A preliminary X-ray study of a refolded PTS EIIBfruc protein from Escherichia coli

The phosphoenolpyruvate-carbohydrate phosphotransferase system (PTS) catalyzes the phosphorylation and transportation of its sugar substrates. A sugar-specific enzyme II complex involved in the PTS finally functions to translocate substrates across the membrane. A PTS EIIB(fruc) protein, a fructose specific EIIB subunit, from Escherichia coli has been cloned, expressed, refolded, purified, and crystallized. The synchrotron data were collected to 2.6 A from the crystal of a selenomethionine substitute PTS EIIB(fruc) protein. The crystal belongs to the primitive trigonal space group P3(1)21, with unit-cell parameters of a = 33.4 A, b = 33.4 A, c = 154.0 A, and beta = 120.0 degrees . A full structure determination is under way to provide insights into the structure-function relationships of this protein.     

Root-tip diameters of woody species in subalpine Abies forest

We clarified the differences in root-tip diameter (RTD) among tree and shrub species in an Abies forest. To evaluate the effects of sampling month and tree size on RTD, we measured the root-tip diameters of mature individuals of nine woody species and sapling individuals of two Abies species in a subalpine Abies forest on Mount Shimagare in central Japan. Species, sampling month, and their interaction affected RTD; however, the differences in RTD between some pairs of species were consistent across sampling months. The woody species fell into two groups, based on RTD size: tree species with larger RTDs (group 1) and shrub species with smaller RTDs (group 2). Seasonal changes in RTD were observed in three species and showed different patterns among species. Tree size did not affect RTD for either Abies species; however, there was an interaction between tree size and sampling month for Abies veitchii. The woody species category had the greatest effect on RTD, followed by sampling month and then tree size.     

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.     

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.     

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.     

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.     

Epstein-Barr virus-encoded latent membrane protein 1 impairs G2 checkpoint in human nasopharyngeal epithelial cells through defective Chk1 activation

Nasopharyngeal carcinoma (NPC) is a common cancer in Southeast Asia, particularly in southern regions of China. EBV infection is closely associated with NPC and has long been postulated to play an etiological role in the development of NPC. However, the role of EBV in malignant transformation of nasopharyngeal epithelial cells remains enigmatic. The current hypothesis of NPC development is that premalignant nasopharyngeal epithelial cells harboring genetic alterations support EBV infection and expression of EBV genes induces further genomic instability to facilitate the development of NPC. The latent membrane protein 1 (LMP1) is a well-documented EBV-encoded oncogene. The involvement of LMP1 in human epithelial malignancies has been implicated, but the mechanisms of oncogenic actions of LMP1, particularly in nasopharyngeal cells, are unclear. Here we observed that LMP1 expression in nasopharyngeal epithelial cells impaired G2 checkpoint, leading to formation of unrepaired chromatid breaks in metaphases after γ-ray irradiation. We further found that defective Chk1 activation was involved in the induction of G2 checkpoint defect in LMP1-expressing nasopharyngeal epithelial cells. Impairment of G2 checkpoint could result in loss of the acentrically broken chromatids and propagation of broken centric chromatids in daughter cells exiting mitosis, which facilitates chromosome instability. Our findings suggest that LMP1 expression facilitates genomic instability in cells under genotoxic stress. Elucidation of the mechanisms involved in LMP1-induced genomic instability in nasopharyngeal epithelial cells will shed lights on the understanding of role of EBV infection in NPC development.