like (RBL) domains. In the fragmented gene sequence analysis, PvRBP1a region II (RII) and RIII (head and tail structure homolog, 152-625 aa.) showed extensive polymorphism caused by random point mutations. The haplotype network of these polymorphic regions was classified into three clusters that converged to independent populations. Antigenicity screening was performed using recombinant proteins PvRBP1a-N (157-560 aa.) and PvRBP1a-C (606-962 aa.), which contained head and tail structure region and sequence conserved region, respectively. Sensitivity against PvRBP1a-N (46.7%) was higher than PvRBP1a-C (17.8%). PvRBP1a-N was reported as a reticulocyte binding domain and this study identified a linear epitope with moderate antigenicity, thus an attractive domain for merozoite invasion-blocking vaccine development. However, our study highlights that a global PvRBP1a-based vaccine design needs to overcome several difficulties due to three distinct genotypes and low antigenicity levels.

Functional characterization of Clonorchis sinensis sodium-bile acid co-transporter (CsSBAT) as a steroid sulfate transporter.

Clonorchis sinensis (Cs) is a common trematode in Asian countries. Infection by Cs can result in many clinical symptoms. Here, a cDNA encoding a Cs apical sodium-dependent bile acid transporter (CsSBAT) was isolated from a Cs cDNA library, and functional characterization was performed using Xenopus laevis oocyte expression system. When expressed in Xenopus laevis oocytes, CsSBAT mediated the transport of radiolabeled estrone sulfate and dehydroepiandrosterone sulfate. No trans-uptake of carnitine, estradiol 17 ß-D glucuronide, prostaglandin E2, p-aminohippuric acid, α-ketoglutaric acid, and tetraethylammonium was observed. CsSBAT-mediated estrone sulfate uptake was in a time- and sodium-dependent manner. CsSBAT showed no exchange properties in efflux experiments. Concentration-dependent results showed saturable kinetics consistent with the Michaelis-Menten equation. Nonlinear regression analyses yielded a Km value of 0.3 ± 0.04 µM for [3H]estrone sulfate. CsSBAT-mediated estrone sulfate uptake was strongly inhibited by sulfate conjugates but not glucuronide conjugates. These findings contribute to our understanding of CsSBAT transport properties and the cascade of estrogen metabolite movement in Cs.

Characterization of Echinostoma cinetorchis endoribonuclease, RNase H.

Echinostoma cinetorchis is an oriental intestinal fluke causing significant pathological damage to the small intestine. The aim of this study was to determine a full-length cDNA sequence of E. cinetorchis endoribonuclease (RNase H; EcRNH) and to elucidate its molecular biological characters. EcRNH consisted of 308 amino acids and showed low similarity to endoribonucleases of other parasites (<40%). EcRNH had an active site centered on a putative DDEED motif instead of DEDD conserved in other species. A recombinant EcRNH produced as a soluble form in Escherichia coli showed enzymatic activity to cleave the 3'-O-P bond of RNA in a DNA-RNA duplex, producing 3'-hydroxyl and 5'-phosphate. These findings may contribute to develop antisense oligonucleotides which could damage echinostomes and other flukes.

Characterization of the Nanog 5'-flanking Region in Bovine.

Bovine embryonic stem cells have potential for use in research, such as transgenic cattle generation and the study of developmental gene regulation. The Nanog may play a critical role in maintenance of the undifferentiated state of embryonic stem cells in the bovine, as in murine and human. Nevertheless, efforts to study the bovine Nanog for pluripotency-maintaining factors have been insufficient. In this study, in order to understand the mechanisms of transcriptional regulation of the bovine Nanog, the 5'-flanking region of the Nanog was isolated from ear cells of Hanwoo. Results of transient transfection using a luciferase reporter gene under the control of serially deleted 5'-flanking sequences revealed that the -134 to -19 region contained the positive regulatory sequences for the transcription of the bovine Nanog. Results from mutagenesis studies demonstrated that the Sp1-binding site that is located in the proximal promoter region plays an important role in transcriptional activity of the bovine Nanog promoter. The electrophoretic mobility shift assay with the Sp1 specific antibody confirmed the specific binding of Sp1 transcription factor to this site. In addition, significant inhibition of Nanog promoter activity by the Sp1 mutant was observed in murine embryonic stem cells. Furthermore, chromatin-immunoprecipitation assay with the Sp1 specific antibody confirmed the specific binding of Sp1 transcription factor to this site. These results suggest that Sp1 is an essential regulatory factor for bovine Nanog transcriptional activity.

Molecular cloning and functional characterization of a glucose transporter (CsGLUT) in Clonorchis sinensis.

A complementary DNA (cDNA) encoding a glucose transporter of Clonorchis sinensis (CsGLUT) was isolated from the adult C. sinensis cDNA library. The open reading frame of CsGLUT cDNA consists of 1653 base pairs that encode a 550-amino acid residue protein. Hydropathy analysis suggested that CsGLUT possess 12 putative membrane-spanning domains. The Northern blot analysis result using poly(A)(+)RNA showed a strong band at ~2.1 kb for CsGLUT. When expressed in Xenopus oocytes, CsGLUT mediated the transport of radiolabeled deoxy-D-glucose in a time-dependent but sodium-independent manner. Concentration-dependency results showed saturable kinetics and followed the Michaelis-Menten equation. Nonlinear regression analyses yielded a Km value of 588.5 ± 53.0 µM and a Vmax value of 1500.0 ± 67.5 pmol/oocyte/30 min for [1,2-(3)H]2-deoxy-D-glucose. No trans-uptakes of bile acid (taurocholic acid), amino acids (tryptophan and arginine), or p-aminohippuric acid were observed. CsGLUT-mediated transport of deoxyglucose was significantly and concentration-dependently inhibited by radio-unlabeled deoxyglucose and D-glucose. 3-O-Methylglucose at 10 and 100 µM inhibited deoxyglucose uptake by ~50 % without concentration dependence. No inhibitory effects by galactose, mannose, and fructose were observed. This work may contribute to the molecular biological study of carbohydrate metabolism and new drug development of C. sinensis.

Polymorphisms of SLC22A9 (hOAT7) in Korean Females with Osteoporosis.

Among solute carrier proteins, the organic anion transporters (OATs) play an important role for the elimination or reabsorption of endogenous and exogenous negatively charged anionic compounds. Among OATs, SLC22A9 (hOAT7) transports estrone sulfate with high affinity. The net decrease of estrogen, especially in post-menopausal women induces rapid bone loss. The present study was performed to search the SNP within exon regions of SLC22A9 in Korean females with osteoporosis. Fifty healthy controls and 50 osteoporosis patients were screened for the genetic polymorphism in the coding region of SLC22A9 using GC-clamped PCR and denaturing gradient gel electrophoresis (DGGE). Six SNPs were found on the SLC22A9 gene from Korean women with/without osteoporosis. The SNPs were located as follows: two SNPs in the osteoporosis group (A645G and T1277C), three SNPs in the control group (G1449T, C1467T and C1487T) and one SNP in both the osteoporosis and control groups (G767A). The G767A, T1277C and C1487T SNPs result in an amino acid substitution, from synonymous vs nonsynonymous substitution arginine to glutamine (R256Q), phenylalanine to serine (F426S) and proline to leucine (P496L), respectively. The Km values and Vmax of the wild type, R256Q, P496L and F426S were 8.84, 8.87, 9.83 and 12.74 µM, and 1.97, 1.96, 2.06 and 1.55 pmol/oocyte/h, respectively. The present study demonstrates that the SLC22A9 variant F426S is causing inter-individual variation that is leading to the differences in transport of the steroid sulfate conjugate (estrone sulfate) and, therefore this could be used as a marker for certain disease including osteoporosis.

Evaluation of the accuracy of the EasyTest™ malaria Pf/Pan Ag, a rapid diagnostic test, in Uganda.

In recent years, rapid diagnostic tests (RDTs) have been widely used for malaria detection, primarily because of their simple operation, fast results, and straightforward interpretation. The Asan EasyTest™ Malaria Pf/Pan Ag is one of the most commonly used malaria RDTs in several countries, including Korea and India. In this study, we tested the diagnostic performance of this RDT in Uganda to evaluate its usefulness for field diagnosis of malaria in this country. Microscopic and PCR analyses, and the Asan EasyTest™ Malaria Pf/Pan Ag rapid diagnostic test, were performed on blood samples from 185 individuals with suspected malaria in several villages in Uganda. Compared to the microscopic analysis, the sensitivity of the RDT to detect malaria infection was 95.8% and 83.3% for Plasmodium falciparum and non-P. falciparum, respectively. Although the diagnostic sensitivity of the RDT decreased when parasitemia was ≤500 parasites/µl, it showed 96.8% sensitivity (98.4% for P. falciparum and 93.8% for non-P. falciparum) in blood samples with parasitemia ≥100 parasites/µl. The specificity of the RDT was 97.3% for P. falciparum and 97.3% for non-P. falciparum. These results collectively suggest that the accuracy of the Asan EasyTest™ Malaria Pf/Pan Ag makes it an effective point-of-care diagnostic tool for malaria in Uganda.

Seroprevalence of Plasmodium vivax in the Republic of Korea (2003-2005) using indirect fluorescent antibody test.

Plasmodium vivax reemerged in the Republic of Korea (ROK) in 1993, and is likely to continue to affect public health. The purpose of this study was to measure levels of anti-P. vivax antibodies using indirect fluorescent antibody test (IFAT) in border areas of ROK, to determine the seroprevalence of malaria (2003-2005) and to plan effective control strategies. Blood samples of the inhabitants in Gimpo-si, Paju-si, and Yeoncheon-gun (Gyeonggi-do), and Cheorwon-gun (Gangwon-do) were collected and kept in Korea Centers for Disease Control and Prevention (KCDC). Out of a total of 1,774 serum samples tested, the overall seropositivity was 0.94% (n=17). The seropositivity was the highest in Paju-si (1.9%, 7/372), followed by Gimpo-si (1.4%, 6/425), Yeoncheon-gun (0.67%, 3/451), and Cheorwon-gun (0.19%, 1/526). The annual parasite incidence (API) in these areas gradually decreased from 2003 to 2005 (1.69, 1.09, and 0.80 in 2003, 2004, and 2005, respectively). The highest API was found in Yeoncheon-gun, followed by Cheorwon-gun, Paju-si, and Gimpo-si. The API ranking in these areas did not change over the 3 years. The seropositivity of Gimpo-si showed a strong linear relationship with the API of 2005 (r=0.9983, P=0.036). Seropositivity data obtained using IFAT may be useful for understanding malaria prevalence of relevant years, predicting future transmission of malaria, and for establishing and evaluating malaria control programs in affected areas.

Evaluation of circumsporozoite protein of Plasmodium vivax to estimate its prevalence in the Republic of Korea: an observational study of incidence.

BACKGROUND: Plasmodium vivax re-emerged in 1993. Although the number of infections has been steadily decreasing, it is likely to continue to affect public health until it is eradicated. The aim of this study is to measure anti-circumsporozoite protein (CSP) antibody and compare malaria prevalence. As to understand the prevalence, an epidemiology study has to be conducted in the Republic of Korea. METHODS: A total of 1,825 and 1,959 blood samples were collected in 2010 and 2011, respectively, from the inhabitants of Ganghwa and Cheorwon counties. The antibody titers of the inhabitants were measured by enzyme-linked immunosorbent assay (ELISA) using recombinant protein purified from Escherichia coli transformed with a CSP gene-inserted pET-28a(+) expression vector. Microscopic examination was performed to identify malaria parasites. RESULTS: The annual parasite incidence (API) in Ganghwa decreased from 4.28 in 2010 to 2.23 in 2011, and that in Cheorwon decreased from 1.88 in 2010 to 1.15 in 2011. The antibody-positive CSP rate in these areas also decreased from 18.14% (331/1825) in 2010 to 15.36% (301/1959) in 2011. Pearson analysis showed a strong correlation between the API and the antibody-positive CSP rate in these areas (r = 1.000, P < 0.01). The intensity of the immune responses of the inhabitants of Cheorwon, as measured by the mean optical density, decreased from 0.9186 ± 0.0472 in 2010 to 0.7035 ± 0.0457 in 2011 (P = 0.034), but increased in Ganghwa from 0.7649 ± 0.0192 in 2010 to 0.8237 ± 0.1970 in 2011 (P = 0.006). The immune response increased according to age (r = 0.686, P = 0.041). CONCLUSIONS: The positive CSP-ELISA rate was closely related to the API in the study areas. This suggests that seroepidemiological studies based on CSP-ELISA may be helpful in estimating the malaria prevalence. Moreover, such studies can be used to establish and evaluate malaria control and eradication programmes in high-risk areas in Korea.

Decreasing incidence of Plasmodium vivax in the Republic of Korea during 2010-2012.

BACKGROUND: After the re-emergence of Plasmodium vivax in 1993, a total of 31,254 cases of vivax malaria were reported between 1993-2012 in the Republic of Korea (ROK). The purpose of this study was to review Korea Centers for Disease Control and Prevention records to investigate the transmission of malaria from 2010-2012. METHODS: Reporting of microscopy-diagnosed cases of malaria is mandatory in the ROK. In this study, all available records of malaria cases and malaria vectors collected from 2010 - 2012 in Cheorwon County, Gangwon Province and Ganghwa County, Incheon Metropolitan City, were reviewed. RESULTS: Although the number of cases of malaria peaked a third time in 2010 (1,772 cases) since the re-emergence of P. vivax, the incidence decreased two-fold to 838 in 2011 and three-fold to 555 in 2012. The number of cases decreased 52.7% in 2011 compared with that in 2010 and 33.8% in 2012 compared with that in 2011. However, the number of cases increased in Incheon Metropolitan City (15.3%) and Gyeongnam Province (23.1%) in 2012 compared with 2011. Of the 3,165 cases of vivax malaria in 2010-2012, 798 (25.2%) were in ROK military personnel, 519 (16.4%) in veterans, and 1,848 (58.4%) in civilians. In total, there were 2,666 male patients and 499 female patients, and the ratio of female to male patients increased from 1:7.9 in 2011 to 1:4.1 in 2012. CONCLUSIONS: A rapid decrease in the incidence of malaria was observed in most areas from 2010 to 2012, but the incidence increased again in the western part of the demilitarized zone. Therefore, more intensive surveillance is needed throughout high risk areas to identify factors responsible for increase/decrease in the incidence of malaria in the ROK.

Development of a polymerase chain reaction applicable to rapid and sensitive detection of Clonorchis sinensis eggs in human stool samples.

Microscopic examination of eggs of parasitic helminths in stool samples has been the most widely used classical diagnostic method for infections, but tiny and low numbers of eggs in stool samples often hamper diagnosis of helminthic infections with classical microscopic examination. Moreover, it is also difficult to differentiate parasite eggs by the classical method, if they have similar morphological characteristics. In this study, we developed a rapid and sensitive polymerase chain reaction (PCR)-based molecular diagnostic method for detection of Clonorchis sinensis eggs in stool samples. Nine primers were designed based on the long-terminal repeat (LTR) of C. sinensis retrotransposon1 (CsRn1) gene, and seven PCR primer sets were paired. Polymerase chain reaction with each primer pair produced specific amplicons for C. sinensis, but not for other trematodes including Metagonimus yokogawai and Paragonimus westermani. Particularly, three primer sets were able to detect 10 C. sinensis eggs and were applicable to amplify specific amplicons from DNA samples purified from stool of C. sinensis-infected patients. This PCR method could be useful for diagnosis of C. sinensis infections in human stool samples with a high level of specificity and sensitivity.

Preventive effects of Eleutherococcus senticosus bark extract in OVX-induced osteoporosis in rats.

Eleutherococcus senticosus (Siberian ginseng), has been used as a powerful tonic herb with an impressive range of health benefits. This medicinal herb has been commonly used to treat bone metabolism diseases due to its traditional Korean medicine use to strengthen muscle and bone. This study was conducted to investigate prevention of bone loss by a standardized extract of dried E. senticosus stem bark in an ovariectomized (OVX) rat model of osteoporosis. The OVX groups were divided into five groups treated with distilled water, 17ß-estradiol (E2 10 µg/kg, once daily, i.p) and dried stem bark of E. senticosus extracts (DES 10, 30, and 100 mg/kg, once daily, p.o) for eight weeks, respectively. After eight weeks of treatments, the femur bone mineral density of the 100 mg/kg DES-treated group was significantly higher than that of the OVX-control group (16.7%, p < 0.01) without affecting the body, organs, and uterus weights, and serum estradiol levels. Additionally, bone markers such as serum ALP, CTx, and OC levels were significantly decreased in the DES 100 mg/kg treated group. These results show that DES is able to prevent OVX-induced in bone loss without the influence of hormones such as estrogen.

Limited sequence polymorphisms of four transmission-blocking vaccine candidate antigens in Plasmodium vivax Korean isolates.

BACKGROUND: Transmission-blocking vaccines (TBVs), which target the sexual stages of malaria parasites to interfere with and/or inhibit the parasite's development within mosquitoes, have been regarded as promising targets for disrupting the malaria transmission cycle. In this study, genetic diversity of four TBV candidate antigens, Pvs25, Pvs28, Pvs48/45, and PvWARP, among Plasmodium vivax Korean isolates was analysed. METHODS: A total of 86 P. vivax-infected blood samples collected from patients in Korea were used for analyses. Each of the full-length genes encoding four TBV candidate antigens, Pvs25, Pvs28, Pvs48/45, and PvWARP, were amplified by PCR, cloned into T&A vector, and then sequenced. Polymorphic characteristics of the genes were analysed using the DNASTAR, MEGA4, and DnaSP programs. RESULTS: Polymorphism analyses of the 86 Korean P. vivax isolates revealed two distinct haplotypes in Pvs25 and Pvs48/45, and three different haplotypes in PvWARP. In contrast, Pvs28 showed only a single haplotype. Most of the nucleotide substitutions and amino acid changes identified in all four TBV candidate antigens were commonly found in P. vivax isolates from other geographic areas. The overall nucleotide diversities of the TBV candidates were much lower than those of blood stage antigens. CONCLUSIONS: Limited sequence polymorphisms of TBV candidate antigens were identified in the Korean P. vivax population. These results provide baseline information for developing an effective TBV based on these antigens, and offer great promise for applications of a TBV against P. vivax infection in regions where the parasite is most prevalent.

A surge in the seroprevalence of toxoplasmosis among the residents of islands in Gangwha-gun, Incheon, Korea.

Seroepidemiological changes of Toxoplasma gondii infection among the residents of the islands of Gangwha-gun, Incheon for 2 years were surveyed and evaluated by ELISA using a crude extract antigen. In 2010, sera of 919 adult residents in Gyodong-myeon and 313 adults in Samsan-myeon were collected and checked for IgG antibody titers, which showed 14.5% (133 sera) and 19.8% (62 sera) positive rates, respectively. In 2011, sera of 955 adults in Gyodong-myeon and 341 adults in Samsan-myeon were examined, which showed an increase of positive rates to 23.8% (227 sera) and 31.7% (108 sera), respectively. Totally, the seroprevalence of the first year was 15.8% and it increased rapidly to 25.8% in the second year. The positive rates of both sexes increased simultaneously with the significant ratio of males to females by 1.7-2.2 fold (P<0.05). In both myeons, 661 sera were collected every year and showed changes in optical density (OD) in 177 sera; newly found as positives in 73 persons (11.0%), negative conversion in 10 persons (1.5%), and maintained or increased in 94 persons (14.2%). This rapid increase in the prevalence of toxoplasmosis in Gangwha islands may be due to in part peculiar changes in the toxoplasmic environment of the islands and presumably the consumption of the pork bred domestically within the islands or imported from high endemic nations. It is necessary to find out symptomatic toxoplasmic patients and confirm the risk factors for further infection in the islands of Gangwha-gun.

Seroprevalence of toxoplasmosis in the residents of Cheorwon-gun, Gangwon-do, Korea.

The seroepidemiological status of toxoplasmosis was surveyed among the residents of Cheorwon-gun, Gangwon-do by means of ELISA using a crude extract antigen of Toxoplasma gondii. The sera of 1,661 adult residents (866 males and 795 females) were collected and checked for IgG antibody titers, which showed 17.0% positive rate (282 sera). The positive rate was significantly different between the sex; 20.6% for males and 13.1% for females (P<0.05). The positive rates were higher in fifties of males (28.7%) and forties of females (20.0%). This positive rate of toxoplasmosis in Cheorwon-gun residents is regarded as the highest among the surveys of different geographical regions of Korea. This high positive rate may due in part to peculiar geographical locality of the surveyed area near the naturally well preserved demilitarized zone (DMZ) or presumably consumption of the pork imported from high endemic nations. Therefore, it is necessary to study further the epidemiology of toxoplasmosis in Cheorwon-gun.

Induction of glioma apoptosis by microglia-secreted molecules: The role of nitric oxide and cathepsin B.

Microglia contributes significantly to brain tumor mass, particularly in astrocytic gliomas. Here, we examine the cytotoxic effects of soluble components secreted from microglia culture on glioma cells. Microglia conditioned culture medium (MCM) actively stimulated apoptotic death of glioma cells, and the effects of MCM prepared from LPS- or IFN-gamma-activated microglia were more pronounced. The cytotoxic effects were glioma-specific in that primary cultured rat astrocytes were not affected by MCM. A donor of peroxynitrite induced glioma-specific cell death. In addition, NO synthase inhibitor suppressed glioma cell death induced by activated MCM, indicating that NO is one of the key molecules responsible for glioma cytotoxicity mediated by activated MCM. However, since unstimulated resting microglia produces low or very limited level of NO, MCM may contain other critical molecule(s) that induce glioma apoptosis. To identify the proteins secreted in MCM, proteomic analysis was performed on control or activated medium. Among over 200 protein spots detected by Coomassie blue staining, we identified 26 constitutive and 28 LPS- or IFN-gamma-regulated MCM proteins. Several cathepsin proteases were markedly expressed, which were reduced upon activation. In particular, suppression of cathepsin B by the chemical inhibitors significantly reversed MCM-induced glioma cell death, implying a critical role of this protease in cytotoxicity. Our findings provide evidence on the functional implications of specific microglial-secreted proteins in glioma cytotoxicity, as well as a basis to develop a proteomic databank of both basal and activation-related proteins in microglia.

Synergistic production of interleukin-23 by dendritic cells derived from cord blood in response to costimulation with LPS and IL-12.

This study was performed to provide insight for the optimization and regulation of immune homeostasis, which should be taken into account in the development of cell therapy using DCs and/or cytokine. Human CBDCs costimulated with LPS and IL-12 were examined for cytokine expression compared with ABDCs. Our results showed that costimulation with IL-12 and LPS in CBDCs resulted in increased expression of IL-23. Concomitantly, the phosphorylation of ERKs and p38 MAPK was increased, suggesting that these kinases are important signaling components for IL-23 induction in CBDC costimulated with LPS and IL-12. Furthermore, production of IL-23 in CBDC costimulated with LPS and IL-12 caused CD4(+)CD45RO(+) memory cells to increase IFN-gamma production. Taken together, CBDCs, costimulated with LPS and IL-12, show a synergistic increase in IL-23 production via enhanced phosphorylation of ERK1/2 and p38 MAPK and consequently, an induction of IFN-gamma production in the memory cells.

Co-localization and interaction of human organic anion transporter 4 with caveolin-1 in primary cultured human placental trophoblasts.

The human organic anion transporter 4 (hOAT4) has been identified as the fourth isoform of OAT family. hOAT4 contributes to move several negatively charged organic compounds between cells and their extracellular milieu. The functional characteristics and regulatory mechanisms of hOAT4 remain to be elucidated. It is well known that caveolin plays a role in modulating proteins having some biological functions. To address this issue, we investigated the co-localization and interaction between hOAT4 and caveolin-1. hOAT4 and caveolin-1 (mRNA and protein expression) were observed in cultured human placental trophoblasts isolated from placenta. The confocal microscopy of immuno-cytochemistry using primary cultured human trophoblasts showed hOAT4 and caveolin-1 were co-localized at the plasma membrane of the cell. This finding was confirmed by Western blot analysis using isolated caveolae-enriched membrane fractions and immune-precipitates from the trophoblasts. When synthesized cRNA of hOAT4 along with scrambled- or antisense-oligodeoxynucleotide (ODN) of Xenopus caveolin-1 were co-injected to Xenopus oocytes, the [3H]estrone sulfate uptake was significantly decreased by the co-injection of antisense ODN but not by scrambled ODN. These findings suggest that hOAT4 and caveolin-1 share a cellular expression in the plasma membrane and caveolin-1 up-regulates the organic anionic compound uptake by hOAT4 under the normal physiological condition.