Hepatitis C virus nonstructural protein 5A modulates the toll-like receptor-MyD88-dependent signaling pathway in macrophage cell lines

Hepatitis C virus (HCV) infection induces a wide range of chronic liver injuries; however, the mechanism through which HCV evades the immune surveillance system remains obscure. Blood dendritic cells (DCs) play a pivotal role in the recognition of viral infection and the induction of innate and adaptive immune responses. Several reports suggest that HCV infection induces the dysfunction of DCs in patients with chronic hepatitis C. Toll-like receptor (TLR) has been shown to play various roles in many viral infections; however, the involvement of HCV proteins in the TLR signaling pathway has not yet been precisely elucidated. In this study, we established mouse macrophage cell lines stably expressing HCV proteins and determined the effect of HCV proteins on the TLR signaling pathways. Immune cells expressing NS3, NS3/4A, NS4B, or NS5A were found to inhibit the activation of the TLR2, TLR4, TLR7, and TLR9 signaling pathways. Various genotypes of NS5A bound to MyD88, a major adaptor molecule in TLR, inhibited the recruitment of interleukin-1 receptor-associated kinase 1 to MyD88, and impaired cytokine production in response to TLR ligands. Amino acid residues 240 to 280, previously identified as the interferon sensitivity-determining region (ISDR) in NS5A, interacted with the death domain of MyD88, and the expression of a mutant NS5A lacking the ISDR partially restored cytokine production. These results suggest that the expression of HCV proteins modulates the TLR signaling pathway in immune cells.     

Nuclease-resistant immunostimulatory phosphodiester CpG oligodeoxynucleotides as human Toll-like receptor 9 agonists

Background

Gardnerella vaginalis is identified as the predominant colonist of the vaginal tract in women with bacterial vaginosis. Vaginolysin (VLY) is a protein toxin released by G. vaginalis. VLY possesses cytolytic activity and is considered as a main virulence factor of G. vaginalis. Inhibition of VLY-mediated cell lysis by antibodies may have important physiological relevance.

Results

Single-chain variable fragments of immunoglobulins (scFvs) were cloned from two hybridoma cell lines producing neutralizing antibodies against VLY and expressed as active proteins in E. coli. For each hybridoma, two variants of anti-VLY scFv consisting of either VL-VH or VH-VL linked with a 20 aa-long linker sequence (G₄S)₄ were constructed. Recovery of scFvs from inclusion bodies with subsequent purification by metal-chelate chromatography resulted in VLY-binding proteins that were predominantly monomeric. The antigen-binding activity of purified scFvs was verified by an indirect ELISA. The neutralizing activity was investigated by in vitro hemolytic assay and cytolytic assay using HeLa cell line. Calculated apparent K_d values and neutralizing potency of scFvs were in agreement with those of parental full-length antibodies. VH-VL and VL-VH variants of scFvs showed similar affinity and neutralizing potency. The anti-VLY scFvs derived from hybridoma clone 9B4 exhibited high VLY-neutralizing activity both on human erythrocytes and cervical epithelial HeLa cells.

Conclusions

Hybridoma-derived scFvs with VLY-binding activity were expressed in E. coli. Recombinant anti-VLY scFvs inhibited VLY-mediated cell lysis. The monovalent scFvs showed reduced affinity and neutralizing potency as compared to the respective full-length antibodies. The loss of avidity could be restored by generating scFv constructs with multivalent binding properties. Generated scFvs is the first example of recombinant single-chain antibodies with VLY-neutralizing activity produced in prokaryote expression system. G. vaginalis caused infections continue to be a world-wide problem, therefore neutralizing recombinant antibodies may provide novel therapeutic agents useful in the treatment of bacterial vaginosis and other diseases caused by G. vaginalis.     

GSK-3 inhibition in vitro and in vivo enhances antitumor effect of sorafenib in renal cell carcinoma (RCC)

Sorafenib is a multikinase inhibitor approved for the systemic treatment of renal cell carcinoma (RCC). However, sorafenib treatment has a limited effect due to acquired chemoresistance of RCC. Previously, we identified glycogen synthase kinase-3 (GSK-3) as a new therapeutic target in RCC. Here, we observed that sorafenib inhibits proliferation and survival of RCC cells. Significantly, we revealed that sorafenib enhances GSK-3 activity in RCC cells, which could be a potential mechanism of acquired chemoresistance. We found that pharmacological inhibition of GSK-3 potentiates sorafenib antitumor effect in vitro and in vivo. Our results suggest that combining GSK-3 inhibitor and sorafenib might be a potential new therapeutic approach for RCC treatment.     

Repair of brain damage size and recovery of neurological dysfunction after ischemic stroke are different between strains in mice: evaluation using a novel ischemic stroke model

In the current study, we established a novel murine ischemic brain damage model using a photochemical reaction to evaluate the recovery of neurological dysfunction and brain repair reactions. In this model, reproducible damage was induced in the frontal lobe of the cortex, which was accompanied by neurological dysfunction. Sequential changes in damage size, microglial accumulation, astrocyte activation, and neurological dysfunction were studied in C57BL/6J and BALB/c mouse strains. Although the initial size of damage was comparable in both strains, the extent of damage was later reduced to a greater extent in C57BL/6J mice than that in BALB/c mice. In addition, C57BL/6J mice showed later edema clearance until day 7, less microglial accumulation, and relatively more astrocyte activation on day 7. Neurologic dysfunction was evaluated by three behavioral tests: the von Frey test, the balance beam test, and the tail suspension test. The behavioral abnormalities evaluated by these tests were remarkable following the induction of damage and recovered by day 21 in both strains. However, the abnormalities were more prominent and the recovery was later in C57BL/6J mice. These findings demonstrate that our novel ischemic stroke model is useful for evaluating brain repair reactions and the recovery of neurological dysfunction in mice with different genetic backgrounds. In addition, we found that both the brain repair reactions and the recovery of neurological dysfunction after comparable ischemic brain damage varied between strains; in that, they both occurred later in C57BL/6J mice.     

BROMI/TBC1D32 together with CCRK/CDK20 and FAM149B1/JBTS36 contributes to intraflagellar transport turnaround involving ICK/CILK1

Primary cilia are antenna-like organelles that contain specific proteins, and are crucial for tissue morphogenesis. Anterograde and retrograde trafficking of ciliary proteins are mediated by the intraflagellar transport (IFT) machinery. BROMI/TBC1D32 interacts with CCRK/CDK20, which phosphorylates and activates the intestinal cell kinase (ICK)/CILK1 kinase, to regulate the change in direction of the IFT machinery at the ciliary tip. Mutations in BROMI, CCRK, and ICK in humans cause ciliopathies, and mice defective in these genes are also known to demonstrate ciliopathy phenotypes. We show here that BROMI interacts not only with CCRK but also with CFAP20, an evolutionarily conserved ciliary protein, and with FAM149B1/ Joubert syndrome (JBTS)36, a protein in which mutations cause JBTS. In addition, we show that FAM149B1 interacts directly with CCRK as well as with BROMI. Ciliary defects observed in CCRK-knockout (KO), BROMI-KO, and FAM149B1-KO cells, including abnormally long cilia and accumulation of the IFT machinery and ICK at the ciliary tip, resembled one another, and BROMI mutants that are defective in binding to CCRK and CFAP20 were unable to rescue the ciliary defects of BROMI-KO cells. These data indicate that CCRK, BROMI, FAM149B1, and probably CFAP20 altogether regulate the IFT turnaround process under the control of ICK.     

Migration of prospindle before the first asymmetric division in germinating spore of Marchantia polymorpha

The development of the plant body starts with spore germination in bryophytes. In many cases, the first division of the spore occurs after germination and cell elongation of the spore. In Marchantia polymorpha, asymmetric division occurs upon spore germination to generate two daughter cells: the larger one retains the ability to divide and develops into the thallus via sporeling or protonema, while the smaller one maintains tip growth and differentiates into the first rhizoid, providing a scaffold for initial development. Although spore germination of M. polymorpha was described in the 19th century, the intracellular processes of the first asymmetric division of the spore have not been well characterized. In this study, we used live-cell imaging analyses to elucidate microtubule dynamics during the first asymmetric division concomitantly with germination. In particular, we demonstrated that the preprophase band was not formed in the spore and that the bipolar prospindle, which is a microtubule structure surrounding the nucleus during prophase, migrated from the center to the periphery in the spore, suggesting that it was the earliest visible sign of cell polarity. We also showed that the occurrence of asymmetric division depended on actin filaments. Our findings regarding the first division of the spore in M. polymorpha will lead to a better model for cell-autonomous asymmetric division in plants.     

Dry matter production, population structure and environmental conditions of the spring ephemeralErythronium japonicum growing in various habitats differing in sunlight exposure in cool temperate Japan

Phenology, dry matter production, population structure and environmental conditions were examined inErythronium japonicum Decne plants growing on the floor of a deciduous broad-leavedQuercus mongolica forest (Q.m. stand), an evergreen coniferousCryptomeria japonica plantation (C. j. stand) and bare ground left for 3 years after the clearing of a forest composed of youngQ. mongolica andPinus densiflora trees (bare stand) in the cool temperate zone of Japan. The average population density of the plants growing at theQ.m. stand was much higher than that observed at the bare stand, whereas the average number of flowering plants at the former stand was less than half of that at the latter. The population density and number of flowering plants growing at theC. j. stand were both less than 30% of those at theQ. m. stand. The number of seedlings at theQ. m. stand was much more than that at the bare andC. j. stands. Their survivorship rate over 1 year at the former stand also seems to be significantly higher than those at the other stands. Their aboveground and belowground parts at the bare stand were exposed to more severe heat and water stress than those at the other two stands. The net production per leaf area of the plants growing at theQ. m. stand was two and six times larger than those at the bare andC. j. stands, respectively. The plants at the bare stand did not use the available solar radiation as efficiently for dry matter production through photosynthesis as those growing at theQ. m. stand, whereas those at theC. j. stand are strongly restricted in their photosynthetic process by the significantly limited light condition on the floor of the evergreen coniferous plantation. The differences in the number of plants reaching sexual maturity, the density and structure of the population and the net production between their three habitats are discussed here from the standpoint of differences in environmental conditions.     

Development of LC-MS/MS analysis of cyclic dipeptides and its application to tea extract

2,5-Diketopiperazines (DKPs), also called cyclic dipeptides, have been known to occur in various foods. Recently, DKPs have attracted attentions as bioactive components. There were some reports on analytical methods for DKPs, but the number of analyzed DKPs was only a part of all DKPs and the quantitative performance was not studied in detail. In this study, we selected 31 kinds of DKPs and developed a quantitative and simultaneous analytical method using LC-MS/MS. This method was applied to DKPs determination in Pu-erh tea, post-fermentation tea, and 18 kinds of DKPs were determined at concentration of 0.0017–0.11 ppm. As a result of spiked test, it was concluded that the developed method using LC-MS/MS was useful for estimating DKPs concentration in tea.     

Adrenomedullin: a possible regulator of germinal vesicle breakdown

Adrenomedullin (ADM) is a multifunctional hormone that regulates processes as diverse as blood pressure and cell growth. Although expressed in the ovary, the role of ADM in this organ is not clear. In the present study, we found the expression of ADM receptor and receptor activity-modifying proteins in mouse cumulus cells but not in the oocytes. We report that germinal vesicle breakdown (GVBD), which is required for oocyte maturation, is not inhibited by ADM alone. However, ADM in the presence of the nitric oxide donor sodium nitroprusside (SNP) significantly inhibited GVBD. Furthermore, the ADM- and SNP-dependent inhibition of GVBD was abrogated by Akt blockade. Additionally, Akt expression and phosphorylation was exhibited by ADM, suggesting that Akt signaling upstream in cumulus cells is responsible. Additionally, immunohistochemical analysis revealed that ADM was localized in the granulosa cells of developed follicles, implying the possibility that ADM physiologically affects oocyte maturation in vivo. Our results provide the evidence that ADM can act as a GVBD regulator.