Overexpression of nicotinamide N-methyltransferase in gastric cancer tissues and its potential post-translational modification

Gastric cancer is one of the most common cancers worldwide. The purpose of this study was to find out potential markers for gastric cancer. Tumor and normal tissues from 152 gastric cancer cases were analyzed by two-dimensional gel electrophoresis (2-DE). The images of silver stained gels were analyzed and statistical analysis of spot intensities revealed that spot 4262 showed higher expression (5.7-fold increase) in cancer tissues than in normal tissues (P < 0.001). It was identified by peptide mass fingerprinting as nicotinamide N-methyltransferase (NNMT). A monoclonal antibody with a detection limit down to 10 ng was produced against NNMT in mouse. Using the prepared monoclonal antibody, western blot analysis of NNMT was performed for gastric tissues from 15 gastric cancer patients and two gastric ulcer patients. The results corroborated those of 2-DE experiments. A single spot was detected in gastric ulcer tissues while four to five spots were detected in gastric cancer tissues. In cancer tissues, two additional spots of acidic and basic form were mainly detected on 2-DE gels. This suggests that NNMT receives a post-translational modification in cancer- specific manner.     

Fluorometric assay of rat tissue N-methyltransferases with nicotinamide and four isomeric methylnicotinamides.

Nicotinamide (NA) and its four isomeric methyl analogs [2-, 4-, 5- and 6-methylnicotinamides (MNs)] were tested as substrates for nicotinamide N-methyltransferase (NNMT) and amine N-methyltransferase (ANMT) using rat liver, kidney, spleen and brain 9000 x g supernatant fluids as model enzyme preparations. The N-methylated products were determined fluorometrically by their reaction with acetophenone or 4-methoxybenzaldehyde to form fluorescent 2,7-naphthyridine derivatives, and the lower limits of the determination were 8-30 pmol/100 microliters. N-Methyltransferase activities were detected in the liver with NA, 4-MN and 5-MN, and in the brain and spleen with 4-MN. On this basis, 5-MN is considered to be a selective substrate for NNMT in addition to NA, which is a known methyl acceptor for this enzyme. Although 4-MN appears to serve as a methyl acceptor for both ANMT and NNMT, it seems to be essentially a selective substrate for brain ANMT because of the absence of NNMT in brain. The fluorometric methods used here are also very useful because of their simplicity, sensitivity and selectivity.     

Long-term and stable correction of uremic anemia by intramuscular injection of plasmids containing hypoxia-regulated system of erythropoietin expression

Relative deficiency in production of glycoprotein hormone erythropoietin (Epo) is a major cause of renal anemia. This study planned to investigate whether the hypoxia-regulated system of Epo expression, constructed by fusing Epo gene to the chimeric phosphoglycerate kinase (PGK) hypoxia response elements (HRE) in combination with cytomegalovirus immediate-early (CMV IE) basal gene promoter and delivered by plasmid intramuscular injection, might provide a long-term physiologically regulated Epo secretion expression to correct the anemia in adenine-induced uremic rats. Plasmid vectors (pHRE-Epo) were synthesized by fusing human Epo cDNA to the HRE/CMV promoter. Hypoxia-inducible activity of this promoter was evaluated first in vitro and then in vivo in healthy and uremic rats (n = 30 per group). The vectors (pCMV-Epo) in which Epo expression was directed by a constitutive CMV gene promoter served as control. ANOVA and Student''s t-test were used to analyze between-group differences. A high-level expression of Epo was induced by hypoxia in vitro and in vivo. Though both pHRE-Epo and pCMV-Epo corrected anemia, the hematocrit of the pCMV-Epo-treated rats exceeded the normal (P < 0.05), but that of the pHRE-Epo-treated rats didn''t. Hypoxia-regulated system of Epo gene expression constructed by fusing Epo to the HRE/CMV promoter and delivered by plasmid intramuscular injection may provide a long-term and stable Epo expression and secretion in vivo to correct the anemia in adenine-induced uremic rats.     

Three of a Kind: Control of the Expression of Liver-Expressed Antimicrobial Peptide 2 (LEAP2) by the Endocannabinoidome and the Gut Microbiome

The endocannabinoidome (expanded endocannabinoid system, eCBome)-gut microbiome (mBIome) axis plays a fundamental role in the control of energy intake and processing. The liver-expressed antimicrobial peptide 2 (LEAP2) is a recently identified molecule acting as an antagonist of the ghrelin receptor and hence a potential effector of energy metabolism, also at the level of the gastrointestinal system. Here we investigated the role of the eCBome-gut mBIome axis in the control of the expression of LEAP2 in the liver and, particularly, the intestine. We confirm that the small intestine is a strong contributor to the circulating levels of LEAP2 in mice, and show that: (1) intestinal Leap2 expression is profoundly altered in the liver and small intestine of 13 week-old germ-free (GF) male mice, which also exhibit strong alterations in eCBome signaling; fecal microbiota transfer (FMT) from conventionally raised to GF mice completely restored normal Leap2 expression after 7 days from this procedure; in 13 week-old female GF mice no significant change was observed; (2) Leap2 expression in organoids prepared from the mouse duodenum is elevated by the endocannabinoid noladin ether, whereas in human Caco-2/15 epithelial intestinal cells it is elevated by PPARγ activation by rosiglitazone; (3) Leap2 expression is elevated in the ileum of mice with either high-fat diet—or genetic leptin signaling deficiency—(i.e., ob/ob and db/db mice) induced obesity. Based on these results, we propose that LEAP2 originating from the small intestine may represent a player in eCBome- and/or gut mBIome-mediated effects on food intake and energy metabolism.     

Selenite Downregulates STAT3 Expression and Provokes Lymphocytosis in the Liver of Chronically Exposed Syrian Golden Hamsters

Arsenic is considered a worldwide pollutant that can be present in drinking water. Arsenic exposure is associated with various diseases, including cancer. Antioxidants as selenite and α-tocopherol-succinate have been shown to modulate arsenic toxic effects. Since changes in STAT3 and PSMD10 gene expression have been associated with carcinogenesis, the aim of this study was to evaluate the effect of arsenic exposure and co-treatments with selenite or α-tocopherol-succinate on the expression of these genes, in the livers of chronically exposed Syrian golden hamsters. Animals were divided into six groups: (i) control, (ii) chronically treated with 100 ppm arsenic, (iii) treated with 6 ppm α-tocopherol-succinate (α-TOS), (iv) treated with 8.5 ppm selenite, (v) treated with arsenic + α-TOS, and (vi) treated with arsenic + selenite. Urine samples and livers were collected after 20 weeks of continuous exposure. The urine samples were analyzed for arsenic species by atomic absorption spectrophotometry, and real-time RT-qPCR analysis was performed for gene expression evaluation. A reduction in STAT3 expression was observed in the selenite-treated group. No differences in PSMD10 expression were found among groups. Histopathological analysis revealed hepatic lymphocytosis in selenite-treated animals. As a conclusion, long-term exposure to arsenic does not significantly alter the expression of STAT3 and PSMD10 oncogenes in the livers of hamsters; however, selenite down-regulates STAT3 expression and provokes lymphocytosis.     

β-Carotene Inhibits Expression of Matrix Metalloproteinase-10 and Invasion in Helicobacter pylori-Infected Gastric Epithelial Cells

Matrix metalloproteinases (MMPs), key molecules of cancer invasion and metastasis, degrade the extracellular matrix and cell–cell adhesion molecules. MMP-10 plays a crucial role in Helicobacter pylori-induced cell-invasion. The mitogen-activated protein kinase (MAPK) signaling pathway, which activates activator protein-1 (AP-1), is known to mediate MMP expression. Infection with H. pylori, a Gram-negative bacterium, is associated with gastric cancer development. A toxic factor induced by H. pylori infection is reactive oxygen species (ROS), which activate MAPK signaling in gastric epithelial cells. Peroxisome proliferator-activated receptor γ (PPAR-γ) mediates the expression of antioxidant enzymes including catalase. β-Carotene, a red-orange pigment, exerts antioxidant and anti-inflammatory properties. We aimed to investigate whether β-carotene inhibits H. pylori-induced MMP expression and cell invasion in gastric epithelial AGS (gastric adenocarcinoma) cells. We found that H. pylori induced MMP-10 expression and increased cell invasion via the activation of MAPKs and AP-1 in gastric epithelial cells. Specific inhibitors of MAPKs suppressed H. pylori-induced MMP-10 expression, suggesting that H. pylori induces MMP-10 expression through MAPKs. β-Carotene inhibited the H. pylori-induced activation of MAPKs and AP-1, expression of MMP-10, and cell invasion. Additionally, it promoted the expression of PPAR-γ and catalase, which reduced ROS levels in H. pylori-infected cells. In conclusion, β-carotene exerts an inhibitory effect on MAPK-mediated MMP-10 expression and cell invasion by increasing PPAR-γ-mediated catalase expression and reducing ROS levels in H. pylori-infected gastric epithelial cells.     

PALB2 as a potential prognostic biomarker for colorectal cancer

Partner and localizer of BRCA2 (PALB2) is regarded as a colorectal cancer (CRC) risk gene, but the prognostic implication of PALB2 in CRC remains unclear. In this study, we evaluate the prognostic value of the gene copy number alteration (CNA) and mRNA expression of PALB2 in The Cancer Genome Atlas (TCGA) database, and then validated with our database. We downloaded the copy number and mRNA data of PALB2 from TCGA database and examined the relationship among the genetic alterations, expression levels and survival outcomes. Gene ontology (GO) analysis was performed to study the function of PALB2. cBioPortal database was used to explore the potential co-expression genes of PALB2. There were 6.3% (37 of 582) CRC patients diagnosed as PALB2 gene deletion. The PALB2 deletion group expressed significantly lower of PALB2 mRNA than the non-deletion group (P < 0.001). Survival analysis showed that PALB2 deletion was significantly associated with shorter disease-free survival (DFS) (P = 0.026) and overall survival (OS) (P = 0.028). Low mRNA expression of PALB2 correlated with shorter OS (P < 0.001). Multivariate analysis also confirmed that PALB2 deletion and low mRNA expression of PALB2 were independent prognostic factors of poor OS in CRC (P = 0.019, 0.034, respectively). In validation cohort, negative expression of PALB2 was associated with shorter OS (P = 0.006) in stage I patients. Multivariate analysis confirmed that negative expression of PALB2 was a poor-prognostic factor (P = 0.002). GO analysis and co-expression analysis investigated that PALB2 is primarily involved in the DNA repair process. These results suggest that PALB2 gene copy number deletion and low mRNA expression could be novel prognostic biomarkers for CRC.     

Enhanced Production of a Thermostable Carbonic Anhydrase in Escherichia coli by Using a Modified NEXT Tag

Carbonic anhydrase (CA) is an ultrafast enzyme that catalyzes the reversible conversion of carbon dioxide (CO₂) to bicarbonate. CA is considered to be a green catalyst for enzyme-based CO₂ capture and utilization. In particular, the CA of Thermovibrio ammonificans (taCA) has attracted increasing attention as a highly stable enzyme. However, the poor solubility and the low expression level in Escherichia coli have hampered further utilization of taCA. In a recent study, these limitations were partly resolved by using a small solubility-enhancing fusion tag named NEXT, which originates from the N-terminal extension of Hydrogenovibrio marinus CA. In this study, the NEXT tag was engineered by adding small peptides to the N terminus to further increase the production yield of NEXT-tagged taCA. The addition of ng3 peptide (His-Gly-Asn) originating from the N-terminal sequence of Neisseria gonorrhoeae CA improved the expression of NEXT-taCA, while the previously developed translation-enhancing element (TEE) and Ser-Lys-Ile-Lys (SKIK) tag were not effective. The expression test with all 16 codon combinations for the ng3 sequence revealed that the change in translation initiation rate brought about by the change in nucleotide sequence was not the primary determinant for the change in expression level. The modified ng3-NEXT tag may be applied to increase the production yields of various recombinant proteins.     

Construction of the ie1-Bacmid Expression System and Its Use to Express EGFP and BmAGO2 in BmN Cells

The presently available expression tools and vectors (e.g., eukaryotic expression vectors and the adenovirus expression system) for studying the functional genes in Bombyx mori are insufficient. The baculovirus expression system is only used as a protein production tool; therefore, recombinant proteins expressed by B. mori using the baculovirus expression system equipped with a polyhedrin promoter cannot be used for in vivo research applications. In this work, we constructed and screened a eukaryotic expression vector for silkworm cells The EGFP and B. mori Argonaute2 proteins were found to be efficiently expressed using the screened pIEx-1 vector with the FuGENE 6 transfection reagent. Additionally, we constructed a novel nucleopolyhedrovirus ie1-Bacmid expression system for the production of recombinant protein; we then used the system to highly express the EGFP and B. mori Argonaute2 proteins. In this system, the protein of interest can be efficiently expressed 13 h after infection by controlling the B. mori nucleopolyhedrovirus immediate early ie1 promoter. The ie1-Bacmid system provides a powerful “adenovirus-like” expression tool; not only can the tool be used to study baculovirus molecular biology for the silkworm but it is also useful in other research applications as well, such as the study of gene functions involved in cellular physiological processes.     

Protective Effect of Potentilla glabra in UVB-Induced Photoaging Process

Maintaining skin homeostasis is one of the most important factors for skin health. UVB-induced skin photoaging is a difficult problem that has negative impacts on skin homeostasis. So far, a number of compounds have been discovered that improve human skin barrier function and hydration, and are thought to be effective ways to protect skin homeostasis. Potentilla glabra var. mandshurica (Maxim.) Hand.-Mazz. Ethanol Extract (Pg-EE) is a compound that has noteworthy anti-inflammatory properties. However, its skin-protective effects are poorly understood. Therefore, we evaluated the capacity of Pg-EE to strengthen the skin barrier and improve skin hydration. Pg-EE can enhance the expression of filaggrin (FLG), transglutaminase (TGM)-1, hyaluronic acid synthase (HAS)-1, and HAS-2 in human keratinocytes. Moreover, Pg-EE down-regulated the expression of pro-inflammatory cytokines and up-regulated the production of FLG, HAS-1, and HAS-2 suppressed by UVB through inhibition of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) pathways. Given the above, since Pg-EE can improve skin barrier, hydration and reduce the UVB-induced inflammation on skin, it could therefore be a valuable natural ingredient for cosmetics or pharmaceuticals to treat skin disorders.     

Glucocorticoids Directly Affect Hyaluronan Production of Orbital Fibroblasts; A Potential Pleiotropic Effect in Graves’ Orbitopathy

Orbital connective tissue expansion is a hallmark of Graves’ orbitopathy (GO). In moderate-to-severe active GO, glucocorticoids (GC) are the first line of treatment. Here we show that hydrocortisone (HC), prednisolone (P), methylprednisolone (MP), and dexamethasone (DEX) inhibit the hyaluronan (HA) production of orbital (OF) and dermal (DF) fibroblasts. HA production of GO OFs (n = 4), NON-GO OFs (n = 4) and DFs (n = 4) was measured by ELISA. mRNA expression of enzymes of HA metabolism and fibroblast proliferation was examined by RT-PCR and BrdU incorporation, respectively. After 24 h of GC treatment (1µM) HA production decreased by an average of 67.9 ± 3.11% (p < 0.0001) in all cell cultures. HAS2, HAS3 and HYAL1 expression in OFs also decreased (p = 0.009, p = 0.0005 and p = 0.015, respectively). Ten ng/mL PDGF-BB increased HA production and fibroblast proliferation in all cell lines (p < 0.0001); GC treatment remained effective and reduced HA production under PDGF-BB-stimulated conditions (p < 0.0001). MP and DEX reduced (p < 0.001, p = 0.002, respectively) PDGF-BB-induced HAS2 expression in OFs. MP and DEX treatment decreased PDGF-BB stimulated HAS3 expression (p = 0.035 and p = 0.029, respectively). None of the GCs tested reduced the PDGF-BB stimulated proliferation rate. Our results confirm that GCs directly reduce the HA production of OFs, which may contribute to the beneficial effect of GCs in GO.     

Genetic and expression alterations in association with the sarcomatous change of cholangiocarcinoma cells

Cholangiocarcinoma (CC) is an intrahepatic bile duct carcinoma with a high mortality rate and a poor prognosis. Sarcomatous change/epithelial mesenchymal transition (EMT) of CC frequently leads to aggressive intrahepatic spread and metastasis. The aim of this study was to identify the genetic alterations and gene expression pattern that might be associated with the sarcomatous change in CC. Previously, we established 4 human CC cell lines (SCK, JCK1, Cho-CK, and Choi-CK). In the present study, we characterized a typical sarcomatoid phenotype of SCK, and classified the other cell lines according to tumor cell differentiation (a poorly differentiated JCK, a moderately differentiated Cho-CK, and a well differentiated Choi-CK cells), both morphologically and immunocytologically. We further analyzed the genetic alterations of two tumor suppressor genes (p53 and FHIT) and the expression of Fas/FasL gene, well known CC-related receptor and its ligand, in these four CC cell lines. The deletion mutation of p53 was found in the sarcomatoid SCK cells. These cells expressed much less Fas/FasL mRNAs than did the other ordinary CC cells. We further characterize the gene expression pattern that is involved in the sarcomatous progression of CC, using cDNA microarrays that contained 18,688 genes. Comparison of the expression patterns between the sarcomatoid SCK cells and the differentiated Choi-CK cells enabled us to identify 260 genes and 247 genes that were significantly over-expressed and under-expressed, respectively. Northern blotting of the 14 randomly selected genes verified the microarray data, including the differential expressions of the LGALS1, TGFBI, CES1, LDHB, UCHL1, ASPH, VDAC1, VIL2, CCND2, S100P, CALB1, MAL2, GPX1, and ANXA8 mRNAs. Immunohistochemistry also revealed in part the differential expressions of these gene proteins. These results suggest that those genetic and gene expression alterations may be relevant to the sarcomatous change/EMT in CC cells.     

Clinicopathological values of NBS1 and DNA damage response genes in epithelial ovarian cancers

Epithelial ovarian cancers (EOCs) are highly lethal gynecological malignancies with a high recurrence rate. Therefore, developing prognostic markers for recurrence after chemotherapy is crucial for the treatment of ovarian cancers. As ovarian cancers frequently respond to DNA-damaging agents, we assessed the clinicopathological significance of key double-strand DNA break (DSB) repair genes, including BRCA1, BRCA2, BARD1, ATM, RAD51 and NBS1 in EOC cell lines and paraffin-embedded tissue sections from 140 EOC patients treated with cytoreductive surgery, followed by platinum-based chemotherapy. These samples were analyzed for the clinicopathological impact of DSB genes by western blot analysis, immunohistochemistry and quantitative real-time PCR. Of the DSB repair genes, BRCA1, ATM and NBS1, which are involved in the homologous recombination-mediated repair pathway, were related to aggressive parameters in EOC. When survival analysis was performed, NBS1 expression exhibited an association with EOC recurrence. Specifically, increased NBS1 expression was found in 107 out of 140 cases (76.0%) and correlated with advanced stage (P=0.001), high grade (P=0.001) and serous histology (P=0.008). The median recurrence-free survival in patients with positive and negative expression of NBS1 was 30 and 78 months, respectively (P=0.0068). In multivariate analysis, NBS1 was an independent prognostic factor for the recurrence of EOC. Together, these results suggest that NBS1 is a marker of poor prognosis for the recurrence of EOC and is associated with aggressive clinicopathological parameters.     

Toll-like Receptors Gene Expression in the Gastrointestinal Tract of Salmonella Serovar Pullorum-Infected Broiler Chicken

Salmonella enterica serovar Pullorum causes substantial mortality in chicks as well as results in persistent infection and vertical transmission in layer birds. An effective innate immune response in the early stages of infection could reduce bacterial colonization and mortality in chicks and persistency of infection in later stages. Toll-like receptors (TLRs), important components of innate immune response, plays a pivotal role in early recognition of pathogen as well as in the initiation of robust and specific adaptive immune response. In the present study, we quantified the expression levels of chicken TLRs (1LA, 1LB, 2A, 2B, 3, 4, 5, 7, 15, and 21) mRNA by quantitative real-time PCR in the gastrointestinal (GI) tissues (duodenum, jejunum, ileum, and cecum) of 3-day-old broiler chicks after 24 h of oral infection with S. enterica serovar Pullorum. We found significant upregulation of TLRs (TLR2, TLR4, TLR21) mRNA expressions in GI tract tissues after S. Pullorum infection. The exceptions were for TLR3 and TLR15 with decrease in the expression levels in the jejunum after infection. TLR4 gene expression was significantly (P?<?0.05) upregulated in the duodenum and ileum of infected chicks. Gene expression for some of the TLRs (TLR1LA, ILB, 2B, and TLR5) remained unchanged after infection with S. Pullorum in all the GI tissues studied. Most substantial change in gene expression was found for TLR21, being significantly (P?<?0.05) upregulated in all the tissues investigated. The differential expression levels of TLRs shed light on tailored innate immune response induced by S. Pullorum during the early stages of infection in chicks.