Iranian Biomedical Journal
Volume:10 Issue: 4, Oct 2006

K+ channel toxins are essential tools for the first purifications, analysis of subunit structures and brain localization of voltage-gated K+ (Kv) channels. The effects of a lot of toxins on Kv are not fully known. Using whole-cell patch clamping technique the action of a series of toxins on Kv3.4 current in rat liver cells with expressed Kv3.4 channels (RLE) cloned cells was investigated. The cells were grown in Williams E medium and after 6-8 days, they were suitable for patch clamping. A family of currents was recorded during voltage-clamp steps to various potentials applied from a holding potential of -60 mV to 60-80 mV in 10 mV increments. Upon depolarization, all channels were opened with a sigmoidal time course, reached to the peak within a few 10th of milliseconds and then slowly inactivated. Bath application of tetraethyl ammonium (TEA) or 3, 4-diaminopyridine (DAP) reduced the current dose dependently and inhibited it completely at 3 mM and 25 mM respectively. The Bunodosoma granulifera (BgK) and Heteractis magnifica (HmK) toxins at concentrations up to 30 and 10 mM respectively could not completely inhibit the current. On the hand, toxins such as b-bungarotoxin, corotoxin, novel toxin and dendrotoxins I (DIP) and K (DPK) even in high concentrations (up to 100 mM) had not any significant effect on Kv3.4 current. Comparison of chemical structures of these effective agents with other reported effective toxins such as blood depressing substances (BDS I and II) show no homology between them, but specially the potency of 3, 4-DAP is comparable with these toxins. These results showed that, the Kv3.4 is more sensitive than other K+ channels to 3, 4-DAP. The sensitivity of this channel to the TEA is low (at mM concentration). More investigation is necessary to find more selective and potent inhibitor of Kv3.4 channels. Iran. Biomed.

Mesenchymal stem cells (MSC) are a very promising transplantable stem cell source for a variety of cell replacement therapies. As the main source of MSC is bone marrow (BM), most of studies have been done on BM-derived MSC (BM-MSC). Umbilical cord (UC)-derived MSC (UC-MSC) which are recently introduced, is one of the good alternative source for these cells. The objective of this study was to isolate and characterize UC-MSC from human UC veins and studying of their potential to differentiate into various cell types such as fat, bone, cartilage and neuronal cells. In this way, a cell population was isolated from 20 UC veins using a solution of 0.1% collagenase type IV. After identification of isolated cells by immunocytochemical and flow cytometry methods, these cells were exposured with adipogenic, osteogenic, chondrogenic and neurogenic agents. Resulted differentiated cells were detected using specific staining for each lineage and room temperature (RT)-PCR. Immunophenotypically, this cell population was positive for CD73, CD166, CD105 markers and a-smooth muscle actin and negative for CD31, CD34, CD49d markers, von Willebrand factor and smooth muscle myosin. Exposure of these cells to adipogenic, osteogenic, chondrogenic and neurogenic agents resulted in morphological changes followed by lineage-specific staining for each differentiated cell type. RT-PCR analysis showed that these differentiated cells express fat, bone, cartilage and neuronal markers, respectively. Altogether, these findings indicate that UC-MSC possess morphological, immunophenotypical and cell differentiation capacities similar to BM-MSC and so they can be used more extensively in cell based therapy protocols and in vitro differentiation study models. Iran. Biomed.

Leukemia inhibitory factor (LIF) is a pleiotropic cytokine of interleukin-6 family with a remarkable range of biological actions such as proliferative effects on the granulosa and theca cells. The aim of this study was to evaluate the effects of LIF on the growth and maturation of mouse fresh and vitrified preantral follicles, an in vitro model was developed. The ovaries of 14-day-old mice were vitrified in a mixture of ethylene glycol, ficoll 70 and sucrose in PB1 for 5 min. The preantral follicles were mechanically isolated from vitrified-warmed and non-vitrified ovaries. They were cultured in α-minimum essential medium supplemented with 5% fetal bovine serum, 100 mIU/ml recombinant follicle stimulating hormone, 1% insulin, transferrin and selenium, 20 ng/ml murine recombinant epidermal growth factor and different concentrations of LIF (25, 50, 100 ng/ml) for 12 days. On day 12, ovulation was induced using 1.5 mIU/ml human chorionic gonadotropin. In this study, the follicle diameter, survival rate and maturation rate were assessed. The mean diameter of fresh and vitrified preantral follicles cultured in 50 ng/ml concentration of murine recombinant LIF was significantly higher than that of other concentrations in each group on day 2 (229.42 ± 30.40, 222.55 ± 33.4) (P<0.001) and on day 4 (340.45 ± 61.05, 299.50 ± 65.55), respectively (P<0.01). The survival rates of follicle in fresh and vitrified groups were 80.56% and 77.78, respectively. There was no significant difference between control and treated groups. The percentage of follicles which released metaphase II (MII(oocyte in fresh groups in the presence of 0, 25, 50, 100 ng/ml of LIF was 16%, 14.28%, 40% and 21.05% (P<0.01) and so in vitrified groups were 11.76%, 14.28%, 28.57% and 13.38%, respectively (P<0.05). There were significant differences between 50 ng/ml LIF-treated groups with other concentrations in each group. Therefore, in vitro growth and maturation of mouse preantral follicles were improved in the presence of 50 ng/ml LIF. Iran. Biomed.

A great deal of evidence has indicated that oxidatively modified LDL plays a critical role in the initiation and progression of atherosclerosis. Antioxidants that can prevent LDL oxidation may act as antiatherogens. Copper is a candidate for oxidizing LDL in atherosclerotic lesions. The binding of copper ions to LDL is usually thought to be a prerequisite for LDL oxidation by copper. The aim of this study was to investigate effect of lycopene on copper bound to LDL and also effect of this binding on the susceptibility of LDL to oxidative modification. In this study, LDL was isolated from EDTA-plasma by ultracentrifugation using a single- step discontinuous gradient. Then lycopene was added to LDL and oxidizability of LDL was estimated by thiobarbitoric acid reactive substances (TBARS) after CuSo4 addition. Finally, the effect of lycopene on formation of LDL-copper complex by gel filtration was studied. Our results showed that lycopene (as dose dependently) was suppressed the formation TBARS and LDL-copper complex. The lycopene with concentrations of 10 µM, 50 µM and 100 µM was reduced susceptibility of LDL to oxidative modification approximately by 31, 67 and 71 percent, respectively. Furthermore, the addition of lycopene to the mixture containing LDL and copper before incubation was prevented the formation LDL-copper complex, approximately by 38 percent. The results of this investigation show that lycopene with inhibition of binding of copper to LDL may decrease the susceptibility of LDL oxidation to this ion and thus may have a role in ameliorating atherosclerosis. Iran. Biomed.

The premature termination of high producer clones, which will be killed due to cell proliferation and proteins production antagonism, is one of the basic drawback in recombinant proteins technology. Furtheremore, it is supposed some toxic proteins like interferon which we intended to clone and express, inhibit host cells’ proliferation. So, it is necessary to tightly control IFN-γ production during growing and selecting of highly producing clones. In the present study, we constructed an expression vector, pMPGB43P2(6)K containing the cumate-regulatable expression cassette to high production of human IFN-γ in Chinese Hamster Ovary- Cumate Transactivator (CHO-CTA). The clones were selected in the cumate and without cumate treated medium. Our results showed, induced IFN-γ expression level was about 5 of magnitude higher than the constitive transgenic system. Application of cumate-regulatable expression cassette, which can be switch off and on by cumate, is useful to production of high producer clones and express toxic proteins in animal cells. Iran. Biomed.

Epigallocatechin gallate (EGCG), a major constituent of green tea, has been introduced as a potent free radical scavenger and can effectively reduce free radical-induced lipid peroxidation. Since free radical injury plays an important role in neuronal damage in Parkinson’s disease (PD), this study examined whether EGCG administration would reduce functional asymmetry in an experimental model of PD in male Wistar rats. For this purpose, unilateral intrastriatal 6-hydroxydopamine-lesioned rats were intraperitoneally pretreated with EGCG (40 mg/Kg) 2 hours before surgery and daily (20 mg/Kg) for a period of 2 weeks post-surgery. Apomorphine- and amphetamine-induced rotations were measured pre- and post-surgery after 2 weeks. The results showed that there are 35.1% (P<0.05) and 33.2% (P<0.05) reductions in controversies apomorphine- and ipsiversive amphetamine-induced rotations in EGCG-treated-lesioned group respectively as compared to the untreated lesioned group at 2nd week post-surgery. Taken together, these results showed that two-week administration of EGCG could attenuate the drug-induced behavioral abnormalities in this model of PD. Iran. Biomed.

The monoclonal antibody, C595, against breast cancer cell line was conjugated with cyclic anhydride gadolinium-diethylenetriaminepenta-acetic acid (Gd-cDTPAa) to produce Gd-DTPA-C595 and used as specific breast cancer cell line (MCF-7) contrast agents in magnetic resonance imaging (MRI). After incubation of breast cancer cell line (MCF-7), with different contrast agents (Gd-DTPA-C595, Gd-DTPA, Gd-H and GdCl3) at 37°C for 12 h and twice washing, the T1 relaxation times and the signal enhancement of washing solutions of different contrast agents are examined by nuclear magnetic resonance and results are compared. The percent of Gd that attached into the cell membrane of MCF-7 was also measured by UV spectrophotometer. The data indicate that the T1 relaxation of washing solutions at 11.4 Tesla (500 MHz) in Gd-DTPA-C595 was greater than in Gd-DTPA solutions and was much greater than in control. These conjugates (Gd-DTPA-C595) show high specificity for breast cancer cell line (MCF-7). The gadolinium concentration in washing solutions measured using UV-spectrophotometer showed no gadolinium attached into the cell membrane in the GdCl3 as control. Good cell membrane uptakes of Gd‑DTPA-C595 indicate selective delivery of this agent into the breast cancer cell membrane and have considerable potential in diagnostic MRI. Iran. Biomed.

engineering new bone tissue with cells and a synthetic extracellular matrix represents a new approach for the regeneration of mineralized tissues compared with the transplantation of bone (autografts or allografts). in this study, to mimic the mineral and organic component of natural bone, hydroxapatite (HA) and gelatin (GEL) composite scaffolds were prepared. The raw materials were first compounded and the resulting composite were molded into cylindrical shape. Using solvent-casting method combined with freeze drying process, it is possible to produce scaffolds with mechanical and structural properties close to natural trabecular bone. Glutaraldehyde (GA) was used as cross linking agent. The chemical bonding and the microstructure were investigated by Fourier Transform Infra Red (FT-IR), Scanning Electron Microscopy (SEM) and Light microscopy. it was observed that the prepared scaffold has an open, interconnected porous structure with a pore size of 80-400, which is suitable for osteoblast cell proliferation. The mechanical properties of different weight fraction of HA (30, 40, and 50 wt%) was assessed and it was found that the GEL/HA with ratio of 50wt% HA has the compressive modulus of ~10 Giga Pascal (GPa), the ultimate compressive stress of ~32 Mega Pascal (MPa) and the elongation of ~3MPa similar to that of trabecular bone. The porosity and the apparent density of 50wt% HA scaffold were calculated and it was found that the addition of HA content can reduce the water absorption and the porosity. Since GA is cytotoxin, sodium bisulfite was used as GA discharger. The biological responses of scaffolds carried out by L929 fibroblast cell culture and it was observed that fibroblast cells partially proliferated and covered scaffold surface, 48h after seeding. these results demonstrate that the manufactured scaffolds are suitable candidate for trabecular bone tissue engineering. Iran. Biomed.