[Cytotoxicity evaluation of taverniera spartea on human cancer cell lines]

For centuries, plants have been a major source for drug discovery. Some examples of anticancer agents developed from plants are the vinblastine, vincristine, taxol and camptothecin. Breast cancer is one of the most commonly diagnosed cancers among women and prostate cancer remains a considerable health problem for men around the world. The purpose of this study was cytotoxicity evaluation of Taverniera spartea on human cancer cell lines. Methods: In the present study, we determined the cytotoxic effects of total methanol extracts and their fractions of Taverniera spartea on MCF-7 and BT-474 human breast cancer cells and also PC-3 and Du-145 prostate cancer cells. Cytotoxicity was evaluated by MTT assay and flow cytometry analysis. The chloroform fraction of Taverniera spartea showed the highest toxicity MTT assay. The IC50 value of this fraction was 70.69 mg/ml for MCF-7 breast cancer cell line after 48 h of exposure. Chloroform fraction showed necrotic effects on MCF-7, BT-474 and PC-3 in contrast apopthotic induction on Du-145 in flow cytometry analysis Taverniera spartea has cytotoxic effects. Further investigation is needed to determine chemical characterization of the active principles and the molecular mechanisms mediated anticancer activities of Taverniera spartea

Production and characterization of anti-Her 2 monoclonal antibodies

Breast cancer is the most common cancer among women in the world. Early diagnosis of this cancer is a key element for its treatment. One of the approaches for diagnosis of breast cancer is detection of its tumour-associated markers. Hence, Her2 has been the main focus of the researches in the field. For diagnosis of Her2 overexpression, monoclonal antibodies [mAb] reacting against Her2 were produced in this study. For this purpose, two peptides from extracellular domain of Her2 were selected and the mAbs reacting against them were produced by hybrodoma technology. Reactivity of these antibodies were then evaluated in different immunological assays including ELISA, Immunoflurescence [IF], western blot [WB] and immunoprecipitation [IP]. Total of 5 clones were produced from two separate fusions, and antibody isotyping revealed that all clones were IgM. These mAbs showed appropriate reactivities in the following assays: ELISA, immunofluresence by staining of breast cancer cell line [SKBR3], WB and IP by detecting the 185 KD band of Her2. In conclusion, it seems that the mAbs are useful diagnostic tools for detection of Her2 expression in patients with breast cancer

Intrafollicular fluid antigamete antibodies in infertile patient candidates for ICSI

Immunologic disturbances must be considered as a major cause of infertility. Antigamete antibodies like antisperm antibodies [ASA] and to anti-zona antibodies [AZA] seem to be implicated in the etiology of infertility. These antibodies affect fertilization and embryo development. It is important to screen these antibodies in infertile women who are candidates for in-vitro fertilization [IVF], because the presence of these antibodies may switch the treatment from IVF to intra-cytoplasmic microinjection [ICSI]. The objective of this study was to determine the presence of ASA and AZA in the follicular fluids [FF] of women who sought candidacy for ICSI. In this prospective study, the follicular fluids of 96 infertile women [20 to 39 years old, mean 31.5 +/- 5.1], who were candidates for ICSI, were evaluated. According to the etiologies, 80 women had explained whereas 16 had unexplained infertility. All the follicular fluids were evaluated for the presence of ASA by ELISA and Sperm MAR test and also for the presence of AZA by ELISA. The data were analyzed by Chi-square test using SPSS soft-ware and the significance level was considered p<0.05. According to the results of ELISA and Sperm MAR test, none of the patients had ASA in their follicular fluids. However, twenty samples [20.8%] were positive for AZA. In patients with unexplained infertility, autoantibodies to zona pellucida were significantly higher in the follicular fluid than the group with proven etiologies for infertility [p=0.001]. The low incidence of ASA and the high incidence of AZA in the infertile women in this study, especially in women with unexplained infertility in Iran have to be considered seriously. Determination of AZA is highly recommended in the evaluation of infertile couples, especially those with unexplained infertility

[Extraction and purification of ferritin from liver tissue]

Ferritin with molecular weight of 450 kDa is the most important iron storage protein and is made of 24 subunits consisting of light and heavy chains. Each ferritin molecule is able to store 4500 Fe[3+] molecules. The aim of this study was to determine the preparation of highly pure ferritin for usage in diagnostic and research systems. In this study, ferritin was extracted and purified by homogenizing liver tissue, heating at 75 degrees centigrade, ammonium sulfate fractionation and gel filtration chromatography on sephadex G-200 column. The purify of ferritin was improved by using recycling chromatography. Resulted protein was electrophoresed on polyacrylamide gel in the presence of sodium dodecylsulfate [SDS-PAGE]. Existence of ferritin was confirmed by ELISA test and potassium ferricyanide staining of gel. Silver nitrate staining of gel was used to confirm the purity of ferritin. Electrophoresis of ferritin under reducing conditions in presence of 2- mercapto ethanol was done to show the subunits [19 and 21 kDa] of ferritin. This purification method resulted in very pure ferritin and the yield was 100 microg/gr of wet liver tissue. Electrophoresis of ferritin under reducing conditions in presence of 2- mercapto ethanol showed the both subunits [19 and 21 kDa] of ferritin. Highly pure ferritin resulted by this method is appropriate for diagnostic and research purpose and the yield is reasonable comparing other studies

[Monoclonal antibody production against human spermatozoal surface antigens]

As monoclonal antibodies are potential tools for characterization of soluble or cellular surface antigens, use of these proteins has always been considered in infertility and reproduction research. Therefore, in this study, monoclonal antibodies against human sperm surface antigens were produced. To produce specific clones against human sperm surface antigens, proteins were extracted using solubilization methods. Balb/c mice were immunized intraperitoneally with the proteins using complete Freund's adjuvant in the first injection and incomplete Adjuvant in the following booster injections. Hybridoma cells producing ASA were cloned by limiting dilution. Five stable ASA producing hybridoma clones were achieved and their antibody isotypes were determined by ELISA. All the isotypes were of IgG class. Their cross reactivity with rat and mice spermatozoa was examined but they did not have any cross reactivity. The produced antibodies can be used in further studies to characterize and evaluate each of the antigens present on human sperm surface and determining their role in fertilization

[Optimization of Dendritic cells purification from mouse spleen a recommended method for purification of Dendritic cells from reproductive organs]

Dendritic cells [DC] are the principal antigen-presenting cells [APC] responsible for induction of primary immune responses by T lymphocytes. Although DCs are present in most lymphoid tissues, they occur in very low frequency accounting for 0.5% or less of nucleated cells in peripheral lymphoid organs. In the present study, we report the purification of DCs from mouse spleen with high yield and purity using a three-step purification technique including: collagenase digestion of tissue, selection of low-density cells using Optiprep density gradient medium and plastic adherence. By using techniques outlined above, we obtained 5-7x10[7] DC/spleen with purity >/= of 97%. Such large numbers of purified DCs enables us to further document their different characteristics including morphology, immunophenotype and to evaluation of their role in immune system. Finally, since DCs have been reported to be present in all reproductive organs, we suggest that this protocol be used for isolation and purification of DCs from those organs for further in vitro studies