Increase in intracellular cAMP is a prerequisite signal for initiation of physiological oocyte meiotic maturation in the hydrozoan Cytaeis uchidae

In medusae of the hydrozoan Cytaeis uchidae, oocyte meiotic maturation and spawning occur as a consequence of dark-light transition. In this study, we investigated the mechanism underlying the initiation of meiotic maturation using in vitro (isolated oocytes from ovaries) and in vivo (ovarian oocytes in medusae) systems. Injection of cAMP derivatives into isolated oocytes induced meiotic maturation in a dose-dependent manner. Meiotic maturation was also achieved in isolated oocytes preloaded with caged cAMP and exposed to UV irradiation. The caged cAMP/UV irradiation-induced meiotic maturation was completely inhibited by blockers of protein kinase A (PKA), H-89, KT5720, and Rp-cAMPS. The medusae from which most parts of the umbrella were removed (umbrella-free medusae) survived for at least 2 weeks, during which time oocyte meiotic maturation and spawning occurred. When H-89 and Rp-cAMPS were injected into ovarian oocytes of umbrella-free medusae within 3 min of dark-light stimulation, meiotic maturation was inhibited or delayed. An increase in intracellular cAMP was confirmed by FlCRhR, a fluorescent cAMP indicator, in ovarian oocytes exposed to dark-light transition as well as in isolated oocytes stimulated by caged cAMP/UV irradiation. These results indicate that the cAMP/PKA signaling pathway positively contributes to light-triggered physiological oocyte meiotic maturation in Cytaeis uchidae.     

Retrovirus-mediated conditional immortalization and analysis of established cell lines of osteoclast precursor cells

Osteoclast precursor cells (OPCs) have previously been established from bone marrow cells of SV40 temperature-sensitive T antigen-expressing transgenic mice. Here, we use retrovirus-mediated gene transfer to conditionally immortalize OPCs by expressing temperature-sensitive large T antigen (tsLT) from wild type bone marrow cells. The immortalized OPCs proliferated at the permissive temperature of 33.5 degrees C, but stopped growing at the non-permissive temperature of 39 degrees C. In the presence of receptor activator of NFkappaB ligand (RANKL), the OPCs differentiated into tartrate-resistant acid phosphatase (TRAP)-positive cells and formed multinucleate osteoclasts at 33.5 degrees C. From these OPCs, we cloned two types of cell lines. Both differentiated into TRAP-positive cells, but one formed multinucleate osteoclasts while the other remained unfused in the presence of RANKL. These results indicate that the established cell lines are useful for analyzing mechanisms of differentiation, particularly multinucleate osteoclast formation. Retrovirus-mediated conditional immortalization should be a useful method to immortalize OPCs from primary bone marrow cells.     

An N-glycan structure correlates with pulmonary metastatic ability of cancer cells

N-Glycan structures on the surface of cancer cells have diverse structures and play significant roles in metastatic process. However, little is known about their roles in organ-selective metastasis. Our study revealed that an alpha1,6-fucosylated biantennary N-glycan structure designated A2G2F is characteristic of lungs, with far more abundant expression in normal human and murine lungs than in other organs. In this study, we further examined the role of A2G2F in pulmonary metastasis. We stained metastatic cancers by alpha1,6-fucose-specific Lens culinaris agglutinin lectin and revealed that pulmonary metastatic nodules more abundantly expressed alpha1,6-fucosylated N-glycans than hepatic metastatic nodules from common primary cancers. The most specific alpha1,6-fucosylated N-glycan structure in pulmonary metastatic cancer was identified to be A2G2F. Using a B16 melanoma cell metastasis model, we showed that A2G2F-rich B16 cells formed more pulmonary metastatic nodules than A2G2F-poor cells. Our results suggest that A2G2F plays a critical role in pulmonary metastasis.     

Enhanced resistance to four species of Clypeorrhynchan pests in Neotyphodium uncinatum infected Italian ryegrass

Particular alkaloids produced by Neotyphodium endophytes show toxicity to invertebrates. Italian ryegrass (Lolium multiflorum Lamarck) cultivars and strains that are symbiotic with Neotyphodium endophytes have been recently established in Japan. N. uncinatum-infected Italian ryegrass lines accumulate N-formylloline, a type of loline alkaloid (1-aminopyrrolizidine) showing neurotoxicity to herbivorous insects. This study investigated the toxicity of N-formylloline and resistance of N. uncinatum-infected Italian ryegrass to vascular-sap feeding Clypeorrhynchan pests. When four vascular-sap feeding insects: Laodelphax striatellus (Fallén) (Homoptera: Delphacidae), Sogatella furcifera (Horváth) (Homoptera: Delphacidae), Cicadulina bipunctata (Melichar) (Homoptera: Cicadellidae), and Nephotettix cincticeps (Uhler) (Homoptera: Cicadellidae) fed on N. uncinatum-infected Italian ryegrass, significant decreases in survival rate were observed for three phloem-sap feeders but not for a xylem-sap feeder, N. cincticeps. This result suggests an uneven distribution of N-formylloline among plant tissues. A potency assay for N-formylloline using a Parafilm feeding sachet and a quantitative analysis of N-formylloline in plant showed a concentration-dependent lethal effect of N-formylloline on all four tested vascular-sap feeders. Our results strongly suggest that N. uncinatum-infected plants can control some Clypeorrhynchan pests in crop fields.     

Inhibition of high glucose-induced VEGF and ICAM-1 expression in human retinal pigment epithelium cells by targeting ILK with small interference RNA

The aim of this study was to investigate the change of Integrin-linked kinase (ILK) expression of human retinal pigment epithelium (RPE) cells in response to high glucose, and the effect of targeting ILK with small interference RNA (siRNA) on the high glucose-induced expression of vascular endothelial growth factor (VEGF) and intercellular adhesion molecule-1 (ICAM-1). The ILK mRNA and protein expression in human RPE cells were analyzed with RT-PCR and western blot after exposure to 5.5, 30, 40, 50 mM glucose, or 5.5 mM glucose + 45.5 mM mannitol for 48 h. The expression of VEGF and ICAM-1 was also determined. Cells were treated with ILK siRNA, to determine the effect of ILK on VEGF and ICAM-1 expression following treatment with high glucose. High concentrations of glucose significantly up-regulated ILK mRNA and protein expression, and the ILK expression increased along with the glucose concentration. The changes of VEGF and ICAM-1 expression were similar to that of ILK expression. Knocking down ILK gene expression with siRNA inhibited the elevation of VEGF and ICAM-1 induced by high glucose treatment. These results suggested that ILK was involved in the response of RPE cells to high glucose and may therefore play a role in the pathogenesis of diabetic ophthalmology.     

Identification of SAMT family proteins as substrates of MARCH11 in mouse spermatids

MARCH11, a RING-finger transmembrane ubiquitin ligase, is predominantly expressed in spermatids and localized to the trans-Golgi network (TGN) and multivesicular bodies (MVBs). Because ubiquitination acts as a sorting signal of cargo proteins, MARCH11 has been postulated to mediate selective protein sorting via the TGN–MVB pathway. However, the physiological substrate of MARCH11 has not been identified. In this study, we have identified and characterized SAMT1, a member of a novel 4-transmembrane protein family, which consists of four members. Samt1 mRNA and its expression product were found to be specific to the testis and were first detected in germ cells 25 days after birth in mice. Immunohistochemical analysis further revealed that SAMT1 was specifically expressed in haploid spermatids during the cap and acrosome phases. Confocal microscopic analysis showed that SAMT1 co-localized with MARCH11 as well as with fucose-containing glycoproteins, another TGN/MVB marker, and LAPM2, a late endosome/lysosome marker. Furthermore, we found that MARCH11 could increase the ubiquitination of SAMT1 and enhance its lysosomal delivery and degradation in an E3 ligase activity-dependent manner. In addition, the C-terminal region of SAMT1 was indispensable for its ubiquitination and proper localization. The other member proteins of the SAMT family also showed similar expression profile, intracellular localization, and biochemical properties, including ubiquitination by MARCH11. These results suggest that SAMT family proteins are physiological substrates of MARCH11 and are delivered to lysosomes through the TGN–MVB pathway by a ubiquitin-dependent sorting system in mouse spermatids.     

Alterations in renal iron metabolism caused by a copper/zinc-superoxide dismutase deficiency

Copper/zinc-superoxide dismutase knockout (SOD1 KO) mice have been extensively used as an experimental animal model of pathology associated with oxidative stress. The mice spontaneously develop mild chronic hemolytic anaemia (HA). We previously reported that the kidneys of these types of mice contain massive amounts of iron. In this study, to clarify the role of the kidney for iron metabolism under HA, changes in the levels of expression and functions of iron-related proteins were examined. In SOD1 KO mice kidneys, protein levels of iron transporters, the iron-responsive element (IRE)-binding activity of IRP1 and the levels of phosphorylation of IRP1 are all increased. These findings indicate that oxidative stress caused by a SOD1 deficiency probably enhances the phosphorylation of and the conversion of IRP1 to the IRE-binding form, which may accelerate the reabsorption of iron by renal tubular cells. Kidney could play an important role in iron homeostasis under conditions of HA.     

Estimation of climatic factors relating to occurrence of the maize orange leafhopper, Cicadulina bipunctata

The maize orange leafhopper, Cicadulina bipunctata is a serious pest of forage maize in East and Southeast Asia. In temperate Japan, the occurrence of this leafhopper fluctuates widely among years. Here, we examined effects of climatic factors (temperature, precipitation and sunlight) on the occurrence of C. bipunctata. Seasonal occurrence of adult C. bipunctata in a census field from July to August, when forage maize was most susceptible to the pest, could be described by a simple exponential function with two parameter: estimated density of C. bipunctata on 1 July (N ₀) and intrinsic rate of natural increase (r) for each year. Forward stepwise multiple regression analysis using seasonal occurrence data from 2004 to 2009 detected positive contributions of average temperatures in the previous December and February and a negative contribution of total precipitation during the previous winter to N ₀. The analysis also indicated that average temperature in July of the current year and N ₀ contributed positively and negatively to r, respectively. These results indicated that high temperature and little precipitation during winter and high temperature in early summer induced high occurrence of C. bipunctata in summer. A prediction model based on these factors supported the actual seasonal occurrence in 2010, suggesting that this prediction model is applicable to C. bipunctata forecasting. The prediction model indicated that further global warming in the future is likely to cause further outbreaks of C. bipunctata.     

25-hydroxycholesterol enhances cytokine release and toll-like receptor 3 response in airway epithelial cells

Background

25-hydroxycholesterol (25-HC) is one of the oxysterols, which are oxidized derivatives of cholesterol, and has been reported to be involved in the pathogenesis of atherosclerosis and Alzheimer’s disease. In lung, the possible involvement of 25-HC in airway diseases has been revealed. In the present study, we examined whether 25-HC affects the release of cytokines and also modulates the responses of toll-like receptor 3 (TLR3) in airway epithelial cells.

Methods

The effect of 25-HC on the release of cytokines from primary human bronchial epithelial cells after stimulation with or without polyinosine-polycytidylic acid [poly(I:C)], a ligand for TLR3, and the signal transduction were examined.

Results

25-HC significantly potentiated the release of interleukin-8 (IL-8) and IL-6 from the cells. This effect was more potent compared with that of other oxysterols, 22-HC and 27-HC. GW3965 and TO901317, synthetic agonists of liver X receptors that are receptors for oxysterols, did not augment the IL-8 release. 25-HC enhanced the nuclear factor-kappa B (NF-κB) DNA binding activity and translocation of phosphorylated c-Jun into the nucleus. The release of IL-8 was inhibited by the NF-κB inhibitor, caffeic acid phenethyl ester (CAPE), an inhibitor of nuclear factor kappa-B alpha (IκBα) inhibitor, BAY 11–7085, and an inhibitor of nuclear factor kappa-B kinase-2 (IKK-2) inhibitor, SC-514, but not by a c-Jun N-terminal kinase (JNK) inhibitory peptide, L-JNKi1. 25-HC significantly potentiated IL-8 release in poly(I:C)-treated cells and the augmentation was inhibited by CAPE, BAY 11–7085, and SC-514. Furthermore, 25-HC potentiated the translocation of interferon regulatory factor 3 into the nucleus and the release of interferon-beta (IFN-β) in poly(I:C)-treated cells.

Conclusions

These data demonstrated that 25-HC augments the release of IL-8 and IL-6 via NF-κB signalling pathway and enhances the release of IL-8 and IFN-β after stimulation of TLR3 in airway epithelial cells. 25-HC may be involved in the neutrophilic airway inflammation through the stimulant effect of IL-8 and IL-6 release and also potentiate the TLR3-mediated innate immunity in airway diseases.