High temperature induces cyp26b1 mRNA expression and delays meiotic initiation of germ cells by increasing cortisol levels during gonadal sex differentiation in Japanese flounder

The Japanese flounder (Paralichthys olivaceus) is a teleost fish with an XX/XY sex determination system. XX flounder can be induced to develop into phenotypic females or males, by rearing them at 18°C or 27°C, respectively, during the sex differentiation period. Therefore, the flounder provides an excellent model to study the molecular mechanisms underlying temperature-dependent sex determination. We previously showed that cortisol, the major glucocorticoid produced by the interrenal cells in teleosts, causes female-to-male sex reversal by directly suppressing mRNA expression of ovary-type aromatase (cyp19a1), a steroidogenic enzyme responsible for the conversion of androgens to estrogens in the gonads. Furthermore, an inhibitor of cortisol synthesis prevented masculinization of XX flounder at 27°C, suggesting that masculinization by high temperature is due to the suppression of cyp19a1 mRNA expression by elevated cortisol levels during gonadal sex differentiation in the flounder. In the present study, we found that exposure to high temperature during gonadal sex differentiation upregulates the mRNA expression of retinoid-degrading enzyme (cyp26b1) concomitantly with masculinization of XX gonads and delays meiotic initiation of germ cells. We also found that cortisol induces cyp26b1 mRNA expression and suppresses specific meiotic marker synaptonemal complex protein 3 (sycp3) mRNA expression in gonads during the sexual differentiation. In conclusion, these results suggest that exposure to high temperature induces cyp26b1 mRNA expression and delays meiotic initiation of germ cells by elevating cortisol levels during gonadal sex differentiation in Japanese flounder.     

Annexin A4 is a possible biomarker for cisplatin susceptibility of malignant mesothelioma cells

Mesothelioma is a highly malignant tumor with a poor prognosis and limited treatment options. Although cisplatin (CDDP) is an effective anticancer drug, its response rate is only 20%. Therefore, discovery of biomarkers is desirable to distinguish the CDDP-susceptible versus resistant cases. To this end, differential proteome analysis was performed to distinguish between mesothelioma cells of different CDDP susceptibilities, and this revealed that expression of annexin A4 (ANXA4) protein was higher in CDDP-resistant cells than in CDDP-susceptible cells. Furthermore, ANXA4 expression levels were higher in human clinical malignant mesothelioma tissues than in benign mesothelioma and normal mesothelial tissues. Finally, increased susceptibility was observed following gene knockdown of ANXA4 in mesothelioma cells, whereas the opposite effect was observed following transfection of an ANXA4 plasmid. These results suggest that ANXA4 has a regulatory function related to the cisplatin susceptibility of mesothelioma cells and that it could be a biomarker for CDDP susceptibility in pathological diagnoses.     

Targeted disruption of exogenous EGFP gene in medaka using zinc-finger nucleases

Zinc-finger nucleases (ZFNs) are artificial enzymes that create site-specific double-strand breaks and thereby induce targeted genome editing. Here, we demonstrated successful gene disruption in somatic and germ cells of medaka (Oryzias latipes) using ZFN to target exogenous EGFP genes. Embryos that were injected with an RNA sequence pair coding for ZFNs showed mosaic loss of green fluorescent protein fluorescence in skeletal muscle. A number of mutations that included both deletions and insertions were identified within the ZFN target site in each embryo, whereas no mutations were found at the non-targeted sites. In addition, ZFN-induced mutations were introduced in germ cells and efficiently transmitted to the next generation. The mutation frequency varied (6-100%) in the germ cells from each founder, and a founder carried more than two types of mutation in germ cells. Our results have introduced the possibility of targeted gene disruption and reverse genetics in medaka.     

CLP36 and RIL recruit α-actinin-1 to stress fibers and differentially regulate stress fiber dynamics in F2408 fibroblasts

CLP36 is a member of the ALP/Enigma protein family and has been shown to be localized to stress fibers in various cells. We previously reported that depletion of CLP36 caused loss of stress fibers in BeWo choriocarcinoma cells, but it remains unclear how CLP36 contributes to stress fiber formation. In this study, we generated CLP36-depleted F2408 fibroblasts and found that stress fibers showed abnormal non-oriented organization in these cells. In addition to CLP36, F2408 cells contained RIL, another ALP/Enigma protein, and we demonstrated that RIL could compensate for the role of CLP36 in stress fiber formation. CLP36 and RIL form a complex with α-actinin-1 and palladin. We found a strong correlation between loss of CLP36/RIL and failure of α-actinin-1 or palladin to localize on stress fibers. In addition, time lapse observation revealed that incorporation of RIL stabilizes stress fibers and that CLP36 influences the dynamic architecture of these fibers. Our findings indicate that CLP36 and RIL have a redundant role in the formation of stress fibers, but have different effects on stress fiber dynamics in F2408 cells.     

Complement C3a-induced IL-17 plays a critical role in an IgE-mediated late-phase asthmatic response and airway hyperresponsiveness via neutrophilic inflammation in mice

Allergen-specific IgE plays an essential role in the pathogenesis of allergic asthma. Although there has been increasing evidence suggesting the involvement of IL-17 in the disease, the relationship between IL-17 and IgE-mediated asthmatic responses has not yet been defined. In this study, we attempted to elucidate the contribution of IL-17 to an IgE-mediated late-phase asthmatic response and airway hyperresponsiveness (AHR). BALB/c mice passively sensitized with an OVA-specific IgE mAb were challenged with OVA intratracheally four times. The fourth challenge caused a late-phase increase in airway resistance associated with elevated levels of IL-17(+)CD4(+) cells in the lungs. Multiple treatments with a C3a receptor antagonist or anti-C3a mAb during the challenges inhibited the increase in IL-17(+)CD4(+) cells. Meanwhile, a single treatment with the antagonist or the mAb at the fourth challenge suppressed the late-phase increase in airway resistance, AHR, and infiltration by neutrophils in bronchoalveolar lavage fluid. Because IL-17 production in the lungs was significantly repressed by both treatments, the effect of an anti-IL-17 mAb was examined. The late-phase increase in airway resistance, AHR, and infiltration by neutrophils in bronchoalveolar lavage fluid was inhibited. Furthermore, an anti-Gr-1 mAb had a similar effect. Collectively, we found that IgE mediated the increase of IL-17(+)CD4(+) cells in the lungs caused by repeated Ag challenges via C3a. The mechanisms leading to the IgE-mediated late-phase asthmatic response and AHR are closely associated with neutrophilic inflammation through the production of IL-17 induced by C3a.     

Gain of function by phosphorylation in Presenilin 1-mediated regulation of insulin signaling

During pregnancy, activation of the maternal immune system results in inflammation in the foetal nervous system. The causative agents are pro-inflammatory cytokines like interleukin-1β (IL-1β), produced by the foetus. In this study, we examine the effect of IL-1β on the proliferation and differentiation of neural progenitor cells (NPCs) to better understand its potential effects on the developing brain. We find that the IL-1β receptor (IL-1R1) is expressed in the ventral mesencephalon of the developing brain. Furthermore, IL-1R1 is expressed on Nestin-positive, Sox-2-positive NPCs. IL-1β treatment reduced the numbers of proliferating NPCs, an effect prevented by the IL-1R1 receptor antagonist. LDH and MTT assays, and western blot analysis for cleaved caspase 3 and poly(ADP-ribose) polymerase, confirmed that this was not due to an increase in cell death but rather an induction of differentiation. To further study the effects of IL-1β on cell fate determination, we differentiated NPCs in the presence and absence of IL-1β. Il-1β promoted gliogenesis and inhibited neurogenesis, an effect that required p38-MAPK kinase signalling. In summary, these data show that exposure of NPCs to IL-1β affects their development. This necessitates an examination of the consequences that maternal immune system activation during pregnancy has on the cellular architecture of the developing brain.     

Long-term expression of glial cell line-derived neurotrophic factor slows, but does not stop retinal degeneration in a model of retinitis pigmentosa

J. Neurochem. (2012) 122, 1047-1053. ABSTRACT: Retinitis pigmentosa is a group of diseases in which one of hundreds of mutations causes death of rod photoreceptor cells and then cones gradually die from oxidative damage. As different mutations cause rod cell death by different mechanisms, mutation-specific treatments are needed. Another approach is to use a neurotrophic factor to promote photoreceptor survival regardless of the mechanism of cell death, and previous studies have demonstrated encouraging short-term results with gene transfer of glial cell line-derived neurotrophic factor (GDNF). We generated rd10 mice with doxycycline-inducible expression of GDNF in photoreceptors (Tet/IRBP/GDNF-rd10 mice) or retinal pigmented epithelial cells (Tet/VMD2/GDNF-rd10 mice). In doxycycline-treated Tet/IRBP/GDNF-rd10 mice, there was a 9.3?×?10(4) -fold increase in Gdnf mRNA at P35 and although it decreased over time, it was still increased by 9.4?×?10(3) -fold at P70. Gdnf mRNA was increased 4.5?×?10(2) -fold in doxycycline-treated Tet/VMD2/GDMF-rd10 mice at P35 and was not significantly decreased at P70. GDNF protein levels were increased about 2.3-fold at P35 and 30% at P70 in Tet/IRBP/GDNF-rd10 mice, and in Tet/VMD2/GDNF-rd10 mice they were increased 30% at P35 and not significantly increased at P70. Despite the difference in expression, Tet/IRBP/GDNF-rd10 and Tet/VMD2/GDNF-rd10 mice had comparable significant increases in outer nuclear layer thickness and mean photopic and scotopic ERG b-wave amplitudes compared with rd10 mice at P35 which decreased, but was still significant at P70. Compared with rd10 mice, Tet/IRBP/GDNF-rd10 and Tet/VMD2/GDNF-rd10 mice had comparable significant improvements in cone density at P50 that decreased, but were still significant at P70. These data indicate that despite a large difference in expression of GDNF, Tet/IRBP/GDNF-rd10 and Tet/VMD2/GDNF-rd10 provide comparable slowing of photoreceptor degeneration, but cannot stop the degeneration.     

Glutamate receptor δ1 induces preferentially inhibitory presynaptic differentiation of cortical neurons by interacting with neurexins through cerebellin precursor protein subtypes

Glutamate receptor (GluR) δ1 is widely expressed in the developing forebrain, whereas GluRδ2 is selectively expressed in cerebellar Purkinje cells. Recently, we found that trans-synaptic interaction of postsynaptic GluRδ2 and pre-synaptic neurexins (NRXNs) through cerebellin precursor protein (Cbln) 1 mediates excitatory synapse formation in the cerebellum. Thus, a question arises whether GluRδ1 regulates synapse formation in the forebrain. In this study, we showed that the N-terminal domain of GluRδ1 induced inhibitory presynaptic differentiation of some populations of cultured cortical neurons. When Cbln1 or Cbln2 was added to cultures, GluRδ1 expressed in HEK293T cells induced preferentially inhibitory presynaptic differentiation of cultured cortical neurons. The synaptogenic activity of GluRδ1 was suppressed by the addition of the extracellular domain of NRXN1α or NRXN1β containing splice segment 4. Cbln subtypes directly bound to the N-terminal domain of GluRδ1. The synaptogenic activity of GluRδ1 in the presence of Cbln subtypes correlated well with their binding affinities. When transfected to cortical neurons, GluRδ1 stimulated inhibitory synapse formation in the presence of Cbln1 or Cbln2. These results together with differential interactions of Cbln subtypes with NRXN variants suggest that GluRδ1 induces preferentially inhibitory presynaptic differentiation of cortical neurons by interacting with NRXNs containing splice segment 4 through Cbln subtypes.