The Role of Endoplasmic Reticulum Stress in Gastroesophageal Reflux Disease Symptoms and Treatment.

BACKGROUND: Gastroesophageal reflux disease is a common condition worldwide. There is no curative treatment for gastroesophageal reflux disease. Endoplasmic reticulum stress leads to the activation of the unfolded protein response and has an important role in inflammation. The aim is to determine the role of endoplasmic reticulum stress in the follow-up of individuals with gastroesophageal reflux disease and the temporal changes of endoplasmic reticulum stress markers with treatment. METHODS: Twenty-four subjects in total were recruited prospectively, of whom 15 had nonerosive reflux disease. Two biopsies from 2 cm above the esophagogastric junction, 2 biopsies from gastric antrum mucosa, and 2 biopsies from gastric corpus mucosa were taken. Simultaneously, 2 tubes of venous blood samples were drawn from each individual (1 tube for studying the genetic markers and 1 tube for analyzing the CYP2C19 polymorphism). RESULTS: The mean age was 42.3 ± 17.6 for women and 34.66 ± 11.2 for men. Pantoprazole, esomeprazole, rabeprazole, and lansoprazole preparations were used for treatment. There was no significant difference between tissue and blood samples for panel genes ATF-6, XBP-1, DDIT-3, DNAJC-10, and EIF-2-AK before treatment. There was a significant decrease in the level of ATF-6, XBP-1, DNAJC-9, EIF2-AK, and NF-2L-2 genes in blood after treatment. In the comparison of proton pump inhibitors, significant decreases in the expression of the ATF-6, XBP-1, and DNAJC-9 mRNAs were detected in blood from individuals after treatment. CONCLUSION: Endoplasmic reticulum stress can be for evaluating the clinical improvement and the effectiveness of treatment in gastroesophageal reflux disease.

Prion protein-dependent regulation of p53-MDM2 crosstalk during endoplasmic reticulum stress and doxorubicin treatments might be essential for cell fate in human breast cancer cell line, MCF-7.

In this study, we investigated the effect of doxorubicin and tunicamycin treatment alone or in combination on MDM-, Cul9-and prion protein (PrP)-mediated subcellular regulation of p53 in the context of apoptosis and autophagy. MTT analysis was performed to determine the cytotoxic effect of the agents. Apoptosis was monitorized by ELISA, flow cytometry and JC-1 assay. Monodansylcadaverine assay was performed for autophagy. Western blotting and immunofluorescence were performed to determine p53, MDM2, CUL9 and PrP levels. Doxorubicin increased p53, MDM2 and CUL9 levels in a dose-dependent manner. Expression of p53 and MDM2 was higher at the 0.25 µM concentration of tunicamycin compared to the control, but it decreased at 0.5 µM and 1 µM concentrations. CUL9 expression was significantly decreased only after treatment of tunicamycin at 0.25 µM. According to its glycosylation status, the upper band of PrP increased only in combination treatment. In combination treatment, p53 expression was higher than control, whereas MDM2 and CUL9 expressions were decreased. Combination treatments may make MCF-7 cells more susceptible to apoptosis rather than autophagy. In conclusion, PrP may be important in determining the fate of cell death through crosstalk between proteins such as p53 and MDM2 under endoplasmic reticulum (ER) stress conditions. Further studies are needed to obtain in-depth information on these potential molecular networks.

Determination of histopathological effects and myoglobin, periostin gene-protein expression levels in Danio rerio muscle tissue after acaricide yoksorrun-5EC (hexythiazox) application.

Although pesticides are essential agrochemicals to annihilate harmful organisms in agriculture, their uncontrolled use has become an important threat to environmental health. Exposure to pesticides can affect many biological systems including immune system, endocrine system, and nervous system. However, the potential side effects of pesticides to skeletal muscle system remain unclear. Present study has focused on the evaluation of this issue by using an acaricide, yoksorrun-5EC (hexythiazox), in an aquatic model organism, Danio rerio. The histological analyses revealed that increased concentrations of the acaricide cause degradation of skeletal muscle along with increased necrosis and atrophy in myocytes, intercellular edema, and increased infiltrations between perimysium sheaths of muscle fibers. The effects of acaricide on myoglobin and periostin, which are associated with oxygen transport and muscle regeneration, respectively, were investigated at the gene and protein levels. RT-PCR results suggested that high concentration yoksorrun-5EC (hexythiazox) can induce myoglobin and periostin genes. Similar results were also obtained in the protein levels of these genes by western blotting analysis. These results suggested that yoksorrun-5EC (hexythiazox)-dependent disruption of skeletal muscle architecture is closely associated with the expression levels of myoglobin and periostin genes in Danio rerio model.

Tunicamycin induced endoplasmic reticulum stress in the small intestine.

Because the small intestine is exposed to variety of foreign substances, it participates in host immune response. We investigated whether the expression levels of intestinal MAdCAM-1, PECAM-1 (CD31) and CAV-1 are affected by endoplasmic reticulum (ER) stress following brief treatment with tunicamycin (TN). We administered a single dose of TN intraperitoneally. Twenty-four hours later, MAdCAM-1, PECAM-1 and CAV-1 expression levels in Peyer's patches and villi were examined using immunohistochemistry (IHC), immunofluorescence (IF) and western blotting. Immunostaining of MAdCAM-1 and CAV-1 in control and TN treated Peyer's patches and villi exhibited similar staining patterns. The immunoreactivity of PECAM-1 was similar for the control and TN treated Payer's patches, whereas staining was decreased significantly in TN treated villi. Our findings suggest that short term TN treatment did not affect leukocyte movement to lymphoid compartments of the small intestine, but it altered villus architecture due to decreased PECAM-1 expression.

Quantitative approach to lectin-based glycoprofiling of thymic tissues in the control- and the dexamethasone-treated mice.

Dexamethasone (DEX) is the most commonly used synthetic glucocorticoid in treatment of various inflammatory conditions. Here we focused on evaluating the effect of DEX on apoptosis and glycan profile in the mouse thymic tissues. Histological examinations revealed that the DEX treatment cause severe alterations in thymus, such as disruption of thymic capsule, impaired epithelial cell-thymocyte contacts, cellular loss and increased apoptosis. The identification of thymic glycans in the control- and the DEX-treated mice was carried out by using a panel of five plant lectins, Maackia amurensis agglutinin (MAA), peanut agglutinin (PNA), Sambucus nigra agglutinin (SNA), Concanavalin A (ConA) and wheat germ agglutinin (WGA). Lectin histochemistry results showed that glycosylation pattern of thymus changes upon DEX treatment. For further detailed quantitative analyses of the binding intensities for each lectin, histochemical data were scored as high positive (HP), mild positive (MP) and low positive (LP) and differences among signaling densities were investigated. The staining patterns of thymic regions observed with lectin histochemistry suggest that DEX can affect the thymic glycan profile as well as thymocyte apoptosis. These results are consistent with the opinion that not only sialic acid, but also other sugar motifs may be responsible for thymocyte development.

Do prion protein gene polymorphisms induce apoptosis in non-mammals?

Genetic variations such as single nucleotide polymorphisms (SNPs) in prion protein coding gene, Prnp, greatly affect susceptibility to prion diseases in mammals. Here, the coding region of Prnp was screened for polymorphisms in redeared turtle, Trachemys scripta. Four polymorphisms, L203V, N205I, V225A and M237V, were common in 15 out of 30 turtles; in one sample, three SNPs, L203V, N205I and M237V, and in the remaining 14 samples, only L203V and N205I polymorphisms, were investigated. Besides, C658T, C664T, C670A and C823A SNPs were silent mutations. To elucidate the relationship between the SNPs and apoptosis, TUNEL assays and active caspase-3 immunodetection techniques in brain sections of the polymorphic samples were performed. The results revealed that TUNEL-positive cells and active caspase-3-positive cells in the turtles with four polymorphisms were significantly increased compared with those of the turtles with two polymorphisms (P less than 0.01 and P less than 0.05, respectively). In conclusion, this study provides preliminary information about the possible relationship between SNPs within the Prnp locus and apoptosis in a non-mammalian species, Trachemys scripta, in which prion disease has never been reported.

Effect of Tunicamycin on Glycosaminoglycans and Laminins in Embryonic and Postnatal Thymic Tissues.

OBJECTIVE: To compare histological and molecular alterations in the embryonic and neonatal thymi following exposure to tunicamycin. STUDY DESIGN: Mouse embryos at gestational days 17 (n = 7) and 18 (n = 7) and newborn animals at post-natal days 1 (n = 5) and 3 (n = 5) were divided into two groups: control and tunicamycin-treated. Combined Alcian blue and Periodic acid-Schiff sequences immunohistochemistry and immunoblotting were performed to determine glycosaminoglycan (GAG) accumulation and laminin expression in control and tunicamycin-treated embryonic and postnatal thymi. The apoptotic effect of tunicamycin was evaluated by TUNEL assay. RESULTS: In the control group acidic GAGs first appeared in medullary cells at postnatal day 3, whereas treatment with tunicamycin promoted the accumulation of acidic GAGs in all treated groups as of embryonic day 17. However, tunicamycin slightly decreased the laminin expression, and the number of apoptotic cells was considerably increased after tunicamycin treatment. CONCLUSION: Results suggest that carboxylated and acidic GAGs are two presumptive candidates to establish the thymic microenvironment during the late fetal development and postnatal periods of mice and that tunicamycin would be implicated in this establishment by increasing the acidic GAG accumulation and by reducing the laminin expression and the thymic stromal cell population.

[Comparison of Immune Responses Elicited by Adjuvanted Tachyzoite Lysate Vaccines Developed from Two Different Toxoplasma gondii Strains Isolated in Turkey]. / Türkiye'de Izole Edilen Iki Farkli Toxoplasma gondii Susundan Üretilen Adjuvanli Takizoit Eriyik Protein Asilarinin Uyardigi Immün Yanitlarin Karsilastirilmasi

Toxoplasma gondii the causative agent of toxoplasmosis is an obligate intracellular parasite with a wide host range including all warm-blooded animals and birds. T.gondii infection causes congenital toxoplasmosis in newborns and this may lead to fetal anomalies, retinochoroiditis leading to blindness, lethal toxoplasmic encephalitis in immune compromised patients, and organ failure in transplantation patients. The pathogenesis of toxoplasmosis change due to differences in the specific immune response elicited by diverse T.gondii strains. The protective immunity against toxoplasmosis is conferred by cellular immune responses. In the present study, two different strains isolated from Turkey named T.gondii Ankara and Ege were used to evaluate the types of humoral and cellular immune responses elicited by adjuvanted tachyzoite protein vaccines in an animal model. In the study, 6-8 weeks old female BALB/c mice were used and six study groups (each contains three mice) were composed for vaccination. The first and second groups were vaccinated with T.gondii Ankara and Ege (TAnkPE and TEgePE, respectively) tacyhzoite lysates, the third and fourth groups were vaccinated by tacyhzoite lysates adjuvanted with Freund's adjuvant (TAnkPE-Freund; TEgePE-Freund, respectively). The fifth and sixth groups were vaccinated with PBS and Freund's adjuvant as controls. Immunization of the animals was performed two times at three weeks intervals. The serum samples were collected before vaccination and after each vaccination to determine the IgG response by Western blotting, and IgG1 and IgG2a responses by ELISA. To determine the cellular immune response, CD8/CD4 cell ratio, intracellular IFN-g and IL-4 levels were determined in stimulated spleen cells grown in cell culture systems by flow cytometry. Toxoplasma IgG antibodies were only detected in TAnkPE-Freund group. IgG1 and IgG2a responses did not increase in any vaccination groups and there was not any polarization towards IgG1 or IgG2a. There was no significant increase in CD8/CD4 ratio of stimulated spleen cells. IFN-g level was increased in only TAnkPE-Freund vaccination group, however IL-4 levels were increased in TAnkPE-Freund, TEgePE-Freund and TEgePE groups. Our data showed that TAnkPE-Freund vaccine led to increase in IgG and IFN-g responses in BALB/c mice, however, tachyzoite lysate vaccines developed in this study did not induce sufficient protective immune response against toxoplasmosis. Thus, use of specific immunogenic proteins must be taken into consideration in the future vaccine development studies against toxoplasmosis.

Regressing amphibian tail as a model for the cadherin/beta-catenin complex disruption and glycosylation alteration during epithelial apoptosis.

Epidermis is one of the many tissues that are resorbed during metamorphosis in the regressing tail of amphibian tadpoles. Apoptotic mechanisms play an important role in this process. In this study, loss of intercellular contacts and alterations in plasma membrane glycosylation were observed during apoptosis. The cadherin/beta-catenin complex represents one of the major adhesive systems in multiple epithelial tissues. Here, we analysed the fate of cadherin/beta-catenin complex and alterations of plasma membrane glycoconjugate compositions in apoptotic epithelial cells. Our results showed that the cadherin molecules were cleaved into extracellular and beta-catenin associated cytosolic domains by an intracellular mechanism. However, the extracellular domains were probably removed completely by matrix metalloproteinases. Lectin histochemistry studies suggested that mannose and alpha(2-->6) linked (but not alpha(2-->3) linked) sialic acids were major sugar motifs in plasma membranes of apoptotic tadpole epithelial cells. Although previous studies indicated reduced levels of sialic acid residues during apoptosis, elevated Sambucus nigra agglutinin (SNA) reactivity might be due to the degradation of high molecular weight glycoproteins (probably including cadherin) that masked the SNA-binding residues of the plasma membrane prior to apoptosis.

The glycosylation status of murine [corrected] postnatal thymus: a study by histochemistry and lectin blotting.

During the intrathymic development, the fate of the thymocytes depends largely on variable expression of CD4/CD8 markers and T cell receptor protein expressions. In addition, changes of cell surface glycosylation status also affect the thymocyte maturation. In this study the glycosylation alterations in thymic tissues from 1, 9, 13 and 16 days old mice were evaluated by histochemical and lectin blotting techniques. With alcian blue (AB) at pH 5.7/periodic acid-Schiff (PAS) stainings, it was shown that thymic microenvironments contained carboxlylated and sulfated glycosaminoglycans (GAGs). Strong positivity to AB at pH 2.5, which specific for sialomucins, was seen in some medullary thymocytes. Similarly, it was shown that with Maackia amurensis agglutinin (MAL) medullary thymocytes, but not cortical ones, contained alpha(2 --> 3) linked sialic acid structures. On the other hand, while reaction with peanut agglutinin (PNA), which specific for core disaccharide galactose beta(1 --> 3) N-acetylgalactosamine, was only seen in cortical thymocytes, reaction with Galanthus nivalis agglutinin (GNA), which specific for terminal mannose residues, was seen in both cortex and medulla. However, Datura stramonium agglutinin (DSA), which recognizes galactose beta(1 --> 4) N-acetylglucosamine, was not only cell-specific, but it was bound some thymic vessels. With lectin blotting studies, five glycoprotein bands of molecular weights approximately 39, approximately 54, 100, approximately 110 and approximately 212 were found which reacted with MAL, PNA and DSA as well as GNA. These results suggest that glycosylation patterns of cell surface glycoconjugates are modified during thymocyte selection processes of postnatal days.

Cell surface glycosylation diversity of embryonic thymic tissues.

In the thymus, glycosylation status of many cell surface molecules changes during the thymocyte maturation and selection processes. In this study, we evaluated the glycosylation changes and possible relationships with programmed cell death in the thymic tissues from mouse embryos at the days 14 (E14), 15 (E15), 16 (E16), 17 (E17) and 18 (E18) of embryonic development. In order to determine glycosylation changes we used three different plant lectins: peanut agglutinin (PNA), Maackia amurensis leucoagglutinin (MAL or MAAI) and Sambucus nigra agglutinin (SNA), which recognize core disaccharide galactose (1-3) N-acetylgalactosamine [Galbeta(1-->3)GalNAc], sialic acid linked (2-->3) to galactose [SAalpha(2-->3)Gal] and sialic acid linked to galactose [SAalpha(2-->6)Gal] structures, respectively. Our lectin histochemistry and lectin blotting studies indicated that glycosylation pattern was modified in thymocytes at the embryonic developmental stages analyzed. The immature cortical thymocytes were labeled by PNA, whereas medullary thymocytes were positive for MAL and SNA binding. Many medullary thymocytes exhibited alpha(2-->6)-linked sialic acid on their surface and this increased throughout the gestational stages. In the lectin blotting studies, different protein bands of various molecular weights were identified in thymocytes. Two of them were putatively identified as CD43 and CD45 glycoproteins. In addition, TUNEL (deoxynucleotdyltransferase-mediated dUDP nick end labeling) indicated that only PNA-positive cortical thymocytes were deleted in all embryonic stages. These results indicate that the glycosylation pattern was modified in thymocytes at all embryonic developmental stages, and these modifications can affect the T cell deletion, probably via the galectin-1 molecule in the embryonic thymus.