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1.
Cells ; 11(7)2022 03 28.
Article in English | MEDLINE | ID: mdl-35406710

ABSTRACT

Kisspeptin (KP) and kisspeptin receptor (KPR) are essential for the onset of puberty, development of gonads, and maintenance of gonadal function in both males and females. Hypothalamic KPs and KPR display a high degree of sexual dimorphism in expression and function. KPs act on KPR in gonadotropin releasing hormone (GnRH) neurons and induce distinct patterns of GnRH secretion in males and females. GnRH acts on the anterior pituitary to secrete gonadotropins, which are required for steroidogenesis and gametogenesis in testes and ovaries. Gonadal steroid hormones in turn regulate the KP neurons. Gonadal hormones inhibit the KP neurons within the arcuate nucleus and generate pulsatile GnRH mediated gonadotropin (GPN) secretion in both sexes. However, the numbers of KP neurons in the anteroventral periventricular nucleus and preoptic area are greater in females, which release a large amount of KPs in response to a high estrogen level and induce the preovulatory GPN surge. In addition to the hypothalamus, KPs and KPR are also expressed in various extrahypothalamic tissues including the liver, pancreas, fat, and gonads. There is a remarkable difference in circulating KP levels between males and females. An increased level of KPs in females can be linked to increased numbers of KP neurons in female hypothalamus and more KP production in the ovaries and adipose tissues. Although the sexually dimorphic features are well characterized for hypothalamic KPs, very little is known about the extrahypothalamic KPs. This review article summarizes current knowledge regarding the sexual dimorphism in hypothalamic as well as extrahypothalamic KP and KPR system in primates and rodents.


Subject(s)
Kisspeptins , Sex Characteristics , Animals , Arcuate Nucleus of Hypothalamus/metabolism , Female , Gonadotropin-Releasing Hormone , Kisspeptins/metabolism , Male , Sexual Maturation
2.
Biol Reprod ; 96(1): 145-158, 2017 01 01.
Article in English | MEDLINE | ID: mdl-28395334

ABSTRACT

Natural killer (NK) cells are the most prevalent leukocyte population in the uterus during early pregnancy. Natural killer cells contribute to uterine vascular (spiral artery) remodeling in preparation for the increased demand on these vessels later in pregnancy. A second wave of spiral artery modification is directed by invasive trophoblast cells. The significance of the initial wave of NK-cell-mediated vascular remodeling in species exhibiting deep trophoblast invasion such as humans and rats is not known. The purpose of this study was to generate a genetic model of NK-cell deficiency in rats, and determine the consequences of NK-cell deficiency on spiral artery remodeling and reproductive outcomes. To accomplish this task, we utilized zinc finger nuclease-mediated genome editing of the rat interleukin-15 (Il15) gene. Il15 encodes a cytokine required for NK-cell lineage development. Using this strategy, a founder rat was generated containing a frameshift deletion in Il15. Uteri of females harboring a homozygous mutation at the Il15 locus contained no detectable NK cells. NK-cell deficiency did not impact fetal growth or viability. However, NK-cell deficiency caused major structural changes to the placenta, including expansion of the junctional zone and robust, early-onset activation of invasive trophoblast-guided spiral artery remodeling. In summary, we successfully generated an NK-cell-deficient rat and showed, using this model, that NK cells dampen the extent of trophoblast invasion and delay trophoblast-directed spiral artery remodeling. This study furthers our understanding of the role of NK cells on uterine vascular remodeling, trophoblast invasion, and placental development.


Subject(s)
Killer Cells, Natural/physiology , Placentation , Animals , Body Weight , Female , Interleukin-15/deficiency , Interleukin-15/genetics , Male , Mutagenesis, Site-Directed , Organ Size , Pregnancy , Pregnancy Outcome , Rats, Sprague-Dawley , Spleen/pathology
3.
Stem Cells Dev ; 22(3): 431-43, 2013 Feb 01.
Article in English | MEDLINE | ID: mdl-22889370

ABSTRACT

Here, we describe a focused microarray for screening rat embryonic stem cells (ESCs) and provide validation data that this array can distinguish undifferentiated rat ESCs from rat trophoblast stem (TS) cells, rat extraembryonic endoderm cells, mouse embryonic fibroblast feeder cells, and differentiated rat ESCs. Using this tool, genuine rat ESC lines, which have been expanded in a conventional rat ESC medium containing two inhibitors (2i), for example, glycogen synthase kinase 3 (GSK3) and mitogen-activated protein kinase (MEK) inhibitors, and leukemia inhibitory factor, and genuine rat ESCs, which have been expanded in rat ESC medium containing four inhibitors (4i), for example, GSK3, MEK, Alk5, and Rho-associated kinase inhibitors were compared; as were genuine rat ESCs from 4 different strains of rats. Expression of Cdx2, a gene associated with trophoblast determination, was observed in genuine, undifferentiated rat ESCs from 4 strains and from both 2i and 4i ESC derivation medium. This finding is in contrast to undifferentiated mouse ESCs that do not express Cdx2. The rat ESC focused microarray described in this report has utility for rapid screening of rat ESCs. This tool will enable optimization of culture conditions in the future.


Subject(s)
Embryonic Stem Cells/metabolism , Oligonucleotide Array Sequence Analysis , Transcriptome , Animals , Biomarkers/metabolism , Cell Differentiation , Cell Line , Feeder Cells , Fibroblasts/metabolism , Genes, Essential , Germ Layers/cytology , Mice , Rats , Rats, Inbred F344 , Rats, Long-Evans , Rats, Wistar , Reverse Transcriptase Polymerase Chain Reaction , Trophoblasts/cytology
4.
J Clin Biochem Nutr ; 49(1): 42-9, 2011 Jul.
Article in English | MEDLINE | ID: mdl-21765606

ABSTRACT

In this study we investigated if peroxisome proliferator-activated receptor ß/δ activation protects from copper-induced acute liver damage. Mice treated with copper had significant body weight loss, serum alanine aminotransferase increase, modest changes in liver histology, increase of tumor necrosis factor α and macrophage inflammatory protein 2 mRNA and 8-hydroxy-2'-deoxyguanosine. Mice treated with copper and peroxisome proliferator-activated receptor ß/δ agonist GW0742 had significantly less body weight loss, less serum alanine aminotransferase increase, less tumor necrosis factor α, macrophage inflammatory protein-2 and 8-hydroxy-2'-deoxyguanosine upregulation than copper treated mice. The opposite effect was observed in mice treated with copper and peroxisome proliferator-activated receptor ß/δ antagonist GSK0660. In vitro, copper induced reactive oxygen species, which was lower in cells treated with GW0742 or transfected with peroxisome proliferator-activated receptor ß/δ expression vector; together, transfection and GW0742 had an additive reactive oxygen species-reducing effect. Copper also upregulated Fas ligand and Caspase 3/7 activity, effects that were significantly lower in cells also treated with GW0742. In conclusion, peroxisome proliferator-activated receptor ß/δ activation reduced copper-induced reactive oxygen species, pro-inflammatory and acute phase reaction cytokines in mice liver. Peroxisome proliferator-activated receptor ß/δ agonists could become useful in the management of copper-induced liver damage.

5.
Cancer Prev Res (Phila) ; 3(8): 940-52, 2010 Aug.
Article in English | MEDLINE | ID: mdl-20682802

ABSTRACT

The erbB family of receptor tyrosine kinases are known to play important roles in normal epithelial development and epithelial neoplasia. Considerable evidence also suggests that signaling through the epidermal growth factor receptor (EGFR) plays an important role in multistage skin carcinogenesis in mice; however, less is known about the role of erbB2. In this study, to further examine the role of both erbB2 and EGFR in epithelial carcinogenesis, we examined the effect of a dual erbB2/EGFR tyrosine kinase inhibitor, GW2974, given in the diet on skin tumor promotion during two-stage carcinogenesis in wild-type and BK5.erbB2 mice. In BK5.erbB2 mice, erbB2 is overexpressed in the basal layer of epidermis and leads to heightened sensitivity to skin tumor development. GW2974 effectively inhibited skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate in wild-type and BK5.erbB2 mice, although a more marked effect was seen in BK5.erbB2 mice. In addition, this inhibitory effect was reversible when GW2974 treatment was withdrawn. GW2974 inhibited 12-O-tetradecanoylphorbol-13-acetate-induced epidermal hyperproliferation, which correlated with reduced activation of both the EGFR and erbB2. These results support the hypothesis that both the EGFR and erbB2 play an important role in the development of skin tumors during two-stage skin carcinogenesis, especially during the tumor promotion stage. Furthermore, the marked sensitivity of BK5.erbB2 mice to the inhibitory effects of GW2974 during tumor promotion suggest greater efficacy for this compound when erbB2 is overexpressed or amplified as an early event in the carcinogenic process.


Subject(s)
ErbB Receptors/antagonists & inhibitors , Neoplasms, Glandular and Epithelial/drug therapy , Quinazolines/therapeutic use , Receptor, ErbB-2/antagonists & inhibitors , Skin Neoplasms/drug therapy , Algorithms , Animals , Antineoplastic Agents/therapeutic use , Cell Line, Tumor , Cell Transformation, Neoplastic/drug effects , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/pathology , Disease Progression , Drug Evaluation, Preclinical , ErbB Receptors/genetics , Gene Expression Regulation, Neoplastic/drug effects , Mice , Mice, Transgenic , Neoplasms, Glandular and Epithelial/genetics , Neoplasms, Glandular and Epithelial/pathology , Protein Kinase Inhibitors/therapeutic use , Receptor, ErbB-2/genetics , Skin Neoplasms/genetics , Skin Neoplasms/pathology , Treatment Outcome
6.
J Immunol ; 182(11): 7222-32, 2009 Jun 01.
Article in English | MEDLINE | ID: mdl-19454719

ABSTRACT

MFG-E8 (milk fat globule-epidermal growth factor 8) deficiency is strongly associated with acquisition of immune-mediated disorders due to the loss of tissue homeostasis. However, comparatively little is known regarding its functions in gastrointestinal tract disorders, in which immune homeostasis is a major concern. Herein, we report altered MFG-E8 expression in inflamed colons during the acute phase of murine experimental colitis and found that treatment with recombinant MFG-E8, but not its arginine-glycine-aspartate mutant counterpart, ameliorated colitis by reducing inflammation and improving disease parameters. To reveal the MFG-E8-mediated antiinflammatory mechanism, we employed an in vitro system, which showed the down-regulation of NF-kappaB in an LPS-dependent manner. Additionally, MFG-E8 altered alpha(v)beta(3) integrin-mediated focal adhesion kinase phosphorylation by impeding the binding of one of its potent ligands osteopontin, which becomes activated during colitis. Taken together, our results indicated that MFG-E8 has a novel therapeutic potential for treatment of colitis.


Subject(s)
Antigens, Surface/pharmacology , Inflammation , Integrin alphaVbeta3/metabolism , Intestines/pathology , Milk Proteins/pharmacology , Osteopontin/metabolism , Animals , Antigens, Surface/analysis , Colitis , Disease Models, Animal , Focal Adhesion Protein-Tyrosine Kinases/metabolism , Mice , Milk Proteins/analysis , NF-kappa B/analysis , Phosphorylation , Signal Transduction/drug effects
7.
Apoptosis ; 13(5): 609-20, 2008 May.
Article in English | MEDLINE | ID: mdl-18392683

ABSTRACT

The lactogenic hormone prolactin (PRL) regulates milk protein gene expression in mammary glands. To maintain homeostatic balance in the body, milk fat globule epidermal growth factor 8 (MFG-E8) is vital for phagocytic clearance of apoptotic cells. We investigated the effects of PRL on MFG-E8 expression in macrophages by evaluating its promoter function. Macrophages were stimulated with PRL, and the expression of MFG-E8 was determined using real-time PCR and Western blotting. The role of MFG-E8 on phagocytosis of apoptotic cells in PRL-treated macrophages was assessed using microscopy, while the response of PRL to MFG-E8 expression was evaluated using luciferase assay. Following treatment with PRL, significant up-regulations of the PRL receptor and MFG-E8 were observed in macrophages, though PRL-treated macrophages more efficiently engulfed apoptotic cells. The results of MFG-E8 promoter analysis showed considerable up-regulation of promoter activity in macrophages following PRL treatment and results from mutation analysis of the MFG-E8 promoter suggested that the C/EBPbeta binding site was responsible for PRL-induced activation of the MFG-E8 promoter. C/EBPbeta activity was found to be up-regulated in PRL-treated cells as revealed by an electrophoretic mobility shift assay (EMSA). In conclusion, PRL is a potent inducer of MFG-E8 expression in macrophages, while its effect is mediated by the presence of a responsive element in the MFG-E8 promoter.


Subject(s)
Antigens, Surface/biosynthesis , Apoptosis/physiology , CCAAT-Enhancer-Binding Protein-beta/physiology , Macrophages, Peritoneal/drug effects , Macrophages, Peritoneal/physiology , Milk Proteins/biosynthesis , Prolactin/pharmacology , Animals , Antigens, Surface/genetics , Base Sequence , Cell Line , Cell Nucleus/metabolism , Mice , Milk Proteins/genetics , Phagocytosis/drug effects , Promoter Regions, Genetic , Protein Transport , Receptors, Prolactin/biosynthesis , Up-Regulation
8.
Infect Immun ; 76(2): 796-811, 2008 Feb.
Article in English | MEDLINE | ID: mdl-17984203

ABSTRACT

The attaching and effacing (A/E) bacterial pathogens enteropathogenic Escherichia coli and enterohemorrhagic E. coli and the related mouse pathogen Citrobacter rodentium colonize their hosts' intestines by infecting the apical surfaces of enterocytes, subverting their function, and they ultimately cause diarrhea. Surprisingly, little is known about the interactions of these organisms with goblet cells, which are specialized epithelial cells that secrete the protective molecules Muc2 and trefoil factor 3 (Tff3) into the intestinal lumen. C. rodentium infection leads to dramatic goblet cell depletion within the infected colon, yet it is not clear whether C. rodentium infects goblet cells or if this pathology is pathogen or host mediated. As determined by immunostaining and PCR, both the number of goblet cells and the expression of genes encoding Muc2 and Tff3 were significantly reduced by day 10 postinfection. While electron microscopy and immunostaining revealed that C. rodentium directly infected a fraction of colonic goblet cells, C. rodentium localization did not correlate with goblet cell depletion. To assess the role of the host immune system in these changes, Rag1 knockout (KO) (T- and B-cell-deficient) mice were infected with C. rodentium. Rag1 KO mice did not exhibit the reduction in the number of goblet cells or in mediator (Muc2 and Tff3) expression observed in infected immunocompetent mice. However, reconstitution of Rag1 KO mice with T and B lymphocytes from C57BL/6 mice restored the goblet cell depletion phenotype during C. rodentium infection. In conclusion, these studies demonstrated that while colonic goblet cells can be subject to direct infection and potential subversion by A/E pathogens in vivo, it is the host immune system that primarily modulates the function of these cells during infection.


Subject(s)
Citrobacter rodentium/physiology , Enterobacteriaceae Infections/microbiology , Enterobacteriaceae Infections/pathology , Goblet Cells/immunology , Goblet Cells/microbiology , Adoptive Transfer , Animals , B-Lymphocytes/immunology , Colon/pathology , Female , Gene Expression , Homeodomain Proteins/genetics , Immunohistochemistry , Mice , Mice, Inbred C57BL , Mice, Knockout , Microscopy, Electron, Transmission , Mucin-2 , Mucins/biosynthesis , Mucins/genetics , T-Lymphocytes/immunology , Trefoil Factor-3
9.
Mol Cancer Ther ; 6(6): 1709-17, 2007 Jun.
Article in English | MEDLINE | ID: mdl-17575102

ABSTRACT

Biliary tract cancer is still challenging to treat and manage due to its poor sensitivity to conventional therapies and the inability to prevent or detect the early tumor formation. The most well known risk factor for gallbladder cancer is the presence of chronic inflammation, usually related to gallstones. It has been suggested that cyclooxygenase-2 (COX-2) plays a variety of roles in the gastrointestinal tract, including pathogenic processes such as neoplasia. Recently, we have generated transgenic mice that overexpress rat ErbB-2 under the control of bovine keratin 5 promoter (BK5.ErbB-2 mice). Homozygous BK5.ErbB-2 mice develop adenocarcinoma of gallbladder with an approximately 90% incidence. In addition to the activation of ErbB-2 and epidermal growth factor receptor, mRNA and protein levels of COX-2 were up-regulated in the gallbladder carcinomas that developed in these transgenic mice. The aim of this study was to examine the effects of a COX-2 inhibitor, CS-706, on the development of gallbladder carcinomas using the BK5.ErbB-2 mouse model. Ultrasound image analysis as well as histologic evaluation revealed a significant therapeutic effect of CS-706 on the gallbladder tumors, either as reversion to a milder phenotype or inhibition of tumor progression. The antitumor effect was associated with inhibition of prostaglandin E(2) synthesis. CS-706 treatment also down-regulated the activation of ErbB-2 and epidermal growth factor receptor, resulting in decreased levels of phosphorylated Akt and COX-2 in gallbladder cancers of BK5.ErbB-2 mice. Based on our results, targeting COX-2 could provide a potentially new and effective therapy alone or in combination with other therapeutic agents for patients with biliary tract cancer.


Subject(s)
Cyclooxygenase Inhibitors/therapeutic use , Gallbladder Neoplasms/drug therapy , Pyrroles/therapeutic use , Sulfonamides/therapeutic use , Administration, Oral , Animals , Cyclooxygenase 2/drug effects , Cyclooxygenase Inhibitors/administration & dosage , Dinoprostone/antagonists & inhibitors , Dinoprostone/biosynthesis , ErbB Receptors/metabolism , Fluorescent Antibody Technique , Gallbladder Neoplasms/metabolism , Mice , Mice, Transgenic , Pyrroles/administration & dosage , Receptor, ErbB-2/metabolism , Sulfonamides/administration & dosage
10.
Curr Pharm Des ; 12(32): 4215-28, 2006.
Article in English | MEDLINE | ID: mdl-17100624

ABSTRACT

Toll-like receptors (TLRs) are sensors of microbial products that initiate host defense responses in multicellular organisms. They are mainly linked to innate immunity and bridging to adaptive immunity, signaling through different TLRs responsible for a wide range of biological responses. The intracellular signaling pathways through Toll/interleukin-1 receptor (IL-1R) domains result in recruitment of the cytoplasmic adaptor molecules, with subsequent activation of a signaling cascade leading to nuclear factor-kappa B (NF-kappaB). TLR-signaling induces host inflammatory response and the inflammation becomes more severe in the absence of several extra and intra cellular negative regulators of TLR-signaling. In the intestine, TLR-dependent activation of NF-kappaB plays a vital role in maintaining epithelial homeostasis as well as regulating infections and inflammation, while dysregulation of TLR-signaling is associated with the pathogenesis of inflammatory bowel diseases (IBD). Recent findings regarding innate immunity-mediated regulation of intestinal pathophysiology prove that development of new drugs targeting TLRs including antagonists of TLR-signaling and agonists of their negative regulators has a potential impact on therapeutic strategies for intestinal inflammatory diseases.


Subject(s)
Gastrointestinal Diseases/drug therapy , Gastrointestinal Diseases/immunology , Inflammation/drug therapy , Toll-Like Receptors/antagonists & inhibitors , Toll-Like Receptors/immunology , Animals , Gastrointestinal Diseases/pathology , Humans , Immunity, Innate , Inflammation/immunology , Inflammation/pathology , Signal Transduction
11.
J Gastroenterol Hepatol ; 21(11): 1714-9, 2006 Nov.
Article in English | MEDLINE | ID: mdl-16984595

ABSTRACT

BACKGROUND AND AIM: The size of radiofrequency ablation (RFA) in the liver can be negatively influenced by the surrounding blood flow. The indocyanine green (ICG) test can be used to evaluate the effective blood flow in the liver, and distance from the hilus can affect local blood flow. The aim of this study was to assess whether the ICG test or distance from the hilus could be used to predict the size of the ablated area in liver by RFA treatment of hepatocellular carcinoma (HCC) nodules. METHODS: The RFA measurements of 44 HCC nodules in 39 patients were retrospectively studied. Cases were included if they met the following criteria: (i) no catheter treatment before RFA; (ii) no movement of the RFA device; (iii) strict ablation time; and (iv) only one ablation. In all patients, ICG-R15 testing was done immediately before RFA and the initial therapeutic efficacy was evaluated by dynamic computed tomography scanning 2-5 days after RFA. The correlation between the maximum size of the RFA area and the ICG test results or the distance of the target area from the hilus (site of first portal vein divergence) were analyzed statistically. RESULTS: The ICG-R15 result was significantly correlated with the maximum diameter of the ablated area both in 2 cm-electrode tip length (R2 = 0.35, P = 0.0012), and in 3 cm-tip length (R2 = 0.26, P = 0.0377). Multiple-regression analysis showed that the electrode tip length (P = 0.0010) and ICG-R15 (P = 0.0046) were independent factors that could predict the maximum diameter of the RFA area. CONCLUSION: The results of ICG testing can be used to predict the size of the area that will be ablated at a target liver site before RFA treatment.


Subject(s)
Carcinoma, Hepatocellular/surgery , Catheter Ablation , Coloring Agents/pharmacology , Indocyanine Green/pharmacokinetics , Liver Neoplasms/surgery , Liver/surgery , Aged , Analysis of Variance , Carcinoma, Hepatocellular/diagnostic imaging , Female , Humans , Liver/blood supply , Liver Neoplasms/diagnostic imaging , Male , Middle Aged , Postoperative Complications , Predictive Value of Tests , Prognosis , Regional Blood Flow , Regression Analysis , Retrospective Studies , Tomography, X-Ray Computed
12.
Clin Vaccine Immunol ; 13(1): 132-8, 2006 Jan.
Article in English | MEDLINE | ID: mdl-16426010

ABSTRACT

We recently demonstrated that the pattern recognition receptors (PRRs) toll-like receptor 2 (TLR2), TLR4, and CD14 are expressed in mouse colonic epithelium in a compartmentalized manner. Here we report the localization of TLR5, the receptor for bacterial flagellin, and its distinctive down-regulation during experimental colitis. Guts from normal BALB/c mice and those with dextran sodium sulfate (DSS)-induced colitis were compared. Each gut was divided into seven segments (stomach, small intestine [three parts], and colon [three parts]), and epithelial cells and crypt units were collected by scraping and EDTA treatment, respectively. Northern blotting showed that TLR5 mRNA was preferentially expressed in the epithelium of the proximal colon in normal mice. Laser capture microdissection coupled to reverse transcriptase PCR confirmed this localization. TLR5 protein expression reflected mRNA expression, as evidenced by Western blotting. In mice with acute colitis, inflammation occurred mainly in the distal colon. Interestingly, while TLR2, TLR4, and CD14 were up-regulated in the inflamed colon, TLR5 was down-regulated at both the mRNA and protein levels. Decreased TLR5 expression was more evident during chronic colitis. Additional in vitro studies using a mouse cell line, Colon-26, showed that gamma interferon (IFN-gamma) time- and dose-dependently down-regulates TLR5. In conclusion, epithelial cells, mainly in the proximal colon, constitutively express TLR5. TLR5 expression is down-regulated in vivo during acute and chronic DSS-induced colitis, in contrast to the expression of TLR2, TLR4, and CD14. The mechanism governing TLR5 regulation may therefore differ from that controlling other PRRs. Finally, IFN-gamma may be involved in down-regulating TLR5 expression.


Subject(s)
Cecum/chemistry , Colitis/metabolism , Down-Regulation , Toll-Like Receptor 5/metabolism , Animals , Cell Line, Tumor , Colitis/chemically induced , Colitis/immunology , Disease Models, Animal , Disease Progression , Epithelium , Humans , Interferon-gamma/metabolism , Lipopolysaccharide Receptors/metabolism , Male , Mice , RNA, Messenger/biosynthesis , Toll-Like Receptor 2/metabolism , Toll-Like Receptor 4/metabolism , Toll-Like Receptor 5/immunology , Up-Regulation
13.
Biochem Biophys Res Commun ; 339(2): 540-7, 2006 Jan 13.
Article in English | MEDLINE | ID: mdl-16300730

ABSTRACT

RNA polymerase III promoters of human ribonuclease P RNA component H1, human U6, and mouse U6 small nuclear RNA genes are commonly used in short hairpin RNA (shRNA) expression vectors due their precise initiation and termination sites. During transient transfection of shRNA vectors, we observed that H1 or U6 promoters also express longer transcripts enough to express several reporter genes including firefly luciferase, green fluorescent protein EGFP, and red fluorescent protein JRed. Expression of such longer transcripts was augmented by upstream RNA polymerase II enhancers and completely inhibited by downstream polyA signal sequences. Moreover, the transcription of firefly luciferase from human H1 promoter was sensitive to RNA polymerase II inhibitor alpha-amanitin. Our findings suggest that commonly used polymerase III promoters in shRNA vectors are also prone to RNA polymerase II mediated transcription, which may have negative impacts on their targeted use.


Subject(s)
Genetic Vectors/genetics , Nucleic Acid Conformation , Promoter Regions, Genetic/genetics , RNA Polymerase III/metabolism , RNA Polymerase II/metabolism , RNA/genetics , Transcription, Genetic/genetics , Animals , Cell Line , Chlorocebus aethiops , Enzyme Inhibitors/pharmacology , Genes, Reporter/genetics , Genetic Vectors/chemistry , Humans , Mice , RNA/chemistry , RNA Polymerase II/antagonists & inhibitors
14.
Am J Gastroenterol ; 100(1): 21-6, 2005 Jan.
Article in English | MEDLINE | ID: mdl-15654776

ABSTRACT

BACKGROUND: Because of a rapid increase in the incidence of Barrett's cancer, the appropriate surveillance method for Barrett's esophagus is of interest. Methylene blue chromoendoscopy has been reported to be an effective and inexpensive method to improve biopsy surveillance of Barrett's epithelium. However, the usefulness of this method in short-segment Barrett's esophagus cases is still controversial. AIMS: This study was undertaken to evaluate the abilities of crystal violet and methylene blue chromoendoscopy to detect potentially dysplastic Barrett's epithelium in cases with short-segment columnar-appearing epithelium of the esophago-gastric junction. PATIENTS AND METHODS: Four hundred patients with endoscopically suspected short-segment Barrett's esophagus were enrolled and randomly assigned to receive chromoendoscopy with 0.05% crystal violet, 0.1% crystal violet, 0.5% methylene blue, or 1.0% methylene blue. During crystal violet and methylene blue chromoendoscopy, biopsy specimens were obtained from stained and unstained columnar-appearing epithelium of the esophago-gastric junction, and the detection rates of Barrett's epithelium were evaluated. The value of pit pattern diagnosis was also evaluated as a possible way to detect dysplastic Barrett's epithelium. RESULTS: Chromoendoscopy with 0.05% crystal violet detected histologically confirmed Barrett's epithelium with the highest sensitivity (89.2%) and specificity (85.7%). Crystal violet clearly stained both dysplastic and nondysplastic Barrett's epithelia and made the surface pit pattern easy to observe without using magnifying endoscopy. CONCLUSIONS: The combination of crystal violet chromoendoscopy and pit pattern diagnosis is considered to be useful for the surveillance of short-segment Barrett's esophagus.


Subject(s)
Barrett Esophagus/pathology , Coloring Agents , Esophagoscopy/methods , Gentian Violet , Mass Screening/methods , Methylene Blue , Adult , Aged , Aged, 80 and over , Female , Humans , Male , Middle Aged , Mucous Membrane/pathology , Sensitivity and Specificity
15.
J Immunol ; 173(2): 1406-16, 2004 Jul 15.
Article in English | MEDLINE | ID: mdl-15240737

ABSTRACT

TLR4, a member of pattern recognition receptors, is the main receptor of LPS. MD-2 physically associates with TLR4 on the cell surface and confers LPS responsiveness. Helicobacter pylori LPS is one of the major virulence factors for induction of gastritis. We demonstrated in this study the role of MD-2 in TLR4-dependent signaling in H. pylori-associated gastritis. Gastric biopsy samples collected from patients with and without H. pylori infection and four gastric cancer cell lines were used for this study. TLR-4 and MD-2 expression in biopsy specimens and the cell lines was examined by using RT-PCR. Localization of TLR-4 in histological sections was evaluated by immunohistochemistry. For in vitro functional assays, we established stable transfectants of AGS cells expressing TLR4 and MD-2. Cellular distribution of TLR4 was examined by flow cytometry. NF-kappaB activation and activation of IL-8 and MD-2 promoters were assessed by reporter gene assay. H. pylori infection up-regulated the TLR4 and MD-2 expression in gastric mucosa. TLR4 staining was observed predominantly in epithelial cells, located in both the cytoplasm and at the apical surface. MD-2 transfection in AGS cells markedly increased cell surface expression of TLR4 and augmented the activation of NF-kappaB and IL-8 promoter upon stimulation with H. pylori LPS. Live H. pylori also stimulated transcriptional activation of MD-2. This study revealed that MD-2 expression is elevated in gastric epithelial cells during H. pylori infection, suggesting that the TLR4/MD-2 system is a potent receptor complex involved in the response to H. pylori LPS in the stomach.


Subject(s)
Antigens, Surface/metabolism , Gastritis/metabolism , Helicobacter Infections/immunology , Membrane Glycoproteins/metabolism , Receptors, Cell Surface/metabolism , Signal Transduction/physiology , Adult , Antigens, Surface/genetics , Female , Gastritis/immunology , Gastritis/microbiology , Genes, Reporter , Helicobacter Infections/metabolism , Helicobacter pylori/immunology , Helicobacter pylori/metabolism , Humans , Interleukin-8/genetics , Lipopolysaccharides/metabolism , Lymphocyte Antigen 96 , Male , Membrane Glycoproteins/genetics , Middle Aged , NF-kappa B/metabolism , Promoter Regions, Genetic , RNA, Messenger/metabolism , Receptors, Cell Surface/genetics , Toll-Like Receptor 4 , Toll-Like Receptors
16.
Am J Physiol Gastrointest Liver Physiol ; 286(3): G508-14, 2004 Mar.
Article in English | MEDLINE | ID: mdl-14563668

ABSTRACT

For the production and vesicle storage of histamine, Enterochromaffin-like (ECL) cells express histidine decarboxylase (HDC) and vesicular monoamine transporter 2 (VMAT2). Although HDC and VMAT2 show dynamic changes during gastric ulcer healing, the control system of their expression has not been fully investigated. In the present study, we investigated the effect of transforming growth factor-alpha (TGF-alpha) and proinflammatory cytokines on HDC and VMAT2 expression in rat ECL cells. Time course changes in the expression of TGF-alpha during the healing of acetic acid-induced ulcers were studied. EGF receptor (EGFR) expression was also examined in ECL cells, whereas the direct effects of TGF-alpha and proinflammatory cytokines on HDC and VMAT2 expression in ECL cells were investigated using in vivo and in vitro models. During the process of ulcer healing, expression of TGF-alpha mRNA was markedly augmented. Furthermore, EGFR was identified in isolated ECL cells. TGF-alpha stimulated HDC and VMAT2 mRNA expression and protein production and also increased histamine release from ECL cells. Selective EGFR tyrosine kinase inhibitor tyrphostin AG1478 almost completely inhibited HDC and VMAT2 gene expression induced by TGF-alpha in vivo and in vitro. During gastric mucosal injury, TGF-alpha was found to stimulate ECL cell functions by increasing HDC and VMAT2 expression.


Subject(s)
Enterochromaffin Cells/metabolism , Histidine Decarboxylase/biosynthesis , Membrane Glycoproteins/biosynthesis , Membrane Transport Proteins , Neuropeptides , Transforming Growth Factor alpha/pharmacology , Acetic Acid , Animals , Blotting, Northern , Cell Separation , Cells, Cultured , Enterochromaffin Cells/drug effects , Gastric Mucosa/cytology , Gastric Mucosa/pathology , Gene Expression Regulation, Enzymologic/drug effects , Histidine Decarboxylase/metabolism , Immunohistochemistry , Kinetics , Male , Rats , Rats, Wistar , Reverse Transcriptase Polymerase Chain Reaction , Stomach Ulcer/chemically induced , Stomach Ulcer/pathology , Vesicular Biogenic Amine Transport Proteins , Vesicular Monoamine Transport Proteins
17.
J Gastroenterol ; 38(9): 830-5, 2003.
Article in English | MEDLINE | ID: mdl-14564627

ABSTRACT

BACKGROUND: Nocturnal gastric acid breakthrough (NAB) is defined as nocturnal intragastric pH less than 4 for more than 1 h during proton pump inhibitor (PPI) administration. A bedtime dose of an H2 receptor antagonist (H2RA) inhibites NAB, but the efficacy of the H2RA decreases with continuous administration. We carried out the present study to investigate the effect of 14-day H2RA administration on NAB. METHODS: Ten male volunteers without Helicobacter pylori infection received four different 14-day regimens of rabeprazole and ranitidine (study a, morning dose of 20 mg rabeprazole; study b, morning dose of 20 mg rabeprazole with a single bedtime dose of 150 mg ranitidine only on the last day; study c, continuous 20 mg morning dose of rabeprazole and 150 mg at bedtime; study d, morning and evening doses of 10 mg rabeprazole). Ambulatory 24-h gastric pH monitoring was conducted on the last day of each regimen. RESULTS: NAB in studies a, b, c, and d was observed in 9, 1, 4, and 4 subjects, respectively, and the longest periods of nocturnal gastric pH at less than 4.0 were 102.5, 14.0, 37.5, and 52.5 min, respectively (study b vs study c, P<0.05). CONCLUSIONS: The continuous inhibitory effect of ranitidine combined with rabeprazole on nocturnal gastric acid secretion declined during 14-day-long administration in H. pylori-negative subjects. Split dosing of rabeprazole was more effective than the single morning dose for inhibiting nocturnal gastric acid secretion.


Subject(s)
Anti-Ulcer Agents/administration & dosage , Benzimidazoles/administration & dosage , Chronotherapy , Gastric Acid/metabolism , Helicobacter pylori/physiology , Ranitidine/administration & dosage , 2-Pyridinylmethylsulfinylbenzimidazoles , Adult , Cross-Over Studies , Drug Therapy, Combination , Humans , Male , Monitoring, Ambulatory , Omeprazole/analogs & derivatives , Rabeprazole , Reference Values
18.
Cancer Lett ; 199(2): 169-73, 2003 Sep 25.
Article in English | MEDLINE | ID: mdl-12969789

ABSTRACT

We investigated the frequency of BRAF mutations in human pancreatic cancer specimens to determine its role in the development of pancreatic cancer. Nine pancreatic cancer samples without a K-ras codon 12 mutation and 19 with a K-ras mutation were included in the study. Analyses of the BRAF sequence revealed mutations in exon 15 (V599E) in two cases, both of which also exhibited a K-ras codon 12 mutation. No BRAF mutation was found in cases without a K-ras mutation. The BRAF V599E mutation was not found to be a major mutation in pancreatic cancers that had no K-ras codon 12 mutation.


Subject(s)
Genes, ras/genetics , Mutation/genetics , Oncogene Proteins/genetics , Pancreatic Neoplasms/genetics , Adenocarcinoma/etiology , Adenocarcinoma/genetics , Adenocarcinoma, Mucinous/etiology , Adenocarcinoma, Mucinous/genetics , Aged , Aged, 80 and over , Carcinoma, Papillary/etiology , Carcinoma, Papillary/genetics , DNA Mutational Analysis , DNA Primers/chemistry , Female , Humans , Male , Middle Aged , Neoplasm Staging , Pancreatic Neoplasms/etiology , Polymerase Chain Reaction , Proto-Oncogene Proteins B-raf , Survival Rate , Tumor Cells, Cultured
19.
J Immunol ; 170(8): 3977-85, 2003 Apr 15.
Article in English | MEDLINE | ID: mdl-12682225

ABSTRACT

Pattern recognition receptors (PRRs), which include the Toll-like receptors (TLRs), are involved in the innate immune response to infection. TLR4 is a model for the TLR family and is the main LPS receptor. We wanted to determine the expression of TLR4 and compare it with that of TLR2 and CD14 along the gastrointestinal mucosa of normal and colitic BALB/c mice. Colitis was induced with 2.5% dextran sodium sulfate (DSS). Mucosa from seven segments of the digestive tract (stomach, small intestine in three parts, and colon in three parts) was isolated by two different methods. Mucosal TLR4, CD14, TLR2, MyD88, and IL-1beta mRNA were semiquantified by Northern blotting. TLR4 protein was determined by Western blotting. TLR4/MD-2 complex and CD14 were evaluated by immunohistochemistry. PRR genes were constitutively expressed and were especially stronger in colon. TLR4 and CD14 mRNA were increased in the distal colon, but TLR2 mRNA was expressed more strongly in the proximal colon, and MyD88 had a uniform expression throughout the gut. Accordingly, TLR4 and CD14 protein levels were higher in the distal colon. TLR4/MD-2 and CD14 were localized at crypt bottom epithelial cells. TLR4/MD2, but not CD14, was found in mucosal mononuclear cells. Finally, DSS-induced inflammation was localized in the distal colon. All genes studied were up-regulated during DSS-induced inflammation, but the normal colon-stressed gut distribution was preserved. Our findings demonstrate that TLR4, CD14, and TLR2 are expressed in a compartmentalized manner in the mouse gut and provide novel information about the in vivo localization of PRRs.


Subject(s)
Intestinal Mucosa/immunology , Intestinal Mucosa/metabolism , Membrane Glycoproteins/metabolism , Receptors, Cell Surface/metabolism , Receptors, Immunologic/metabolism , Adaptor Proteins, Signal Transducing , Animals , Antigens, Differentiation/biosynthesis , Antigens, Differentiation/genetics , Antigens, Differentiation/metabolism , Antigens, Ly/biosynthesis , Antigens, Ly/genetics , Antigens, Ly/metabolism , Blotting, Northern , Colitis/chemically induced , Colitis/immunology , Colitis/pathology , Colon/immunology , Colon/metabolism , Colon/pathology , Dextran Sulfate/toxicity , Disease Models, Animal , Gene Expression Regulation/immunology , Intestinal Mucosa/cytology , Intestinal Mucosa/pathology , Lipopolysaccharide Receptors/biosynthesis , Lipopolysaccharide Receptors/genetics , Lipopolysaccharide Receptors/metabolism , Lymphocyte Antigen 96 , Macromolecular Substances , Male , Membrane Glycoproteins/biosynthesis , Membrane Glycoproteins/genetics , Mice , Mice, Inbred BALB C , Models, Animal , Myeloid Differentiation Factor 88 , Receptors, Cell Surface/biosynthesis , Receptors, Cell Surface/genetics , Receptors, Immunologic/biosynthesis , Receptors, Immunologic/genetics , Reverse Transcriptase Polymerase Chain Reaction , Toll-Like Receptor 2 , Toll-Like Receptor 4 , Toll-Like Receptors , Up-Regulation/genetics , Up-Regulation/immunology
20.
J Lab Clin Med ; 141(2): 102-5, 2003 Feb.
Article in English | MEDLINE | ID: mdl-12577045

ABSTRACT

Lafutidine is a novel histamine H(2)-receptor antagonist with a potent and long-lasting anti-acid secretory effect that has also been found to have a potent gastroprotective effect. We investigated the effect of lafutidine on gastric mucosal injury induced in rats with the use of water-immersion restraint stress (WRS) by examining serum calcitonin gene-related peptide (CGRP) concentrations, which we measured with the use of an enzyme immunometric assay. WRS-induced mucosal erosive injury in the stomach was reduced significantly by both lafutidine and famotidine pretreatment (from 7.79 +/- 2.02 mm(2) to 3.09 +/- 0.74 mm(2) and 4.05 +/- 1.18 mm(2), respectively). A single administration of lafutidine or famotidine did not change the serum CGRP concentration from the control value when these drugs were administered without WRS. Lafutidine pretreatment before WRS caused a significant increase in serum CGRP concentration compared with famotidine (lafutidine, 86.64 +/- 9.52 pg/mL; famotidine, 47.55 +/- 4.35 pg/mL; control, 58.43 +/- 6.07 pg/mL). Our results suggest that lafutidine augments CGRP release from the rat stomach when administered before the induction of WRS.


Subject(s)
Acetamides/pharmacology , Calcitonin Gene-Related Peptide/blood , Histamine H2 Antagonists/pharmacology , Immersion , Piperidines/pharmacology , Pyridines/pharmacology , Restraint, Physical , Stress, Physiological/blood , Animals , Famotidine/pharmacology , Male , Rats , Rats, Sprague-Dawley , Stomach Ulcer/etiology , Stomach Ulcer/prevention & control , Stress, Physiological/complications , Water
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