BACKGROUND: Gastric carcinomas (GCs) have recently been reclassified according to the mucin phenotypes. We aimed to characterize the relationship between the mucin phenotypes and the genetic alterations or the clinicopathologic parameters of GCs. METHODS: Immunohistochemistry was performed for MUC1, MUC5AC, MUC6, MUC2, CD10, p53, hMLH1, CerbB2 and E-cadherin in 150 GCs. The mucin phenotypes of the GCs were classified as 4 phenotypes: gastric, intestinal, mixed and unclassified. RESULTS: MUC1, MUC5AC, MUC6, MUC2 and CD10 were expressed in 63.3%, 42.7%, 14.0%, 24.7% and 14.0% of the GCs, respectively. The mucin phenotypes of the GCs corresponded to the gastric type in 31.3%, the intestinal type in 20.0%, the mixed type in 15.3% and the unclassified type in 33.3%. The incidence of a p53 overexpression was higher in the gastric or mixed phenotype than in the intestinal or unclassified phenotype. MUC5AC expression, p53 overexpression and the gastric or mixed phenotype were associated with poor patient survival by multivariate analysis. CONCLUSION: This study suggests the gastric or mixed mucin phenotype may more likely go through the p53 pathway in carcinogenesis and the mucin phenotype may be considered as a prognostic indicator.
Cadherins ; Carcinogenesis ; Humans ; Immunohistochemistry ; Incidence ; Mucins* ; Multivariate Analysis ; Phenotype* ; Stomach ; Tumor Suppressor Protein p53

Previously, we have reported that p-chlorophenylalanine (PCPA), a serotonin depletor, profoundly increased pancreatic fluid and bicarbonate secretion but remarkably inhibited pancreatic amylase secretion in anesthetized rats. The present study was performed to verify the detailed effects of PCPA on pancreatic amylase synthesis that is directly related to amylase exocrine secretion. PCPA significantly decreased pancreatic RNA and protein contents as well as the amylase activity. However, pancreatic DNA content, trypsin and chymotrypsin activities were not influenced by the treatment of PCPA. The rate of pancreatic amylase synthesis, which was assessed by the amount of incorporated (35S)-methionine into amylase for 1 h, was also significantly decreased by 44% in PCPA-treated rats. In order to determine whether the PCPA-induced decrease of amylase synthesis resulted from change in the level of amylase mRNA, Northern blot analysis was performed. The mRNA expression level of amylase was also decreased by 48% in the PCPA-treated rats, indicating that the inhibitory effect of PCPA on the synthesis of pancreatic amylase was mainly regulated at a step prior to translation. It was also revealed in SDS-polyacrylamide gel electrophoresis that the qualitative change of amylase was induced by PCPA. The 54 KDa amylase band seems to be degraded into small molecular weight protein bands in PCPA-treated rats, suggesting that the PCPA-induced decrease of amylase may be partly attributed to the degradation of synthesized amylase.
Amylases* ; Animals ; Blotting, Northern ; Chymotrypsin ; DNA ; Electrophoresis ; Fenclonine* ; Molecular Weight ; Pancreas ; Rats* ; RNA ; RNA, Messenger ; Serotonin ; Trypsin

On page 173, the incorrect image which was not submitted by the author was mistakenly printed for Fig. 5 by a system error of the editing company.

Dexamethasone converts pluripotent pancreatic AR42J cells into exocrine cells expressing digestive enzymes. In order to address molecular mechanism of this differentiation, we have investigated the role of mitogen-activated protein (MAP) kinase pathway and gene expressions of p21(waf1/cip1)and nuclear oncogenes (c-fos and c-myc) during AR42J cell differentiation. Dexamethasone markedly increased the intracellular and secreted amylase contents as well as its mRNA level. However, cell growth and DNA content were significantly decreased. With these phenotypic changes, AR42J cells induced transient mRNA expression of p21(waf1/cip1)gene, which reached maximal level by 6 h and then declined gradually toward basal state. In contrast to p21(waf1/cip1), c-fos gene expression was transiently inhibited by 6 h and then recovered to basal level by 24 h. Increased c-myc expression detected after 3 h, peaked by 12 h, and remained elevated during the rest of observation. Dexamethasone inhibited epidermal growth factor-induced phosphorylation of extracellular signal regulated kinase. Inhibition of MAP kinase pathway by PD98059 resulted in further elevation of the dexamethasone-induced amylase mRNA and p21(waf1/cip1)gene expression. These results suggest that p21(waf1/cip1)and nuclear oncogenes are involved in dexamethasone-induced differentiation and inhibition of MAP kinase pathway accelerates the conversion of undifferentiated AR42J cells into amylase-secreting exocrine cells.
Amylases/genetics ; Animals ; Cell Differentiation/*drug effects ; Cell Division/drug effects ; Cell Line, Tumor ; Cyclins/genetics/*metabolism ; Dexamethasone/*pharmacology ; Gene Expression Regulation/drug effects ; Genes, fos/genetics ; Genes, myc/genetics ; MAP Kinase Signaling System/*drug effects ; Mitogen-Activated Protein Kinases/*metabolism ; Pancreas/cytology/*drug effects/enzymology/metabolism ; RNA, Messenger/genetics/metabolism ; Rats ; Support, Non-U.S. Gov't

Inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) have been known to be involved in various pathophysiological processes such as inflammation. This study was performed to determine the regulatory function of superoxide dismutase (SOD) on the LPS-induced expression of iNOS, and COX-2 in RAW 264.7 cells. When a cell-permeable SOD, Tat-SOD, was added to the culture medium of RAW 264.7 cells, it rapidly entered the cells in a dose-dependent manner. Treatment of RAW 264.7 cells with Tat-SOD led to decrease in LPS-induced ROS generation. Pretreatment with Tat-SOD significantly inhibited LPS-induced expression of iNOS and NO production but had no effect on the expression of COX-2 and PGE2 production in RAW 264.7 cells. Tat-SOD inhibited LPS-induced NF-kappaB DNA binding activity, IkappaBalpha degradation and activation of MAP kinases. These data suggest that SOD differentially regulate expression of iNOS and COX-2 in LPS-stimulated RAW 264.7 cells.
Animals ; Cell Line ; Cyclooxygenase 2/*genetics/metabolism ; Cytokines/immunology ; *Gene Expression Regulation ; Lipopolysaccharides/immunology/metabolism ; Mice ; Mitogen-Activated Protein Kinase Kinases/metabolism ; NF-kappa B/metabolism ; Nitric Oxide/metabolism ; Nitric Oxide Synthase Type II/*genetics/metabolism ; Reactive Oxygen Species/metabolism ; Superoxide Dismutase/*metabolism

HIV-1 Tat is considered to be one of key players to facilitate monocyte entry into the CNS, which is characteristic feature of AIDS-related encephalitis and dementia. This study was performed to determine the regulatory function of superoxide dismutase (SOD) on the HIV-1 Tat-induced signaling pathways leading to NF-kappaB activation, expression of adhesion molecules, and monocyte adhesion in CRT-MG human astroglioma cells by using cell-permeable SOD. When cell-permeable SOD was added to the culture medium of CRT-MG cells, it rapidly entered the cells in dose- and time-dependent manners. Treatment of astrocytes with cell-permeable SOD led to decrease in Tat-induced ROS generation as well as NF-kappaB activation. Cell-permeable SOD inhibited the activation of MAP kinases including ERK, JNK and p38 by HIV-1 Tat. Treatment of CRT-MG cells with cell-permeable SOD significantly inhibited protein and mRNA levels of ICAM-1 and VCAM-1 up-regulated by HIV-1 Tat, as measured by Western blot analysis and RT-PCR. Furthermore, enhanced adhesiveness of monocyte to astrocyte by HIV-1 Tat was significantly abrogated by pretreatment with cell-permeable SOD fusion proteins. These data indicate that SOD has a regulatory function for HIV-1 Tat-induced NF-kappaB activation in astrocytes and suggest that cell-permeable SOD can be used as a feasible therapeutic agent for regulation of ROS-related neurological diseases.
Astrocytes/*enzymology ; Cell Adhesion/*physiology ; Cell Membrane Permeability ; Gene Products, tat/*pharmacology ; HIV Infections/metabolism ; HIV-1/*chemistry ; Humans ; Monocytes/cytology/*drug effects ; Signal Transduction ; Superoxide Dismutase/genetics/*physiology

The reactive oxygen species (ROS) are considered to be an important mediator in pancreatic beta cell destruction, thereby triggering the development of insulin-dependent diabetes mellitus. In the present study, HIV-1 Tat-mediated transduction of Cu, Zn-superoxide dismutase (SOD) was investigated to evaluate its protective potential against streptozotocin (STZ) -induced cytotoxicity in insulin-producing MIN6N cells. Tat-SOD fusion protein was successfully delivered into MIN6N cells in a dose-dependent manner and the transduced fusion protein was enzymatically active for 48 h. The STZ induced-cell destruction, superoxide anion radical production, and DNA fragmentation of MIN6N cells were significantly decreased in the cells pretreated with Tat-SOD for 1 h. Furthermore, the transduction of Tat-SOD increased Bcl-2 and heat shock protein 70 (hsp70) expressions in cells exposed to STZ, which might be partly responsible for the effect of Tat-SOD. These results suggest that an increased of free radical scavenging activity by transduction of Tat-SOD enhanced the tolerance of the cell against oxidative stress in STZ-treated MIN6N cells. Therefore, this Tat-SOD transduction technique may provide a new strategy to protect the pancreatic beta cell destruction in ROS-mediated diabetes.
Diabetes Mellitus, Type 1 ; DNA Fragmentation ; HIV-1 ; HSP70 Heat-Shock Proteins ; Insulin-Secreting Cells ; Oxidative Stress ; Reactive Oxygen Species ; Streptozocin ; Superoxide Dismutase ; Superoxides

Essential fatty acid (EFA) is known to be required for the body to function normally and healthily. However, the effect of EFA on glucose uptake in skeletal muscle has not yet been fully investigated. In this study, we examined the effect of two EFAs, linoleic acid (LA) and alpha-linolenic acid (ALA), on glucose uptake of C2C12 skeletal muscle cells and investigated the mechanism underlying the stimulatory effect of polyunsaturated EFAs in comparison with monounsaturated oleic acid (OA). In palmitic acid (PA)-induced insulin resistant cells, the co-treatment of EFAs and OA with PA almost restored the PA-induced decrease in the basal and insulin-stimulated 2-NBDG (fluorescent D-glucose analogue) uptake, respectively. Two EFAs and OA significantly protected PA-induced suppression of insulin signaling, respectively, which was confirmed by the increased levels of Akt phosphorylation and serine/threonine kinases (PKCtheta and JNK) dephosphorylation in the western blot analysis. In PA-untreated, control cells, the treatment of 500 microM EFA significantly stimulated 2-NBDG uptake, whereas OA did not. Phosphorylation of AMP-activated protein kinase (AMPK) and one of its downstream molecules, acetyl-CoA carboxylase (ACC) was markedly induced by EFA, but not OA. In addition, EFA-stimulated 2-NBDG uptake was significantly inhibited by the pre-treatment of a specific AMPK inhibitor, adenine 9-beta-D-arabinofuranoside (araA). These data suggest that the restoration of suppressed insulin signaling at PA-induced insulin resistant condition and AMPK activation are involved at least in the stimulatory effect of EFA on glucose uptake in C2C12 skeletal muscle cells.
Acetyl-CoA Carboxylase ; Adenine ; alpha-Linolenic Acid ; AMP-Activated Protein Kinases* ; Blotting, Western ; Fatty Acids, Essential* ; Glucose* ; Insulin ; Linoleic Acid ; Muscle, Skeletal* ; Oleic Acid ; Palmitic Acid ; Phosphorylation ; Phosphotransferases

OBJECTIVE: Vitrification requires a high concentration of cyroprotectant (CPA) and an elevated cooling speed to avoid ice crystal formation. We have evaluated the effect of different combinations of cooling rate and CPA on embryonic integrity (developmental competence) in order to increase the efficiency of vitrification without impairing embryo viability. We hypothesized that the combination of CPA or the increase of cooling rates can reduce the concentration of toxic CPA for vitrification. As consequently, we performed experiments to evaluate the effect of various composition of CPA or slush nitrogen (SN2) on the mouse embryonic development following vitrification using low CPA concentration. METHODS: Vitrification of mouse embryos was performed with EM grid using liquid nitrogen (LN2) or SN2 and different composition of CPAs, ethylene glycol (EG) and dimethylsulfoxide (DMSO). After vitrification-warming process, their survival and blastocyst formation rates were examined. For analyzing long-term effect, these blastocysts were transferred into the uterus of foster mothers. RESULTS: Survival and blastocyst formation rates of vitrified embryos were higher in EG+DMSO group than those in EG only. Furthermore, the group using SN2 with a lower CPA concentration showed a higher survival of embryos and developmental rates than group using LN2. CONCLUSION: The combination of EG and DMSO as CPAs may enhance the survival of mouse embryos and further embryonic development after vitrification. SN2 can generate high survival and developmental rate of vitrified/warmed mouse embryos when a lower concentration of CPA was applied. Therefore, these systems may contribute in the improvement of cryopreservation for fertility preservation.
Animals ; Blastocyst ; Cryopreservation ; Dimethyl Sulfoxide ; Embryonic Development ; Embryonic Structures ; Ethylene Glycol ; Ethylenes ; Female ; Fertility Preservation ; Ice ; Mental Competency ; Mice ; Nitrogen ; Pregnancy ; Uterus ; Vitrification

Neuraminidase (NA) cleaves the glycosidic bond linkages of sialic acids to release the mature virions from infected cells and has been an attractive therapeutic target for anti-influenza agents. In our ongoing investigation of NA inhibitors in mushroom extracts, we found that the extract the fruiting body of Glaziella splendens potently inhibited neuraminidase. The fruiting bodies of G. splendens were extracted and partitioned successively with hexane, ethyl acetate, and butanol. The ethyl acetate soluble-layer was subjected to silica gel and Sephadex LH-20 column chromatographies, and MPLC to obtain five compounds (1–5). Their structures were determined by spectroscopic methods. NA inhibitory activity of these compounds was evaluated using NAs from recombinant rvH1N1, H3N2, and H5N1 influenza A viruses. One compound (1) was elucidated as a new azaphilone derivative, and four compounds (2–5) were identified as entonaemin A, comazaphilone D, rubiginosin A, and entonaemin B, respectively. Compounds 3 and 4 showed considerable inhibitory activity against three types of neuraminidases with the IC₅₀ values of 30.9, 41.8, and 35.7 µM for 3 and 46.5, 50.4, and 29.9 µM for 4, respectively. This study reveals that the fruiting bodies of G. splendens possess azaphilone derivatives with the NA inhibitory activity. This is the first report on the isolation of neuraminidase inhibitors from the fruiting bodies of G. splendens.
Agaricales ; Chromatography ; Fruit ; Influenza A virus ; N-Acetylneuraminic Acid ; Neuraminidase ; Sialic Acids ; Silica Gel ; Virion