Partial silencing of fucosyltransferase 8 gene expression inhibits proliferation of Ishikawa cells,a cell line of endometrial cancer

Endometrial cancer is the most common gynecologic malignancy and is associated with increased morbidity each year, including young people. However, its mechanisms of proliferation and progression are not fully elucidated. It is well known that abnormal glycosylation is involved in oncogenesis, and fucosylation is one of the most important types of glycosylation. In particular, fucosyltransferase 8 (FUT8) is the only FUT responsible for α1, 6-linked fucosylation (core fucosylation), and it is involved in various physiological as well as pathophysiological processes, including cancer biology. Therefore, we aimed to identify the expression of FUT8 in endometrial endometrioid carcinoma and investigate the effect of the partial silencing of the FUT8 gene on the cell proliferation of Ishikawa cells, an epithelial-like endometrial cancer cell line. Quantitative real-time PCR analysis showed that FUT8 gene expression was significantly elevated in the endometrial endometrioid carcinoma, compared to the normal endometrium. The immunostaining of FUT8 and Ulex europaeus Agglutinin 1 (UEA-1), a kind of lectin family specifically binding to fucose, was detected endometrial endometrioid carcinoma. The proliferation assay showed FUT8 partial knockdown by transfection of siRNA significantly suppressed the proliferation of Ishikawa cells, concomitant with the upregulation in the gene expressions associated with the interesting pathways associated with de-ubiquitination, aspirin trigger, mesenchymal-epithelial transition (MET) et al. It was suggested that the core fucosylation brought about by FUT8 might be involved in the proliferation of endometrial endometrioid carcinoma cells.     

Serine 216 Phosphorylation of?Estrogen Receptor α in Neutrophils: Migration and Infiltration into the Mouse Uterus

Background

Whereas estrogen receptors are present in immune cells, it is not known if they are phosphorylated to regulate immune cell functions. Here we determined the phosphorylation status of estrogen receptor α (ERα) at residue serine 216 in mouse neutrophils and examined its　role in migration and infiltration. Serine 216 is the conserved phosphorylation site within the DNA binding domains found in the majority of nuclear receptors.

Methodology/Principal Findings

A phospho-peptide antibody specific to phosphorylated serine 216 and ERα KO mice were utilized in immunohistochemistry, double immuno-staining or Western blot to detect phosphorylation of ERα in peripheral blood as well as infiltrating neutrophils in the mouse uterus. Transwell assays were performed to examine migration of neutrophils. An anti-Ly6G antibody identified neutrophils. About 20% of neutrophils expressed phosphorylated ERα at serine 216 in peripheral white blood cells (WBC) from C3H/HeNCrIBR females. Phosphorylation was additively segregated between C3H/HeNCrIBR and C57BL/6 females. Only neutrophils that expressed phosphorylated ERα migrated in Transwell assays as well as infiltrated the mouse uterus during normal estrous cycles.

Conclusions/Significance

ERα was phosphorylated at serine 216 in about 20% of mouse peripheral blood neutrophils. Only those that express phosphorylated ERα migrate and infiltrate the mouse uterus. This phosphorylation was the first to be characterized in endogenous ERα found in normal tissues and cells. Phosphorylated ERα may have opened a novel research direction for biological roles of phosphorylation in ERα actions and can be developed as a drug target for treatment of immune-related diseases.     

Methamphetamine Increases Locomotion and Dopamine Transporter Activity in Dopamine D5 Receptor-Deficient Mice

Dopamine regulates the psychomotor stimulant activities of amphetamine-like substances in the brain. The effects of dopamine are mediated through five known dopamine receptor subtypes in mammals. The functional relevance of D5 dopamine receptors in the central nervous system is not well understood. To determine the functional relevance of D5 dopamine receptors, we created D5 dopamine receptor-deficient mice and then used these mice to assess the roles of D5 dopamine receptors in the behavioral response to methamphetamine. Interestingly, D5 dopamine receptor-deficient mice displayed increased ambulation in response to methamphetamine. Furthermore, dopamine transporter threonine phosphorylation levels, which regulate amphetamine-induced dopamine release, were elevated in D5 dopamine receptor-deficient mice. The increase in methamphetamine-induced locomotor activity was eliminated by pretreatment with the dopamine transporter blocker GBR12909. Taken together, these results suggest that dopamine transporter activity and threonine phosphorylation levels are regulated by D5 dopamine receptors.     

Vitamin D2 interacts with Human PrPc (90–231) and breaks PrPc oligomerization in vitro

PrP^sc, the pathogenic isoform of PrP^c, can convert PrP^c into PrP^sc through direct interactions. PrP^c oligomerization is a required processing step before PrP^sc formation, and soluble oligomers appear to be the toxic species in amyloid-related disorders. In the current study, direct interactions between vitamin D₂ and human recombinant PrP^c (90–231) were observed by Biacore assay, and 3F4 antibody, specific for amino acid fragment 109–112 of PrP^c, inhibited this interaction. An ELISA study using3F4 antibody showed that PrP^c (101–130), corresponding sequence to human PrP, was affected by vitamin D₂, supporting the results of Biacore studies and suggesting that the PrP^c sequence around the 3F4 epitope was responsible for the interaction with vitamin D₂. Furthermore, the effects of vitamin D₂ on disruption of PrP^c (90–231) oligomerization were elucidated by dot blot analysis and differential protease k susceptibilities. While many chemical compounds have been proposed as potential therapeutic agents for the treatment of scrapie, most of these are toxic. However, given the safety and blood brain barrier permeability of vitamin D₂, we propose that vitamin D₂ may be a suitable agent to target PrP^c in the brain and therefore is a potential therapeutic candidate for prion disease.     

Chronic Alterations in Monoaminergic Cells in the Locus Coeruleus in Orexin Neuron-Ablated Narcoleptic Mice

Narcolepsy patients often suffer from insomnia in addition to excessive daytime sleepiness. Narcoleptic animals also show behavioral instability characterized by frequent transitions between all vigilance states, exhibiting very short bouts of NREM sleep as well as wakefulness. The instability of wakefulness states in narcolepsy is thought to be due to deficiency of orexins, neuropeptides produced in the lateral hypothalamic neurons, which play a highly important role in maintaining wakefulness. However, the mechanism responsible for sleep instability in this disorder remains to be elucidated. Because firing of orexin neurons ceases during sleep in healthy animals, deficiency of orexins does not explain the abnormality of sleep. We hypothesized that chronic compensatory changes in the neurophysiologica activity of the locus coeruleus (LC) and dorsal raphe (DR) nucleus in response to the progressive loss of endogenous orexin tone underlie the pathological regulation of sleep/wake states. To evaluate this hypothesis, we examined firing patterns of serotonergic (5-HT) neurons and noradrenergic (NA) neurons in the brain stem, two important neuronal populations in the regulation of sleep/wakefulness states. We recorded single-unit activities of 5-HT neurons and NA neurons in the DR nucleus and LC of orexin neuron-ablated narcoleptic mice. We found that while the firing pattern of 5-HT neurons in narcoleptic mice was similar to that in wildtype mice, that of NA neurons was significantly different from that in wildtype mice. In narcoleptic mice, NA neurons showed a higher firing frequency during both wakefulness and NREM sleep as compared with wildtype mice. In vitro patch-clamp study of NA neurons of narcoleptic mice suggested a functional decrease of GABAergic input to these neurons. These alterations might play roles in the sleep abnormality in narcolepsy.     

Developmental and Visual Input-Dependent Regulation of the CB1 Cannabinoid Receptor in the Mouse Visual Cortex

The mammalian visual system exhibits significant experience-induced plasticity in the early postnatal period. While physiological studies have revealed the contribution of the CB1 cannabinoid receptor (CB1) to developmental plasticity in the primary visual cortex (V1), it remains unknown whether the expression and localization of CB1 is regulated during development or by visual experience. To explore a possible role of the endocannabinoid system in visual cortical plasticity, we examined the expression of CB1 in the visual cortex of mice. We found intense CB1 immunoreactivity in layers II/III and VI. CB1 mainly localized at vesicular GABA transporter-positive inhibitory nerve terminals. The amount of CB1 protein increased throughout development, and the specific laminar pattern of CB1 appeared at P20 and remained until adulthood. Dark rearing from birth to P30 decreased the amount of CB1 protein in V1 and altered the synaptic localization of CB1 in the deep layer. Dark rearing until P50, however, did not influence the expression of CB1. Brief monocular deprivation for 2 days upregulated the localization of CB1 at inhibitory nerve terminals in the deep layer. Taken together, the expression and the localization of CB1 are developmentally regulated, and both parameters are influenced by visual experience.     

Rates of Serious Intracellular Infections in Autoimmune Disease Patients Receiving Initial Glucocorticoid Therapy

Background/Aims

The Japanese National Hospital Organization evidence-based medicine (EBM) Study group for Adverse effects of Corticosteroid therapy (J-NHOSAC) is a Japanese hospital-based cohort study investigating the safety of the initial use of glucocorticoids (GCs) in patients with newly diagnosed autoimmune diseases. Using the J-NHOSAC registry, the purpose of this observational study is to analyse the rates, characteristics and associated risk factors of intracellular infections in patients with newly diagnosed autoimmune diseases who were initially treated with GCs.

Methodology/Principal Findings

A total 604 patients with newly diagnosed autoimmune diseases treated with GCs were enrolled in this registry between April 2007 and March 2009. Cox proportional-hazards regression was used to determine independent risk factors for serious intracellular infections with covariates including sex, age, co-morbidity, laboratory data, use of immunosuppressants and dose of GCs. Survival was analysed according to the Kaplan-Meier method and was assessed by the log-rank test. There were 127 serious infections, including 43 intracellular infections, during 1105.8 patient-years of follow-up. The 43 serious intracellular infections resulted in 8 deaths. After adjustment for covariates, diabetes (Odds ratio [OR]: 2.5, 95% confidence interval [95% CI] 1.1–5.9), lymphocytopenia (≦1000/μl, OR: 2.5, 95% CI 1.2–5.2) and use of high-dose (≧30 mg/day) GCs (OR: 2.4, 95% CI 1.1–5.3) increased the risk of intracellular infections. Survival curves showed lower intracellular infection-free survival rate in patients with diabetes, lymphocytopaenia and high-dose GCs treatments.

Conclusions/Significance

Patients with newly diagnosed autoimmune diseases were at high risk of developing intracellular infection during initial treatment with GCs. Our findings provide background data on the risk of intracellular infections of patients with autoimmune diseases. Clinicians showed remain vigilant for intracellular infections in patients with autoimmune diseases who are treated with GCs.     

Sall4 Is Transiently Expressed in the Caudal Wolffian Duct and the Ureteric Bud,but Dispensable for Kidney Development

The kidney, the metanephros, is formed by reciprocal interactions between the metanephric mesenchyme and the ureteric bud, the latter of which is derived from the Wolffian duct that elongates in the rostral-to-caudal direction. Sall1 expressed in the metanephric mesenchyme is essential for ureteric bud attraction in kidney development. Sall4, another member of the Sall gene family, is required for maintenance of embryonic stem cells and establishment of induced pluripotent stem cells, and is thus considered to be one of the stemness genes. Sall4 is also a causative gene for Okihiro syndrome and is essential for the formation of many organs in both humans and mice. However, its expression and role in kidney development remain unknown, despite the essential role of Sall1 in the metanephric mesenchyme. Here, we report that mouse Sall4 is expressed transiently in the Wolffian duct-derived lineage, and is nearly complementary to Sall1 expression. While Sall4 expression is excluded from the Wolffian duct at embryonic (E) day 9.5, Sall4 is expressed in the Wolffian duct weakly in the mesonephric region at E10.5 and more abundantly in the caudal metanephric region where ureteric budding occurs. Sall4 expression is highest at E11.5 in the Wolffian duct and ureteric bud, but disappears by E13.5. We further demonstrate that Sall4 deletion in the Wolffian duct and ureteric bud does not cause any apparent kidney phenotypes. Therefore, Sall4 is expressed transiently in the caudal Wolffian duct and the ureteric bud, but is dispensable for kidney development in mice.     

Mother–calf interactions and social behavior development in Commerson’s dolphins (Cephalorhynchus commersonii)

Mother–calf interactions and the behaviors of mothers during separation from their calves were examined in four Commerson’s dolphin (Cephalorhynchus commersonii) mother–calf pairs. Four infants were observed: 56.8 h over 30 days from birth to 263 days of age, 36.9 h over 20 days from birth to 149 days of age, 10.4 h over 3 days from birth to 2 days of age, and 15.0 h over 3 days from birth to 2 days of age. All four pairs shared common characteristics in the rate and frequencies of mother–calf interactions and the behaviors of mothers during the first week of life. After the first week, individual differences in changes in the frequency of each behavior were observed. The three behaviors considered representative of maternal care (parallel swimming, synchronous breathing, and body-to-body contact) were frequently performed in the first week; thereafter, the frequencies declined. Separate behaviors of mothers were infrequent during the first week and increased with an increase in infants’ age. Bumping by infants increased with time, suggesting an increase in soliciting by calves and conflict between mothers and calves. The frequency of flipper-to-body rubbing also changed but in a complex manner, probably because the calves needed to learn how to perform this behavior from their mothers and because initiator and recipient of this behavior can be changed quickly.