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This study investigated the effect of nano-Ag/TiO2 composite antibacterial agent on bacterial vaginosis and trichomonas vaginitis. Bacterial vaginosis and trichomonas vaginitis were treated with nano-Ag/TiO2 composite antibacterial agent (composite treatment group) and ornidazole suppository (traditional treatment group). Vaginal secretions from both treatment groups were analyzed to determine the effectiveness of the methods. The effective rate of the nano-Ag/TiO2 composite antibacterial agent was 97.0% for bacterial vaginosis and 94.4% for trichomonas vaginitis. The traditional treatment method, ornidazole suppository, showed an effective rate of 79.5% for bacterial vaginitis and 88.9% for trichomonas vaginitis. Therefore, the nano-Ag/TiO2 composite antibacterial agent was determined to be an effective treatment method for bacterial vaginosis and trichomonas vaginitis.

Gene regulation in embryonic stem cells (ESCs) has been extensively studied at the epigenetic-transcriptional level, but not at the posttranscriptional level. Pumilio (Pum) proteins are among the few known translational regulators required for stem-cell maintenance in invertebrates and plants. Here we report the essential function of two murine Pum proteins, Pum1 and Pum2, in ESCs and early embryogenesis. Pum1/2 double-mutant ESCs display severely reduced self-renewal and differentiation, and Pum1/2 double-mutant mice are developmentally delayed at the morula stage and lethal by embryonic day 8.5. Remarkably, Pum1-deficient ESCs show increased expression of pluripotency genes but not differentiation genes, whereas Pum2-deficient ESCs show decreased pluripotency markers and accelerated differentiation. Thus, despite their high homology and overlapping target messenger RNAs (mRNAs), Pum1 promotes differentiation while Pum2 promotes self-renewal in ESCs. Pum1 and Pum2 achieve these two complementary aspects of pluripotency by forming a negative interregulatory feedback loop that directly regulates at least 1,486 mRNAs. Pum1 and Pum2 regulate target mRNAs not only by repressing translation, but also by promoting translation and enhancing or reducing mRNA stability of different target mRNAs. Together, these findings reveal distinct roles of individual mammalian Pum proteins in ESCs and their essential functions in ESC pluripotency and embryogenesis.

This study investigated the effect of nano-Ag/TiO₂ composite antibacterial agent on bacterial vaginosis and trichomonas vaginitis. Bacterial vaginosis and trichomonas vaginitis were treated with nano-Ag/TiO₂ composite antibacterial agent (composite treatment group) and ornidazole suppository (traditional treatment group). Vaginal secretions from both treatment groups were analyzed to determine the effectiveness of the methods. The effective rate of the nano-Ag/TiO₂ composite antibacterial agent was 97.0% for bacterial vaginosis and 94.4% for trichomonas vaginitis. The traditional treatment method, ornidazole suppository, showed an effective rate of 79.5% for bacterial vaginitis and 88.9% for trichomonas vaginitis. Therefore, the nano-Ag/TiO₂ composite antibacterial agent was determined to be an effective treatment method for bacterial vaginosis and trichomonas vaginitis.

Gene regulation in embryonic stem cells (ESCs) has been extensively studied at the epigenetic-transcriptional levels, but not at the post-transcriptional levels. Pumilio (Pum) proteins are among the few known translational regulators required for stem cell maintenance in invertebrates and plants. Here we report the essential function of two murine Pum proteins, Pum1 and Pum2, in ESCs and early embryogenesis. Pum1/2 double mutants are developmentally delayed at the morula stage and lethal by embryonic day 8.5 (e8.5). Correspondingly, Pum1/2 double mutant ESCs display severely reduced self-renewal and differentiation, revealing the combined function of Pum1 and Pum2 in ESC pluripotency. Remarkably, Pum1-deficient ESCs show increased expression of pluripotency genes but not differentiation genes, indicating that Pum1 mainly promote differentiation; whereas Pum2-deficient ESCs show decreased expression of pluripotency genes and accelerated differentiation, indicating that Pum2 promotes self-renewal. Thus, Pum1 and Pum2 each uniquely contributes to one of the two complementary aspects of pluripotency. Furthermore, we show that Pum1 and Pum2 achieve ESC functions by forming a negative auto- and inter-regulatory feedback loop that directly regulates at least 1,486 mRNAs. Pum1 and Pum2 regulate target mRNAs not only by repressing translation as expected but also by promoting translation and enhancing or reducing mRNA stability of different target mRNAs. Together, these findings reveal the distinct roles of individual mammalian Pum proteins in ESCs and their collectively essential functions in ESC pluripotency and embryogenesis. Moreover, they demonstrate three novel modes of regulation of Pum proteins towards target mRNAs.SIGNIFICANCE STATEMENTThis report demonstrates the essential functions of mammalian Pumilio (Pum) proteins for embryonic stem cells (ESCs) pluripotency and embryogenesis. Moreover, it reveals the contrasting but complementary function of individual Pum proteins in regulating distinct aspects of ESC pluripotency, despite their largely overlapping expression and extremely high homology. Furthermore, it unravels a complex regulatory network in which Pum1 and Pum2 form a negative auto- and inter-regulatory feedback loop that regulates 1,486 mRNAs not only by translational repression as expected but also by promoting translation and enhancing or reducing stability of different target mRNAs, which reveals novel modes of post-transcriptional regulation mediated by Pum.

Argonaute/Piwi proteins can regulate gene expression via RNA degradation and translational regulation using small RNAs as guides. They also promote the establishment of suppressive epigenetic marks on repeat sequences in diverse organisms. In mice, the nuclear Piwi protein MIWI2 and Piwi-interacting RNAs (piRNAs) are required for DNA methylation of retrotransposon sequences and some other sequences. However, its underlying molecular mechanisms remain unclear. Here, we show that piRNA-dependent regions are transcribed at the stage when piRNA-mediated DNA methylation takes place. MIWI2 specifically interacts with RNAs from these regions. In addition, we generated mice with deletion of a retrotransposon sequence either in a representative piRNA-dependent region or in a piRNA cluster. Both deleted regions were required for the establishment of DNA methylation of the piRNA-dependent region, indicating that piRNAs determine the target specificity of MIWI2-mediated DNA methylation. Our results indicate that MIWI2 affects the chromatin state through base-pairing between piRNAs and nascent RNAs, as observed in other organisms possessing small RNA-mediated epigenetic regulation.

SummaryThe Piwi-piRNA pathway is well known for its germline function, yet its somatic role remains elusive. We show here that Piwi is required autonomously not only for germline stem cell (GSC) but also for somatic cyst stem cell (CySC) maintenance in the Drosophila testis. Reducing Piwi activity in the testis caused defects in CySC differentiation. Accompanying this, GSC daughters expanded beyond the vicinity of the hub but failed to differentiate further. Moreover, Piwi deficient in nuclear localization caused similar defects in somatic and germ cell differentiation, which was rescued by somatic Piwi expression. To explore the underlying molecular mechanism, we identified Piwi-bound piRNAs that uniquely map to a gene key for gonadal development, Fasciclin 3, and demonstrate that Piwi regulates its expression in somatic cyst cells. Our work reveals the cell-autonomous function of Piwi in both somatic and germline stem cell types, with somatic function possibly via its epigenetic mechanism.

We have used a yeast two-hybrid interaction assay to identify Asator, a tau-tubulin kinase homolog in Drosophila that interacts directly with the spindle matrix protein Megator. Using immunocytochemical labeling by an Asator-specific mAb as well as by transgenic expression of a GFP-labeled Asator construct, we show that Asator is localized to the cytoplasm during interphase but redistributes to the spindle region during mitosis. Determination of transcript levels using qRT-PCR suggested that Asator is expressed throughout development but at relatively low levels. By P-element excision, we generated a null or strong hypomorphic Asator(exc) allele that resulted in complete adult lethality when homozygous, indicating that Asator is an essential gene. That the observed lethality was caused by impaired Asator function was further supported by the partial restoration of viability by transgenic expression of Asator-GFP in the Asator(exc) homozygous mutant background. The finding that Asator localizes to the spindle region during mitosis and directly can interact with Megator suggests that its kinase activity may be involved in regulating microtubule dynamics and microtubule spindle function.

Yb regulates the proliferation of both germline and somatic stem cells in the Drosophila melanogaster ovary by activating piwi and hh expression in niche cells. In this study, we show that Yb protein is localized as discrete cytoplasmic spots exclusively in the somatic cells of the ovary and testis. These spots, which are different from all known cytoplasmic structures in D. melanogaster, are evenly electron-dense spheres 1.5 µm in diameter (herein termed the Yb body). The Yb body is frequently associated with mitochondria and a less electron-dense sphere of similar size that appears to be RNA rich. There are one to two Yb bodies/cell, often located close to germline cells. The N-terminal region of Yb is required for hh expression in niche cells, whereas the C-terminal region is required for localization to Yb bodies. The entire Yb protein is necessary for piwi expression in niche cells. A double mutant of Yb and a novel locus show male germline loss, revealing a function for Yb in male germline stem cell maintenance.

Piwi proteins associate with piRNAs and functions in epigenetic programming, post-transcriptional regulation, transposon silencing, and germline development. However, it is not known whether the diverse functions of these proteins are molecularly separable. Here we report that Piwi interacts with Tudor-SN (Tudor staphylococcal nuclease, TSN) antagonistically in regulating spermatogenesis but synergistically in silencing transposons. However, it is not required for piRNA biogenesis. TSN is known to participate in diverse molecular functions such as RNAi, degradation of hyper-edited miRNAs, and spliceosome assembly. We show that TSN colocalizes with Piwi in primordial germ cells (PGCs) and embryonic somatic cells. In adult ovaries and testes, TSN is ubiquitously expressed and enriched in the cytoplasm of both germline and somatic cells. The tsn mutants display a higher mitotic index of spermatogonia, accumulation of spermatocytes, defects in meiotic cytokinesis, a decreased number of spermatids, and eventually reduced male fertility. Germline-specific TSN-expression analysis demonstrates that this function is germline-dependent. Different from other known Piwi interters, TSN represses Piwi expression at both protein and mRNA levels. Furthermore, reducing piwi expression in the germline rescues tsn mutant phenotype in a dosage-dependent manner, demonstrating that Piwi and TSN interact antagonistically in germ cells to regulate spermatogenesis. However, the tsn deficiency has little, if any, impact on piRNA biogenesis but displays a synergistic effect with piwi mutants in transposon de-silencing. Our results reveal the biological function of TSN and its contrasting modes of interaction with Piwi in spermatogenesis, transposon silencing, and piRNA biogenesis., Author Summary Piwi proteins bind to a large class of small noncoding RNAs called Piwi-interacting RNAs (piRNAs). These proteins have emerged as major players in germline development, stem cell self-renewal, transposon silencing, and gene regulation. However, it is not known whether these functions of Piwi proteins represent separate molecular mechanisms. Furthermore, although multiple Piwi interactors have been identified, including Tudor-domain-containing proteins, none of them regulates Piwi expression or interacts with Piwi antagonistically, or only impact on a subset of Piwi functions. Here we show that Drosophila Piwi interacts with a special Tudor-domain-containing protein called Tudor-SN (Tudor staphylococcal nuclease, TSN). TSN is drastically different from the known Piwi interactors because it represses Piwi mRNA and protein expression and interacts with Piwi antagonistically in spermatogenesis but synergistically in transposon silencing. However, this interaction is not required for piRNA biogenesis. Our study represents the first demonstration that different functions of Piwi are mediated by different molecular mechanisms. In addition, this is the first in vivo study that reveals the biological function of TSN protein in an organism.

In this study we have generated two new hypomorphic Chro alleles and analyzed the consequences of reduced Chromator protein function on polytene chromosome structure. We show that in Chro71/Chro612 mutants the polytene chromosome arms were coiled and compacted with a disruption and misalignment of band and interband regions and with numerous ectopic contacts connecting non-homologous regions. Furthermore, we demonstrate that Chromator co-localizes with the JIL-1 kinase at polytene interband regions and that the two proteins interact within the same protein complex. That both proteins are necessary and may function together is supported by the finding that a concomitant reduction in JIL-1 and Chromator function synergistically reduces viability during development. Overlay assays and deletion construct analysis suggested that the interaction between JIL-1 and Chromator is direct and that it is mediated by sequences in the C-terminal domain of Chromator and by the acidic region within the C-terminal domain of JIL-1. Taken together these findings indicate that Chromator and JIL-1 interact in an interband-specific complex that functions to establish or maintain polytene chromosome structure in Drosophila.

WehaveusedimmunocytochemistrytodemonstratethattheEASTproteininDrosophila,whichformsan expandablenuclearendoskeletonatinterphase,redistributesduringmitosistocolocalizewiththespindlematrixproteins, Megator and Skeletor. EAST and Megator also colocalize to the intranuclear space surrounding the chromosomes at interphase. EAST is a novel protein that does not have any previously characterized motifs or functional domains. However, we show by immunoprecipitation experiments that EAST is likely to molecularly interact with Megator which hasalargeNH2-terminalcoiled-coildomainwiththecapacityforselfassembly.Onthebasisofthesefindings,wepropose that Megator and EAST interact to form a nuclear endoskeleton and as well are important components of the putative spindle matrix complex during mitosis. J. Cell. Biochem. 95: 1284-1291, 2005. ! 2005 Wiley-Liss, Inc.

We have used a yeast two-hybrid interaction assay to identify Chromator, a novel chromodomain containing protein that interacts directly with the putative spindle matrix protein Skeletor. Immunocytochemistry demonstrated that Chromator and Skeletor show extensive co-localization throughout the cell cycle. During interphase Chromator is localized on chromosomes to interband chromatin regions in a pattern that overlaps that of Skeletor. However, during mitosis both Chromator and Skeletor detach from the chromosomes and align together in a spindle-like structure. Deletion construct analysis in S2 cells showed that the COOH-terminal half of Chromator without the chromodomain was sufficient for both nuclear as well as spindle localization. Analysis of P-element mutations in the Chromator locus shows that Chromator is an essential protein. Furthermore, RNAi depletion of Chromator in S2 cells leads to abnormal microtubule spindle morphology and to chromosome segregation defects. These findings suggest that Chromator is a nuclear protein that plays a role in proper spindle dynamics during mitosis.

The nuage is a germline-specific perinuclear structure that remains functionally elusive. Recently, the nuage in Drosophila was shown to contain two of the three PIWI proteins – Aubergine and Argonaute 3 (AGO3) – that are essential for germline development. The PIWI proteins bind to PIWI-interacting RNAs (piRNAs) and function in epigenetic regulation and transposon control. Here, we report a novel nuage component, PAPI (Partner of PIWIs), that contains a TUDOR domain and interacts with all three PIWI proteins via symmetrically dimethylated arginine residues in their N-terminal domain. In adult ovaries, PAPI is mainly cytoplasmic and enriched in the nuage, where it partially colocalizes with AGO3. The localization of PAPI to the nuage does not require the arginine methyltransferase dPRMT5 or AGO3. However, AGO3 is largely delocalized from the nuage and becomes destabilized in the absence of PAPI or dPRMT5, indicating that PAPI recruits PIWI proteins to the nuage to assemble piRNA pathway components. As expected, papi deficiency leads to transposon activation, phenocopying piRNA mutants. This further suggests that PAPI is involved in the piRNA pathway for transposon silencing. Moreover, AGO3 and PAPI associate with the P body component TRAL/ME31B complex in the nuage and transposon activation is observed in tral mutant ovaries. This suggests a physical and functional interaction in the nuage between the piRNA pathway components and the mRNA-degrading P-body components in transposon silencing. Overall, our study reveals a function of the nuage in safeguarding the germline genome against deleterious retrotransposition via the piRNA pathway.

Cytogenetic features of three ascidian species were studied with specimens obtained from Korean coastal areas. The gonadal tissue was processed in order to obtain mitotic and meiotic chromosomes. The chromosome numbers of Amaroucium pliciferum turned out to be 2n=26 and n=13 with 2sm, 3st, 8t and Halocynthia roretzi to be 2n=16 and n=8 with 6m, 2t. Dipioid number of chromosomes in Dendrodoa aggregata was 64 with 32t. The karyotypes of these Korean ascidians were reported for the first time.

We have used immunocytochemistry and cross-immunoprecipitation analysis to demonstrate that Megator (Bx34 antigen), a Tpr ortholog in Drosophila with an extended coiled-coil domain, colocalizes with the putative spindle matrix proteins Skeletor and Chromator during mitosis. Analysis of P-element mutations in the Megator locus showed that Megator is an essential protein. During interphase Megator is localized to the nuclear rim and occupies the intranuclear space surrounding the chromosomes. However, during mitosis Megator reorganizes and aligns together with Skeletor and Chromator into a fusiform spindle structure. The Megator metaphase spindle persists in the absence of microtubule spindles, strongly implying that the existence of the Megator-defined spindle does not require polymerized microtubules. Deletion construct analysis in S2 cells indicates that the COOH-terminal part of Megator without the coiled-coil region was sufficient for both nuclear as well as spindle localization. In contrast, the NH2-terminal coiled-coil region remains in the cytoplasm; however, we show that it is capable of assembling into spherical structures. On the basis of these findings we propose that the COOH-terminal domain of Megator functions as a targeting and localization domain, whereas the NH2-terminal domain is responsible for forming polymers that may serve as a structural basis for the putative spindle matrix complex.

To study the relationship between HLA-DRB1 alleles and idiopathic thrombocytopenic purpura (ITP) in children.PCR-SSO was used to identify DRB1 alleles of 42 children with ITP. Among them, anti-GPIIb/IIIa and anti-GPIb/IX autoantibody were detected in 36 cases by modified monoclonal antibody specific immobilization of platelet antigens (MAIPA).(1) Compared with healthy controls, HLA-DRB1 * 17 was significantly increased (relative risk = 2.76, P0.05, etiologic factor = 0.106 4) and HLA-DRB1 * 1202 decreased (relative risk = 0.20, P0.025, prophylactic factor = 0.761 6) in children with ITP. (2) In comparison with patients with good response to steroids and IgG therapy, HLA-DRB1 * 11 was significantly increased (P0.025) in patients with a poor response, furthermore, most (5/6) of HLA-DRB1 * 11-positive patients were female teen-ager. (3) Twenty-seven patients (75%) had anti-GPIIb/IIIa and seventeen (47.22%) had anti-GPIb/IX autoantibodies, the positivity rates of both anti-GPIIb/IIIa (P = 0.02) and anti-GPIb/IX (P = 0.01) were associated with HLA-DRB1 * 02. However, the pos./itivity rates of autoantibodies between refractory and non-refractory patients showed no significant difference.(1) The DRB1 * 17 seems to predict susceptibility to ITP in children, while DRB1 * 1202 appears to be protective to against ITP. (2) The DRB1 * 11 plays an important role in resistance to steroid and IgG therapy in children with ITP. (3) It seems that the response to the antigenic epitope of GPIIb/IIIa and GPIb/IX is restricted by DRB1 * 02, while the presence of the autoantibodies couldn't predict prognosis. Our preliminary findings indicate that genetic factors influence the clinical course of ITP, but its exact mechanism needs to be further investigated.

To gain an insight into the relations between human leukocyte antigen-DRB1 (HLA-DRB1) alleles and idiopathic thrombocytopenic purpura (ITP) in children.Polymerase chain reaction-sequence specific oligonucleotide (PCR-SSO) was used to identify DRB1 alleles of 42 children with ITP. Among them, 36 were identified for anti-GPIIb/IIIa and anti-GPIb/Ix autoantibody by modified monoclonal antibody specific immobilization of platelet antigens.Compared with health controls, the frequency of HLA-DRB1*17 significantly increased (P0.05, relative risk=2.76, etiologic factor=0.1064) and the frequency of HLA-DRB1*1202 significantly decreased (P0.025, relative risk=0.20, prophylactic factor=0.7616) in children with ITP. In comparison with patients of good response to steroids and IVIgG therapy, the frequency of HLA DRB1*11 significantly increased (Chi-square=6.091, P0.025) in patients with a poor response, furthermore, the most of HLA-DRB1*11 positive patients were female teen-agers. Twenty-seven patients (75%) had anti GPIIb/IIIa and seventeen (47.22%) had anti_GPIb/Ix autoantibodies. The positivities of both anti_GP IIb/IIIa (P=0.02) and anti-GPIb/Ix (P=0.01) were associated with HLA-D RB1*02. However, the positivity of autoantibodies between refractory and non-refractory patients showed no significant difference.The allele of HLA-DRB1*17 seems to predict susceptibility of ITP in children, while HLA-DRB1*1202 appears to be protective to ITP. The allele of HLA DRB1*11 plays an important role in resistance to steroid and IgG therapy in children with ITP. It seems that the response to the antigenic epitope of GPIIb/IIIa and GPIb/Ix is restricted by HLA-DRB1*02, while the presence of the antibodies could not predict prognosis. In conclusion, the above preliminary findings indicate that genetic factors influence the clinical course of ITP, but the exact mechanism needs to be investigated further.

The chromosome numbers of two sea anemones, Anthopleura midori and A. kurogane, were determined to be 2n=18 in both by the air‐drying method. The chromosomes of A. midori are all telocentric, while the first pair of chromosomes of A. kurogane is subtelocentric and the remainders are all telocentric.