Cellular Auxin Homeostasis under High Temperature Is Regulated through a SORTING NEXIN1–Dependent Endosomal Trafficking Pathway

High-temperature-mediated adaptation in plant architecture is linked to the increased synthesis of the phytohormone auxin, which alters cellular auxin homeostasis. The auxin gradient, modulated by cellular auxin homeostasis, plays an important role in regulating the developmental fate of plant organs. Although the signaling mechanism that integrates auxin and high temperature is relatively well understood, the cellular auxin homeostasis mechanism under high temperature is largely unknown. Using the Arabidopsis thaliana root as a model, we demonstrate that under high temperature, roots counterbalance the elevated level of intracellular auxin by promoting shootward auxin efflux in a PIN-FORMED2 (PIN2)-dependent manner. Further analyses revealed that high temperature selectively promotes the retrieval of PIN2 from late endosomes and sorts them to the plasma membrane through an endosomal trafficking pathway dependent on SORTING NEXIN1. Our results demonstrate that recycling endosomal pathway plays an important role in facilitating plants adaptation to increased temperature.     

DNA methylation profiles provide a viable index for porcine pluripotent stem cells

Porcine induced pluripotent stem cells (iPSCs) provide useful information for translational research. The quality of iPSCs can be assessed by their ability to differentiate into various cell types after chimera formation. However, analysis of chimera formation in pigs is a labor‐intensive and costly process, necessitating a simple evaluation method for porcine iPSCs. Our previous study identified mouse embryonic stem cell (ESC)‐specific hypomethylated loci (EShypo‐T‐DMRs), and, in this study, 36 genes selected from these were used to evaluate porcine iPSC lines. Based on the methylation profiles of the 36 genes, the iPSC line, Porco Rosso‐4, was found closest to mouse pluripotent stem cells among 5 porcine iPSCs. Moreover, Porco Rosso‐4 more efficiently contributed to the inner cell mass (ICM) of blastocysts than the iPSC line showing the lowest reprogramming of the 36 genes (Porco Rosso‐622‐14), indicating that the DNA methylation profile correlates with efficiency of ICM contribution. Furthermore, factors known to enhance iPSC quality (serum‐free medium with PD0325901 and CHIR99021) improved the methylation status at the 36 genes. Thus, the DNA methylation profile of these 36 genes is a viable index for evaluation of porcine iPSCs. genesis 51:763–776. © 2013 Wiley Periodicals, Inc.     

Mouse arylamine N-acetyltransferase 2 (Nat2) expression during embryogenesis: a potential marker for the developing neuroendocrine system

Arylamine N-acetyltransferase (NAT) genes in humans and in rodents encode polymorphic drug metabolizing enzymes. Human NAT1 (and the murine equivalent mouse Nat2) is found early in embryonic development and is likely to have an endogenous role. We report the detailed expression of the murine gene (Nat2) and encoded protein in mouse embryos, using a transgenic mouse model bearing a lacZ transgene inserted into the coding region of mouse Nat2. In mouse embryos, the transgene was expressed in sensory epithelia, epithelial placodes giving rise to visceral sensory neurons, the developing pituitary gland, sympathetic chain and urogenital ridge. In Nat2^+/+ mice, the presence and activity of Nat2 protein was detected in these tissues and their adult counterparts. Altered expression of the human orthologue in breast tumours, in which there is endocrine signalling, suggests that human NAT1 should be considered as a potential biomarker for neuroendocrine tissues and tumours.     

Therapeutic Efficacy of C-Kit-Targeted Radioimmunotherapy Using 90Y-Labeled Anti-C-Kit Antibodies in a Mouse Model of Small Cell Lung Cancer

Small cell lung cancer (SCLC) is an aggressive tumor and prognosis remains poor. Therefore, the development of more effective therapy is needed. We previously reported that high levels of an anti-c-kit antibody (12A8) accumulated in SCLC xenografts. In the present study, we evaluated the efficacy of two antibodies (12A8 and 67A2) for radioimmunotherapy (RIT) of an SCLC mouse model by labeling with the ⁹⁰Y isotope.

Methods

¹¹¹In- or ¹²⁵I-labeled antibodies were evaluated in vitro by cell binding, competitive inhibition and cellular internalization assays in c-kit-expressing SY cells and in vivo by biodistribution in SY-bearing mice. Therapeutic efficacy of ⁹⁰Y-labeled antibodies was evaluated in SY-bearing mice upto day 28 and histological analysis was conducted at day 7.

Results

[¹¹¹In]12A8 and [¹¹¹In]67A2 specifically bound to SY cells with high affinity (8.0 and 1.9 nM, respectively). 67A2 was internalized similar to 12A8. High levels of [¹¹¹In]12A8 and [¹¹¹In]67A2 accumulated in tumors, but not in major organs. [¹¹¹In]67A2 uptake by the tumor was 1.7 times higher than for [¹¹¹In]12A8. [⁹⁰Y]12A8, but not [⁹⁰Y]67A2, suppressed tumor growth in a dose-dependent manner. Tumors treated with 3.7 MBq of [⁹⁰Y]12A8, and 1.85 and 3.7 MBq of [⁹⁰Y]67A2 (absorbed doses were 21.0, 18.0 and 35.9 Gy, respectively) almost completely disappeared approximately 2 weeks after injection, and regrowth was not observed except for in one mouse treated with 1.85 MBq [⁹⁰Y]67A2. The area of necrosis and fibrosis increased depending on the RIT effect. Apoptotic cell numbers increased with increased doses of [⁹⁰Y]12A8, whereas no dose-dependent increase was observed following [⁹⁰Y]67A2 treatment. Body weight was temporarily reduced but all mice tolerated the RIT experiments well.

Conclusion

Treatment with [⁹⁰Y]12A8 and [⁹⁰Y]67A2 achieved a complete therapeutic response when SY tumors received an absorbed dose greater than 18 Gy and thus are promising RIT agents for metastatic SCLC cells at distant sites.     

Interferon-β Produces Synergistic Combinatory Anti-Tumor Effects with Cisplatin or Pemetrexed on Mesothelioma Cells

Interferons (IFNs) have been tested for the therapeutic effects in various types of malignancy, but mechanisms of the anti-tumors effects and the differential biological activities among IFN members are dependent on respective cell types. In this study, we examined growth inhibitory activities of type I and III IFNs on 5 kinds of human mesothelioma cells bearing wild-type p53 gene, and showed that type I IFNs but not type III IFNs decreased the cell viabilities. Moreover, growth inhibitory activities and up-regulated expression levels of the major histocompatibility complexes class I antigens were greater with IFN-β than with IFN-α treatments. Cell cycle analyses demonstrated that type I IFNs increased S- and G2/M-phase populations, and subsequently sub-G1-phase fractions. The cell cycle changes were also greater with IFN-β than IFN-α treatments, and these data collectively showed that IFN-β had stronger biological activities than IFN-α in mesothelioma. Type I IFNs-treated cells increased p53 expression and the phosphorylation levels, and activated apoptotic pathways. A combinatory use of IFN-β and cisplatin or pemetrexed, both of which are the current first-line chemotherapeutic agents for mesothelioma, produced synergistic anti-tumor effects, which were also evidenced by increased sub-G1-phase fractions. These data demonstrated firstly to our knowledge that IFN-β produced synergistic anti-tumor effects with cisplatin or pemetrexed on mesothelioma through up-regulated p53 expression.     

An In Vitro ES Cell-Based Clock Recapitulation Assay Model Identifies CK2α as an Endogenous Clock Regulator

We previously reported emergence and disappearance of circadian molecular oscillations during differentiation of mouse embryonic stem (ES) cells and reprogramming of differentiated cells, respectively. Here we present a robust and stringent in vitro circadian clock formation assay that recapitulates in vivo circadian phenotypes. This assay system first confirmed that a mutant ES cell line lacking Casein Kinase I delta (CKIδ) induced ∼3 hours longer period-length of circadian rhythm than the wild type, which was compatible with recently reported results using CKIδ null mice. In addition, this assay system also revealed that a Casein Kinase 2 alpha subunit (CK2α) homozygous mutant ES cell line developed significantly longer (about 2.5 hours) periods of circadian clock oscillations after in vitro or in vivo differentiation. Moreover, revertant ES cell lines in which mutagenic vector sequences were deleted showed nearly wild type periods after differentiation, indicating that the abnormal circadian period of the mutant ES cell line originated from the mutation in the CK2α gene. Since CK2α deficient mice are embryonic lethal, this in vitro assay system represents the genetic evidence showing an essential role of CK2α in the mammalian circadian clock. This assay was successfully applied for the phenotype analysis of homozygous mutant ES cells, demonstrating that an ES cell-based in vitro assay is available for circadian genetic screening.     

Role of Musclin in the Pathogenesis of Hypertension in Rat

Musclin is a novel skeletal muscle-derived secretory factor found in the signal sequence trap of mouse skeletal muscle cDNAs. Musclin possesses a region homologous to the natriuretic peptide family. Thus, musclin is found to bind with the natriuretic peptide clearance receptors. However, the role of musclin in vascular regulation remains unclear. In this study, we aim to investigate the direct effect of musclin on vascular tone and to analyze its role in hypertension using the spontaneously hypertensive rats (SHR). In aortic strips isolated from SHR, musclin induced contractions in a dose-dependent manner. We found that the musclin-induced vasoconstriction was more marked in SHR than in normal rats (WKY). Moreover, this contraction was reduced by blockade of natriuretic peptide receptor C using the ab14355 antibody. Therefore, mediation of the natriuretic peptide receptor in musclin-induced vasoconstriction can be considered. In addition, similar to the natriuretic peptide receptor, expression of the musclin gene in blood vessels was higher in SHR than in WKY. Injection of musclin markedly increased the blood pressure in rats that can be inhibited by anti-musclin antibodies. Musclin-induced vasoconstriction was more pronounced in SHR than in WKY as in its expression. Taken together, these results suggest that musclin is involved in blood pressure regulation. The higher expression of musclin in hypertension indicates that musclin could be used as a new target for the treatment of hypertension in the future.     

Helicobacter pylori Cholesteryl α-Glucosides Contribute to Its Pathogenicity and Immune Response by Natural Killer T Cells

Approximately 10–15% of individuals infected with Helicobacter pylori will develop ulcer disease (gastric or duodenal ulcer), while most people infected with H. pylori will be asymptomatic. The majority of infected individuals remain asymptomatic partly due to the inhibition of synthesis of cholesteryl α-glucosides in H. pylori cell wall by α1,4-GlcNAc-capped mucin O-glycans, which are expressed in the deeper portion of gastric mucosa. However, it has not been determined how cholesteryl α-glucosyltransferase (αCgT), which forms cholesteryl α-glucosides, functions in the pathogenesis of H. pylori infection. Here, we show that the activity of αCgT from H. pylori clinical isolates is highly correlated with the degree of gastric atrophy. We investigated the role of cholesteryl α-glucosides in various aspects of the immune response. Phagocytosis and activation of dendritic cells were observed at similar degrees in the presence of wild-type H. pylori or variants harboring mutant forms of αCgT showing a range of enzymatic activity. However, cholesteryl α-glucosides were recognized by invariant natural killer T (iNKT) cells, eliciting an immune response in vitro and in vivo. Following inoculation of H. pylori harboring highly active αCgT into iNKT cell-deficient (Jα18^−/−) or wild-type mice, bacterial recovery significantly increased in Jα18^−/− compared to wild-type mice. Moreover, cytokine production characteristic of Th1 and Th2 cells dramatically decreased in Jα18^−/− compared to wild-type mice. These findings demonstrate that cholesteryl α-glucosides play critical roles in H. pylori-mediated gastric inflammation and precancerous atrophic gastritis.