CD8 T-cell activation in mice injected with a plasmid DNA vaccine encoding AMA-1 of the reemerging Korean Plasmodium vivax

Relatively little has been studied on the AMA-1 vaccine against Plasmodium vivax and on the plasmid DNA vaccine encoding P. vivax AMA-1 (PvAMA-1). In the present study, a plasmid DNA vaccine encoding AMA-1 of the reemerging Korean P. vivax has been constructed and a preliminary study was done on its cellular immunogenicity to recipient BALB/c mice. The PvAMA-1 gene was cloned and expressed in the plasmid vector UBpcAMA-1, and a protein band of approximately 56.8 kDa was obtained from the transfected COS7 cells. BALB/c mice were immunized intramuscularly or using a gene gun 4 times with the vaccine, and the proportions of splenic T-cell subsets were examined by fluorocytometry at week 2 after the last injection. The spleen cells from intramuscularly injected mice revealed no significant changes in the proportions of CD8(+) T-cells and CD4(+) T-cells. However, in mice immunized using a gene gun, significantly higher (P<0.05) proportions of CD8(+) cells were observed compared to UB vector-injected control mice. The results indicated that cellular immunogenicity of the plasmid DNA vaccine encoding AMA-1 of the reemerging Korean P. vivax was weak when it was injected intramuscularly; however, a promising effect was observed using the gene gun injection technique.     

Efficient expression and characterization of hepatitis B virus preS2 antigen in Escherichia coli

The complete (encoding 55 amino acids, aa) or partial (encoding aa 1–26) preS2 region gene of hepatitis B virus (HBV) was fused to the 3-end of glutathion-S-transferase (GST) gene and expressed under the control of the inducible tac promoter in Escherichia coli at 37 °C. The fusion protein with the complete preS2 region was moderately expressed (8%) while the protein with the N-terminal 26 aa was expressed at a higher level, yielding about 20% of the total cellular proteins. The GST-preS2 (aa 1–26) protein, which contains the immunodominant epitope, was produced form the soluble protein fraction of the recombinant bacteria and purified by affinity chromatography using glutathione-agarose column. The purified preS2 fusion protein showed the antigenicity of preS2, as assessed by indirect and competitive ELISAs.     

The multivalent PDZ domain-containing protein PDZK1 regulates transport activity of renal urate-anion exchanger URAT1 via its C terminus

The urate-anion exchanger URAT1 is a member of the organic anion transporter (OAT) family that regulates blood urate level in humans and is targeted by uricosuric and antiuricosuric agents. URAT1 is expressed only in the kidney, where it is thought to participate in tubular urate reabsorption. We found that the multivalent PDZ (PSD-95, Drosophila discs-large protein, Zonula occludens protein 1) domain-containing protein, PDZK1 interacts with URAT1 in a yeast two-hybrid screen. Such an interaction requires the PDZ motif of URAT1 in its extreme intracellular C-terminal region and the first, second, and fourth PDZ domains of PDZK1 as identified by yeast two-hybrid assay, in vitro binding assay and surface plasmon resonance analysis (K(D) = 1.97-514 nM). Coimmunoprecipitation studies revealed that the wild-type URAT1, but not its mutant lacking the PDZ-motif, directly interacts with PDZK1. Colocalization of URAT1 and PDZK1 was observed at the apical membrane of renal proximal tubular cells. The association of URAT1 with PDZK1 enhanced urate transport activities in HEK293 cells (1.4-fold), and the deletion of the URAT1 C-terminal PDZ motif abolished this effect. The augmentation of the transport activity was accompanied by a significant increase in the V(max) of urate transport via URAT1 and was associated with the increased surface expression level of URAT1 protein from HEK293 cells stably expressing URAT1 transfected with PDZK1. Taken together, the present study indicates the novel role of PDZK1 in regulating the functional activity of URAT1-mediated urate transport in the apical membrane of renal proximal tubules.     

HO-1 and JAK-2/STAT-1 signals are involved in preferential inhibition of iNOS over COX-2 gene expression by newly synthesized tetrahydroisoquinoline alkaloid, CKD712, in cells activated with lipopolysacchride

We found that CKD712, an S enantiomer of YS49, strongly inhibited inducible nitric oxide synthase (iNOS) and NO induction but showed a weak inhibitory effect on cyclooxygenase-2 (COX-2) and PGE(2) induction in LPS-stimulated RAW 264.7 cells. We, therefore, investigated the molecular mechanism(s) responsible for this by using CKD712 in LPS-activated RAW264.7 cells. Treatment with either SP600125, a specific JNK inhibitor or TPCK, a NF-kappaB inhibitor, but neither ERK inhibitor PD98059 nor p38 inhibitor SB203580, significantly inhibited LPS-mediated iNOS and COX-2 induction. CKD712 inhibited NF-kappaB (p65) activity and translocation but failed to prevent JNK activation. However, AG490, a specific JAK-2/STAT-1 inhibitor, efficiently prevented LPS-mediated iNOS induction but not the induction of COX-2, and CKD712 completely blocked STAT-1 phosphorylation by LPS, suggesting that the NF-kappaB and JAK-2/STAT-1 pathways but not the JNK pathway are important for CKD712 action. Interestingly, CKD712 induced heme oxygenase 1 (HO-1) gene expression in LPS-treated cells. LPS-induced NF-kappaB and STAT-1 activation was partially prevented by HO-1 overexpression. Furthermore, HO-1 siRNA partly reversed not only the LPS-induced NF-kappaB activation and STAT-1 phosphorylation but also inhibition of these actions by CKD 712. Additionally, silencing HO-1 by siRNA prevented CKD712 from inhibiting iNOS expression but not COX-2. When examined plasma NO and PGE(2) levels and iNOS and COX-2 protein levels in lung tissues of mice injected with LPS (10 mg/kg), pretreatment with CKD712 greatly prevented NO and iNOS induction in a dose-dependent manner and slightly affected PGE(2) and COX-2 production as expected. Taken together, we conclude that inhibition of JAK-2/STAT-1 pathways by CKD 712 is critical for the differential inhibition of iNOS and COX-2 by LPS in vitro and in vivo where HO-1 induction also contributes to this by partially modulating JAK-2/STAT-1 pathways.     

NMR studies on novel antitumor drug candidates, deoxoartemisinin and carboxypropyldeoxoartemisinin

Artemisinin and its derivatives, which have been known as antimalarial drugs, have also demonstrated their cytotoxicity against tumor cells. It has been proposed that antitumor activity depends on the lipophilicity of functional group on artemisinin derivatives. Solution structures of two artemisinin derivatives as antitumor drug candidates, deoxoartemisinin and carboxypropyldeoxoartemisinin, were determined by NMR spectroscopy to elucidate structure-activity relationship. According to biological assay, antitumor efficiencies are not dependent upon lipophilicity. Instead, these compounds demonstrated their distinctive structural features of boat/chair conformation and capability to interact with receptors, as they have different efficiencies on antitumor activity. Especially, carboxypropyl moiety or carbonyl moiety in artemisinin derivatives influences the conformation and stability of ring structure. Although the detailed mechanism of antitumor activity by artemisinin derivatives has not been addressed, we suggest that antitumor activity is not determined only with lipophilicity and that artemisinin derivatives have specific target proteins in each type of cancer.     

Solid cultures of thrips-pathogenic fungi Isaria javanica strains for enhanced conidial productivity and thermotolerance

Entomopathogenic fungi have great potential to control agricultural and horticultural insect pests, however optimizing conidial production systems to demonstrate high productivity and stability still needs additional efforts for successful field application and industrialization. Although many virulent entomopathogenic fungal isolates have been viewed as potential candidates in a laboratory environment, very few of the isolates are being used in practice for application in agricultural fields as commercial products. I. javanicus is an entomopathogenic fungus that is parasitic to various diverse coleopteran and lepidopteran insects and thought good candidate as biopesticdes. In this work, the basic characteristics of two entomopathogenic fungi, I. javanica FG340 and Pf04, were investigated in morphological examinations, genetic identification, and virulence against Thrips palmi, and then the feasibility of various grains substrates for conidial production was assessed, particularly focusing on conidial productivity and thermotolerance. Isaria javanica FG340 and Pf04 conidia were solid-cultured on 12 grains for 14?days in a Petri dish. Of the tested Italian millet, perilla seed, millet and barley-based cultures showed high conidial production. The four-grain media yielded >1?×?10⁹ conidia/g of I. javanica FG340 and Pf04. Pf04 strain had enhanced thermotolerance up to 45?°C when cultured on Italian millet. In application, it was easy to make a conidial suspension using the cultured grains, and several surfactants were tested to release the conidia. This work suggests several possible inexpensive grain substrates by which to promote conidial production combined with enhanced stability against exposure to high temperature.     

ROS-Mediated ABA Signaling

In plants, reactive oxygen species (ROS) are short-lived molecules produced through various cellular mechanisms in response to biotic and abiotic stimuli. ROS function as second messengers for hormone signaling, development, oxygen deprivation, programmed cell death, and plant–pathogen interactions. Recent research on ROS-mediated responses has produced stimulating findings such as the specific sources of ROS production, molecular elements that work in ROS-mediated signaling and homeostasis, and a ROS-regulated gene network (Neill et al., Curr Opin Plant Biol 5:388–395, 2002a; Apel and Hirt, Annu Rev Plant Biol 55:373–399, 2004; Mittler et al., Trends Plant Sci 9:490–498, 2004; Mori and Schroeder, Plant Physiol 135:702–708, 2004; Kwak et al., Plant Physiol 141:323–329, 2006; Torres et al., Plant Physiol 141:373–378, 2006; Miller et al., Physiol Plant 133:481–489, 2008). In this review, we highlight new discoveries in ROS-mediated abscisic acid (ABA) signaling. Drs. Daeshik Cho and June M. Kwak are the corresponding authors for this paper.     

Effective immunotherapy of cancer by DNA vaccination.

Direct injection of naked plasmid DNA either intramuscularly or intradermally induces strong, long-lived cell-mediated and humoral immune responses to the antigen encoded by the gene vaccine. In the present study, we used gene vaccination with naked plasmid DNA to induce prophylactic immune responses to tumor associated antigens. MAGE-1 (melanoma antigen 1) is an ideal candidate for cancer vaccines because it belongs to a family of genes that are expressed in a number of human tumors of various histological types but not in normal adult tissues except for the testis, and because both humoral and cell-mediated immune responses against MAGE-1 antigen were detected in tumor patients. Intradermal administration of plasmid DNA encoding MAGE-1 (pcMAGE1) induced anti-MAGE-1-specific antibody in BALB/c mice. In contrast, no detectable level of anti-MAGE-1 antibody was induced by intramuscular injection of pcMAGE1. Also, intradermal injection of pcMAGE1 was capable of generating CTLs reactive with MAGE-1-transfected murine tumor cells, M-MSV-MAGE1. Most of the mice (8 out of 10) immunized with pcMAGE1 rejected the challenge of M-MSV-MAGE1 tumor cells, compared with control animals most of which developed tumors. This suggests that intradermal DNA vaccination could provide a novel immunotherapy of cancer.     

Hydrogen sulfide removal by immobilized Thiobacillus novellas on SiO2 in a fluidized bed reactor

The removal of hydrogen sulfide (H2S) from aqueous media was investigated using Thiobacillus novellas cells immobilized on a SiO2 carrier (biosand). The optimal growth conditions for the bacterial strain were 30 degrees C and initial pH of 7.0. The main product of hydrogen sulfide oxidation by T. novellus was identified as the sulfate ion. A removal efficiency of 98% was maintained in the three-phase fluidized-bed reactor, whereas the efficiency was reduced to 90% for the two-phase fluidized-bed reactor and 68% for the two-phase reactor without cells. The maximum gas removal capacity for the system was 254 g H2S/m3/h when the inlet H2S loading was 300 g/m3/h (1,500 ppm). Stable operation of the immobilized reactor was possible for 20 days with the inlet H2S concentration held to 1,100 ppm. The fluidized bed bioreactor appeared to be an effective means for controlling hydrogen sulfide emissions.