Research in Molecular Medicine
Volume:3 Issue: 2, Jun 2015

Lactoferrin is a glycoprotein with a molecular weight of 80 kDa, as a member of the transferring family. In recent investigations the important biological properties such as anti microbial, antiviral and anticancer activity of lactoferrin were studied. In the present study, the effect of antitumor induced by lactoferin was evaluated on stomach cancer cell AGS. Bovine milk were collected after parturition. After isolation of fat and casein, the other milk’s proteins were separated by ammonium sulfate precipitation.Next step, lactoferrin was purified using CM-Sephadex-C50 cation exchange chromatography by FPLC system.The purified protein identified by SDS-PAGE electrophoresis.The concentration of lactoferrin from milk and colostrum by Bradford assay obtained 0.6 and 2 mg/ml, respectively. Stomach cancer cell line, AGS and normal cell lines, HEK-293 and HFF were cultured in 37 °C and 5 percent CO2. After appropriate cell growth, different concentrations of purified lactoferrin added to cells and incubated for 20, 36 and 48 hours. The cell viability and inhibition of cell growth were determined by MTT assay also apoptosis assay was evaluated using propidium iodide statining through flow cytometry. The results indicated that more inhibitory effect of lactoferrin with 500 µg/ml during 20,36 and 48 h were 55, 71 and 80percent, respectively for AGS but had not notable inhibitory effect on normal cell lines. The apoptosis level of AGS in analysis with flow cytometry was indicated 55 and 70 % in 20 and 48 h, respectively but not effective in normal cells.The isolated lactoferrin from bovine milk had inhibitory effect on stomach cancer cell line whereas, it didn’t has observable effect on normal cells.

The organochlorine DDT has estrogenic activity but the mechanism underlying the estrogenic activity of this pesticide remains unclear. In the present investigation here, we studied the transcriptional effects of a synthetic organochlorine pesticide o,p’-DDT [1.1.1.-trichloro-2-(o-chlorophenyl)-2-p-chloriphenyl ethane] and its metabolite p,p''-DDE (2-2-bis(4/chlorophenyl)-1-1-dichloroethyl) on the bovine oxytocin and the thymidine kinase-ERE promoter by estrogen receptor α in MDA-MB 231 and MCF-7 breast cancer cell line. Cells were seeded for transfections into 12- well plates at a density of 100000 cells per well and were transfected with a total of 3 μg of plasmid DNA using calcium phosphate coprecipitation. o,p’-DDT and p,p''-DDE were used for stimulation of transfected MDA-MB 231 and MCF-7 breast cancer cell lines. The results showed o,p’-DDT has no agonistic activity in MDA-MB 231 cells transfected with the oxytocin promoter construct (OTwt) or the thymidine kinase-ERE promoter construct (TK ERE) and estrogen receptor α. While, The results obviously show that o,p’-DDT has an agonistic effect on both the oxytocin and the thymidine kinase-ERE promoter in MCF-7 leading to a more than two-fold stimulation of transcription at 10-5 M. In addition, there is no agonistic effect with p,p''-DDE on transfected MCF-7 cells and MDA-MB 231 cell line. In conclusion, our results revealed that o,p’-DDT has not estrogenic activity in a classical mechanism in transfected MDA-MB 231 breast cancer cells while has estrogenic activity in a classical mechanism in transfected MCF-7 human breast cancer cell line.

Breast cancer is one of the most prevalent malignancies among women in various countries. HER2 overexpression, which is due to different reasons, occurs in 20-30% of breast cancers. HER2 gene encodes an 185kDa transmembrane glycoprotein with 1255 amino acids. This active product triggers downstream intracellular signaling pathways inducing cell proliferation and cell survival. These activities can be done in an uncontrolled manner in the cases which HER2 expression undergoes up-regulation. The aim of this study was optimization of Real Time PCR condition. RNA purification, cDNA synthesis and then optimization of Real Time PCR method performed respectively. In this study, total RNA was extracted from fresh tissue samples, first strand of total cDNA was synthesized and in the following steps, Real Time PCR was performed to be optimized. Although altering the protocol, annealing temperature and concentration of MgCl2 did not make any improvement and beneficial effects on reactions, changing the concentration of primers to 0.24 pm/μl was influential to eliminate primer dimers of Real Time PCR reactions. It demonstrated that the copy number of GAPDH transcripts is more than HER2 transcripts in normal breast tissues. Therefore, deviation in 2.5 differences between the Ct value of HER2 and GAPDH indicated that the copy number of HER2 transcripts was increased; therefore, HER2 underwent overexpression in these cases. Under these optimized conditions, this technique can be applied as a powerful method in clinical laboratories.

Gamma -aminobutyric acid (GABA), a non-protein amino acid acts as an inhibitory neurotransmitter in the central nervous system of mammalians. The glutamate decarboxylase (GAD) is responsible for the conversion of L-glutamate to GABA. The human brain has two isoforms of this enzyme, GAD65 and GAD67 that differ in molecular weight, amino acid sequence, antigenicity, cellular location and interaction by factor of pyridoxal phosphate. The purpose of this study was cloning of gene encoding the human glutamate decarboxylase. Total cellular RNA was extracted from human brain tissue and then converted to cDNA. PCR was performed using exclusive primers for gad gene amplification. After purification of PCR product, it was partially cloned successfully in pJET1.2 blunt t-vector and was sent for sequencing. The outcomes indicate that only gad gene was cloned partially. The length of human gad gene isoform 65 is 1759 base pair that encodes 585 amino acids. The length of partially cloned gad gene in this study was 385 base pair. Because obtaining fresh human brain is difficult and amount of mRNA is low, it may not be easy to clone full length of human gad gene. The approach described in this paper may be useful in cloning of other genes for which the corresponding mRNA is present at low levels.

This study aimed at investigating the association between alleles and genotypes of APOE and developing Age-related macular degeneration (AMD). After ophthalmological examination, 120 patients with confirmed AMD and 120 healthy controls were enrolled. The polymorphic segment of APOE gene was PCR-amplified and sequenced to determine the frequency distribution of polymorphic alleles and genotypes of this gene in sample population. The frequency distribution of APOE alleles and genotypes differed significantly between the patients and control groups (P<0.05). The frequency of APOEε2 was higher in patients than that of the controls (P = 0.00) and this variant allele showed a significant association with AMD even after removal of the effects of age, sex and smoking in logistic regression analysis (P = 0.00, OR= 3.439; CI 95% 1.664-7.108). On the other hand, the frequency distribution of APOEε4 was not statistically different between patients and healthy controls. The results showed a moderate positive association between APOE ε2 and AMD, but no specific role was found for APOE ε4 in protection against AMD. However, more studies are required to clarify the possible role of APOE in the pathogenesis of AMD.

Genetic polymorphisms of cytochrome p450 in humans are the main cause of differences in the metabolism. The allele and genotype frequencies of CYP2C19 and CYP2C9 have been studied in some Iranian populations. The aim of present study was to examine the frequencies of CYP2C18m1, and CYP2C18m2, alleles in the Mazandarani ethnic group among Iranian Population. In this study, genomic DNA was extracted from leucocytes of one hundred unrelated healthy volunteers. The prevalence of the common variants CYP2C18 m1 and m2 alleles were studied by using high fidelity polymerase chain reaction (HF-PCR) - DHPLC methods. The frequency of CYP2C18 m1 and m2 alleles were 0.0% and 3.0%, respectively. CYP2C18 genotypes wt/wt, wt/m1, wt/m2, m1/m1, m1/m2, and m2/m2 frequencies were 97 %, 0.0 %, 3.0%, 0.0 %, 0.0%, and 0.0%, respectively CONCLUTION: The result of the current study shows that impaired CYP2C18 activity in 03% of our sample population may decrease extra hepatic metabolism of some important drugs such as Phenytoin. It may also affect transdemal delivery of drug substrate for this isoenzyme.

Bachground: Endometriosis is a disease of female genital system, which is defined by the presence of ectopic endometrial tissue outside the uterine cavity. IL-8 is an autocrine growth factor in the endometrium that contributes to the pathogenesis of endometriosis. The aim of this study was to investigate the association between -251T/A polymorphism in the IL-8 gene and endometriosis risk in Iranian population. This case-control study was performed on 100 endometriosis patients and 100 healthy individuals. The IL-8 -251T/A genotypes were determined using PCR-RFLP method. The association between genotypes of the -251T/A polymorphism and the endometriosis risk was examined by use of odds ratios (OR) and 95% of confidence intervals (CIs). No statistically significant associations were observed between IL-8 -251 variants and the risk of endometriosis development (χ2: 1.02, P: 0.63). In addition, subgroup analysis according to severity of disease, were unable to identify any association between IL-8 -251T/A polymorphism and endometriosis (P>0.05), and there was no significant association between IL-8 -251T/A and the severity of endometriosis. This is the first study regard to association of -251T/A polymorphism with endometriosis risk. Our results indicate that the presence of the -251T/A polymorphism in IL-8 gene is not associated with the risk of endometriosis.

MPNs including a heterogeneous group of clonal or oligoclonal hamtopathies characterized by proliferation and accumulation of mature myeloid cells. JAK2 tyrosine kinase mutation is the most common molecular lesion identified in 90% of cases. JAK2 is involved in EPO signaling pathway, and mutations in it lead to EPO-independent spontaneous phosphorylation. Most tyrosine kinase inhibitors (TKI) are small molecules that compete with ATP for binding the ATP-binding site in tyrosine kinase domains, since ATP is a source of phosphate groups used by Tks to phosphorylate the target protein.there are many TKI agent that are studing for treatment of the MPNs with JAK2 tyrosine kinase mutation.the most important TKI drugs including CEP701, CYT387, LY2784544, SB1518, TG101348, XL019, INCB18424. Most important mechanism of them are reduse the splenomegaly, improvement of constitutional symptoms(improvement of bone marrow fibrosis and anemia). Although this drugs are useful but they have some side effect that common of them including Gastrointestinal disease (GI), diarrhea, nausea and vomiting, anemia, thrombocytopenia, thrombosis, leukocytosis, thrombocytosis, peripheral neuropathy, transient loss of blood pressure and lightheadedness.