A Survey of Cryptosporidium Oocysts in Water Supplies during a 10-Year Period (2000-2009) in Seoul

This study has been conducted to estimate the occurrence of Cryptosporidium oocysts in water supplies in the Metropolitan area of Seoul, South Korea, for 10 years from 2000 to 2009. Water samples were collected quarterly at 6 intakes in the Han River and its largest stream and 6 conventional Water Treatment Plants (WTPs) serving drinking water for 10 million people of Seoul. Cryptosporidium oocysts were found in 22.5% of intake water samples and arithmetic mean was 0.65 oocysts/10 L (range 0-22 oocysts/10 L). Although the annual mean of oocyst number was as low as 0.04-1.90 oocysts/10 L, 3 peaks in 2004 and 2007 were observed and the pollution level was a little higher in winter. The lowest density was observed at Paldang intake and the pollution level increased at Kuui and Jayang intakes. At the end of the largest stream, oocysts were found in 70% of collected samples (mean 5.71 oocysts/10 L) and it seemed that its joining the Han River resulted in the increase at Kuui intake and downstream. Oocyst removal by physical process exceeded 2.0-2.3 log and then all finished water samples collected at 6 WTPs were negative for Cryptosporidium in each 100 L sample for 10 years. These results suggested that domestic wastewater from the urban region could be a source of Cryptosporidium pollution and separating sewage systems adjacent to the intakes could be meaningful for some intakes having weakness related to parasitological water quality.     

Suppressed CD31 Expression in Sarcoma-180 Tumors after Injection with Toxoplasma gondii Lysate Antigen in BALB/c Mice

The anti-tumorigenic effects of Toxoplasma gondii (RH) antigens were studied in a murine sarcoma-180 tumor model. To determine the anti-tumor effects, the reduction in tumor size and expression of CD31 (an angiogenesis marker in the tumor tissue) were examined after injection of BALB/c mice with T. gondii lysate antigen (TLA) or formalin-fixed, proliferation-inhibited, T. gondii tachyzoites. Tumors were successfully produced by an intradermal injection of sarcoma-180 cells with plain Matrigel in the mid-backs of mice. After injection with TLA or formalin-fixed T. gondii tachyzoites, the increase in tumor size and weight nearly stopped while tumor growth continued in control mice that were injected with PBS. CD31 expression in TLA-treated or formalin-fixed T. gondii-injected mice was lower than the control mice. Accordingly, the present study shows that the treatment of mice with formalin-fixed T. gondii or TLA in the murine sarcoma-180 tumor model results in a decrease of both tumor size and CD31 expression.     

Differential Auxin-Transporting Activities of PIN-FORMED Proteins in Arabidopsis Root Hair Cells

The Arabidopsis (Arabidopsis thaliana) genome includes eight PIN-FORMED (PIN) members that are molecularly diverged. To comparatively examine their differences in auxin-transporting activity and subcellular behaviors, we expressed seven PIN proteins specifically in Arabidopsis root hairs and analyzed their activities in terms of the degree of PIN-mediated root hair inhibition or enhancement and determined their subcellular localization. Expression of six PINs (PIN1–PIN4, PIN7, and PIN8) in root hair cells greatly inhibited root hair growth, most likely by lowering auxin levels in the root hair cell by their auxin efflux activities. The auxin efflux activity of PIN8, which had not been previously demonstrated, was further confirmed using a tobacco (Nicotiana tabacum) cell assay system. In accordance with these results, those PINs were localized in the plasma membrane, where they likely export auxin to the apoplast and formed internal compartments in response to brefeldin A. These six PINs conferred different degrees of root hair inhibition and sensitivities to auxin or auxin transport inhibitors. Conversely, PIN5 mostly localized to internal compartments, and its expression in root hair cells rather slightly stimulated hair growth, implying that PIN5 enhanced internal auxin availability. These results suggest that different PINs behave differentially in catalyzing auxin transport depending upon their molecular activity and subcellular localization in the root hair cell.Auxin plays a critical role in plant development and growth by forming local concentration gradients. Local auxin gradients, created by the polar cell-to-cell movement of auxin, are implicated in primary axis formation, root meristem patterning, lateral organ formation, and tropic movements of shoots and roots (for recent review, see Vanneste and Friml, 2009). The cell-to-cell movement of auxin is achieved by auxin influx and efflux transporters such as AUXIN-RESISTANT1 (AUX1)/LIKE-AUX1 for influx and PIN-FORMED (PIN) and the P-glycoprotein (PGP) of ABCB (ATP-binding cassette-type transporter subfamily B) for efflux. Since diffusive efflux of the natural auxin indole-3-acetic acid (IAA; pKa = 4.75) is not favorable and PINs are localized in the plasma membrane in a polar manner, PINs act as rate-limiting factors for cellular auxin efflux and polar auxin transport through the plant body. These PINs'' properties explain why representative physiological effects of auxin transport are associated with PINs.Auxin flows from young aerial parts all the way down to the root tip columella in which an auxin maximum is formed for root stem cell maintenance and moves up toward the root differentiation zone through root epidermal cells, where a part of it travels back to the root tip via cortical cells (Blilou et al., 2005). This directional auxin flow is supported by the polar localization of PINs: PIN1, PIN3, and PIN7 at the basal side of stele cells (Friml et al., 2002a, 2002b; Blilou et al., 2005), PIN4 at the basal side in root stem cells (Friml et al., 2002a), and PIN2 at the upper side of root epidermis and at the basal side of the root cortex (Luschnig et al., 1998; Müller et al., 1998). Another interesting aspect of PIN-mediated auxin transport is the dynamics in directionality of auxin flow due to environmental stimuli-directed changes of subcellular PIN polarity, as exemplified for PIN3, whose subcellular localization changes in response to the gravity vector (Friml et al., 2002b).An intriguing question is how different PIN proteins have different subcellular polarities, which might be attributable to PIN-specific molecular properties, cell-type-specific factors, or both. The different PIN subcellular polarities in different cell types seemingly indicate that cell-type-specific factors are involved in polarity. In the case of PIN1, however, both classes of factors appear to affect its subcellular localization because when expressed under the PIN2 promoter, PIN1 localizes to the upper or basal side of root epidermal cells, depending on the GFP insertion site of the protein (Wiśniewska et al., 2006). A recent study demonstrated that the polar targeting of PIN proteins is modulated by phosphorylation/dephosphorylation of the central hydrophilic loop of PINs, which is mediated by PINOID (PID; a Ser/Thr protein kinase)/PP2A phosphatase (Michniewicz et al., 2007). The central hydrophilic domain of PINs might provide the molecule-specific cue for PIN polarity, together with as yet unknown cell-specific factors. Different recycling behaviors of PINs, which show variable sensitivities to brefeldin A (BFA), also imply different molecular characters among PIN species. Most PIN1 proteins are internalized by BFA treatment, whereas considerable amounts of PIN2 remain in the plasma membrane in addition to internal accumulation after BFA treatment. Recycling and basal polar targeting of PIN1 is dependent on the BFA-sensitive guanine nucleotide exchange factor for adenosyl ribosylation factors (ARF GEFs), GNOM, which is the major target of BFA. In contrast, apical targeting and recycling of PIN2 is independent of GNOM and controlled by BFA-resistant ARF GEFs (Geldner et al., 2003; Kleine-Vehn and Friml, 2008).In contrast to their distinct subcellular localizations, the differential auxin-transporting activities of PINs remain to be studied. The divergent primary structures of PIN proteins are not only indicative of differential subcellular polarity, but also would represent their differential catalytic activities for auxin transport. The auxin efflux activities of Arabidopsis (Arabidopsis thaliana) PINs have been demonstrated using Arabidopsis and heterologous systems: PIN1 and PIN5 in Arabidopsis cells (Petrásek et al., 2006; Mravec et al., 2009); PIN2, PIN3, PIN4, PIN6, and PIN7 in tobacco (Nicotiana tabacum) Bright Yellow-2 (BY-2) cells (Lee and Cho, 2006; Petrásek et al., 2006; Mravec et al., 2008); PIN1, PIN2, PIN5, and PIN7 in yeast (Saccharomyces cerevisiae) cells (Petrásek et al., 2006; Blakeslee et al., 2007; Mravec et al., 2009; Yang and Murphy, 2009); and PIN1, PIN2, and PIN7 in HeLa cells (Petrásek et al., 2006; Blakeslee et al., 2007). Among the eight Arabidopsis PIN members, PIN1, PIN2, PIN3, PIN4, PIN6, and PIN7, which share a similar molecular structure in terms of the presence of a long central loop (hereafter called long-looped PINs; Fig. 1A; Supplemental Fig. S1), have been shown to catalyze auxin efflux at the cellular level. On the other hand, PIN5 and PIN8 possess a very short putative central loop (hereafter called short-looped PINs). Although PIN5 was recently shown to be localized in the endoplasmic reticulum (ER) and proposed to transport auxin metabolites into the ER lumen, its cellular function regarding its intracellular auxin-transporting activity has not been shown, and the auxin-transporting activity of PIN8 has yet to be demonstrated. In spite of the same transport directionality (auxin efflux) and similar molecular structures, the long-looped PINs exhibit sequence divergence not only in their central loop, but also in certain residues of the transmembrane domains. This structural divergence of long-looped PINs might be indicative of their differential auxin-transporting activities, which have not yet been quantitatively compared.Open in a separate windowFigure 1.Differential activities of PINs in the Arabidopsis root hair. A, Two distinctive PIN groups with different central hydrophilic loop sizes. Topology of PIN proteins was predicted by four different programs as described in Supplemental Figure S1. Numbers above indicate the number of transmembrane helices for each N- and C-terminal region, and numbers below indicate the number of amino acid residues of the central hydrophilic domain. B, Representative root images of control (Cont; Columbia-0) and root-hair-specific PIN-overexpressing (PINox; ProE7:PIN-GFP or ProE7:PIN [−]) plants. Bar = 100 μm for all. C, Root hair lengths of control and PINox plants. Six to 12 independent transgenic lines (average = 8.3), and 42 to 243 roots (average = 86.8) and 336 to 2,187 root hairs (average = 727.8) per construct, were observed for the estimation of root hair length. Data represent means ± se. The root hair lengths of PIN5ox lines were significantly longer than those of the control (P = 0.016 for PIN5ox; P < 0.0001 for PIN5-GFP1ox and PIN5-GFP2ox).To comparatively assess the cytological behaviors and molecular activities of different PIN members, it would be favorable to use a single assay system that provides a consistent cellular environment and enables quantitative estimation of PIN activity. In previous studies, we adopted the root hair single cell system to quantitatively assay auxin-transporting or regulatory activities of PINs, PGPs, AUX1, and PID (Lee and Cho, 2006; Cho et al., 2007a). Root hair growth is proportional to internal auxin levels in the root hair cell. Therefore, auxin efflux inhibits and auxin influx enhances root hair growth (Cho et al., 2007b; Lee and Cho, 2008). In addition, the use of a root-hair-specific promoter (Cho and Cosgrove, 2002; Kim et al., 2006) for expression of auxin transporters enables the transporters'' biological effect to be pinpointed to only the root hair cell, thus excluding probable non-cell-autonomous effects that could be caused by the general expression of auxin transporters.In this study, we expressed five long-looped PINs (PIN1, PIN2, PIN3, PIN4, and PIN7) and two short-looped PINs (PIN5 and PIN8) in root hair cells and compared their auxin-transporting activities and cytological dynamics. To directly measure the radiolabeled auxin-transporting activities of PIN5 and PIN8, we used an additional assay system, tobacco suspension cells. Our data revealed that PINs have differential molecular activities and pharmacological responses and that the short-looped and long-looped PINs have different subcellular localizations.     

The C-terminal zinc finger domain of Arabidopsis cold shock domain proteins is important for RNA chaperone activity during cold adaptation

Among the four cold shock domain proteins (CSDPs) identified in Arabidopsis thaliana, it has recently been shown that CSDP1 harboring seven CCHC-type zinc fingers, but not CSDP2 harboring two CCHC-type zinc fingers, function as a RNA chaperone during cold adaptation. However, the structural features relevant to this differing RNA chaperone activity between CSDP1 and CSDP2 remain largely unknown. To determine which structural features are necessary for the RNA chaperone activity of the CSDPs, the importance of the N-terminal cold shock domain (CSD) and the C-terminal zinc finger glycine-rich domains of CSDP1 and CSDP2 were assessed. The results of sequence motif-swapping and deletion experiments showed that, although the CSD itself harbored RNA chaperone activity, the number and length of the zinc finger glycine-rich domains of CSDPs were crucial to the full activity of the RNA chaperones. The C-terminal domain itself of CSDP1, harboring seven CCHC-type zinc fingers, also has RNA chaperone activity. The RNA chaperone activity and nuclei acid-binding property of the native and chimeric proteins were closely correlated with each other. Collectively, these results indicate that a specific modular arrangement of the CSD and the zinc finger domain determines both the RNA chaperone activity and nucleic acid-binding property of CSDPs; this, in turn, contributes to enhanced cold tolerance in plants as well as in bacteria.     

An Arabidopsis F-box protein regulates tapetum degeneration and pollen maturation during anther development

The Arabidopsis anther has a bilateral symmetry with four lobes, each consisting of four distinct layers of somatic cells from the outer to inner side: epidermis, endothecium, middle layer and tapetum. The tapetum is a layer of cells comprising the inner surface of the pollen wall. It plays an important role in anther development by providing enzymes, materials and nutrients required for pollen maturation. Genes and molecular mechanisms underlying tapetum formation and pollen wall biosynthesis have been studied in Arabidopsis. However, tapetum degeneration and anther dehiscence have not been well characterized at the molecular level. Here, we report that an Arabidopsis gene, designated reduced male fertility (RMF), regulates degeneration of tapetum and middle layer during anther development. The Arabidopsis dominant mutant rmf-1D overexpressing the RMF gene exhibited pleiotropic phenotypes, including dwarfed growth with small, dark-green leaves and low male fertility. Tapetum development and subsequent degeneration were impaired in the mutant. Accordingly, pollen maturation was disturbed, reducing the male fertility. In contrast, tapetum degeneration was somewhat accelerated in the RMF RNAi plants. The RMF gene was expressed predominantly in the anther, particularly in the pollen grains. Notably, the RMF protein contains an F-box motif and is localized to the nucleus. It physically interacts with the Arabidopsis-Skp1-like1 protein via the F-box motif. These observations indicate that the RMF gene encodes an F-box protein functioning in tapetum degeneration during anther development.     

Spatiotemporal Expression of tmie in the Inner Ear of Rats during Postnatal Development

The circling (cir/cir) mouse is a murine model for human nonsyndromic deafness DFNB6. Transmembrane inner ear (tmie) is the causative gene and its mutation through deletion of a 40-kilobase genomic region including tmie leads to deafness. The function of Tmie is unknown. To better understand the function of Tmie, we focused on the spatiotemporal expression of tmie in the rat cochlea by using a Tmie-specific antibody. Results showed that tmie expression was prominent in early postnatal rat cochleas in the stereocilia bundles of hair cells. The Tmie signal spread from the stereocilia to the hair cell body region and on to organ of Corti cells. No Tmie signal was observed in cell nuclei; Tmie was localized to the cytoplasm. Because Tmie is predicted to have 1 or 2 transmembrane domains, we postulate that it is localized to membrane-based organelles or the plasma membrane. Our results imply that Tmie exists in the cytoplasm and may have a key role in the maturation and structure of stereocilia bundles in developing hair cells. After hair cell maturation, Tmie is thought to be involved in the maintenance of organ of Corti cells.Circling is often observed in mouse and rat deafness mutants and is commonly suggested to be a consequence of inner ear defects that impair vestibular systems.^3,12,14 The circling (cir/cir) mouse is a murine model for human nonsyndromic deafness DFNB6; these mice have abnormal circling behavior, suggesting a balance disorder, and profound deafness.^6,7 The most notable pathologic phenotypes of circling mice are the almost completely degenerated cochlea and remarkably reduced cellularity in spiral ganglion neurons. The causative gene for circling is transmembrane inner ear (tmie), with a 40-kilobase genomic deletion including tmie.¹ tmie is also the causative gene of the spinner (sr/sr) mouse, which has phenotypes similar to circling mice, although the mutation patterns are different.⁸ Spinner mice also show circling behavior, hearing loss, imbalance, and swimming inability. In addition, spinner mice have 2 mutations in the tmie gene: the 40-kb genomic deletion including tmie and a point mutation that leads to a truncated protein.⁸In humans, 7 different homozygous recessive mutations in TMIE currently are known to exist in affected members of consanguineous families segregating severe-to-profound prelingual deafness, consistent with linkage to DFNB6.^9,10 Although the functions of murine Tmie and human TMIE are unknown, this protein appears to be important for normal hearing and vestibular function.In a previous study, we produced transgenic mice overexpressing tmie that resulted in phenotypic rescue of circling.¹¹ Normal expression of transgenic tmie induced phenotypic rescue in circling homozygous mutants, although some mice did not show amelioration of abnormal behavior, hearing ability, or tissue morphology in the inner ear. Therefore the Tmie protein is required for normal inner ear function in mouse.¹¹To better understand the function of Tmie, we focused on the spatiotemporal expression of tmie. Knowing when, where, and to what extent this protein is produced in the developing inner ear will provide important clues to protein function. In adult mouse and rat, tmie is expressed in various tissues.^2,13 Whether Tmie plays an important role in those tissues is uncertain, because circling mice that lack the entire tmie gene have no noteworthy problems in any tissues except those of the inner ear systems.⁶In this study, we were interested in the postnatal stages before and after the onset of hearing (around postnatal day [P] 12) in rats; therefore, the postnatal period P0 to19 was studied. Although all the cells that form the mature cochlea are present at birth, important conformational changes occur during this period, including the formation of the tunnel of Corti and the establishment or retraction of neuronal connections. The expression pattern of tmie in the developing inner ear during early postnatal development has not been investigated previously. Here we document our use of a Tmie-specific antibody to elucidate the spatial and temporal expression of tmie in the rat inner ear during postnatal development.     

Differential immunogenicity and clinical relevance of kidney compartment specific antigens after renal transplantation

To evaluate the pathogenic role of non-HLA antibodies after organ transplantation, 81 unique serum samples from renal transplant patients were analyzed by protein array technology on integrative genomics approach (Li, L.; et al. Proc. Natl. Acad. Sci. U.S.A. 2009, 106 (11), 4148-53; Higgins, J. P.; et al. Mol. Biol. Cell 2004, 15 (2), 649-56), validated by ELISA, and the results correlated with clinical relevance with time post-transplantation or post-transplant graft function. There was a significant association of de novo non-HLA antibodies with time post-transplantation (n = 1,785) and decline in graft function over the subsequent year (n = 105). There was an enrichment of immunogenic antigens in the renal cortex (p = 0.01) with post-transplant time, and for glomerular specific targets (p = 0.02) with decline in graft function. Two targets with very strong correlation in each category (AGT and SPDYA) were validated by customized ELISA assays in independent patient sera and their localization confirmed by immunohistochemistry. In conclusion, defined profiles of these non-HLA antibodies to renal cortical proteins develop with increasing length of engraftment, and may reflect the increasing recognition of altered localization or exposure of renal tubular and interstitial proteins, affected by advancing chronic nonimmune graft injury. The panel of non-HLA antibodies to glomerular targets is most interesting, as these corresponding antigenic targets may be important pathways in functional graft injury and could provide novel targets for drug design.