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Gastric cancer (GC) is a major of cause of cancer-related death and is characterized by its heterogeneity and molecular complexity. FOXO1 is a transcription factor that plays a key role in GC growth and metastasis. However, the implication of FOXO1 in GC cell stemness has been elusive. This study, for the first time, demonstrates that FOXO1 regulates GC cell stemness in association with LGR5. FOXO1 expression was significantly lower in GC tumorsphere cells than in adherent GC cells. FOXO1 silencing and overexpression promoted and inhibited the tumorsphere formation capacity of GC cells, respectively. Additionally, there was an inverse correlation between FOXO1 and GC stem cell marker LGR5 in human GC specimens. Further in vitro and in vivo experiments showed that negative crosstalk between these two molecules exists and that LGR5 silencing reversed the FOXO1 shRNA-induced tumorsphere formation even without FOXO1 restoration. Taken together, our results suggest that FOXO1 inhibits the self-renewal capacity of GC cells through interaction with LGR5. Thus, FOXO1/LGR5 signaling pathway may provide a novel targeted therapy for GC., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Interleukin (IL)-10 is an anti-inflammatory factor that suppresses renal fibrosis and improves renal function in CKD rats. IL-20 belongs to the IL-10 family; therefore, we sought to determine whether IL-20 is involved in CKD. CKD patients at stage five expressed significantly higher IL-20 in serum than controls. Immunohistochemical staining demonstrated that more IL-20 protein was expressed in the kidney tubular-epithelial cells, mesangial cells, and immune cells of CKD rats with a 5/6 nephrectomy. The lung, liver, and heart tissue of CKD rats also overexpressed IL-20. Thus, we treated two tubular epithelial cells, TKPTS and M-1 cells, with IL-20 to study its effects on CKD. IL-20 treatment induced apoptosis in these cells via caspase-3 activation. Incubating IL-20 with rat interstitial fibroblasts, NRK-49F cells, upregulated TGF-beta1 production, one key inducer for renal fibrogenesis. Therefore, IL-20 injured renal epithelial cells and induced fibroblasts to produce TGF-beta1 that hastened the progression of CKD.

Thiazolidinediones (TZDs) have recently been proposed as a therapy for PPARgamma-expressing tumors. Pheochromocytoma (PHEO) is associated with high morbidity and mortality due to excess catecholamine production, and few effective drug therapies currently exist. We investigated the effects of TZDs on PHEO both in vitro and in vivo. PPARgamma protein was expressed in human adrenal PHEO tissues as well as in rat PHEO cells, PC12. TZDs, including rosiglitazone (RGZ) and pioglitazone (PGZ), inhibited proliferation of PC12 cells in a dose-dependent manner and increased casapse-3 expression of PC12 cells. TZDs also reduced expression of cyclin E and cyclin-dependent kinase2. RGZ inhibited nerve growth factor-induced neurite outgrowth and reduced expression of catecholamine-synthesizing enzymes. Finally, rat PHEO growth generated by subcutaneous injection of PC12 cells was slowed in an RGZ-treated mouse. These data suggest that TZDs may be a promising therapeutic approach for medical treatment for PHEO.

Interleukin (IL)-19 belongs to the IL-10 family which includes IL-19, IL-20, IL-22, AK155, and MDA-7. IL-10 is a potent immunomodulatory cytokine with implications for pathogenesis in various autoimmune diseases. Polymorphism of the IL-10 promoter region correlates with disease outcome. To understand the gene regulation of IL-19, we analyzed the IL-19 promoter region. A regulatory region (PE), 148bp upstream of exon 1 of IL-19 and linked to a luciferase reporter gene, supported luciferase activity 13 times greater than that supported by a negative promoterless control. An electrophoretic mobility shift assay (EMSA) showed specific binding sites for the transcription factors of the oligonucleotides PE1 (-148 to -98) derived from PE. We identified the sequence TGTGGT (-142 to -138) on PE1 as the binding site for the transcription factor AML1, and crucial for the promoter activity of IL-19 because substituting 1bp in the PE region (-139G-->T) abolished IL-19 promoter activity.

Astrin is a microtubule-associated protein and localizes with mitotic spindles in the M-phase. We silenced the expression of astrin protein and tested the cell viability in response to paclitaxel treatment in paclitaxel-sensitive and paclitaxel-resistant cells. We found that the absence of astrin by siRNA resulted in the activation of a p53-dependent apoptosis, which elevated pro-apoptotic Bax expression and increased the activity of caspase-3 in astrin-depleted cells. The HPV18 E6 transcription was found to be inhibited along with the increase expression of p53. Intriguingly, the expression of astrin decreased in paclitaxel-sensitive HeLa cells but remained steady in paclitaxel-resistant cells in response to paclitaxel treatment. Furthermore, we identified that the depletion of astrin caused more cell death both in paclitaxel-sensitive and -resistant cells in combination with paclitaxel treatment. These findings suggest that the silencing of astrin induce a p53-dependent apoptosis and has an additive effect on paclitaxel treatment.

Early detection and identification of SARS-CoV-infected patients and actions to prevent transmission are absolutely critical to prevent another SARS outbreak. Antibodies that specifically recognize the SARS-CoV spike and nucleocapsid proteins may provide a rapid screening method to allow accurate identification and isolation of patients with the virus early in their infection. For this reason, we raised peptide-induced polyclonal antibodies against SARS-CoV spike protein and polyclonal antibodies against SARS-CoV nucleocapsid protein using 6x His nucleocapsid recombinant protein. Western blot analysis and immunofluorescent staining showed that these antibodies specifically recognized SARS-CoV.

Human astrin is a newly identified microtubule-associated protein, which is highly expressed in the testis. Silencing of astrin has resulted in growth arrest and apoptotic cell death. In this study, we describe the cloning and genomic structure of mastrin, the mouse counterpart to astrin. The overall mouse mastrin amino-acid sequence is 66% identical to human astrin. Mastrin protein was demonstrated to localize to mitotic spindles during mitosis. Genomic clones containing mastrin gene were isolated; the gene was found to have 24 exons spanning 24kb of genomic DNA. Deletion analysis of 5(')-flanking sequences demonstrated that the first 120bp proximal to the TATA-less promoter region is necessary for minimal transcription of the mouse mastrin gene.

The BH3-only proapoptotic protein, BAD, was cloned from zebrafish embryos and its properties were characterized. Zebrafish BAD (zBAD) is a protein with 147 amino acids that contains a BH3 domain and a putative 14-3-3 binding site with the sequence of RPRSRS(84)AP, corresponding to S(136) in mouse BAD (mBAD). zBAD shares 34%, 28%, and 29% amino acid sequence identity to the human, mouse, and rat BAD, respectively. RT-PCR analysis revealed that the expression of zBAD gene is found in various parts of zebrafish tissues. The treatment with the z-VAD fmk, a broad-range caspase inhibitor, in COS-1 cells significantly increased the expression of zebrafish BAD fusion proteins (GFP-zBAD and HA-zBAD), indicating that zebrafish BAD fusion proteins may be cleaved by caspase(s). zBAD was shown to induce apoptosis when it was overexpressed in COS-1 cells. In addition, zBAD was also expressed in muscle cells under the muscle-specific promoter from zebrafish alpha-actin gene. Abnormality in the skeletal muscles and the loss of green fluorescence signal in the same region were observed. Taken together, our results indicate that zBAD could induce apoptosis in vitro and in vivo and may have biological implications in apoptosis during zebrafish development.

Human p29 has been demonstrated in the yeast two-hybrid method and in vitro GST pull-down assay to associate with GCIP, a cyclin D interacting protein. In this study, we describe the cloning and genomic structure of the mouse homologue, mp29. The overall mouse mp29 amino acid sequence is highly identical (91%) to human p29. Polyclonal antibody against mp29 was raised and the subcellular localization of mp29 was identified to be in the nucleus. Genomic clones containing mp29 gene were isolated and this gene was divided into seven exons spanning 9kb of genomic DNA. The transcription initiation site of mp29 gene was determined to be 94bp upstream of the translation initiation codon and the first 140bp proximal TATA-less promoter region is required to activate minimal transcription of mouse mp29 gene in mammalian cells.

One novel gene product, hMAP126, was demonstrated to interact with p29 in the yeast two-hybrid assay. The full-length cDNA of hMAP126 has been obtained and encodes a protein of 1120 amino acids. Multiple tissue Northern blot analysis showed that hMAP126 was abundantly expressed in the testis. Polyclonal antiserum against hMAP126 was raised and affinity-purification of anti-hMAP126 antibodies was performed. The subcellular distribution of hMAP126 was localized to the mitotic spindle. Furthermore, hMAP126 was identified to be post-translationally modified and phosphorylated by p34(cdc2) kinase in vitro. Taken together, we have isolated a novel protein, hMAP126, which may be involved in the functional and dynamic regulation of mitotic spindles., (Copyright 2001 Academic Press.)

GCIP, a newly identified cyclin D-interacting protein, was found to reduce the phosphorylation of retinoblastoma protein and inhibit E2F1-mediated transcriptional activity. To explore more GCIP interacting proteins, the yeast two-hybrid screening using GCIP as a bait protein was performed. One novel gene, p29, was demonstrated to associate with GCIP in the yeast two-hybrid method and in vitro GST pull-down assay. Multiple tissue Northern blot analysis showed that p29 was abundantly expressed in the heart, skeletal muscle, and kidney relative to other tissues. The transient expression of HA-tagged p29 in HeLa cells localized in the nucleus. Taken together, we have isolated a novel protein, p29, which may be involved in the functional regulation of GCIP., (Copyright 2000 Academic Press.)

Cu/Zn superoxide dismutase (SOD1) catalyzes the dismutation of superoxide radicals produced from biological oxidation and environmental stresses. A number of xenobiotics are toxic because they generate free radicals, such as superoxide and hydroxyl radicals, through a redox cycle. The xenobiotic responsive element (XRE) was located between the nt -268 and -262 region of the 5'-flanking sequence of the SOD1 gene. Functional analyses of this element by deletion, mutations, and heterologous promoter systems confirmed that the expression of the SOD1 gene was induced by a xenobiotic through the XRE. Gel mobility shift assays showed the xenobiotic inducible binding of the receptor-ligand complex to XRE. The cytoplasmic fraction from nontreated HepG2 cells also contains the factor as a cryptic form and prominently reveals its DNA-binding activity by incubation with betaNF in vitro. These results suggest that the XRE participates in the induction of the rat SOD1 gene by xenobiotics., (Copyright 1999 Academic Press.)

The presence of L-arginine methylester (AME), L-arginine ethylester (AEE), or N-nitro-L-arginine methylester (NAME) in the growth media of Staphylococcus aureus increased the nitric oxide synthase (NOS) activity approximately 5- to 14-fold. The increase of NOS activity was confirmed by two assay methods, namely assaying the formation of L-[3H] citrulline from L-[3H] arginine and NO formation. The increase of NOS activity was most likely due to increased de novo synthesis, demonstrated by Western immunoblot analysis. The addition of methanol to the culture medium also increased the NOS activity as much as that found with the above three compounds. Evidence is presented to show that AME, AEE, or NAME gave rise to the formation of methanol in vivo by the action of intracellular esterase(s) and that methanol is subsequently involved in the induction of NOS in this bacterial system.

The presence of nitric oxide synthase (NOS) in Staphylococcus aureus ATCC6538P was established by western blot analysis. The identity of citrulline formed from L-arginine by the NOS was confirmed by both TLC and HPLC and the other product, NO, by directly measuring the production of nitrogen oxides (NOx) in the reaction mixture. The activity was inhibited by typical NOS inhibitors such as N-nitro-L-arginine methylester and NG,NG-dimethyl-L-arginine with the IC50 of 4.9 x 10(-4) and 2.5 x 10(-4) M, respectively. The NOS activity was maximum at pH 6.5 and at 47.5 degrees C. These results indicate that the NOS of S. aureus ATCC6538P is an isoform distinct from mammalian NOS.

With one-suction electrode voltage-clamp technique, we demonstrated that genistein, a tyrosine kinase (TK) inhibitor, could directly activate cystic fibrosis transmembrane regulator (CFTR) chloride current in guinea pig ventricular myocytes. The activation showed concentration-dependent effect with the estimated IC50 of 39.7 microM. Tyrphostin 51, another TK inhibitor, had no effect, suggesting that genistein's effect might be unrelated to TK inhibition. After the chloride current had been activated by the maximally elevated intracellular cAMP content by saturating concentration of isoproterenol, forskolin and IBMX, genistein could further enhance the current. Pre-treatment with saturating concentration of a specific protein kinase A (PKA) inhibitor, H-89, or other protein kinase inhibitors H-8 and H-9 in the perfusate or intracellularly could not prevent the activation of the current by genistein, suggesting a PKA-independent activity. Furthermore, saturating concentration of calyculin A, a specific inhibitor of phosphotase 1 and 2A, in the perfusate or intracellularly could not block genistein's action. It is possible that genistein opens the channels directly or inhibits the dephosphorylation process of CFTR, which is not sensitive calyculin A.

We have examined the possible modulatory effects of genistein, a specific tyrosine kinase inhibitor, on cardiac L-type calcium currents and cAMP-dependent chloride currents in guinea pig ventricular myocytes. With one-suction electrode voltage-clamp technique, genistein dose-dependently and reversibly inhibited L-type calcium currents in cardiomyocytes (Km = 17.5 microM). Neither threshold potential nor the peak potential of current-voltage relationship was affected. Interestingly, daidzein (an inactive analogue of genistein) also depressed L-type calcium currents. When L-type calcium currents were directly activated by Bay K 8644, genistein was able to exert an inhibitory action. In contrast, genistein potentiated cardiac cAMP-dependent chloride currents activated by either isoproterenol or 3-isobutyl-1-methylxanthine. These results suggest that genistein may directly inhibit L-type calcium currents but may potentiate cAMP-dependent chloride currents in the heart.