Hulya Ayar Kayali

EpCAM Controls Actomyosin Contractility and Cell Adhesion by Direct Inhibition of PKC

Epithelial cell adhesion molecule (EpCAM) is a cell-surface protein highly expressed in embryonic tissues and in malignant carcinomas. We report that EpCAM acts as a potent inhibitor of novel protein kinase C (nPKC) in both embryos and cancer cells. We observed dramatic effects of loss of EpCAM on amphibian embryonic tissues, which include sequentially strong overstimulation of PKC activity and of the Erk pathway, leading to exacerbated myosin contractility, loss of cadherin-mediated adhesion, tissue dissociation, and, ultimately, cell death. We show that PKC inhibition is caused by a short segment of the EpCAM cytoplasmic tail. This motif resembles the pseudosubstrate inhibitory domains of PKCs and binds nPKCs with high affinity. A bioinformatics search reveals the existence of similar motifs in other plasma membrane proteins, most of which are cell-cell adhesion molecules. Thus, direct inhibition of PKC by EpCAM represents a general mode of regulation of signal transduction by cell-surface proteins.

Role of pyruvate and ascorbate production in regulation of antioxidant enzymes and membrane LPO levels in Fusarium acuminatum.

The role of pyruvate and ascorbate in the regulation of superoxide dismutase (SOD); catalase (CAT); glutathione peroxidase enzymes; and, therefore, membrane lipid peroxidation (LPO) levels in Fusarium acuminatum was investigated in media containing either glycerin or glucose as a carbon source, depending on the incubation period, in the range of 5–25 g/L. Increasing SOD activity between d 9 and 16 of the incubation period showed a positive correlation with a significant increase in pyruvate production up to 15 g/L of glycerin and glucose. In addition, maximum ascorbate production was observed at 15 g/L of glycerin as 82.5 ± 2.1 and 20 g/L of glucose as 54±1.51, whereas CAT activity decreased with an increased concentration of both carbon sources. When compared with the LPO levels determined in media supplemented with glycerin and glucose, the minimum LPO level was 1.88±0.028 nmol of malondialdehyde/g wet wt at 15 g/L of glycerin on d 16, at which it was also observed to have a maximum pyruvate and ascorbate production and SOD, CAT, and GSH-Px activities of 75±1.42 µg/mL, 82.5±2.1 µg/mL, 32.5±0.634 µg/mL, 86.8±2.58 IU/mg, and 1.867 IU/mg, respectively. These results indicate that the biosynthesis of pyruvate and ascorbate may be involved in the regulation of antioxidant enzymes, depending on the glycerin and glucose concentrations, and also this defense network was effective in preventing membrane damage from oxidative stress.

Functions of Antioxidant Enzyme Activities on the Membrane Bound Total Sialic Acid and Lipid Peroxidation Level in F. equiseti and F. acuminatum

The variations of membrane bound total sialic acid (TSA) and lipid peroxidation level dependent on the antioxidant enzyme activities such as Superoxide Dismutase (SOD), Catalase (CAT), Glutathione peroxidase (GSH-Px) have been studied in yeast extract supplemented medium. The maximum SOD and CAT activities of F. equiseti tended to increase with raises of yeast extract concentration up to 25 g/L where they were determined to be 78.6±0.96 and 312.7±5.6 IU/mg. On the other hand, SOD and CAT activities in F. acuminatum significantly increased with the rise of yeast extract concentration up to 10 g/L (p < 0.01) and maximum activities were observed at this concentration as 36.3±0.54 and 115.3±2.19 IU/mg on the 12th day incubation. Other H2O2 scavenger enzyme, GSH-Px activities of F. equiseti and F. acuminatum were reached the maximum at 5 and 25 g/L yeast extract and determined as 5.06±0.04 and 4.74±0.09 IU/mg, respectively. TSA level showed positive correlation with SOD and CAT activities while LPO levels variations negatively correlated. The results may indicate that these antioxidant enzymes also appeared to be involved in protecting membrane bound sialic acids as well as membrane lipid of the fungus from exogenous reactive oxygen species.

Screening organometallic thiophene containing thiosemicarbazone ruthenium (II/III) complexes as potential anti-tumour agents

The new ruthenium (III) complex has been synthesized and characterized by elemental analysis, FT-IR, UV–Vis, EI-Mass, EPR spectroscopy, and magnetic susceptibility measurement. Cytotoxic effects of organoruthenium (II/III) complexes 1a, 1b, and 2a, and their ligands (TSC1 and TSC2) in cultured human ovarian (A2780, SKOV-3, and OVCAR-3) and colon (DLD, CCD18Co, and Caco-2) cells have been investigated comparing reactivity of the Ru (II/III) complexes and their free TSC ligands. The complexes exhibit higher cytotoxicity in three cancer cell lines than in normal cells. The binding with CT-DNA and BSA of the all complexes were weak compared with their ligand in spite of the cellular uptake of these complexes into the cytoplasm and then nucleus while their cytotoxic effects were vice versa. All the results showed that Complex 1b has more efficient cytotoxicity on the colon cancer cells than ovarian cancer cells. However, Complex 2a is a better drug candidate especially for antitumor therapy of metastasized ovarian cancer.

Protein kinase C Inhibitors selectively modulate dynamics of cell adhesion molecules and cell death in human colon cancer cells

ABSTRACT During development of colon cancer, Protein Kinase Cs (PKCs) are involved in regulation of many genes controlling several cellular mechanisms. Here, we examined the changes in cell adhesion molecules and PKCs for colorectal cancer progression. We identified that PKCs affected expression of EpCAM, claudins, tetraspanins. Treatment with low concentrations of PKC inhibitors resulted in decreased cell viability. In addition, immunoblotting and qRT-PCR analysis showed that apoptosis was inhibited while autophagy was induced by PKC inhibition in colon cancer cells. Furthermore, we observed decreased levels of intracellular Reactive Oxygen Species (ROS), lipid peroxidation and protein carbonyl, confirming the ROS-induced apoptosis. Taken together, our results reveal that PKC signalling modulates not only cell adhesion dynamics but also cell death-related mechanisms. Abbreviations: PKC: Protein Kinase C; EpCAM: Epithelial cell adhesion molecule; FBS: fetal bovine serum; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide); CAM: cell adhesion molecule; ROS: reactive oxygen species

Phenylpropanoid Pathway Response to Cadmium and Lead Stress in Phaselous vulgaris Roots and Leaves

A are toxic and carcinogenic secondary metabolites produced by Aspergillus species. Due to their effects on health, its limits in food and feed are regulated by authorities. In determination of Aflatoxin (AFL) levels in food and feed, HPLC analysis upon clean-up with immunoaffinity column (IAC) is the mostly used method. IACs are used to concentrate and purify toxin content of the food or feed sample by the use of antibodies. In this study, 8G8 monoclonal antibody that recognizes AFL B1, B2, G1, G2 and M1 with high affinity was developed, produced in cell culture and immobilized on CNBr activated Sepharose resin without purification of antibody for cost effective and labor saving production of IACs. Antibody immobilized column matrix was filled within column and AFL spiked samples were applied to the columns. Aflatoxin content of eluates was analyzed with HPLC. Recovery % and relative standard deviation of developed IACs is calculated as 99.17% and 2.4 % relatively. These results show developed columns can be used for high performance clean-up of AFL B1, B2, G1, G2 and M1 containing food and feed samples. In addition, we developed an HPLC analysis method to discriminate AFL B1, B2, G1, G2 and M1 peaks for simultaneous detection of these aflatoxin types. By the use of IAC and HPLC analysis method developed in this study, easier and quicker analysis of the samples that have the risk of having multiple aflatoxin types can be achieved.T expressions of cell adhesion molecules (CAMs) that are located on the cell surface involved in binding with other cells or with the extracellular matrix (ECM) alter depending on the cancer types and stages. Especially, Epithelial Cell Adhesion Molecule (EpCAM) and claudin family members of tight junctions are really important in the cancer progression, invasion, metastasis, angiogenesis and apoptosis resistance to the chemotherapeutic drugs. In our study, we determined expression profile of EpCAM and claudin-3, 4 and 7 in both ovarian and colon cancer cell lines by comparing with the normal epithelium cell lines. Significantly, EpCAM and claudin levels up-regulated in the primary cancer lines (A2780 and Caco-2) compared to the normal epithelial cell lines (OSE and CCD-18Co). Also, OVCAR-3, SKOV-3 and DLD-1 cell lines that were isolated from metastatic regions showed higher EpCAM and claudin expression compared to the primary cancer and normal epithelium cell lines. These results evince that EpCAM and claudin molecules can be used for diagnosis of ovarian and colon cancers.

Improvement of ramoplanin production by submerged fermentation of Actinoplanes sp. ATCC 33076 using animal origin protein meals

A are toxic and carcinogenic secondary metabolites produced by Aspergillus species. Due to their effects on health, its limits in food and feed are regulated by authorities. In determination of Aflatoxin (AFL) levels in food and feed, HPLC analysis upon clean-up with immunoaffinity column (IAC) is the mostly used method. IACs are used to concentrate and purify toxin content of the food or feed sample by the use of antibodies. In this study, 8G8 monoclonal antibody that recognizes AFL B1, B2, G1, G2 and M1 with high affinity was developed, produced in cell culture and immobilized on CNBr activated Sepharose resin without purification of antibody for cost effective and labor saving production of IACs. Antibody immobilized column matrix was filled within column and AFL spiked samples were applied to the columns. Aflatoxin content of eluates was analyzed with HPLC. Recovery % and relative standard deviation of developed IACs is calculated as 99.17% and 2.4 % relatively. These results show developed columns can be used for high performance clean-up of AFL B1, B2, G1, G2 and M1 containing food and feed samples. In addition, we developed an HPLC analysis method to discriminate AFL B1, B2, G1, G2 and M1 peaks for simultaneous detection of these aflatoxin types. By the use of IAC and HPLC analysis method developed in this study, easier and quicker analysis of the samples that have the risk of having multiple aflatoxin types can be achieved.T expressions of cell adhesion molecules (CAMs) that are located on the cell surface involved in binding with other cells or with the extracellular matrix (ECM) alter depending on the cancer types and stages. Especially, Epithelial Cell Adhesion Molecule (EpCAM) and claudin family members of tight junctions are really important in the cancer progression, invasion, metastasis, angiogenesis and apoptosis resistance to the chemotherapeutic drugs. In our study, we determined expression profile of EpCAM and claudin-3, 4 and 7 in both ovarian and colon cancer cell lines by comparing with the normal epithelium cell lines. Significantly, EpCAM and claudin levels up-regulated in the primary cancer lines (A2780 and Caco-2) compared to the normal epithelial cell lines (OSE and CCD-18Co). Also, OVCAR-3, SKOV-3 and DLD-1 cell lines that were isolated from metastatic regions showed higher EpCAM and claudin expression compared to the primary cancer and normal epithelium cell lines. These results evince that EpCAM and claudin molecules can be used for diagnosis of ovarian and colon cancers.

EpCAM and claudins as cancer biomarkers in ovarian and colon cancer

A are toxic and carcinogenic secondary metabolites produced by Aspergillus species. Due to their effects on health, its limits in food and feed are regulated by authorities. In determination of Aflatoxin (AFL) levels in food and feed, HPLC analysis upon clean-up with immunoaffinity column (IAC) is the mostly used method. IACs are used to concentrate and purify toxin content of the food or feed sample by the use of antibodies. In this study, 8G8 monoclonal antibody that recognizes AFL B1, B2, G1, G2 and M1 with high affinity was developed, produced in cell culture and immobilized on CNBr activated Sepharose resin without purification of antibody for cost effective and labor saving production of IACs. Antibody immobilized column matrix was filled within column and AFL spiked samples were applied to the columns. Aflatoxin content of eluates was analyzed with HPLC. Recovery % and relative standard deviation of developed IACs is calculated as 99.17% and 2.4 % relatively. These results show developed columns can be used for high performance clean-up of AFL B1, B2, G1, G2 and M1 containing food and feed samples. In addition, we developed an HPLC analysis method to discriminate AFL B1, B2, G1, G2 and M1 peaks for simultaneous detection of these aflatoxin types. By the use of IAC and HPLC analysis method developed in this study, easier and quicker analysis of the samples that have the risk of having multiple aflatoxin types can be achieved.T expressions of cell adhesion molecules (CAMs) that are located on the cell surface involved in binding with other cells or with the extracellular matrix (ECM) alter depending on the cancer types and stages. Especially, Epithelial Cell Adhesion Molecule (EpCAM) and claudin family members of tight junctions are really important in the cancer progression, invasion, metastasis, angiogenesis and apoptosis resistance to the chemotherapeutic drugs. In our study, we determined expression profile of EpCAM and claudin-3, 4 and 7 in both ovarian and colon cancer cell lines by comparing with the normal epithelium cell lines. Significantly, EpCAM and claudin levels up-regulated in the primary cancer lines (A2780 and Caco-2) compared to the normal epithelial cell lines (OSE and CCD-18Co). Also, OVCAR-3, SKOV-3 and DLD-1 cell lines that were isolated from metastatic regions showed higher EpCAM and claudin expression compared to the primary cancer and normal epithelium cell lines. These results evince that EpCAM and claudin molecules can be used for diagnosis of ovarian and colon cancers.

Improvement of ramoplanin production by submerged fermentation of Actinoplanes sp. ATCC 33076 using metal sulfates

A are toxic and carcinogenic secondary metabolites produced by Aspergillus species. Due to their effects on health, its limits in food and feed are regulated by authorities. In determination of Aflatoxin (AFL) levels in food and feed, HPLC analysis upon clean-up with immunoaffinity column (IAC) is the mostly used method. IACs are used to concentrate and purify toxin content of the food or feed sample by the use of antibodies. In this study, 8G8 monoclonal antibody that recognizes AFL B1, B2, G1, G2 and M1 with high affinity was developed, produced in cell culture and immobilized on CNBr activated Sepharose resin without purification of antibody for cost effective and labor saving production of IACs. Antibody immobilized column matrix was filled within column and AFL spiked samples were applied to the columns. Aflatoxin content of eluates was analyzed with HPLC. Recovery % and relative standard deviation of developed IACs is calculated as 99.17% and 2.4 % relatively. These results show developed columns can be used for high performance clean-up of AFL B1, B2, G1, G2 and M1 containing food and feed samples. In addition, we developed an HPLC analysis method to discriminate AFL B1, B2, G1, G2 and M1 peaks for simultaneous detection of these aflatoxin types. By the use of IAC and HPLC analysis method developed in this study, easier and quicker analysis of the samples that have the risk of having multiple aflatoxin types can be achieved.T expressions of cell adhesion molecules (CAMs) that are located on the cell surface involved in binding with other cells or with the extracellular matrix (ECM) alter depending on the cancer types and stages. Especially, Epithelial Cell Adhesion Molecule (EpCAM) and claudin family members of tight junctions are really important in the cancer progression, invasion, metastasis, angiogenesis and apoptosis resistance to the chemotherapeutic drugs. In our study, we determined expression profile of EpCAM and claudin-3, 4 and 7 in both ovarian and colon cancer cell lines by comparing with the normal epithelium cell lines. Significantly, EpCAM and claudin levels up-regulated in the primary cancer lines (A2780 and Caco-2) compared to the normal epithelial cell lines (OSE and CCD-18Co). Also, OVCAR-3, SKOV-3 and DLD-1 cell lines that were isolated from metastatic regions showed higher EpCAM and claudin expression compared to the primary cancer and normal epithelium cell lines. These results evince that EpCAM and claudin molecules can be used for diagnosis of ovarian and colon cancers.

The glutathione-related detoxication responses to juvenile and ecdysone hormones in Galleria mellonella

The effect of 20-hydroxyecdysone (20E) and juvenile hormone (JH) on the glutathione pathway of the greater wax moth Galleria mellonella (Lepidoptera: Pyralidae) was determined by investigating glutathione peroxidase (GSH-Px), glutathione S-transferases (GST), and glutathione reductase (GR) activities as well as reduced and oxidized glutathione (GSH and GSSG) content with respect to developmental stage. The continuous decreases of GSH-Px and GST activities dependent on the growth period of G. mellonella occurred in JH and 20E groups over and under their controls, respectively. While the GR activities of G. mellonella showed increases in young pupa (YP) for both control and in old larvae (OL) for the 20E groups after the minimum at these periods, they also increased after old pupa (OP) for the JH group with a maximum in OL period. Although GR activity levels in the JH group were significantly higher compared with controls and 20E groups up to OP period, the activity levels for the control and 20E groups were higher than those of the JH group at adult (AD) and old pupa (OP) periods, respectively. In spite of increases in the GR activity of 20E and control groups of G. mellonella, decreased GSH and increased GSSG levels were observed at aging period. GSH levels in the JH group reached a maximum at prepupa (PP) and then decreased with non-significant changes from OL to AD period. According to the results, GSH and GSSG levels, as well as GSH/GSSG ratios, were below and over control levels in 20E and JH groups, respectively, during all of the investigated developmental stages. On the contrary, the LPO levels were higher than the control for 20E and lower for the JH groups during the developmental period. These results show that while ecdysone hormone has a negative effect on the glutathione-related detoxication capacity of G. mellonella, the juvenile hormone has a positive effect on this process.