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The sialic glycoprotein, MUC1, is known to be involved in the pathogenesis of various types of cancers. KL-6 is one of the surface antigens of MUC1 and also a marker of interstitial pneumonitis. A fraction of patients with myeloma (3.9%) have elevated serum KL-6 levels without any evidence of interstitial pneumonitis and their myeloma cells have high MUC1 expression. We established a myeloma cell line designated EMM1 from a patient with multiple myeloma accompanied with elevated serum KL-6. EMM1 cells expressed high levels of MUC1 compared with other myeloma cell lines. Knockdown of MUC1 in EMM1 cells induced cell cycle arrest during S phase and apoptosis, suggesting that the MUC1 expression is involved in accelerated growth of EMM1 cells. RNA-seq analysis suggests that MUC1 expression activates k-ras and TNFα-induced NFκB pathways in EMM1 cells. We injected EMM1 cells subcutaneously into Rag2 -/- Jak3 -/- Balb/c mice to establish a mouse xenograft model. These mice had aggressive tumor growth that was accompanied by high serum KL-6 levels. In addition, MUC1 knockdown in EMM1 cells led to inhibited tumor growth. These findings demonstrate that MUC1 serves as a potential target for developing drugs for treatment of patients with KL-6 + myeloma, and EMM1 cells and EMM1-engrafted mice are useful tools for the development of such novel agents., (Copyright © 2018 Elsevier Inc. All rights reserved.)

We previously demonstrated that PU.1 expression is down-regulated in the majority of myeloma cell lines and primary myeloma cells from patients. We introduced the tet-off system into the human myeloma cell lines U266 and KMS12PE that conditionally express PU.1 and demonstrated that PU.1 induces cell cycle arrest and apoptosis in myeloma cells in vitro. Here, we established a mouse xenograft model of myeloma using these cell lines to analyze the effects of PU.1 on the phenotype of myeloma cells in vivo. When doxycycline was added to the drinking water of mice engrafted with these myeloma cells, all mice had continuous growth of subcutaneous tumors and could not survived more than 65 days. In contrast, mice that were not exposed to doxycycline did not develop subcutaneous tumors and survived for at least 100 days. We next generated mice engrafted with subcutaneous tumors 5-10 mm in diameter that were induced by exposure to doxycycline. Half of the mice stopped taking doxycycline-containing water, whereas the other half kept taking the water. Although the tumors in the mice taking doxycycline continued to grow, tumor growth in the mice not taking doxycycline was significantly suppressed. The myeloma cells in the tumors of the mice not taking doxycycline expressed PU.1 and TRAIL and many of such cells were apoptotic. Moreover, the expression of a cell proliferation marker Ki67 was significantly decreased in tumors from the mice not taking doxycycline, compared with that of tumors from the mice continuously taking doxycycline. The present data strongly suggest that PU.1 functions as a tumor suppressor of myeloma cells in vivo., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Immunomodulatory drugs (IMiDs) such as thalidomide, lenalidomide, and pomalidomide are efficacious in the treatment of multiple myeloma and significantly prolong their survival. However, the mechanisms of such effects of IMiDs have not been fully elucidated. Recently, cereblon has been identified as a target binding protein of thalidomide. Lenalidomide-resistant myeloma cell lines often lose the expression of cereblon, suggesting that IMiDs act as an anti-myeloma agent through interacting with cereblon. Cereblon binds to damaged DNA-binding protein and functions as a ubiquitin ligase, inducing degradation of IKZF1 and IKZF3 that are essential transcription factors for B and T cell development. Degradation of both IKZF1 and IKZF3 reportedly suppresses myeloma cell growth. Here, we found that IMiDs act as inhibitors of DNA methyltransferases (DMNTs). We previously reported that PU.1, which is an ETS family transcription factor and essential for myeloid and lymphoid development, functions as a tumor suppressor in myeloma cells. PU.1 induces growth arrest and apoptosis of myeloma cell lines. In this study, we found that low-dose lenalidomide and pomalidomide up-regulate PU.1 expression through inducing demethylation of the PU.1 promoter. In addition, IMiDs inhibited DNMT1, DNMT3a, and DNMT3b activities in vitro. Furthermore, lenalidomide and pomalidomide decreased the methylation status of the whole genome in myeloma cells. Collectively, IMiDs exert demethylation activity through inhibiting DNMT1, 3a, and 3b, and up-regulating PU.1 expression, which may be one of the mechanisms of the anti-myeloma activity of IMiDs., (Copyright © 2015 Elsevier Inc. All rights reserved.)

MER5 (also called peroxiredoxin III, PrxIII) is a member of peroxiredoxin family that has antioxidant activity. The present study was performed to investigate its in vivo function using MER5 knockout mice. MER5 knockout mice were born in normal frequency and could grow to maturity, but we found that intracellular ROS levels are significantly higher in the macrophages of the knockout mice. We examined roles of MER5 function for the oxidative stress responses by intratracheal inoculation of lipopolysaccharide (LPS) to the mice. Lung inflammation such as inflammatory cell infiltration and airway wall thickening was more severely detected in the knockout mice. At the same time, oxidative damage on DNA and proteins was more strongly detected in lung tissues of the knockout mice, including 8-hydroxy-2'-deoxyguanosine (8-OHdG) formation and protein carbonylation. The degrees of lung inflammation and oxidative damage were positively related with LPS doses. Our results indicate that MER5 knockout mice accumulated higher intracellular ROS levels, which cause LPS-induced lung injury more severely, and thus, suggested that MER5 acts as an important scavenger of reactive oxygen species (ROS) under oxidative stress.

Wild watermelon (Citrullus lanatus sp.) has the ability to tolerate severe drought/high light stress conditions despite carrying out normal C3-type photosynthesis. Here, mRNA differential display was employed to isolate drought-responsive genes in the leaves of wild watermelon. One of the isolated genes, CLMT2, shared significant homology with type-2 metallothionein (MT) sequences from other plants. The second-order rate constant for the reaction between a recombinant CLMT2 protein and hydroxyl radicals was estimated to be 1.2 x 10(11) M(-1) s(-1), demonstrating that CLMT2 had an extraordinary high activity for detoxifying hydroxyl radicals. Moreover, hydroxyl radical-catalyzed degradation of watermelon genomic DNA was effectively suppressed by CLMT2 in vitro. This is the first demonstration of a plant MT with antioxidant properties. The results suggest that CLMT2 induction contributes to the survival of wild watermelon under severe drought/high light stress conditions., (Copyright 2004 Elsevier Inc.)

The Rab3 family small G proteins (Rab3A-D) are involved in the regulated secretory pathway of brain and secretory tissues. Among Rab3-interacting proteins, Rabphilin-3, Rim, and Noc2, all of which contain a conserved Rab3-binding domain (RBD3), are generally recognized Rab3 effector proteins in neurons and secretory cells. Although Rab3B was also detected in epithelial cells, its function remained unknown. We isolated cDNA sequences from human epithelial Caco2-cell mRNA by degenerate RT-PCR based on the conserved amino acid sequence of RBD3. Multiple cDNA clones were identified as encoding Noc2. Northern blot analysis revealed that Noc2 mRNA was expressed not only in secretory tissues but also in epithelial tissues and cell lines. A pull-down assay demonstrated that Noc2 bound to Rab3B in a GTP-dependent manner. When Noc2 was co-expressed with the GTP-bound form of Rab3B, it was recruited from the cytosol to perinuclear membranes. Furthermore, overexpression of Noc2 inhibited the cell-surface transport of basolateral vesicular stomatitis virus glycoprotein. These results suggest that Noc2 functions as a potential Rab3B effector protein in epithelial cells.

Regulated transport of proteins to distinct plasma membrane domains is essential for the establishment and maintenance of cell polarity in all eukaryotic cells. The Rab family small G proteins play a crucial role in determining the specificity of vesicular transport pathways. Rab3B and Rab13 localize to tight junction in polarized epithelial cells and cytoplasmic vesicular structures in non-polarized fibroblasts, but their functions are poorly understood. Here we examined their roles in regulating the cell-surface transport of apical p75 neurotrophin receptor (p75NTR), basolateral low-density lipoprotein receptor (LDLR), and tight junctional Claudin-1 using transport assay in non-polarized fibroblasts. Overexpression of Rab3B mutants inhibited the cell-surface transport of LDLR, but not p75NTR and Claudin-1. In contrast, overexpression of Rab13 mutants impaired the transport of Claudin-1, but not LDLR and p75NTR. These results suggest that Rab3B and Rab13 direct the cell-surface transport of LDLR and Claudin-1, respectively, and may contribute to epithelial polarization.

To clarify the therapeutic effects of 6R-L-erythro-5,6,7,8-tetrahydrobiopterin (6R-BH(4)) on the abnormal behaviors induced by neonatal 5,7-dihydroxytryptamine (5,7-DHT, 100 microg; i.c.v.) treatment in immature rats, 6R-BH(4) (10-40 mg/kg) was administered intraperitoneally from 22nd to 28th days or only once on the 28th day. The locomotion activities decreased dramatically in 5,7-DHT-treated rats (p<0.01; as compared to controls) on the 28th day. The reduced locomotion was recovered dose-dependently by repeated administration of 6R-BH(4), whereas it was not altered after a single injection of 6R-BH(4). In addition, repeated administration of 6R-BH(4) significantly facilitated 5-HT turnover ratio (5-HIAA/5-HT) in the striatum, cerebral cortex, and cerebellum. These findings suggest that the behavioral restoration by 6R-BH(4) might be due to the enhancement of 5-HT turnover by accumulated but not a single dose of 6R-BH(4).

Although the existence of the receptor for secretin in the brain was suggested, the localization of secretin receptor and the neuronal function of secretin have not been clarified yet. In the present study, the localization of secretin receptor was investigated in the rat brain by using an in vitro autoradiography technique. Frozen section autoradiography with (125)I-secretin showed intense binding in the nucleus of solitary tract, laterodorsal thalamic nucleus, and accumbens nucleus; moderate binding in the hippocampus, caudate/putamen, cerebellum, cingulate and orbital cortices. Scatchard plot analysis gave the Kd value of 125 pM with Bmax of 134 fmol/mg tissue in the hippocampus. The binding specificity was confirmed with secretin and its analogs, VIP, PACAP, and glucagon. These results indicate the secretin receptor system might have some neural functions in the brain, which could give the basis for therapeutic use of secretin in autistic children., ((C)2002 Elsevier Science (USA).)