Glycan analysis of Lamin A/C protein at G2/M and S phases of the cell cycle.

During mitosis, phosphorylation and dephosphorylation of lamins triggers the nuclear envelope disassembly/assembly. However, it hasn't been known whether lamin proteins undergo any modification other than phosphorylation during the cell cycle. Glycosylation of lamin proteins is one of the less studied post-translational modification. Glycosylation and phosphorylation compete for the same positions and interplay between two modifications generate a post-translational code in the cell. Based on this, we hypothesized that glycosylation of lamin A/C protein may be important in the regulation of the structural organization of the nuclear lamina during interphase and mitosis. We analysed the glycan units of lamin A/C protein in lung carcinoma cells synchronized at G2/M and S phases via CapLC-ESI-MS/MS. Besides, the outermost glycan units were determined using lectin blotting and gold-conjugated antibody and lectin staining. TEM studies also allowed us to observe the localization of glycosylated lamin A/C protein. With this study, we determined that lamin A/C protein shows O-glycosylation at G2/M and S phases of the cell cycle. In addition to O-GlcNAcylation and O-GalNAcylation, lamin A/C is found to be contain Gal, Fuc, Man, and Sia sugars at G2/M and S phases for the first time. Having found the glycan units of the lamin A/C protein suggests that glycosylation might have a role in the nuclear organization during the cell cycle.

Histochemical and ultramorphological visualization for storage molecules in trophocytes at postembryonic developmental stages of Bombyx mori (Lepidoptera: Bombycidae).

The fat body originates from mesoderm during embryogenesis and it exists through the developmental stages in insects. It is equivalent to vertebrate adipose tissue and liver because it has multiple metabolic and storage functions. The fat body controls the synthesis, storage and metabolism of glycogen, lipid and protein, and it plays a major role in immune and endocrine systems and detoxification processes. Main cells of fat body, which accomplish these vital functions are trophocytes. In this study, we aimed to determine the reserve molecules like glycogen, lipid, protein and uric acid accumulated in the fat body at postembryonic developmental stages of Bombyx mori. For this purpose, we used specific histochemical techniques to determine glycogen, lipid, protein and uric acid molecules. We determined that glycogen contents are stored from the 3rd larval stage while proteins and uric acids are stored from the 4th larval stage. We also detected that the amount of glycogen, lipid, protein and uric acid increase gradually throughout the larval stage and then these molecules decrease gradually as they are used in the pupal stage. Fat body biology may require further investigations on the underlying function of the fat body formation throughout the developmental stages. It can also be used as a model in research of metabolic disorders and immune diseases.

Demonstrating the general structure and cell types of the fat body in Blatta orientalis (Oriental Cockroach).

The fat body is a tissue that originates from mesoderm in insects. It consists of several cell types. The basic cell of the fat body is trophocyte. Glycogen, protein and lipid which are required for energy are stored in these cells. Mycetocyte, urocyte, chromotocyte and haemoglobin cells are the other cell types which originate from differentiated trophocytes. Of the cells found in cockroaches, mycetocytes contain an endosymbiont species of bacteria while urocytes are specialized cells for storing and discharging uric acid. Oenocyte, which is not the fat body cell type but associated with epidermis and the fat body cells, is also found in cockroaches. In this research, the fat body distribution was shown for the first time in three selected sections (thorax, beginning and end of abdomen) in all stages of Blatta orientalis (Linnaeus, 1758). In addition, the fat body cell types and distribution were determined by histological, histochemical and ultrastructural studies. As a result, trophocytes, mycetocytes, urocytes of the fat body and oenocytes which are related to the fat body were determined in B. orientalis. Also, it was revealed that the fat body content increased in the selected regions of the stages depending on the development. We hope that these findings will contribute to data about the fat body and give some directions to insecticide studies.

Computational analysis of the structure, glycosylation and CMP binding of human ST3GAL sialyltransferases.

Sialyltransferases (STs) are the fundamental enzymes which are related to many biological processes such as cell signalling, cellular recognition, cell-cell and host-pathogen interactions and metastasis of cancer. All STs catalyse the terminal sialic acid addition from CMP donor to the glycan units. ST3GAL family is one of the most important STs and divided into the six subfamily in mouse and humans which are ST3Gal I, ST3Gal II, ST3Gal III, ST3Gal IV, ST3Gal V, and ST3Gal VI. The members of the ST3GAL family transfer sialic acid to the terminal galactose residues of glycochains through an α2,3-linkage. There are many reports on the ST3GAL function in mammals but, there is a paucity of information about structure of human ST3GAL family. Herein, we investigated the structure, glycosylation and CMP binding site of human ST3GAL family using computational methods. We found for the first time N-glycosylation positions in ST3Gal IV and VI, mucin type glycosylation in ST3Gal III and O-GlcNAcylation in ST3Gal V and their relation with sialylmotifs. In addition, we predicted CMP binding positions of human ST3GAL enzyme family on three-dimensional structure using molecular docking and first demonstrated the sialylmotifs relation with the CMP binding positions in ST3Gal III-VI subfamilies.

Evaluation of Pyrethroid Susceptibility in Culex pipiens of Northern Izmir Province, Turkey.

BACKGROUND: Mosquitoes, being a nuisance species, are considered as one of the most important species in public health control programs due to their role as a vector in mosquito-borne diseases observed in humans and animals. We evaluated the susceptibility status of Culex pipiens collected from northern Izmir, Turkey in 2011-16. METHODS: Mosquito larvae, collected from three different locations in northern Izmir, were reared in the laboratory. Adult susceptibility bioassays were performed using the WHO insecticide-impregnated papers including deltamethrin 0.05%, permethrin 0.75%, α-cypermethrin 0.05% and cyfluthrin 0.15%. In addition, adult bioassays were performed after the pre-exposure to piperonyl butoxide (PBO) to determine the contribution of P450 detoxification enzymes to the phenotypic resistance. RESULTS: In all of the three populations, high levels of resistance were observed (mortalities<63%) to all of the four pyrethroids. Different pyrethroids but with the same mode of action can exhibit significantly different phenotypic resistance in a single population. PBO bioassays also showed that P450 detoxification enzymes can have diverse effects on different pyrethroids. CONCLUSION: Using just one chemical in a class of insecticide can be misleading for resistance studies.

Morphological and ultrastructural characterization of sea urchin immune cells.

The free circulating coelomocytes in the coelomic cavity of echinoderms are considered to be immune effectors by phagocytosis, encapsulation, cytotoxicity, and by the production of antimicrobial agents. Although echinoderms (especially sea urchin embryo) have been used as a model organisms in biology, no uniform criteria exist for classification of coelomocytes in echinoderms, and few studies have reported about the biological functions of their coelomocytes. Hence, we study the coelomocytes in the echinoid sea urchin, Paracentrotus lividus, and describe their morphological and ultrastructural features using light and transmission electron microscopes. We classify the coelomocytes of P. lividus into red spherule and colorless spherule cells, small cells, vibratile cells, and phagocytic cells; petaloid and filopodial cells. To our knowledge, this is the first report describing ultrastructural details of the coelomocytes of P. lividus.

Determination of sialic acids in immune system cells (coelomocytes) of sea urchin, Paracentrotus lividus, using capillary LC-ESI-MS/MS.

Coelomocytes are considered to be immune effectors of sea urchins. Coelomocytes are the freely circulating cells in the body fluid contained in echinoderm coelom and mediate the cellular defence responses to immune challenges by phagocytosis, encapsulation, cytotoxicity and the production of antimicrobial agents. Coelomocytes have the ability to recognize self from non-self. Considering that sialic acids play important roles in immunity, we determined the presence of sialic acid types in coelomocytes of Paracentrotus lividus. Homogenized coelomocytes were kept in 2 M aqueous acetic acid at 80 °C for 3 h to liberate sialic acids. Sialic acids were determined by derivatization with 1,2-diamino-4,5-methylenediaoxy-benzene dihydrochloride (DMB) followed by capillary liquid-chromatography-electrospray ionization/tandem mass spectrometry (CapLC-ESI-MS/MS). Standard sialic acids; Neu5Ac, Neu5Gc, KDN and bovine submaxillary mucin showing a variety of sialic acids were used to confirm sialic acids types. We found ten different types of sialic acids (Neu5Gc, Neu5Ac, Neu5Gc9Ac, Neu5Gc8Ac, Neu5,9Ac2, Neu5,7Ac2, Neu5,8Ac2, Neu5,7,9Ac3, Neu5Gc7,9Ac2, Neu5Gc7Ac) isolated in limited amounts from total coelomocyte population. Neu5Gc type of sialic acids in coelomocytes was the most abundant type sialic acid when compared with other types. This is the first report on the presence of sialic acid types in coelomocytes of P. lividus using CapLC-ESI-MS/MS-Ion Trap system (Capillary Liquid Chromatography-Electrospray Ionization/Tandem Mass Spectrometry).