Recombinant Human Soluble Thrombomodulin Suppresses Arteritis in a Mouse Model of Kawasaki Disease.

INTRODUCTION AND OBJECTIVE: Kawasaki disease (KD) is associated with diffuse and systemic vasculitis of unknown aetiology and primarily affects infants and children. Intravenous immunoglobulin (IVIG) treatment reduces the risk of developing coronary aneurysms, but some children have IVIG-resistant KD, which increases their risk of developing coronary artery injury. Here, we investigated the effect of recombinant human soluble thrombomodulin (rTM), which has anticoagulant, anti-inflammatory, and cytoprotective properties on the development of coronary arteritis in a mouse model of vasculitis. METHODS: An animal model of KD-like vasculitis was created by injecting mice with Candida albicans water-soluble fraction (CAWS). This model was used to investigate the mRNA expression of interleukin (IL)-10, tumour necrosis factor alpha (TNF-α), and tissue factor (TF), in addition to histopathology of heart tissues. RESULTS: rTM treatment significantly reduces cardiac vascular endothelium hypertrophy by 34 days after CAWS treatment. In addition, mRNA expression analysis revealed that rTM administration increased cardiac IL-10 expression until day 27, whereas expression of TNF-α was unaffected. Moreover, in the spleen, rTM treatment restores IL-10 and TF expression to normal levels. CONCLUSION: These findings suggest that rTM suppresses CAWS-induced vasculitis by upregulating IL-10. Therefore, rTM may be an effective treatment for KD.

CDK-dependent complex formation between replication proteins Dpb11, Sld2, Pol (epsilon}, and GINS in budding yeast.

Eukaryotic chromosomal DNA replication requires cyclin-dependent kinase (CDK) activity. CDK phosphorylates two yeast replication proteins, Sld3 and Sld2, both of which bind to Dpb11 when phosphorylated. These phosphorylation-dependent interactions are essential and are the minimal requirements for CDK-dependent activation of DNA replication. However, how these interactions activate DNA replication has not been elucidated. Here, we show that CDK promotes the formation of a newly identified fragile complex, the preloading complex (pre-LC) containing DNA polymerase epsilon (Pol epsilon), GINS, Sld2, and Dpb11. Formation of the pre-LC requires phosphorylation of Sld2 by CDK, but is independent of DNA replication, protein association with replication origins, and Dbf4-dependent Cdc7 kinase, which is also essential for the activation of DNA replication. We also demonstrate that Pol epsilon, GINS, Dpb11, and CDK-phosphorylated Sld2 form a complex in vitro. The genetic interactions between Pol epsilon, GINS, Sld2, and Dpb11 suggest further that they form an essential complex in cells. We propose that CDK regulates the initiation of DNA replication in budding yeast through formation of the pre-LC.

6-(4-Amino-2-butyl-imidazoquinolyl)-norleucine: Toll-like receptor 7 and 8 agonist amino acid for self-adjuvanting peptide vaccine.

Generally, small peptides by themselves are weak to induce antibody responses. Toll-like receptor (TLR) ligands are attractive candidates of vaccine adjuvants to improve their antigenicity. The covalent conjugation of TLR ligands with antigens to produce self-adjuvanting peptide vaccine is a promising approach. Based on the structure of TLR7/8 ligands, a series of synthetic amino acids 6-imidazoquinolyl-norleucines were synthesized, wherein an imidazoquinoline structure as the TLR7/8 agonistic pharmacophores was constructed on the ε-NH2 group of Lys. Of them, 6-(4-amino-2-butyl-imidazoquinolyl)-norleucine showed the most potent TLR7 and TLR8 agonistic activities with EC50 values of 8.55 and 106 µM, respectively. Subsequently, mice were immunized with the influenza A virus M2e antigen mixed with or covalently conjugated to the TLR7/8 agonist amino acid, which led to induction of M2e specific antibody productions in the absence of other adjuvant. We successfully developed a novel efficient tool for self-adjuvanting peptide vaccines targeting TLR7/8.

CDK-dependent phosphorylation of Sld2 and Sld3 initiates DNA replication in budding yeast.

In eukaryotic cells, cyclin-dependent kinases (CDKs) have an important involvement at various points in the cell cycle. At the onset of S phase, active CDK is essential for chromosomal DNA replication, although its precise role is unknown. In budding yeast (Saccharomyces cerevisiae), the replication protein Sld2 (ref. 2) is an essential CDK substrate, but its phospho-mimetic form (Sld2-11D) alone neither affects cell growth nor promotes DNA replication in the absence of CDK activity, suggesting that other essential CDK substrates promote DNA replication. Here we show that both an allele of CDC45 (JET1) and high-copy DPB11, in combination with Sld2-11D, separately confer CDK-independent DNA replication. Although Cdc45 is not an essential CDK substrate, CDK-dependent phosphorylation of Sld3, which associates with Cdc45 (ref. 5), is essential and generates a binding site for Dpb11. Both the JET1 mutation and high-copy DPB11 by-pass the requirement for Sld3 phosphorylation in DNA replication. Because phosphorylated Sld2 binds to the carboxy-terminal pair of BRCT domains in Dpb11 (ref. 4), we propose that Dpb11 connects phosphorylated Sld2 and Sld3 to facilitate interactions between replication proteins, such as Cdc45 and GINS. Our results demonstrate that CDKs regulate interactions between BRCT-domain-containing replication proteins and other phosphorylated proteins for the initiation of chromosomal DNA replication; similar regulation may take place in higher eukaryotes.

Mitotic progression and dual spindle formation caused by spindle association of de novo-formed microtubule-organizing centers in parthenogenetic embryos of Drosophila ananassae.

Facultative parthenogenesis occurs in many animal species that typically undergo sexual reproduction. In Drosophila, such development from unfertilized eggs involves diploidization after completion of meiosis, but the exact mechanism remains unclear. Here we used a laboratory stock of Drosophila ananassae that has been maintained parthenogenetically to cytologically examine the initial events of parthenogenesis. Specifically, we determined whether the requirements for centrosomes and diploidization that are essential for developmental success can be overcome. As a primal deviation from sexually reproducing (i.e. sexual) strains of the same species, free asters emerged from the de novo formation of centrosome-like structures in the cytosol of unfertilized eggs. Those microtubule-organizing centers had distinct roles in the earliest cycles of parthenogenetic embryos with respect to mitotic progression and arrangement of mitotic spindles. In the first cycle, an anastral bipolar spindle self-assembled around a haploid set of replicated chromosomes. Participation of at least one microtubule-organizing center in the spindle was necessary for mitotic progression into anaphase. In particular, the first mitosis involving a monastral bipolar spindle resulted in haploid daughter nuclei, one of which was associated with a microtubule-organizing center whereas the other was not. Remarkably, in the following cycle, biastral and anastral bipolar spindles formed that were frequently arranged in tandem by sharing an aster with bidirectional connections at their central poles. We propose that, for diploidization of haploid nuclei, unfertilized parthenogenetic embryos utilize dual spindles during the second mitosis, as occurs for the first mitosis in normal fertilized eggs.

Inhibitory effect of traditional herbal (kampo) medicines on the replication of human parainfluenza virus type 2 in vitro.

Thirteen herbal medicines, Kakkonto (TJ-001), Kakkontokasenkyushin'i (TJ-002), Hangekobokuto (TJ-016), Shoseiryuto (TJ-019), Maoto (TJ-027), Bakumondoto (TJ-029), Hochuekkito (TJ-041), Goshakusan (TJ-063), Kososan (TJ-070), Chikujountanto (TJ-091), Gokoto (TJ-095), Saibokuto (TJ-096), and Ryokankyomishingeninto (TJ-119) were tested for human parainfluenza virus type 2 (hPIV-2) replication. Eight (TJ-001, TJ-002, TJ-019, TJ-029, TJ-041, TJ-063, TJ-095 and TJ-119) out of the thirteen medicines had virus growth inhibitory activity. TJ-001 and TJ-002 inhibited virus release, and largely inhibited genome, mRNA and protein syntheses. TJ-019 slightly inhibited virus release, inhibited gene and mRNA syntheses, and largely inhibited protein synthesis. TJ-029 slightly inhibited virus release, largely inhibited protein synthesis, but gene and mRNA syntheses were unaffected. TJ-041 only slightly inhibited virus release, the gene and mRNA syntheses, but largely inhibited protein synthesis. TJ-091 largely inhibited gene, mRNA and protein syntheses. TJ-095 largely inhibited gene synthesis, but NP and HN mRNAs were slightly detected, and protein syntheses were observed. TJ-119 inhibited gene, mRNA and protein syntheses. TJ-001, TJ-002, TJ-091, TJ-095 and TJ-119 inhibited multinucleated giant cell formation derived from cell-to-cell spreading of virus. However, in TJ-019, TJ-029 and TJ-041 treated infected cells, only small sized fused cells with some nuclei were found. TJ-019 and TJ-041 slightly disrupted actin microfilaments, and TJ-001 and TJ-002 destroyed them. TJ-041 slightly disrupted microtubules, and TJ-001 and TJ-002 disrupted them. In general, the medicines effective on common cold and bronchitis inhibited hPIV-2 replication.

Passive oral immunization by egg yolk immunoglobulin (IgY) to Vibrio cholerae effectively prevents cholera.

In an attempt to prepare egg yolk immunoglobulin (IgY) to treat and prevent cholera, hens were immunized by a mixture of heat- or formalin-killed Vibrio cholerae O1 and O139 organisms, or by the recombinant cholera toxin B subunit (CTB). The IgYs were partially purified from egg yolk and orally administered to suckling mice before or after challenge with live O1 or O139 cells. The anti-O1 and O139 IgYs and the mixture of either IgY with anti-CTB IgY significantly protected the occurrence of cholera caused by both O1 and O139 infection. Since large amounts of IgY can be prepared very easily and at low cost, this seems to be a useful procedure for preventing and treating cholera.

Detection of Helicobacter hepaticus in human bile samples of patients with biliary disease.

BACKGROUND: Since the discovery of Helicobacter pylori, various enterohepatic Helicobacter spices have been detected in the guts of humans and animals. Some enterohepatic Helicobacters have been associated with inflammatory bowel disease or liver disease in mice. However the association of these bacteria with human diseases remains unknown. MATERIALS AND METHODS: We collected 126 bile samples from patients with cholelithiasis, cholecystitis, gallbladder polyp, and other nonbiliary diseases. Samples were screened for the presence of enterohepatic Helicobacter spp. using cultures, nested PCR, or in situ hybridization. We tested for antibodies to H. pylori and H. hepaticus by Western blot analysis. RESULTS: Attempts at cultivation were unsuccessful. However, H. hepaticus was detected in bile samples with nested PCR whereas H. bilis was not. Helicobacter hepaticus in the bile was confirmed by in situ hybridization, but H. hepaticus from bile samples was coccoid in appearance. We detected immunoglobulin G antibodies to H. hepaticus in bile samples by Western blotting. Helicobacter hepaticus was detected in 40 (32%) of total 126 samples as H. hepaticus positive if at least one of the three methods with nested PCR, in situ, or Western blotting. Patients with cholelithiasis (41%) and cholecystitis with gastric cancer (36%) had significantly higher (p = .029) prevalence of H. hepaticus infection than samples from patients with other diseases. CONCLUSION: Helicobacter hepaticus may closely associate with diseases of the liver and biliary tract in humans.

Immune reactions against elongation factor 2 kinase: specific pathogenesis of gastric ulcer from Helicobacter pylori infection.

Helicobacter pylori (H. pylori) infection is a definite causative factor for gastric ulcers (GUs). In the present study we detected a specific antigen of gastric epithelial cells (HGC-27) using cell ELISA, which was recognized by the sera of GU patients (n = 20) but not in patients with chronic gastritis (CG; n = 20) or in healthy volunteers (HC; n = 10). This antigen was over-expressed by a stressful (heat-stressed) environment, and was identified as elongation factor 2 kinase (EF-2K) by western blotting. The GU patients' lymphocytes stimulated by H. pylori specifically disrupted heat-stressed HGC-27 cells in a cytotoxic assay. In flow cytometry, the effector cells (lymphocytes) from GU patients were significantly differentiated to T helper type 1 lymphocyte (Th1) and cytotoxic T lymphocyte (CTL) as opposed to those from CG patients. The target cells (HGC-27) expressed EF-2K and MHC-class I together with costimulatory molecules from heat stress. This antigen specific immune mechanism could have a prominent role in the pathogenesis of GU.

Investigation of the Effective Concentration of Ozonated Water for Disinfection in the Presence of Protein Contaminants.

Ozonated water (OW) is presently used as a chemical disinfectant in many fields, due to its versatile antimicrobial properties. As ozone rapidly decomposes to oxygen, especially in the presence of organic matter, it is important to estimate the authentic antimicrobial activity of OW in the presence of contaminants. However, the effect of contaminants on the antimicrobial activity of OW has not been fully investigated. To address this, we evaluated the effect of protein contaminants on the antimicrobial activity of OW. The survival rate of each tested microorganism excluding Bacillus subtilis spores was reduced to less than 0.1%, when the microorganism suspension was exposed to 9.1 ppm of OW for 15 s in the presence of 0.0045% protein. Our study therefore suggests that approximately 10 ppm of OW can reduce the survival rates of almost all microorganisms in the presence of proteins.

Inhibitions of human parainfluenza virus type 2 replication by ribavirin and mycophenolate mofetil are restored by guanosine and S-(4-nitrobenzyl)-6-thioinosine.

The antiviral activities of a nucleoside analog antiviral drug (ribavirin) and a non-nucleoside drug (mycophenolate mofetil) against human parainfluenza virus type 2 (hPIV-2) were investigated, and the restoration of the inhibition by guanosine and S-(4-nitrobenzyl)-6-thioinosine (NBTI: equilibrative nucleoside transporter 1 inhibitor) were also investigated. Ribavirin (RBV) and mycophenolate mofetil (MMF) inhibited cell fusion induced by hPIV-2. Both RBV and MMF considerably reduced the number of viruses released from the cells. Virus genome synthesis was inhibited by RBV and MMF as determined by polymerase chain reaction (PCR) and real time PCR. mRNA syntheses were also reduced. An indirect immunofluorescence study showed that RBV and MMF largely inhibited viral protein syntheses. Using a recombinant green fluorescence protein (GFP)-expressing hPIV-2 without matrix protein (rhPIV-2ΔMGFP), it was found that virus entry into the cells and multinucleated giant cell formation were almost completely blocked by RBV and MMF. RBV and MMF did not disrupt actin microfilaments or microtubules. Both guanosine and NBTI completely or partially reversed the inhibition by RBV and MMF in the viral replication, syntheses of genome RNA, mRNA and protein, and multinucleated giant cell formation. NBTI caused a little damage in actin microfilaments, but had no effect on microtubules. Both RBV and MMF inhibited the replication of hPIV-2, mainly by inhibiting viral genome RNA, mRNA and protein syntheses. The inhibition was almost completely recovered by guanosine. These results indicate that the major mechanism of the inhibition is the depletion of intracellular GTP pools.

Genetic Analyses of Elys Mutations in Drosophila Show Maternal-Effect Lethality and Interactions with Nucleoporin Genes.

ELYS determines the subcellular localizations of Nucleoporins (Nups) during interphase and mitosis. We made loss-of-function mutations of Elys in Drosophila melanogaster and found that ELYS is dispensable for zygotic viability and male fertility but the maternal supply is necessary for embryonic development. Subsequent to fertilization, mitotic progression of the embryos produced by the mutant females is severely disrupted at the first cleavage division, accompanied by irregular behavior of mitotic centrosomes. The Nup160 introgression from D. simulans shows close resemblance to that of the Elys mutations, suggesting a common role for those proteins in the first cleavage division. Our genetic experiments indicated critical interactions between ELYS and three Nup107-160 subcomplex components; hemizygotes of either Nup37, Nup96 or Nup160 were lethal in the genetic background of the Elys mutation. Not only Nup96 and Nup160 but also Nup37 of D. simulans behave as recessive hybrid incompatibility genes with D. melanogaster An evolutionary analysis indicated positive natural selection in the ELYS-like domain of ELYS. Here we propose that genetic incompatibility between Elys and Nups may lead to reproductive isolation between D. melanogaster and D. simulans, although direct evidence is necessary.

Promising Therapies for Fungal Infection Based on the Study to Elucidate Mechanisms to Cope with Stress in Candida Species.

In recent years, the incidence of fungal infections has been increasing, particularly among patients with immune systems compromised by human immunodeficiency virus infection, organ transplantation, and/or chemotherapy for cancer. Current therapies for treating systemic fungal infection have limited effectiveness and have created problems of adverse reactions and drug resistance. These issues therefore motivate us to develop novel antifungals. Elucidation of stress response mechanisms and virulence factors in pathogenic fungi is required in developing an effective antifungal strategy. There are actually numerous studies concerning various stress responses in several important fungal pathogens. Among these responses, we focused on stress response for iron starvation to identify potential targets for novel antifungals because iron starvation occurs in blood, where pathogenic fungi often infect. Here we show recent progress of studies on iron homeostasis in Candida species, especially focusing on Candida glabrata, and propose novel antifungal targets.

Isolation and cytogenetic characterization of male meiotic mutants of Drosophila melanogaster.

Proper segregation of homologous chromosomes in meiosis I is ensured by pairing of homologs and maintenance of sister chromatid cohesion. In male Drosophila melanogaster, meiosis is achiasmatic and homologs pair at limited chromosome regions called pairing sites. We screened for male meiotic mutants to identify genes required for normal pairing and disjunction of homologs. Nondisjunction of the sex and the fourth chromosomes in male meiosis was scored as a mutant phenotype. We screened 2306 mutagenized and 226 natural population-derived second and third chromosomes and obtained seven mutants representing different loci on the second chromosome and one on the third. Five mutants showed relatively mild effects (<10% nondisjunction). mei(2)yh149 and mei(2)yoh7134 affected both the sex and the fourth chromosomes, mei(2)yh217 produced possible sex chromosome-specific nondisjunction, and mei(2)yh15 and mei(2)yh137 produced fourth chromosome-specific nondisjunction. mei(2)yh137 was allelic to the teflon gene required for autosomal pairing. Three mutants exhibited severe defects, producing >10% nondisjunction of the sex and/or the fourth chromosomes. mei(2)ys91 (a new allele of the orientation disruptor gene) and mei(3)M20 induced precocious separation of sister chromatids as early as prometa-phase I. mei(2)yh92 predominantly induced nondisjunction at meiosis I that appeared to be the consequence of failure of the separation of paired homologous chromosomes.

The Drosophila misfire gene has an essential role in sperm activation during fertilization.

The male sterile mutation, misfire (mfr), of Drosophila melanogaster is a novel paternal effect, fertilization defective mutant that effects sperm head decondensation. mfr sperm were motile, appeared normal morphologically and were transferred to the female during copulation. However, less than 0.1% of eggs laid by females mated to mfr males hatched. Although mfr sperm entered eggs at a high frequency (93%), 99% of the inseminated eggs did not initiate the first nuclear division. Unlike wild type fertilizing sperm, the position and shape of mfr sperm tails within the egg were not constant, but varied in a seemingly random manner. The heads of inseminating mutant sperm were always located near the surface of eggs just underlying the egg plasma membrane, and maintained their needle-like shape indicating the failure of nuclear decondensation. Further observations revealed that plasma membrane of inseminating sperm appeared intact, including the head region. These phenotypes were equivalent to those of sneaky (snky), another fertilization defective male sterile mutation. Our observations strongly suggest that mfr mutant males are sterile because their inseminating sperm fail to form a male pronucleus due to the inability of the sperm to properly respond to egg factors responsible for the breakdown of the plasma membrane. Although mfr and snky mutations were phenotypically identical, they mapped to cytologically distinct genetic loci and no genetic interactions were observed, suggesting that at least two distinct paternal gene products are involved in the early stages of pronuclear formation.