effect of sortilin silencing on ovarian carcinoma cells

Our preliminary data on the protein expression of SORT1 in ovarian carcinoma tissues showed that sortilin was overexpressed in ovarian carcinoma patients and cell lines, while non-malignant ovaries expressed comparably lower amount of this protein. In spite of diverse ligands and also different putative functions of sortilin [NTR3], the function of overexpressed sortilin in ovarian carcinoma cells is an intriguing subject of inquiry. The aim of this study was, therefore, to investigate the functional role of sortilin in survival of ovarian carcinoma cell line. Expression of sortilin was knocked down using RNAi technology in the ovarian carcinoma cell line, Caov-4. Silencing of SORT1 expression was assessed using real-time qPCR and Western blot analyses. Apoptosis induction was evaluated using flow cytometry by considering annexin-V FITC binding. [3H]-thymidine incorporation assay was also used to evaluate cell proliferation capacity. Real-time qPCR and Western blot analyses showed that expression of sortilin was reduced by nearly 70-80% in the siRNA transfected cells. Knocking down of sortilin expression resulted in increased apoptosis [27.5 +/- 0.48%] in siRNA-treated ovarian carcinoma cell line. Sortilin silencing led to significant inhibition of proliferation [40.1%] in siRNA-transfected Caov-4 cells as compared to mock control-transfected counterpart [p<0.05]. As it was suspected from overexpression of sortilin in ovarian tumor cells, a cell survival role for sortilin can be deduced from these results. In conclusion, the potency of apoptosis induction via silencing of sortilin expression in tumor cells may introduce sortilin as a potential candidate for developing a novel targeted therapy in patients with ovarian carcinoma

Evaluation of the adequacy of general anesthesia in cesarean section by bispectral index

Background: Awareness and recall, though not common, are the major hazards of general anesthesia, especially in Cesarean section [C/S] because of the absence of benzodiazepine and opioids for a significant time during anesthesia. In this study, the Bispectral Index [BIS], end-tidal isoflurane, and hemodynamic parameters were examined to evaluate the depth of the routine general anesthetic technique in C/S

Methods: This study was carried out on 60 parturient patients undergoing elective C/S. A standardized anesthetic technique was applied: induction with Thiopental [4-5 mg/kg] and Succinylcholine [1.5-2 m g/kg] a s w ell a s m aintenance w ith O2, N2O, and isoflurane. Electrocardiogram, heart rate, blood pressure, Spo2, end-tidal isoflurane concentration, BIS, and any clinical signs of inadequate depth of anesthesia such as movement, sweating, lacrimation, coughing, and jerking were continuously monitored and recorded at 16 fixed time points during anesthesia

Results: A median BIS of less than 70 [range: 42-68] was obtained on all occasions during surgery; however, at each milestone, at least 20% of the patients had BIS values above 60. Hemodynamic parameters increased significantly in some patients, especially during laryngoscopy and intubation. No patient experienced recall or awareness

Conclusion: The currently used general anesthetic technique in our center appears inadequate in some milestones to reliably produce BIS values less than 60, which are associated with lower risk of awareness. Therefore, with respect to such desirable outcomes as good Apgar and clinical status in neonates, we would recommend the application of this method [if confirmed by further studies] through larger dosages of anesthetic agents

Construction and stable expression of a truncated human receptor tyrosine kinase ror1 [Ror1-ECD]

Expression of receptor tyrosine kinase Ror1 in a wide variety of cancers has emerged as a new era focusing on targeting this receptor in cancer therapy. Our preliminary results indicate the presence of a truncated transcript of Ror1 in tumor cells. The truncated Ror1 encompasses extracellular and transmembrane domains, lacking catalytic kinase domain [Ror1-ECD]. As enzyme activity is highly dependent on the catalytic domain, we were wondering how this transcript and its encoded protein could play a possible role in tumorigenesis. To understand the function of this truncated transcript and whether or not the encoded protein translocates to the cell surface, we construct-ed a mammalian expression vector containing exon 1 to exon 8 of human Ror1 gene as a model system. The encoded protein by this construct covers the entire extracellular and transmembrane domains of Ror1. The Chinese Hamster Ovary Cell line [CHO] was used for transfection. Our results showed that this construct could express Ror1-ECD at protein level and also the protein could effectively translocate to the surface of transfected cells. Such model may suggest that a proportion of Ror1 molecules ex-pressed by tumor cells are not full-length Ror1. This notion may be considered when applying flow cytometry using antibodies against Ror1 for screening of tumor cells in order to avoid any miscalculation in the number of Ror1 molecules expressed by tumor cells. Furthermore, such expression may bring about assumptions on functional roles of Ror1-ECD in tumorigenesis, which requires extensive functional studies

Combination of thrombophilic gene polymorphisms as a cause of increased the risk of recurrent pregnancy loss

Recurrent pregnancy loss is [RPL] a heterogeneous condition. While the role of acquired thrombophilia has been accepted as an etiology for RPL, the contribution of specific inherited thrombophilic gene polymorphisms to the disorder has been remained controversial. One hundred women with a history of two or more consecutive abortions and 100 women with at least two live births and no miscarriages were included in the study and evaluated for the presence of 11 thrombophilic gene polymorphisms [Factor V LEIDEN, Factor V 4070 A/G, Factor V 5279 A/G, Factor XIII 103 G/T, Factor XIII 614 A/T, Factor XIII 1694 C/T, PAI-1-675 4G/5G, ITGB3 1565 T/C, ?-Fibrinogen-455G/A, MTHFR 677 C/T, MTHFR 1298 A/C] using PCR-RFLP technique. The data were statistically analyzed using Mann-Whitney test and logistic regression model. There was no relation between factor XIII 103G/T gene polymorphism with increased risk of RPL. However, the other 10 gene polymorphisms were found to be associated with increased/decreased risk of RPL. Multiple logistic regression model for analyzing the simultaneous effects of these polymorphisms on the risk of RPL showed that six of these 11 polymorphisms [Factor V 1691G/A, Factor V 5279A/G, Factor XIII 614A/T, ?-Fibrinogen-455G/A, ITGB3 1565T/C, and MTHFR 1298A/C] were associated with RPL. It is possible to calculate the risk of abortion in a patient with RPL by determining only six of the 10 polymorphisms that are individually associated with RPL

Comparison of the photobleaching and photostability traits of alexa fluor 568- and fluorescein isothiocyanate- conjugated antibody

Synthetic fluorescent dyes that are conjugated to antibodies are useful tools to probe molecules. Based on dye chemical structures, their photobleaching and photostability indices are quite diverse. It is generally believed that among different fluorescent dyes, Alexa Fluor family has greater photostability than traditional dyes like fluorescein isothiocyanate [FITC] and Cy5. Alexa Fluor 568 is a member of Alexa Fluor family presumed to have superior photostability and photobleahing profiles than FITC. In this experimental study, we conjugated Alexa Fluor 568 and FITC dyes to a mouse anti-human nestin monoclonal antibody [ANM] to acquire their photobleaching profiles and photostability indices. Then, the fluorophore/antibody ratios were calculated using a spectrophotometer. The photobleaching profiles and photostability indices of conjugated antibodies were subsequently studied by immunocytochemistry [ICC]. Samples were continuously illuminated and digital images acquired under a fluorescent microscope. Data were processed by ImageJ software. Alexa Fluor 568 has a brighter fluorescence and higher photostability than FITC. Alexa Fluor 568 is a capable dye to use in photostaining techniques and it has a longer photostability when compared to FITC

Optimization of gene transfection in murine myeloma cell lines using different transfection reagents

Purification and isolation of cellular target proteins for monoclonal antibody [MAb] production is a difficult and time-consuming process. Immunization of mice with murine cell lines stably transfected with genes coding for xenogenic target molecules is an alternative method for mouse immunization and MAb production. Here we present data on transfection efficiency of some commercial reagents used for transfection of murine myeloma cell lines. Little is known about transfectability of murine myeloma cell lines by different transfection reagents. Mouse myeloma cell lines [SP[2]/O, NSO, NS1, Ag8, and P3U1] were transfected with pEGFP-N1 vector using Lipofectamine 2000, jetPEI and LyoVec commercial transfection reagents in different combinations. The transfection permissible HEK293-FT cell line was used as a control in transfection procedure. Transfected cells, expressing the Enhanced Green Fluorescent Protein [EGFP], were analyzed by flow cytometry 48 hrs post transfection. Our results showed transfection efficiency of 71%, 57% and 22% for HEK293-FT, 5.5%, 3.4% and 1% for SP[2]/O, 55.7%, 21.1% and 9.3% for NSO, 8.2%, 6% and 5.5% for NS1, 22%, 49.2% and 5.5% for Ag8 and 6.3%, 21.5% and 4.6% for P3U1 cell lines after transfection with Lipofectamine 2000, jetPEI and LyoVec reagents, respectively. Our data indicate that NSO and Ag8 are efficiently transfected by Lipofectamine 2000 and jetPEI reagents. Finally, we propose Ag8 and NSO cell lines as suitable host cells for efficient expression of target genes which can be used for mouse immunization and MAb production

Production of monoclonal antibody against human nestin

We have employed a peptid-based antibody generation protocol for producing antibody against human nestin. Using a 12-mer synthetic peptide from repetitive region of human nestin protein devoid of any N-or O-glycosylation sequences, we generated a mouse monoclonal antibody capable of recognizing human, mouse, bovine, and rat nestin. A wide variety of nestin proteins ranging from 140-250 kDa was detected by this antibody. This antibody is highly specific and functional in applications such as ELISA, flow cytometry, immunocytochemistry, and Western blot assays

Producing recombinant mTEX101; a murine testis specific protein

Production of antibodies against specific proteins of testis germ cells is of great significance for the investigation of processes involved in spermatogenesis, study of infertility problems and determination of the probable role of these proteins as cancer-testis antigens. Murine Testis Specific Recombinant Protein 101 [mTEX101] is a 38kDa, GPI-anchored protein which is expressed in testis germ cells of adult mice but it seems to be absent in other tissues. The structure and function of mTEX101 is not completely understood yet, but it is speculated that it may transduce biochemical signals into the cytoplasm since mTEX101 does not have an intracellular domain but the precise mechanisms are still ambiguous. RNA was extracted from three adult mice testis. The RNA was used in RT-PCR, employing a pair of specific primers for mTEX101 ORF region. TA-cloning technique was performed by the insertion of mTEX101 into a pGEM-T Easy Vector, followed by its subcloning into a His-tagged expression vector, pET-28a [+]. The recombinant mTEX101 was then produced by transfection of the expression vector into BL 21 [DE3] E. coli strain. A recombinant protein, weighing 27kDa, was produced upon IPTG-induction of the bacterial host. The presence of mTEX101 protein was detected through Western blot analysis by anti-mTEX101 peptide antibodies. We produced mTEX101 recombinant protein that could be used for the production of mono and polyclonal antibodies