Ethanol-induced chromosomal abnormalities in human fibroblast cells

Introduction: ethanol consumption may impose cytotoxic effects on the human body, by producing toxic metabolites through different pathways. Furthermore, some studies have also linked alcohol consumption to various types of diseases. Therefore, this in vitro study is aimed to assess the ethanol toxic effects on the human fibroblastic chromosome structure. Materials and Methods: G banding staining method was used to determine the karyotype of chromosomal abnormalities of cultured fibroblast cells. Karyogram of ethanol-treated cells after 48 hours of incubation with 54 and 108 mmol of ethanol were assessed against the untreated cultured cells. Results: the comparison between two karyogram models confirmed that lower ethanol concentration (54mmol) caused breakage in chromosome type C and number 1, while with higher concentration (108mmol) breakage was observed in chromosome 9, chromatids as well as endoreplication in some other cells. Conclusion: this study indicates that specific concentrations of ethanol can cause vast alterations in chromosomal structure. Therefore, in general, more care is suggested in consuming this substance. In addition, to confirm the cytotoxic alterations in chromosomal structure in relation to alcohol consumption, using other cell lines and /or in vivo studies, more investigations are warrant. © 2014, Semnan University of Medical Sciences. All rights reserved

Isolation and cloning of the β subunit of human follicle stimulating Hormone (hFSHβ) with its native gene's signal sequence in methylotroph yeast Pichia pastoris pPIC9 shuttle vector

Introduction: Follicle Stimulating Hormone (FSH) is one of the pituitary glycoproteines that it consists of two subunits; alpha and beta. The beta subunit is responsible for the biological activity of FSH. The aim of present study was isolation of the β subunit coding sequence containing its signal sequence from human genome and then cloning of the isolated sequence in pPIC9 shuttle vector under the control of AOX1 promoter and α factor signal sequence. Material and Methods: the gene sequence of interest was isolated as a 2kb DNA fragment and cloned in pTZ57R vector resulting to pTV-2019 plasmid. The construct was used as template for modification of 5΄ region of gene upstream to ATG codon using PCR. Finally, amplicon was cloned in pPIC9 and the new construct named pPIC9F1. Results: The sequence of FSHβ gene in pTV-2019 was confirmed by restriction analysis and DNA sequencing. In addition, restriction analysis and AOX1 primer-mediated PCR showed that pPIC9F1 has correct construction. Conclusion: The new construct, pPIC9F1, contains the coding sequence of FSHβ gene and its signal sequence (E2-IVS2-E3). Therefore, this construct can be used for integration of FSHβ gene into yeast genome exactly downstream to AOX1 promoter. Under this condition, a fusion protein is produced that it contains two signal peptides, α factor and FSH signal peptides. Yeast expression system is able to cleavage α factor. It seems this is the first attempt for cloning of human FSHβ in yeast expression system

Anticancer Activity of Ipomoea purpurea Leaves Extracts in Monolayer and Three-Dimensional Cell Culture

Cancer is a leading cause of death and a vital health care challenge in the world. Hence, this work was conducted to determine the in vitro anticancer property and also the molecular mechanism of aqueous and organic extracts of Ipomoea purpurea leaves in three human cancer cell lines, including A-549 (human lung cancer), HepG-2 (human liver cancer), MDA-MB-231 (human breast cancer), and MCF-10A (breast normal cell line). In vitro cytotoxic potential of organic extracts, such as hexane, chloroform, ethyl-acetate, methanol, and aqueous extract was examined using a standard (3-(4,5-dimethylthiazole)-2,5-diphenyltetrazolium bromide) MTT method in both monolayer two-dimensional (2D) and spheroids multicellular three-dimensional (3D) cultures. The MTT assay data showed that methanol and chloroform extracts of I. purpurea leaves had the antiproliferative effect on lung and breast cancer cells with IC50 of 53.62 ± 0.07 and 124.5 ± 0.01 µg/mL, respectively. The results of further examinations, such as dual acridine orange/ethidium bromide, Annexin V-FITC/PI, and caspase-3 colorimetric assay, confirmed that methanol and chloroform extracts of I. purpurea as the most potent cytotoxic extracts might contain a variety of phytochemicals, promoting apoptosis in lung and breast cancer cells. The present research findings suggested that methanolic extract of I. purpurea leaves induced S-phase cell cycle arrest and intrinsic pathway of apoptosis in A-549 lung cancer cells. The study further showed that I. purpurea could be a helpful candidate for cancer treatment