Using several complimentary strategies, BCFMT was found to inhibit cancer cell proliferation at G2/M phase of the cell cycle apparently by targeting microtubules. In addition, BCFMT strongly suppressed the dynamics of individual microtubules in live MCF-7 cells. At its half maximal proliferation inhibitory concentration (10 µM), BCFMT reduced the rates of growing and shortening phases of microtubules in MCF-7 cells by 37 and 40%, respectively. Further, it increased the time microtubules spent in the pause (neither growing nor shortening detectably) state by 135% and reduced the dynamicity (dimer exchange per unit time) of microtubules by 70%. In vitro, BCFMT bound to tubulin with a dissociation constant of 8.3±1.8 µM, inhibited tubulin assembly and suppressed GTPase activity of microtubules. BCFMT competitively inhibited the binding of BODIPY FL-vinblastine to tubulin with an inhibitory concentration (Ki) of 5.2±1.5 µM suggesting that it binds to tubulin at the vinblastine site. In cultured cells, BCFMT-treatment depolymerized interphase microtubules, perturbed the spindle organization and accumulated checkpoint proteins (BubR1 and Mad2) at the kinetochores. BCFMT-treated MCF-7 cells showed enhanced nuclear accumulation of p53 and its downstream p21, which consequently activated apoptosis in these cells. The results suggested that BCFMT inhibits proliferation of several types of cancer cells including drug resistance cells by suppressing microtubule dynamics and indicated that the compound may have chemotherapeutic potential.Using cell based screening assay, we identified a novel anti-tubulin agent (Z)-5-((5-(4-bromo-3-chlorophenyl)furan-2-yl)methylene)-2-thioxothiazolidin-4-one (BCFMT) that inhibited proliferation of human cervical carcinoma (HeLa) (IC The development of resistance to the existing anticancer drugs and tumor metastasis are among the major obstacles in cancer chemotherapy Microtubules are structural components of the mitotic spindle and the dynamic microtubules play an important role in several cellular processes including intracellular trafficking, cell migration and cell division Rhodanine derived compounds are acquiring attention in chemotherapy because of the presence of heterocyclic ring (2-thioxothiazolidin-4-one) in the parent scaffolds in vitro and in cultured cells while MNFMT and DHBPT did not show any significant effect on tubulin assembly. We obtained several lines of evidence suggesting that BCFMT exerts its antiproliferative and antimitotic activities by dampening dynamic instability of individual microtubules in cultured cells through binding at the vinblastine site in tubulin. In addition, BCFMT potently inhibited the proliferation of drug resistant namely cisplatin resistant human ovarian carcinoma A2780-cis and multi-drug resistant mouse mammary tumor EMT6/AR1 cells and highly metastatic MDA-MB-231 cells suggesting that it may have chemotherapeutic potential.We found that three compounds namely (E)-5-((5-(2-methyl-5-nitrophenyl) furan-2-yl)methylene)-2-thioxothiazolidin-4-one (MNFMT), (E)-5--3-phenyl-2-thioxothiazolidin-4-one (DHBPT) and (Z)-5-((5-(4-bromo-3-chlorophenyl) furan-2-yl) methylene)-2-thioxothiazolidin-4-one (BCFMT) inhibited the proliferation of HeLa and MCF-7 cells in culture. Among these compounds, BCFMT was found to increase the mitotic cell population of HeLa and MCF-7 cells more potently than the other two compounds. BCFMT inhibited the assembly of purified tubulin Sulforhodamine B, bovine serum albumin, mouse anti-α-tubulin IgG, mouse anti-β-actin IgG, FITC conjugated anti-rabbit IgG were obtained from Sigma, St. Louis, MO, USA. Alexa fluor 568 goat anti-mouse IgG was purchased from Molecular Probes, Invitrogen, CA, USA. Mouse anti-cyclin B1, rabbit anti-p-Histone H3 (Ser 10), mouse anti-p53 IgG, mouse anti-p21 IgG antibodies and apoptosis detection Kit (Annexin V-Propidium Iodide) were purchased from Santa Cruz Biotechnology, CA, USA. Mouse anti-BubR1 IgG was obtained from BD Biosciences, CA, USA. Rabbit anti-Mad2 IgG was purchased from Bethyl laboratories, Montgomery, USA. Mouse anti-Hec1 IgG was purchased from Abcam, Cambridge, MA, USA. Fetal bovine serum was obtained from Biowest, Nuaille, France. All other reagents were of analytical grade and obtained from Sigma, MO, USA and Himedia, Mumbai, India. All compounds tested were obtained from Chembridge Corporation, San Diego, CA, USA.Human cervical carcinoma (HeLa), human breast adenocarcinoma (MCF-7) and metastatic breast adenocarcinoma (MDA-MB-231) cells were obtained from cell repository of National Centre for Cell Science, (NCCS) Pune, India. NCCS characterized the cells by mt-rDNA sequence to confirm the species. These cell lines were found to be free of mycoplasma. Cisplatin-resistant human ovarian carcinoma (A2780-cis) cells and multi-drug resistant mouse mammary tumor (EMT6/AR1) cells were purchased from Sigma, St. Louis, MO, USA. Cell line authentication was done by short tandem repeat profiling and isoenzyme analysis by the supplier and was also reported negative for the presence of mycoplasma.2 and 95% air.HeLa and MCF-7 cells were cultured in Eagle’s Minimal Essential Medium (MEM). MDA-MB-231 cells were grown in Leibovitz's L-15 Medium. A2780-cis cells were maintained in RPMI-1640 media containing 1 µM cisplatin. EMT6/AR1 cells were grown in MEM medium containing 1 µg/ml doxorubicin. Media were supplemented with 10% fetal bovine serum, 2.2 g/l sodium bicarbonate and 1% antibiotic-antimycotic solution containing streptomycin, amphotericin B and penicillin. Cells were grown and maintained at 37°C incubator in humidified atmosphere of 5% CO×105 cells/ml) were seeded in 96-well cell culture plates. Stocks of compounds were prepared in DMSO. After 24 h of seeding, the media was replaced with fresh media containing either vehicle (0.1% DMSO) or 2 µM of each of the rhodanine compounds. After 24 h of incubation with different compounds, cells were fixed with 10% TCA and processed for sulforhodamine B assay The antiproliferative potential of 156 rhodanine derived compounds against HeLa cells was determined by sulforhodamine B assay 50) of MNFMT, DHBPT and BCFMT, 1×105 cells/ml HeLa and MCF-7 cells were seeded in 96 well cell culture plates. Different concentrations of compounds were diluted in media and added in the wells after 24 h of cell seeding. HeLa and MCF-7 cells were grown in the absence and presence of compounds for 24 h and 48 h, respectively. Inhibition of cell proliferation in the presence of compounds was determined using standard sulforhodamine B assay. Data were an average of three independent experiments.To determine the half maximal inhibitory concentration  and 1 M glutamate was incubated without and with different concentrations of MNFMT, DHBPT or BCFMT on ice for 10 min. Then, 1 mM GTP was added to the reaction mixtures and the assembly kinetics was monitored at 37°C using a FP-6500 spectrofluorometer JASCO, Tokyo, Japan.The effects of MNFMT, DHBPT or BCFMT on the assembly of purified tubulin were monitored by light scattering at 400 nm. Tubulin was purified as described previously 212, FEI, Eindhoven, Netherlands).Tubulin (10 µM) was polymerized without or with 25 and 50 µM BCFMT at 37°C for 15 min as described above. The samples were fixed with 0.5% glutaraldehyde, transferred to carbon-formvar coated grids  and negatively stained with 2% uranyl acetate. Samples were visualized under electron microscope (Tecnai G2 and 1 mM GTP was polymerized at 37°C for 30 min. Microtubule seeds were generated by shearing the polymers through a 23 gauge needle of a 5 ml syringe. Tubulin (15 µM) in PEM buffer and 1 mM GTP was polymerized by adding 20% (v/v) microtubule seeds at 37°C for 10 min. After 10 min of polymerization, different concentrations of BCFMT were added into the reaction mixtures and further polymerized for 30 min. The hydrolysis reaction was stopped by adding 10% (v/v) of 7 M perchloric acid and the amount of inorganic phosphate released was determined by malachite green assay Tubulin (25 µM) in 4 M glycerol, 5 mM MgClTubulin (2 µM) in 25 mM PIPES buffer pH 6.8 was incubated without and with different concentrations of BCFMT at 25°C for 20 min. The fluorescence intensity was monitored by exciting the reaction mixture at 295 nm and the emission spectrum was recorded in the range of 310 nm to 370 nm. A fluorescence cell of 0.3 cm path length was used and the fluorescence intensities were corrected for inner filter effect using the formulamax is the maximum change in the fluorescence intensity of tubulin when it is saturated with BCFMT and C is the concentration of BCFMT. The dissociation constant (Kd) for BCFMT binding to tubulin was estimated using the Graph Pad Prism 5 software .Fluorescence data were fitted in the following equation;Tubulin (2 µM) in 25 mM PIPES buffer pH 6.8 was incubated without and with 10, 25 and 50 µM BCFMT for 20 min at 25°C. BODIPY FL-vinblastine (2 µM) was added in the reaction mixtures and incubated at 25°C for an additional 20 min in dark. Tubulin-BODIPY FL-vinblastine complex was excited at 490 nm and the emission spectrum was taken in the range of 500–550 nm ×104 cells/ml on polylysine-coated glass coverslip in 24-well cell culture plate. After 24 h of seeding, different concentrations of BCFMT were added in the wells. Control cells were treated with vehicle (0.1% DMSO). After 24 or 48 h of incubation with BCFMT, immunostaining was performed using antibody against α-tubulin, p53, p21, BubR1, cyclin B1, β-actin , phosphohistone–H3 (Ser 10) , Hec 1  and Mad2  as described earlier MCF-7 or HeLa cells were seeded at a density of 5Transfection of EGFP-α tubulin construct in MCF-7 cells was done using lipofectamine-2000 MCF-7 cells were incubated in the absence and presence of 20 and 40 µM of BCFMT for 36 h. The effect on the polymerized amount of microtubules in the cells was determined by western blotting using monoclonal antibody for α-tubulin as described previously MCF-7 cells were blocked in M phase of cell cycle after 24 h of treatment with 1 µM nocodazole. To remove nocodazole, cells were cytospinned and washed carefully three times with fresh media. After nocodazole removal, cells were incubated in the absence and presence of 40 µM BCFMT at 37°C. Cells were fixed at 0, 1, 2 and 4 h of incubation with BCFMT at 37°C incubator. Fixed cells were stained with Hoechst 33258 and the mitotic cells were scored .×104 cells/ml) were seeded on polylysine-coated glass coverslip in a 24-well cell culture plate. After 24 hours of seeding, cells were incubated without and with different concentrations of BCFMT for 48 hours. Cells were collected by cytospinning at 2400 rpm at 30°C for 10 min. Annexin V/PI staining was performed as described earlier MCF-7 cells (550) of 16.8±1, 7.3±0.4, 7.2±1.8 µM, and 12.2±0.3, 4.9±0.3 and 10.0±0.5 µM, respectively.We examined the antiproliferative potential of 156 rhodanine compounds using HeLa cells. Among 156 compounds, three compounds, namely MNFMT, DHBPT and BCFMT , were fo50 concentration of MNFMT, DHBPT and BCFMT, respectively (50 concentration of MNFMT (50 µM), DHBPT (20 µM) and BCFMT (40 µM) the mitotic indices were found to be 4±0.5, 6±1 and 12±1 , respectively. The findings together suggested that BCFMT blocked the cells in mitosis more strongly than the other two agents; therefore, we further explored the antimitotic activity of BCFMT.To determine the antimitotic potential, we checked the effect of these agents on cyclin B1 which is a marker specific for G2/M phase ectively . In the In mitotic cells histone H3 gets phosphorylated at serine 10 Further, BCFMT treatment was found to delay the kinetics of the release of nocodazole- induced mitotic block in MCF-7 cells. For example, 60% of the cells were found to be in mitosis at the time of nocodazole washout, while 30%, 15% and 5% cells were in the mitotic phase after 1, 2 and 4 h release of the nocodazole block. In the presence of 40 µM BCFMT, 46%, 38% and 30% cells were found to be in the mitotic phase after 1, 2 and 4 h of block release suggesting that BCFMT can suppress mitotic progression.in vitro and in cultured cells. BCFMT inhibited the assembly of purified tubulin in a concentration dependent manner  of 5.2±1.5 µM  treated cells showed a significant depolymerization of the microtubules . MicrotuMCF-7 or HeLa cells were incubated with different concentrations of BCFMT for one cell cycle. BCFMT depolymerized interphase microtubules in MCF-7 cells in a concentration dependent manner. For example, the microtubule network was not visibly perturbed in the presence of 10 µM BCFMT while 20 µM BCFMT induced a significant depolymerization of the interphase microtubules and a strong depolymerization of microtubules was observed in the presence of 30 µM BCFMT . WesternMicrotubules were depolymerized by incubating MCF-7 cells on ice for 1 h and subsequently, the growth kinetics of interphase microtubules were monitored by incubating the cells at 37°C. Microtubules of the vehicle-treated cells grew rapidly and attained normal interphase network within 30 min while BCFMT (40 µM) treatment strongly suppressed the growth of microtubules  decreased the rates of growing and shortening of individual microtubules by 37% and 40%, respectively  images of vehicle-treated MCF-7 cells showed well defined morphology while BCFMT-treatment induced apoptotic body formation in the cells. Annexin V/PI staining clearly indicated that BCFMT treatment caused apoptosis in MCF-7 cells .A short exposure of BCFMT also inhibited MCF-7 cells proliferation. MCF-7 cells were incubated in the absence and presence of 50 µM BCFMT or with 500 nM nocodazole for 5 h. After the incubation, BCFMT or nocodazole containing media was removed; cells were washed with fresh media and were further incubated in fresh media for 40 h. The short exposure of 50 µM BCFMT inhibited MCF-7 cell proliferation by 43%, whereas 13% inhibition was observed in the presence of 500 nM nocodazole. Further, Annexin V/PI staining indicated that a brief treatment of BCFMT caused cell death in MCF-7 cells. For example, 1%, 32%, 15% and 35% of MCF-7 cells showed PI staining in the absence and presence of 50 µM BCFMT, 500 nM nocodazole and 100 nM vinblastine, respectively.Microtubule targeting agents are known to induce the apoptosis via activation and nuclear translocation of p53 50 value of 5.8±0.3, 6.5±1 µM and 6±1 µM, respectively , EMT6/AR1 (a multi-drug resistant cell line known to be cross resistant against vincristine and colchicine) and MDA-MB-231 (a highly metastatic breast cancer cell line) cells were determined in culture. BCFMT inhibited the proliferation of A2780-cis, EMT6/AR1 and MDA-MB-231 cells in dose dependent manner with an ICin vitro than that of BCFMT. These three compounds contain 2-thioxothiazolidin-4-one as a common moiety and have substitutions at different positions of the heterocyclic ring  d = 30 µM) d = 30 µM) d = 0.5 µM) d = 47 nM) 50 values of 7.2±1.8 µM, 10.0±0.5 µM, 6±1 µM, 5.8±0.3 µM and 6.5±1.0 µM, respectively. The IC50 values of BCFMT are comparable with that of several other known tubulin targeting agents like estramustine (5 µM) BCFMT bound to tubulin with a KLike several other microtubule targeting anticancer agents In some instances, microtubule targeting agents such as paclitaxel and vinflunine have been found to induce cell death without arresting cells in mitosis BCFMT treatment induced apoptotic death in HeLa and MCF-7 cells. Microtubule targeting agents are shown to induce apoptosis by different pathways The current limitations of the existing anticancer drugs include the development of resistance against them and tumor metastasis In conclusion, we identified a novel anti-tubulin agent BCFMT that inhibits proliferation of several types of cancer cells including drug resistance cells by suppressing microtubule dynamics and the results indicated that the compound may have chemotherapeutic potential.Figure S1BCFMT treatment increased the number of phosphohistone positive MCF-7 cells. MCF-7 cells were incubated without and with different concentrations of BCFMT for 48 h. Cells were fixed and then, immunostained using antibody against phosphohistone. Phosphohistone positive cells were counted. Data were an average of three independent experiments. In each set 1000 cells were counted. Bars represent ± SD.(TIF)Click here for additional data file.Figure S2BCFMT suppressed the mitotic progression in HeLa cells. HeLa cells were incubated without and with different concentrations of BCFMT for 24 h. (A) Cells were stained with Hoechst 33258 and mitotic indices were counted in the absence and presence of different concentrations of BCFMT. (B) Cells were fixed and then, immunostained using antibody against phosphohistone. Phosphohistone positive cells were visually counted. (C) Metaphase/anaphase ratio was calculated in the absence and presence of 10 and 20 µM of BCFMT. Data were an average of three independent experiments. In each set 1000 cells were counted. Bars represent ± SD.(TIF)Click here for additional data file.Figure S3BCFMT did not inhibit the binding of colchicine to tubulin. Tubulin (5 µM) in 25 mM PIPES buffer pH 6.8 was incubated without (♦) and with 10 (▪) and 20 (▴) µM of BCFMT for 20 min at 25°C. Colchicine (10 µM) was added to the reaction milieu and samples were incubated for an additional 1 h at 37°C. Fluorescence of tubulin-colchicine complex was examined by exciting the samples at 350 nm and emission was recorded in the range of 390–470 nm.(TIF)Click here for additional data file.Figure S4BCFMT reduced the polarization of tubulin-BODIPY FL-vinblastine complex. Tubulin (4 µM) in 25 mM PIPES buffer (pH 6.8) was incubated without and with different concentrations  of BCFMT at 25°C for 20 min. BODIPY FL-vinblastine (2 µM) was added in the reaction mixtures and incubated at 25 °C for an additional 20 min in dark. Polarization of tubulin-BODIPY FL-vinblastine complex at 515 nm in the absence and presence of BCFMT was measured using JASCO FDP 200/210 polarization accessories in a FP-6500 spectrofluorometer. Polarization was calculated using the equation P = (Ivv–GIvh)/(Ivv + GIvh) h and v represent horizontal and vertical positioning of the excitation and emission polarizer, respectively. G is the correction factor for the transmission efficiency and calculated by Ihv/Ihh. Data were an average of three independent experiments. Bars represent ± SD.(TIF)Click here for additional data file.Figure S5BCFMT depolymerized interphase microtubules in HeLa cells. HeLa cells were incubated without and with different concentrations of BCFMT for 24 h. Cells were fixed and then, immunostained using antibody against α-tubulin (red). DNA stained in blue. Scale bar is 10 µm.(TIF)Click here for additional data file.Figure S6BCFMT did not perturb the organization of actin network in MCF-7 cells. MCF-7 cells were incubated without and with 15 and 30 µM of BCFMT for 40 h. Cells were fixed and immunostaining was performed using antibody against β-actin (red). Scale bar is 10 µm.(TIF)Click here for additional data file.Figure S7BCFMT suppressed the growth of interphase and mitotic microtubule. (A) MCF-7 cells (8×104 cells/ml) were incubated without and with 40 µM BCFMT on ice for 1 h. Subsequently, cells were incubated in a CO2 incubator at 37°C for different durations and fixed with 3.7% formaldehyde. Upper panel and lower panel show the kinetics of microtubule growth in control and 40 µM BCFMT treated MCF-7 cells, respectively. (B) MCF-7 cells were synchronized in mitosis by incubating the cells with 1 µM nocodazole for 24 h. Nocodazole containing media was removed; cells were carefully washed 4 times with fresh media and further incubated on ice for 30 min without and with 40 µM BCFMT. Cells were incubated for different durations at 37°C and fixed with 3.7% formaldehyde. Upper panel and lower panel show the assembly kinetics of spindle microtubules in the absence and presence of 40 µM BCFMT, respectively. Scale bar is 10 µm.(TIF)Click here for additional data file.Figure S8BCFMT inhibited the proliferation of cisplatin-resistant A2780-cis, multi-drug resistant EMT6/AR1 and MDA-MB-231 cells. Cells were incubated without and with different concentrations of BCFMT for 24 hours. The inhibitory effect of BCFMT on the proliferation of A2780-cis (▴), EMT6/AR1 (•) and MDA-MB-231 (□) cells was determined by sulforhodamine B assay. Data were an average of three independent experiments. The bars represent ± SD.(TIF)Click here for additional data file.


Dear Editor,2O3) is a standard medication for relapsed acute promyelocytic leukemia (APL). However, high blood arsenic levels lead to potentially fatal arrhythmias. As2O3 is renal-excreted and considered contraindicated in renal failure.Arsenic trioxide (As2/day) and oral As2O3 (10 mg/day) [2O3 to 5 mg/day. On the third day, the leukocyte count increased to 8.9 × 109/L, associated with bilateral pulmonary infiltrates. Features were consistent with the APL differentiation syndrome, which with the underlying septicemia led to anuric acute renal failure. ATRA was stopped. Idarubicin (6 mg/m2/day × 5), dexamethasone (12 mg/day × 7), and alternate daily hemodialysis were administered. Oral As2O3 at 5 mg was given after each hemodialysis and was stopped after 9 days. At 4 weeks, marrow examination confirmed complete remission. He remained anuric, and hemodialysis was continued. Oral As2O3 was re-commenced at 2 mg after each hemodialysis. Two months later, continuous ambulatory peritoneal dialysis (CAPD) was started. A maintenance regimen for APL, comprising oral As2O3 (5 mg/day) and ATRA (20 mg twice daily) [PML-RARA fusion gene characteristic of APL. No cardiac arrhythmias were observed at any time.A 42-year-old man was referred for treatment of APL in first relapse, presenting with pancytopenia, impaired renal function, and septicemia. He was treated with all-trans retinoic acid  As2O3, used in most reports, may lead to lethal arrhythmias due to QT prolongation, which is directly proportional to blood arsenic levels [2O3 leads to a rapid surge of blood arsenic levels, arrhythmia related to QT prolongation is an important adverse effect. Oral As2O3 is slowly absorbed and results in lower blood arsenic levels. Its bioavailability, estimated by area-under-the-curve pharmacokinetically, is comparable to that of i.v. As2O3 [2O3 has the same efficacy, but little risk of arrhythmia [2O3 were measured in an APL patient on hemodialysis [2O3 therapy. It was concluded that i.v. As2O3 was contraindicated in renal failure [2O3 and institution of hemodialysis prevented further increases in arsenic levels. Subsequently, with careful dose adjustment and meticulous monitoring, oral As2O3 was safely administered during hemodialysis. The blood arsenic levels were also kept within the therapeutic range. We have also previously demonstrated the safety of oral As2O3 during CAPD. Therefore, patients on renal replacement therapy are still eligible for oral As2O3 treatment.Asc levels . Becausev. As2O3 . Hence, rhythmia . In one dialysis . The pea failure . In our


Although bicuspid aortic valve (BAV) is the commonest congenital cardiac defect, longitudinal studies investigating the natural history of its associated aortic dilatation are scarce. We aim to retrospectively investigate the progression of ascending aortic (Asc Ao) dilatation in BAV patients.A retrospective analysis of serial Asc Ao dimension was made of 31 patients  with BAV who underwent at least 2 cardiovascular magnetic resonance imaging study (CMR) > 4 months apart from January 2002 to September 2012 at the Royal Brompton Hospital. Patients with associated aortic coarctation or aortic valvular dysfunction were excluded. Images were acquired with either a Siemens Avanto or Siemens Sonata scanner. Maximal intraluminal Asc Ao diameters at the level of right pulmonary artery (RPA) were measured in end diastole using the axial single-shot spin-echo (HASTE) sequence. The rate of change in aortic dimension per year was calculated.2) and 47.3±4.4 mm (indexed 24.3±2 mm/m2). The average time between CMR studies was 28.2±19 months (4 to 80.4 months). The annual rate of progression of Asc Ao dilatation was 0.6±0.72 mm/year in group A and 0.5±0.3 4 mm/year in group B, p=0.2.There were 19 BAV patients with baseline Asc Ao dimension of <40 mm (Group A) and 12 BAV patients with ≥40 mm (Group B). The mean baseline Asc Ao diameters in group A was 32±3.4 mm (indexed 17±2.4 mm/mhttp://www.nature.com/clinicalpractice/cardio].The annual progression rate of Asc Ao dilatation in BAV patients with baseline dimension of <40 mm was 0.6mm±0.72 mm/year while those with ≥40 mm was 0.5±0.34 mm/year. The difference in progression between these 2 groups of BAV patients are not statistically significant and are comparable with previous longitudinal studies [Thanassoulis et al., Nat Clin Prac Cardio Med, 2008 V5, No Funding


Phyllosticta sp. TG78. Seeds treated with 20 µg/mL CC at 4°C expressed the higher germination rate (77.78%), potential (23.46%), index (3.44) and the shorter germination time (6.19 d); root growth was also significantly improved by 13.76% in length, 13.44% in fresh weight and 6.88% in dry mass compared to controls. During the cultivation process at 4°C for three days and the followed 24 h at 25°C, lipid peroxidation, expressed by malondialdehyde (MDA) content and relative membrane permeability (RMP) was significantly reduced in CC-treated roots; activities of lipoxygenase (LOX), phospholipid C (PLC) and phospholipid D (PLD) were inhibited by 13.62–62.26%, 13.54–63.93% and 13.90–61.17%, respectively; unsaturation degree of fatty acids was enhanced through detecting the contents of CC-induced linoleic acid, linolenic acid, palmitic acid and stearic acid using GC-MS; capacities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) were individually increased by 7.69–46.06%, 3.37–37.96%, and −7.00–178.07%. These results suggest that increased chilling tolerance may be due, in part, to the reduction of lipid peroxidation and alternation of lipid composition of roots in the presence of CC.Chilling tolerance was increased in seed germination and root growth of wheat seedlings grown in media containing 20 µg/mL cerebroside C (CC), isolated from the endophytic  Low temperature, an important environmental factor, constitutes one of the major yield and quality limitations to cereal productivity et al.Fusarium, Penicillium, Phoma and Trichoderma could be designated as plant growth-promoting fungi and enhance the growth of a variety of crop plants. Many endophytes have been discovered to produce phenolic compounds to counteract ROS that escape the enzymatic antioxidant systems Endophytes are microorganisms that commonly reside within the plant tissues without causing any disease symptoms and have been recognized as a potential source of various novel active secondary metabolites with anticancer, antimicrobial, antiviral, antioxidant and other biological activities Phyllosticta sp. (strain number TG78) was isolated from the stem of Ginkgo biloba which has long been used as a traditional medicine for various ailments in China Phylosticta sp. TG78 increases wheat seeds germination under cold stress. Based on the bioactivity-guided approach, two cerebrosides were ultimately isolated from TG78 for the first time and one of them  was isolated from the healthy sterilized leaf of et al.3-CH3OH gradient (1∶1 v/v) for 24 h at room temperature, and then filtered and evaporated under reduced pressure for three times.The preparation of liquid inoculum and the subsequent solid fermentation of the fungus TG78 were accomplished according to Song n-butanol for three times respectively at room temperature to give a petroleum ether fraction , ethyl acetate fraction  and a n-butanol fraction . Based on the activity-guided approach, the petroleum ether phase precipitated the salts and lipids was chromatographed on silica gel  using a stepwise gradient of CHCl3/CH3OH (100 : 0→0 : 100 v/v) to give five fractions  based on TLC at 254 nm, spraying with 10% H2SO4-EtOH followed by heating at 100°C for 10 min. Fraction A2 was then separated on a Sephadex LH-20 column eluted with CHCl3/CH3OH (1∶1 v/v), followed by recrystallization to yield a metabolite (80 mg), which was established as -1-O-β-D-glucopyranosyl-2-N-(2′-hydroxy-3′- octadecenoyl) -3-hydroxy-9-methyl-4,8-sphingadienine    based onA stock solution of CC at 500 µg/mL in 5% dimethyl sulfoxide (DMSO) was serially diluted up to the desired final concentrations . The vehicle (0.5% DMSO in water) was used as control treatment. All the final DMSO concentration of the diluted solution was kept at 0.5% (v/v) to ensure that DMSO at the concentration did not interfere with the experiments.Triticum aestivum, cv. Huaimai 25), obtained from Huaiyin Institute of Agricultural Sciences, Huai'an, Jiangsu Province, P. R. China, were surface sterilized for 10 min in 5% NaClO solution, rinsed thoroughly and germinated on filter papers with distilled water in Petri dishes for 24 h at 25°C in dark. Thereafter, three replicates of 28 equal-sized and plump germinating seeds were selected to place on new filter paper moistened with CC aqueous solution and sprayed only 0.5% DMSO for control seedlings (CK) for 10 days at 4°C under cold stress treatment. All the seeds were watered daily with appropriate aqueous solution of CC or 0.5% DMSO to keep the filter papers moistened. Data on germinant seeds of different treatments was recorded at 24 h intervals as radical emergence to 3–5 mm and the effects on wheat growth under low temperature were determined by calculating germination rate (GR), germination potential (GP), germination index (GI) and the mean germination time (MGT) 1: the total number of germinated seeds at the tenth day; n2: the total number of germinated seeds at the third day; N: the whole number of tested seeds; Gi: the number of germinated seeds on day Ti; Ti: the corresponding germinant days.Seeds of wheat  Before cold stress, all germinated wheat seeds were cultured in the growth chamber at approximately 25°C for 3 days ensuring sufficient available sample of roots. (2) Cold stress, that is, on the 4th day, the wheat seedlings of control were evenly divided into two groups. One group of CK (CK+4°C) and CC treatments (CC+4°C) were transferred to a temperature of 4°C for another 3 days, while the other seedlings of CK and CC were still kept in the growth chamber as room temperature treatment (CC+25°C) and room temperature control (CK+25°C) for the same time. (3) Recovering room temperature, after chilling stress treatment, the plants at 4°C were recovered to the same conditions of the last three days with those at 25°C. A 12/12 h (day/night) photo-period and a light intensity of 6000 lx were maintained during the nine days. Then some groups grown of the three periods  were measured for the morphological characters and growth parameters of roots and shoots, while the other samples at the time of 0, 6, 12, 24, 48, and 72 h in cold stress for three days, together with those recovering to room temperature for 24 h  were harvested for physiological targets analysis. Three replicates (n = 3) were done for each condition, and commonly 5–10 seedlings were used to provide enough amounts of plant material to measure growth parameters and physiological targets analysis in each replicate.Lipid peroxidation was determined as the amount of malondialdehyde (MDA) based on the spectrophotometer measurement of a red-complex produced during the reaction of thiobarbituric acid (TBA) with MDA as described earlier et al.0) using a conductivity meter . After immersed for 10 h at 15°C, the solution was then assayed for EC1. The same samples containing roots were autoclaved at 100°C for 30 min to determine EC2. Percent RMP was calculated as:The relative membrane permeability (RMP) was determined based on Yang et al.2, and then 5 mL of 50 mM tris-HCl (pH 8) was added, containing 10 mM KCl, 500 mM sucrose and 0.5 mM phenylmethylsulfonylfluoride. The extracts were then homogenised and centrifuged at 12000× g for 10 min at 4°C. One unit of LOX was defined as the amount of enzyme causing an increase in absorption of 0.01 per min at 234 nm and 25°C when linoleic acid is employed as the substrate. PLC and PLD assay was determined according to Munishwar and Finn The enzyme extract of lipoxygenase , phospholpase C  and phospholpase D  were extracted according to the method described by Rui et al.2. Methylation of fatty acids was carried out by adding 5 mL of 0.5 M KOH methanol for 40 min at 50°C under N2. The resultant fatty acid methyl esters (FAME) were extracted twice with high performance liquid chromatography-grade hexane and determined by GC-MS.Fatty acids were extracted, methanol etherified and analyzed according to Hassan GC-MS analyses were performed by an Aligent series 6890 capillary gas chromatography - mass spectrometry (GC-MS) equipped with a J&W DB-1701 MS fused-silica capillary column (30.0 m length×0.25 mm i.d.×0. 25 µm film thickness) under programmed conditions: 140°C for 1 min then rising at 4°C per min to 164°C and then at 15°C per min to 180°C, and finally at 5°C per min to 235°C for 1 min. Helium was used as a carrier gas which flowed at a rate of 1 mL/min. The injection volume was 1 µl, and the temperature of the detector and injector was 280°C and 250°C, respectively. The FAME determination was identified by MS  and valuFor enzyme extracts and assays, fresh roots (0.1 g) were ground in liquid nitrogen, and then suspended in 0.9 mL solution containing 10 mM phosphate buffer (pH 7.4). The homogenate was centrifuged at 4°C, 2500 rpm for 10 min and the resulting supernatant was collected for determination of the activities of superoxide dismutase , catalase , peroxidase  and glutathione peroxidase  using commercial assay kits purchased from Nanjing Jiancheng Bioengineering Institute . All enzymes above were detected using a microplate reader , and 5 to 10 seedlings were used to provide enough amounts of root tissues in each experimental replicate (n = 3).2−· produced by the xanthine morpholine with xanthine oxidase using the SOD assay kit. Each endpoint assay was detected the red substances of the reaction system by absorbance at 550 nm after 40 min of reaction time at 37°C. And one unit SOD activity (U) was defined as the quantity of SOD required to produce 50% inhibition of reduction of nitrite in 1 mL reaction solution by measuring the change of absorbance at 550 nm.The activity of SOD was determined by measuring the inhibiting rate of the enzyme to O2O2) with CAT, which could be terminated by molybdenum acid (MA) to produce yellow MA-H2O2 complex. CAT activity was calculated by the decrease in absorbance at 405 nm due to the degradation of H2O2, and one unit is defined as the amount of enzyme that will cause the decompose of 1 µmol hydrogen peroxide (H2O2) per second at 37°C in 1.0 g fresh tissue according to CAT assay kit.The CAT activity was measured based on the hydrolysis reaction of hydrogen peroxide  could be catalyzed by GSH-Px to produce oxidized glutathione (GSSG) and H2O. In addition GSH reacts with 5, 5′-dithiobis (2-nitrobenzoic acid) (DTNB) to produce stable yellow substances and the decrease of GSH at 412 nm during the reaction is indicative of GSH-Px activity in tissues. One GSH-Px unit of GSH-Px activity (U) was calculated as the amounts of enzyme that will oxidize 1 µmol/L GSH in reaction system at 37°C per minute in 1.0 g fresh tissue according to the assay kit. All of the enzymes were expressed as in U/g FW.The GSH-Px activity was also measured using the assay kit based on the principle that oxidation of glutathione (GSH) and hydrogen peroxide (HAll the experiments were conducted in triplicate (n = 3) and statistical analysis of the data from the control and the chill-stressed plants was performed by analysis of variance (One-Way ANOVA), using SPSS 16.0 software. A probability value of p≤0.05 was considered to denote a statistically significance difference. Data are presented as mean ± standard deviation (SD) of three replicates.At low temperatures, germination percentage of wheat seed was lower. To investigate whether CC could promote germination under chilling stress, a preliminary experiment at 4°C with CC at 0, 5, 10, 20, and 50 µg/mL was carried out. It was also to determine the optimal dosage showing a maximal effect. As demonstrated in To investigate the elicitation of CC on the wheat seedlings growth, an experiment with three culturing procedures, before cold stress, cold stress and recovering room temperature, were carried out. As shown in Membrane injury, expressed as the MDA content and the relative membrane permeability (RMP) value, worsened as low temperature of 4°C went on. CC treatments at chilling stress considerably affected the MDA content and relative membrane permeability (RMP) in roots of wheat seedlings, which are given in In addition, this study attempted to associate the alleviation of chilling injury by CC with the inhibited activities of LOX, PLC and PLD. As we all known, PLD, PLC together with LOX, related to lipid unsaturation level and cell membrane integrity, plays an important role in generating phospholipd degradation and peroxidative damage in membrane lipids under cold stress Significant changes were observed both in saturated and unsaturated fatty acids during 4°C stress and recovering 25°C for 24 h . The linAntioxidant enzyme activities of SOD, CAT, POD, and GSH-Px in CC treatments were increased significantly in roots under cold stress and generally presented to be firstly increased and then decreased in roots of stressed plants . Plus, tFusarium oxysporum, Pythium sp. and Botrytis sp. possessed potent elicitor activity Brassica campestris L. Previous studies have demonstrated that cerebrosides isolated from a range of fungal pathogens including Chilling stress has effects ranging from morphological to molecular levels and the effects are evident at all physiological stages of plant growth. Wheat is sensitive to prolonged exposure to low temperature particularly at earlier seedlings growth, which can result in significant chilling injury and mortality Chilling stress usually causes injury such as the ROS formation in plant cells, which seriously damage membrane lipids, proteins and nucleic acids and disturb the homeostasis of the organism 2−.), and the SOD activity was induced firstly by CC in our study at the early time (6–24 h) under chilling stress and expressed the similar variable trend with the results of Ferrareze et al.2O2) could be metabolized into oxygen and water by CAT 2O2, could also repair other peroxides such as fatty acids and phospholipids peroxides Interestingly, CC induced long-term protection against cold stress in wheat seedlings by affecting activities of the main antioxidant enzymes such as SOD, CAT, POD, and GSH-Px , which aOn the other hand, more lipolytic cascades achieved by the concerted membranous phospholipid-catabolizing enzymes including LOX, PLC, and PLD In this sense, the decrease in the level of MDA at earlier time of cold stress (before 12 h) might result from improved activities of SOD, CAT, and GSH-Px by CC, leading to the alleviation in relative membrane permeability later (from 24–96 h). At the same time, the increase in contents of C18:2, C18:3 and the degree of unsaturated fatty acids might attributed in inhibition of activities of LOX, PLC and PLD induced by CC, and finally increasing the tolerance of wheat seedling under cold stress.Phyllosticta sp. TG78 synergically promoting the germination of wheat seeds under cool conditions are required to be fully elucidated.In conclusion, the promotion of CC on the seed germination and growth of wheat seedlings under cold stress might due to maintain the integrity and stability of membrane under chilling stress by inhibiting the hydrolyzing enzymes of LOX, PLC and PLD, enhancing the unstauration degree of fatty acids, and increasing the antioxidant enzymes activities of SOD, CAT, and GSH-Px. Our present study suggested that CC may be employed as a kind of natural plant growth regulators. Further investigations about the mechanism of CC on the wheat seedlings and whether cerebroside C together with other cerebrosides, including cerebroside B, in extract from Figure S1Detection of fatty acids  in roots of wheat seedlings at 48 h under cold stress by GC-MS.a. GC-MS total ion chromatogram (TIC); b. mass spectrum of the methyl esters of palmitic acid C16:0 (1), linoleic acid C18:2 (2), stearic acid C18:0 (3) and linolenic acid C18:3 (4) in roots of wheat seedlings at 48 h of cold stress. CC+4°C, pretreated with 20 µg/mL CC under cold stress; CK+4°C, treated with 0.5% DMSO under cold stress; CK+25°C, treated with 0.5% DMSO in growth chamber at 25°C. (TIF)Click here for additional data file.Table S1Effects of cerebroside C (20 µg/mL) on MDA content in roots of wheat seedlings under cold stress (4°C).(DOC)Click here for additional data file.Table S2Effects of cerebroside C (20 µg/mL) on RMP value in roots of wheat seedlings under cold stress (4°C).(DOC)Click here for additional data file.Table S3Inhibition of cerebroside C (20 µg/mL) on activity of LOX in roots of wheat seedlings under cold stress (4°C).(DOC)Click here for additional data file.Table S4Inhibition of cerebroside C (20 µg/mL) on activity of PLC in roots of wheat seedlings under cold stress (4°C).(DOC)Click here for additional data file.Table S5Inhibition of cerebroside C (20 µg/mL) on activity of PLD in roots of wheat seedlings under cold stress (4°C).(DOC)Click here for additional data file.Table S6Effects of cerebroside C (20 µg/mL) on activity of C16:0 in roots of wheat seedlings under cold stress (4°C).(DOC)Click here for additional data file.Table S7Effects of cerebroside C (20 µg/mL) on contents of C18:2 in roots of wheat seedlings under cold stress (4°C).(DOC)Click here for additional data file.Table S8Effects of cerebroside C (20 µg/mL) on contents of C18:0 in roots of wheat seedlings under cold stress (4°C).(DOC)Click here for additional data file.Table S9Effects of cerebroside C (20 µg/mL) on contents of C18:3 in roots of wheat seedlings under cold stress (4°C).(DOC)Click here for additional data file.Table S10Effects of cerebroside C (20 µg/mL) on activity of SOD in roots of wheat seedlings under cold stress (4°C).(DOC)Click here for additional data file.Table S11Effects of cerebroside C (20 µg/mL) on activity of CAT in roots of wheat seedlings under cold stress (4°C).(DOC)Click here for additional data file.Table S12Effects of cerebroside C (20 µg/mL) on activity of GSH-Px in roots of wheat seedlings under cold stress (4°C).(DOC)Click here for additional data file.Table S13Effects of cerebroside C (20 µg/mL) on activity of POD in roots of wheat seedlings under cold stress (4°C).(DOC)Click here for additional data file.


Cryopreservation of human semen for assisted reproduction is complicated by cryodamage to spermatozoa caused by excessive reactive oxygen species (ROS) generation.2O2) to simulate cryopreservation and examined DNA damage repair in embryos fertilized with sperm with H2O2-induced DNA damage. Sperm samples were collected from epididymis of adult male KM mice and treated with capacitation medium  or cryopreservation. The model of DNA-damaged sperm was based on sperm motility, viability and the expression of γH2AX, the DNA damage-repair marker. We examined fertility rate, development, cell cleavage, and γH2AX level in embryos fertilized with DNA-damaged sperm. Cryopreservation and 1-mM H2O2 treatment produced similar DNA damage. Most of the one- and two-cell embryos fertilized with DNA-damaged sperm showed a delay in cleavage before the blastocyst stage. Immunocytochemistry revealed γH2AX in the one- and four-cell embryos.We used exogenous ROS (HγH2AX may be involved in repair of preimplantation embryos fertilized with oxygen-stressed spermatozoa. Such cryopreservation provides patients with “fertility insurance” for intrauterine insemination, especially with cryopreserved donor semen, conventional Cold shock during sperm cryopreservation is associated with oxidative stress and reactive oxygen species (ROS) generation The marker of DNA damage is the formation of γH2AX focus, which is phosphorylated by ataxia telangiectasia mutated kinase. H2AX is one of the H2A variants with a conserved Ser-Gln-Glu motif at the C terminus; its phosphorylation plays a key role in DNA damage 2O2) or cryopreservation, only sperm motility variables and intracellular ROS and nitric oxide levels were examined 2O2 increases intracellular ROS levels in spermatozoa Although numerous reports have described DNA damage to sperm caused by exogenous ROS (H2O2) to simulate cryopreservation and observe the repair of DNA damage by the DNA-damage marker γH2AX in embryos fertilized with sperm with oxygen-induced DNA damage.Double-strand breaks are the most deleterious DNA lesions, which, if left unrepaired, may have severe consequences for cell survival, because they lead to chromosome aberrations, genomic instability, or cell death. The formation of DSBs activates many factors, including phosphorylation of the histone variant H2AX, producing γH2AX. Analysis of γH2AX expression can be used to detect the genotoxic effect of substances 2O2. The percentage motility and viability of sperm was lower for cryopreserved than fresh sperm  and decreased with increasing H2O2.Sperm was exposed to capacitation medium containing 0, 0.1, 0.5 or 1 mM H P<0.05; . The per2O2  to simulate cryopreservation and examined DNA damage repair in mouse embryos fertilized with mouse sperm with H2O2-induced DNA damage. Most of the one- and two-celled embryos fertilized with sperm treated with H2O2 showed a delay in cleavage before the blastocyst stage. Immunocytochemistry revealed the expresison of the DNA-repair marker γH2AX in the one- and four-celled embryos. γH2AX may be involved in DNA repair of preimplantation embryos fertilized with oxygen-stressed spermatozoa.Cryopreservation of human semen for assisted reproduction is complicated by cryodamage to spermatozoa caused by excess ROS generation. We used exogenous ROS  and semen leukocytes 2O2-treated sperm. In our previous study, the fertilized egg rate with cryopreserved sperm was lower than with fresh spermatozoa . The reasons for the loss of fertility are various, including factors affecting the proportion of survivors  and factors influencing the functional status of survivors  2O2-treated sperm. However, the DNA damage was the same between cryopreserved and 1-mM H2O2–treated sperm on immunocytochemistry. DNA damage in sperm is negatively related with natural conception. The possibility of natural conception is zero with sperm DNA damge >30% in vitro fertilisation We found no difference in fertilized egg rate and two- or four-celled embryos fertilized with fresh spermatozoa or 1-mM H2O2-treated sperm. However, the time of cleavage to the two- and four-cell stage was delayed with 1-mM H2O2 treatment as compared with fresh sperm from 12 to 48 h after insemination. The cell checkpoint mechanism may play a role in embryo development. After sperm had been treated with 1 mM H2O2, phosphorylation of γH2AX was detected in one- and four-celled embryos, which suggests that the G2/M checkpoint and DNA repair mechanisms function in these embryos. The presence of γH2AX suggests that DNA repair mechanisms are functional in one- and four-celled embryos. γH2AX is required to maintain repair proteins at sites of DNA damage We found no difference in fertilization rate or percentage of fertilized two- and four-celled embryos with fresh sperm and 1-mM H2O2 was repaired in part before ZGA. However, other DNA damage was not repaired, so the DNA damage was not enough to begin embryonic DNA damage repair. The unrepaired DNA damage signal was amplifed during embryonic development and activated the repair pathway after ZGA. The cleavage delay of the one-celled embryos to the two-celled stage and the two-celled embryos to the four-celled stage suggested that the G2/M checkpoint mechanism is operative.Mature sperm cannot repair DNA damage. So the DNA damage must be repaired after fertilization to ensure the integrity of the DNA transferred to offspring 2O2 in sperm was repaired in part before ZGA. Other DNA damage is repaired during the early pre-zygotic stage of life when DNA repair pathways are activated because we found γH2AX expression in four-celled embryos fertilized with oxygen-stressed sperm.The repair of DNA damage that originates from oxidative-stressed sperm after fertilization depends on the mRNA and protein stored in the maternal gamete. The DNA damage caused by 1-mM H2, 0.5 mM MgCl2, 7 mM Na2HPO4, 1.25 mM NaH2PO4, 1.5 mM KH2PO4, and 1 g/l PVA . R18S3 was prepared with 1.8 g skim milk powder soluted into milli-Q water for 6% solution, then centrifuged at 4°C 15,000 g for 1 h. For 20 ml supernatant, 12.5 ml milli-Q water and 7.2 g raffinose was added, then milli-Q water was added to 40 ml; the mixture was agitated to solute raffinose, filtered, subpackaged and stored at –20°C. Pancreatin solution was prepared by the addition of 0.1% pancreatin to PBS, adjusting pH to 3.0 with HCl, filtering, subpackaging and storage at 4°C. Sperm capacitation liquid was prepared by adding 1.5% bovine serum albumin (BSA) to HTF solution. For fertilization liquid, 0.4% BSA was added to HTF solution. For embryo culture medium, 0.4% HTF and 10% fetal bovine serum was added to HTF solution. H2O2 sperm capacitation liquid was prepared by adding H2O2 to a final concentration of 0.1, 0.5 and 1 mM. T-PBS involved adding 0.05% Tween20 to PBS. Stationary liquid involved adding 4% paraformaldehyde to PBS. Membrane liquid was prepared by adding 0.2% Triton X-100 to PBS. Blocking buffer was prepared by adding 0.05% Tween20, 3% BSA and 10% goat serum to PBS. Sperm capacitation liquid, fertilization liquid and embryo culture medium were placed at 37°C in a 5% CO2 incubator for 4 h.All chemicals were of analytical grade. The mouse antibody for anti-phospho-histone H2AX  was from Millipore (USA) and goat anti-mouse Alexa Fluor 488 secondary antibody was from Invitrogen . Human tubal fluid-HEPES (HTF-HEPES) was from SAGE (USA). Pancreatin was from Sigma . Phosphate buffered saline (PBS) was prepared in milli-Q water: 136.9 mM NaCl, 2.7 mM KCl, 0.9 mM CaClwere obtained from the animal center of Shantou University Medical College and treated in compliance with The Guide for the Care of Use of Laboratory Animals by the US National Institutes of Health  and the rules of the National Animal Protection of China. All experimental protocols were approved by the Laboratory Animal Ethics Committee of our institution (SUMC2011-107). The study was approved by the Institutional Animal Care and Use Committee of Shantou University Medical College. Sperm was obtained from the caudae epididymis of mice and exposed to capacitation medium  at 37? in a 5% CO2 incubator for 1.5 h.Adult male KM mice (4–6 weeks old) 2 incubator. Cauda epididymis tissue was put in 1-ml warming cryoprotectant and extruded slightly to ensure no sperm. Spermatozoa were collected from the cauda epididymis and cultured at 37°C in a 5% CO2 incubator for 10 to 15 min. Sperm variables  were recorded by light microscopy (×400 magnification). Spermatozoa were mixed with cryoprotectant and 100 µl was packed into 1.8-ml cryovials  stored in a refrigerator at 4°C for 30 min. The samples were then frozen as follows: aliquots were suspended in liquid nitrogen vapor (8–10 cm above the level of liquid nitrogen at –80°C) for 10 min, plunged into liquid nitrogen (–196°C) completely, and stored in liquid nitrogen until use The cryoprotectant was thawed, mixed, and added to 35-mm culture dishes and warmed at 37°C in a 5% COSperm motility was assessed by phase-contrast microscopy (100× magnification) with a warm stage maintained at 37°C. A 5-µl drop of semen was wet-mounted on a microscope slide and covered with a coverslip. Sperm motility was estimated in 3 different microscopy fields for each semen sample. The mean of the 3 successive estimations was recorded as the final motility score Fully grown germinal vesicle oocytes were collected from the ovaries of mice 13 to 15 h after injecting 10 IU human chorionic gonadotropin and 10 IU of the mate’s serum gonadotropin ahead 48 h. Cumulus oocytes were collected in PBS, then moved to fertilization liquid with use of a pipette and washed. The oocytes were imseminated with spermatozoa that had been preincubated for 1.5 h in sperm capacitation liquid. At 4 h after insemination in HTF medium, the embryos were washed 3 to 5 times, then incubated.A 5-µl sperm suspension was placed on slides, covered with polylysine and fixed with 4% paraformaldehyde for 15 min, washed with T-PBS 3 times, permeabilized with 0.2% Triton X-100 at room temperature for 30 min, washed with T-PBS 3 times, covered with goat serum working solution at room temperature for 2 h, incubated with primary antibody anti-γH2AX (1∶1000 dilution) for at 4°C for 12 to 15 h, then with secondary FITC-conjugated goat anti-mouse IgG antibody  at 37°C for 1 h. The immunostained sperm were washed with T-PBS, counterstained with 4′,6-diamidino-2-phenylindole (DAPI), and observed under an Olympus FluoView FV1000 confocal microscope .Embryos were washed with PBS and the zona pellucida was removed by digestion with pancreatin (0.1%) for 30 s, then embryos were fixed with 4% paraformaldehyde in T-PBS for 30 min, permeabilized for 30 min at room temperature with T-PBS containing 0.1 Triton X-100 and 3 mg/ml BSA, stained with anti-phospho-γH2AX antibody for 1 h at 37°C, then secondary FITC-conjugated antibody. The immunostained embryos were washed with T-PBS containing 0.1% Triton X, counterstained with propidium iodide, and observed under an Olympus FluoView FV1000 confocal microscope.P<0.05 was considered statistically significant.Results are expressed as mean±SD. Data analysis involved use of SPSS 13.0 . Comparisons between 2 groups involved chi-square test and more than 2 groups one-way ANOVA.


Nyctanthes arbor-tristis  has been traditionally used in Ayurveda, Unani and other systems of medicine in India. The juice of its leaves has been used by various tribal populations of India in treatment of fevers resembling malaria.This work reports the antiplasmodial activity guided fractionation of Harshringar leaves extract.P.falciparum and subsequently subjected to bioassay guided fractionation using reverse phase chromatography to pursue the isolation of active fractions.Crude ethanolic Harshringar leaves extract and its RPHPLC purified fractions were studied for antiplasmodial potency against 3D7 (CQ sensitive) and Dd2 (CQ resistant) strains of P.falciparum.Harshringar crude leaves extract and some of its RPHPLC purified fractions exhibited promising antiplasmodial potency against 3D7 and Dd2 strains of P.falciparum strains.The present study has provided scientific validity to the traditional use of leaves extract of Harshringar against malaria leading to the conclusion that this plant holds promise with respect to antimalarial phytotherapy. This is the first scientific report of antiplasmodial activity of RPHPLC fractions of Harshringar leaves extract against  Plasmodium that affect humans, malaria due to P. falciparum is the most deadly Malaria has been reported to be endemic in 106 countries, infecting 216 million and killing 655000 persons in 2010, mostly pregnant women and children under five. Among five species of Natural products, particularly medicinal plants, have been important sources of new drugs, new drug leads, and new chemical entities throughout the history and continue to serve as the basis for many pharmaceuticals used today Nyctanthes arbor tristis (Family: Oleaceae), commonly known as Harshringar (English- Night Jasmine) is one of them Nyctanthes arbor-tristis is a large shrub with fragrant flowers found wild in forests of central Indian, sub-Himalayan regions and commonly cultivated in gardens in many parts of India P.falciparum.India, with its wealth and variety of medicinal plants has accumulated over generations a great mass of popular remedies. P.falciparum.Although our centre  has an ethics committee {Institutional Animal Ethics Committee (IAEC)}, we did not seek the approval of this committee for the experiments reported in our manuscript. Such an approval was not felt necessary since our study does not have any human subjects. We are associated with the a registered blood bank  at New Delhi, India for supply of human red blood cells used in culture of Harshringar leaves were collected from International Centre for Genetic Engineering and Biotechnology (ICGEB) campus, New Delhi, India. The leaves were identified, authenticated in Raw Materials Herbarium and Museum (RHMD), NISCAIR, India by Dr. H. B. Singh (Voucher specimen No. 2043). The voucher specimen was deposited in RHMD for further reference.After washing, draining of water, leaves were air-dried in shade at room temperature (25±5°C) for 7–10 days on laboratory bench, stalks removed and the dried leaves were powdered with hammer mill to yield 840 grams (g) of powder. Eight hundred forty grams of powdered leaves were extracted with 5.65 liters of milli-Q water in 10 liter plastic beaker under agitation on shaker at room temperature for 19 hrs. The extract was filtered with help of muslin cloth. ∼50 mL of filtered water extract was lyophilized to a dry powder (CWE). Residual leaves so obtained were air-dried in shade and subjected to soxhlet ethanol extraction.2O5). The dark brown sticky extract {2.1% (∼17 g)} was stored at 4°C for further use. CWE and CEE were assayed for antiplasmodial potency against P.falciparum 3D7 using CQ as positive control.827 g of residual leaves were consecutively extracted with ethanol (1 litre×6) in a Soxhlet apparatus at 65°C. The crude ethanol extract (CEE) was evaporated to dryness under reduced pressure on rotary evaporator  at 37°C and further dried in vacuum dessicator under phosphorus pentoxide (P18 resin  were mixed together. The excess methanol was evaporated on rotary evaporator at 37°C. The sample adsorbed resin was packed in glass column. First dimension reverse phase chromatographic elution was done by step gradient, starting with milli-Q water (220 mL), followed by Methanol-water volume/volume (v/v): 20% (200 mL), 40% (200 mL), 60% (300 mL), 80% (250 mL), 100% (250 mL), acetonitrile-water v/v: 50% (200 mL), 75% (200 mL) and isopropanol (200 mL) as eluent. Eluates were concentrated to dryness under reduced pressure on rotary evaporator at 37°C, further dried in vacuum dessicator under P2O5. This fractionation resulted in nine fractions {Water Fraction, 20% Methanol Fraction (MF), 40% MF, 60% MF, 80% MF, 100% MF, 50% Acetonitrile Fraction (ACNF), 75% ACNF, and Isopropanol Fraction}. Each fraction was weighed and stored at 4°C for further use. All nine fractions were tested for antiplasmodial potency against P.falciparum 3D7. The two most active fractions namely 60% MF (150 mg) and 80% MF (300 mg) were pooled together, dissolved in dimethyl sulfoxide (DMSO) (15 mL) and subjected to bioassay guided 2nd dimension RPHPLC.The active CEE was fractionated by reverse phase chromatography on a glass column. Briefly 6.5 grams of CEE (dissolved in methanol) and 6 grams of coarse Bondapak C18 column  using methanol-water, linear gradient 20–95% at a flow rate of 2 mL/min over 48 minutes. Dual wavelength detector was set at 214/280 nm. An aliquot of 200 µL  was injected for analytical RPHPLC. An aliquot of 1 mL  was injected on semi-prep reverse phase C18 column  using methanol-water linear gradient 20–95% at a flow rate of 10 mL/min over 90 minutes. Fifty five fractions (14 mL each) were collected using automated fraction collector. These 55 fractions were judiciously pooled together to total of 10 pools (P1–P10), each of which was tested for antiplasmodial potency against P.falciparum 3D7.Analytical reverse phase chromatographic separations were performed on analytical Crd dimension RPHPLC on an analytical C18 column  using acetonitrile-water as eluent, respective peaks (P3E–P9E) were collected and assayed for antiplasmodial potency against P.falciparum 3D7. Pools (P6 and P9) were rechromatographed to collect three sub-fractions from each peak . These six sub-fractions were assayed for antiplasmodial potency against 3D7 and Dd2 strains of P.falciparum. Pools (P3–P9) were further purified by 3P.falciparum were used in vitro blood stage culture to test the antiplasmodial efficacy of Harshringar leaves extract and its RPHPLC fractions. The culture was maintained at the Malaria Research Laboratory, International Centre for Genetic Engineering and Biotechnology (ICGEB), New Delhi, India. P.falciparum culture was maintained according to the method described by Trager and Jensen P.falciparum 3D7 and Dd2 cultures were maintained in fresh O+ve human erythrocytes suspended at 4% hematocrit in RPMI1640 (Sigma) containing 0.2% sodium bicarbonate, 0.5% Albumax, 45 µg/L hypoxanthine, and 50 µg/L gentamicin and incubated at 37°C under a gas mixture 5% O2, 5% CO2, and 90% N2. After every 24 hrs, infected erythrocytes were transferred into fresh complete medium to propagate the culture.CQ-sensitive (3D7) and CQ-resistant (Dd2) strains of Chloroquine was from Sigma. Stock solution of CQ was prepared in water (milli-Q grade) and test compounds were in DMSO. All stocks were diluted with culture medium to achieve the required concentrations . Drugs and test compounds were then placed in 96-well flat bottom tissue culture grade plates (Corning).P.falciparum strains 3D7 and Dd2. For drug screening, SYBR green I-based fluorescence assay was setup as described 50) was determined by analysis of dose-response curves.Crude extracts and purified fractions were evaluated for their antiplasmodial potency against Spectral studies were carried by spectrophotometer (Perkin-Elmer). All solvents used for spectral studies were of analytical grade. 3 mL quartz cuvettes were used for all studies. 110 µg/mL, 90 µg/mL, 180 µg/mL, 80 µg/mL and 40 µg/mL of 40%, 60%, 80%, 100% MF and pool of 60%–80% MF respectively were used for spectral studies against methanol as blank.Pf3D7) and chloroquine resistant (PfDd2) strains of blood stage P.falciparum in red blood cell culture.Harshringar holds a reputed position as a medicinal plant in different systems of medicine in India including Ayurveda. The juice of its leaves in different forms has been advocated for acute, chronic as well as intermittent fever 50 77±7 µg/mL) against P.falciparum 3D7. We surmised that this modest potency may be related to the fact that crude plant extracts contain a number of complex molecules, only a few of which may be active. In such a scenario, the resultant activity of crude extract may increase/decrease/abolish depending on synergistic/antagonistic interactions among/between various types of complex molecules present in extract 50) of ethanol extract of Harshringar leaves against CQ sensitive strain of P.falciparum in the range of 1000–1200 µg/mL. Mishra et al., P.falciparum by the 50% ethanolic extract of Harshringar leaves both in vivo and in vitro. Even as the crude extract (IC50 77±7 µg/mL) was found to be largely inactive, we subjected it to activity guided RPHPLC fractionation in the hope of finding some molecules which may be present in trace amounts and yet be quite potent.Crude ethanolic extract (CEE) of Harshringar leaves showed only modest potency (IC50 14.5±0.7 µg/mL) and 80% MF (IC50 13±4.2 µg/mL) displayed promising antiplasmodial potency (Glass column reverse phase chromatography of CEE was used to fractionate the crude extract. The yields obtained were 14.6% (Water Fraction), 5% (20% MF), 2.5% (40% MF), 3.5% (60% MF), 6% (80% MF), 24% (100% MF), 0.6% (50% ACNF), 0.2% (75% ACNF) of the extract loaded on column. However among the nine fractions, only two fractions 60% MF , P6 (IC50 12±2.8 µg/mL) and P7 (IC50 11.25±1.8 µg/mL) showed promising activity; P4 (IC50 19.75±1.1 µg/mL), P5 (IC50 15.75±5.3 µg/mL), P8 (IC50 17.5±2.1 µg/mL), P9 (IC50 32 µg/mL) and P10 (IC50 20 µg/mL) showed moderate activity, while P1 (IC50>50 µg/mL) and P2 (IC50>50 µg/mL) were considered inactive  values of P3 (13.7) and P9 (32) dropped to 12.5 and 22.5 respectively after 3rd dimensional separation. However in contrast, the 2nd dimensional potencies of P4–P8 showed a significant decrease in potency following the 3rd dimensional chromatography  associated with the subfractions of P6 were 38, 6 and 6. Similarly the IC50 (µg/mL) associated with the subfractions of P9 were 11, 39 and 39 respectively (Pf 3D7 IC50 38 µg/mL) showed an IC50 7.5 µg/mL against the CQ resistant Pf Dd2  of Nyctanthes arbor tristis leaves. However the most probable phytoconstituents that may be responsible for the observed antiplasmodial activity may belong to the class of iridoid glycosides found in Nyctanthes arbor tristis leaves. This class of molecules has been reported to inhibit trypanothione reductase (TryR), a validated drug target enzyme of the Leishmania parasite P.falciparumP.falciparum. Our hypothesis is consistent with the fact that inhibitors of Trypanothione Reductase are known to inhibit the growth of P. falciparum in culture The antiplasmodial activity in fractions could possibly have its origin in the alkaloids, phytosterols, phenolic compounds and iridoid glycosides that are found in Here we have for the first time fractionated the Harshringar leaf extract and located antiplasmodial activity in specific fractions. The results achieved with this work constitute a proof that Harshringar is a promising plant with regard to anti-malarial phytotherapy and support continuous investigation of this plant to discover new anti-malarials.


We investigated whether Ca2+ channels at bipolar cell ribbon synapses are fixed in position or capable of moving in the membrane.Efficient and precise release of glutamate from retinal bipolar cells is ensured by the positioning of L-type Ca2+ channels in bipolar cell terminals after labeling channels with quantum dots (QDs) attached to α2δ4 accessory Ca2+ channel subunits via intermediary antibodies.We tracked the movements of individual L-type Ca2+ channels moved within a confined domain of 0.13–0.15 μm2 in bipolar cell terminals, similar to ultrastructural estimates of the surface area of the active zone beneath the ribbon. Disruption of actin expanded the confinement domain indicating that cytoskeletal interactions help to confine channels at the synapse, but the relatively large diffusion coefficients of 0.3–0.45 μm2/s suggest that channels are not directly anchored to actin. Unlike photoreceptor synapses, removing membrane cholesterol did not change domain size, indicating that lipid rafts are not required to confine Ca2+ channels at bipolar cell ribbon synapses.We found that individual Ca2+ channels to move within the presynaptic active zone suggests that regulating channel mobility may affect release from bipolar cell terminals.The ability of Ca To study Ca2+ channel movements, we labeled individual L-type Ca2+ channels by attaching a photostable quantum dot (QD) to the α2δ4 accessory Ca2+ channel subunit through intermediary primary and secondary antibodies [2δ subunit because its large extracellular domain makes it accessible to antibodies applied in living tissue. α2δ4 is the principal α2δ subunit in photoreceptors and is strongly expressed in the inner plexiform layer (IPL) suggesting it is also an accessory subunit at L-type Ca2+ channels in bipolar cell terminals [The transmission of light responses to retinal ganglion and amacrine cells is regulated by the entry of Caar cells ,2. Releachannels ,4, and oar cells . This hic ribbon ,7, a proc ribbon ,9. Tightsynapses -14. The nd CaBP4 ,15-18. De ribbon . In the tibodies . We targerminals ,20.2+ channels move within a small confined domain in bipolar cell terminals similar to the size of the active zone beneath the ribbon determined from ultrastructural analysis [2+ channels are mobile suggests that, as in photoreceptors [The results of this study showed that Caanalysis . Disrupteceptors , changesAmbystoma tigrinum, 18–25 cm long; Kons Scientific, Germantown, WI; Charles Sullivan Co., Nashville, TN) were maintained on a 12 h:12 h light-dark cycle and were sacrificed 1–2 h after the beginning of subjective night. Salamanders were decapitated with heavy shears and immediately pithed.Experimental procedures were approved by the University of Nebraska Medical Center Institutional Animal Care and Use Committee. Aquatic tiger salamanders  and resolved on 10% Bis-Tris polyacrylamide gels . Gels were transferred to nitrocellulose membranes and blotted with antibodies to α2δ4 (1:1000) with an anti-β-actin antibody serving as a housekeeping protein . Blots were visualized using horseradish peroxidase-conjugated secondary antibodies and SuperSignal West Pico Chemiluminescent Substrate .2, 0.5 MgCl2, 10 HEPES, and 5 glucose (pH 7.8). The retinal pigment epithelium, choroid, and sclera were peeled away, leaving the retina adhered to the filter paper. The retina was sliced into 125 μm sections with a razor blade tissue chopper  fitted with a #121–6 razor blade . Slices were separated from one another and rotated 90° to view the retinal layers under a water immersion objective.After enucleation, each eye was opened along the ora serrata, and the cornea, lens, and vitreous body were removed. For experiments on retinal slices, the eyecup was sectioned into thirds, and a piece of eyecup was pressed vitreous side down onto a 5 × 10 mm piece of filter paper . The filter paper and eyecup were then immersed in cold amphibian saline solution consisting of (in mM) 111 NaCl, 2.5 KCl, 1.8 CaCl2+ amphibian extracellular saline solution containing 0.2 mg/ml cysteine and 10 U/ml papain (Sigma-Aldrich) for about 35 min at 20 °C. Tissue was washed twice in a low Ca2+ saline solution containing 1% BSA and 1 mg/ml DNase  followed by two additional washes in low Ca2+ saline. To isolate individual neurons, retinal tissue was gently triturated using a fire-polished glass Pasteur pipette and plated onto 18 mm coverslips  coated with Cell Tak .For experiments on dissociated cells, a salamander retina was isolated and incubated in 0.5 mM Ca2+ channels at bipolar cell synapses was conducted as described previously for photoreceptors [2δ4 subunit antibody [2+ saline solution for 3 h at 4 °C. After incubation with the primary antibody, cells were washed 3× with standard amphibian saline solution, and then incubated with secondary biotinylated goat-anti-rabbit immunoglobulin G  for 1 h at 4 °C. In some experiments, the actin disruptor cytochalasin D (20 μM) or cholesterol-depleting agent methyl-β-cyclodextrin  was included with the secondary antibody incubation. After this step, retinal tissue was washed another 3X and then incubated with streptavidin-coated 525 or 655 nm emission QDs (20 nM) for 15 min. Tissue was washed eight more times before visualization. As a control for baseline noise, we also visualized and quantified the movements of QDs immobilized in vacuum grease. Indirect immunofluorescence with fluorescein isothiocyanate (FITC)–conjugated secondary antibodies showed strong labeling by the α2δ4 antibody throughout the IPL where bipolar cell terminals reside [QD attachment to individual Caeceptors . Brieflyantibody   with a 60X, 1.45 NA oil-immersion objective (Olympus) and cooled EMCCD camera . Epifluorescence was provided by an X-Cite 120Q light source  with a FITC filter cube and Model D122 shutter . Videos were captured using MetaMorph imaging software . For retinal slices, QDs were visualized through a 60X, 1.2 NA water-immersion objective  on an upright microscope . Images were acquired using an FITC filter cube , EMCCD camera , and Nikon Elements software.QDs were selected for tracking analysis if they were localized to terminals of isolated bipolar cells or within the IPL of the retinal slices, exhibited a small size (≤4 pixels), and showed intermittent blinking; these criteria are consistent with labeling by a single QD . The loc2/s) [Before tracking, images were smoothed by convolving with a 5 × 5 pixel Gaussian and adjusted to optimize QD contrast. QD position was tracked using NIS-Elements software (Nikon). From the x and y coordinates, we calculated the mean squared displacement   using thThe diffusion coefficient (D) was calculated from the initial slope of the MSD versus time (t) from the origin through the first two data points using data acquired at a rate of 16 ms/frame in dissociated bipolar cells. The value for D was determined with the following equation:2+ channel traverses within the presynaptic plasma membrane (L2) by fitting the data with Equation 3:We calculated the confinement area that a given CaFor a particle moving freely in solution, all three planes of movement are independent, but movements in a lipid bilayer are constrained to the plane of the membrane. Because z-axis movements could not be tracked, calculated confinement areas and diffusion coefficients represent lower-bound estimates of true values . All datFor the illustrations in the paper, post-image processing was performed in Adobe Photoshop  to add color and optimize the brightness and contrast.2+ channels in bipolar cell terminals using QD-based single particle tracking techniques. As described in Methods, streptavidin-coated QDs were attached to individual L-type Ca2+ channels by binding a biotinylated secondary antibody to the Fc region of a primary antibody against the accessory α2δ4 Ca2+ channel subunit. The antibody was originally developed and shown to be specific for α2δ4 protein in humans [2δ4 amino acid sequence (Ac-KVSDRKFLTPEDEASVC-amide) to which the antibody was raised exhibits excellent homology across vertebrate species, and thus, we predicted that the rabbit anti-α2δ4 antibody would also bind selectively to α2δ4 subunits in the tiger salamander retina. Consistent with this, the α2δ4 antibody labeled photoreceptor terminals in the outer plexiform layer (OPL) and bipolar cell terminals in the IPL [1 subunit of CaV1.4 [2δ4 antibody produced a band at 150 kDa, as expected for the α2δ4 subunit. The reducing conditions of the western blot buffer should break the disulfide bond linking the α2 and δ4 components of the α2δ4 subunit, and we observed a second band at about 130 kDa, consistent with dissociated α2 subunits [2δ4 in salamander retina.We tracked the movements of individual L-type Can humans . However the IPL . This laf CaV1.4 . Labelinf CaV1.4 . We alsosubunits ,28. Thessubunits , the ant2+ channel was measured at 30 ms intervals for 400 frames. 2+ channels diffuse within a confined domain [2/3 with L=0.366±0.00483 μm (n=24), suggesting a confinement area (L2) of 0.134 μm2. This is smaller than the confinement areas for Ca2+ channels at the rod and cone synapses [In d domain . The heisynapses , consist2/s. Omitting the origin from the linear fit, as was done in an earlier study on photoreceptor Ca2+ channels [2/s.We measured the D by analyzing movements over short time intervals before the channel was confined . The MSDchannels , reduced2+ channels ON bipolar cell terminals embedded in the IPL. 2δ4 subunits in the IPL (arrows). The MSD versus time interval plot yielded a confinement area similar to that found in isolated cells with an average value of L=0.393±0.00237 μm . Neither the corrected nor the uncorrected values differed significantly from the confinement length L found for isolated bipolar cells (p>0.05).In addition to isolated cells, we also examined the mobility of Caμm n=34; . Subtrac2+ channels at the photoreceptor ribbon synapses [2+ channels in the IPL  and never saw QDs depart from localized confinement zones, consistent with the idea that α2δ4 subunits remain associated with other subunits of synaptic L-type Ca2+ channels.To attach QDs to Ca domains , and thuantibody  used in subunits  and spec subunit . We foun2+ channels in the bipolar cell terminals were 0.3–0.45 μm2/s, similar to the diffusion coefficients for the Ca2+ channels at the photoreceptor ribbon synapses (0.1–0.2 μm2/s) [a), 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid (AMPA) and ether-à-go-go (EAG) channels in the extrasynaptic membrane [The diffusion coefficients for the Ca2 μm2/s) . These vmembrane -40.2+ channels at bipolar cell terminals were confined within an area of 0.13–0.15 μm2. Assuming an active zone width of 200–300 nm [2+ channel confinement of about 400–750 nm, similar to ultrastructural estimates of the length of the bipolar cell ribbon base (about 400 nm) [2+ channel confinement domains measured in rods (0.29–0.36 μm2) and cones (0.18–0.22 μm2) were slightly larger than those of bipolar cells [2+ channel confinement domains in photoreceptors were similar to ultrastructural estimates of the size of the active zone beneath the ribbons [V1.3 channel immunostaining revealed an oblong rectangular pattern beneath ribbon-style active zones [2+ channels are also organized to mirror dimensions along the base of the synaptic ribbon at a non-retinal synapse.Ca0–300 nm , this su 400 nm) . Movemen 400 nm) . Consistar cells ,22. Simi ribbons -44. At hve zones  indicati2+ channel confinement domain suggesting that protein networks are stabilized at the synapse by interactions with the cytoskeleton [2+ channels bind directly to actin at bipolar cell synapses, and consistent with this, the size of the channel confinement domain and diffusion coefficients were much larger than values obtained for other ion channels that interact directly with actin [2+ channel confinement domains, suggesting that cholesterol-rich lipid rafts are not required to confine Ca2+ channels near ribbon synapses.Disrupting actin expanded the size of the Caskeleton ,45-47. Hth actin ,49. Unlith actin , removin2+ channels and the size of the ribbon determined from ultrastructure in rods, cones, and bipolar cells suggests that single particle tracking techniques may provide a means of measuring the size of the active zone at ribbon synapses in living cells. In cones, fusion of nearby synaptic vesicles increased Ca2+ channel displacements and moved channels further away from the center of the confinement area consistent with a brief expansion of the active zone [2+ channel confinement zone by depleting membrane cholesterol reduced peak release efficiency  in cones from three Ca2+ channel openings per vesicle fusion event to 4.5 channel openings per fusion event [2+ channels are close to bipolar cell release sites [2+ channel confinement domain caused by actin disruption or an imbalance between exocytosis and endocytosis might alter release efficiency. Brief treatment with cytochalasin D for 10 min did not significantly alter the total amount of exo- and endocytosis measured with FM1–43 at bipolar cell synapses [2+ channel mobility thus appear to have less influence on the total amount of release and more on the speed and precision of the transmission of light signals through the retina.The agreement between measurements of the confinement area made by tracking QD-labeled Caive zone . Expandion event . Similaron event ,50, Ca2+se sites . We thersynapses . Althougsynapses . Changes


Important reactions of drug metabolism, including UGT mediated glucuronidation and steroidsulfatase mediated hydrolysis of sulfates, take place in the endoplasmic reticulum (ER) of hepatocytes. Consequently, UGT generated glucuronides, like estradiol-17β-glucuronide, have to be translocated back into the cytoplasm to reach their site of excretion. Also steroidsulfatase substrates, including estrone-3-sulfate, have to cross the ER membrane to reach their site of hydrolysis. Based on their physicochemical properties such compounds are not favored for passive diffusion and therefore likely necessitate transport system(s) to cross the ER membrane in either direction.− rat liver.The current study aims to investigate the transport of taurocholate, estradiol-17β-glucuronide, and estrone-3-sulfate in smooth (SER) and rough (RER) endoplasmic reticulum membrane vesicles isolated from Wistar and TRTime-dependent and bidirectional transport was demonstrated for taurocholate, showing higher uptake rates in SER than RER vesicles. For estradiol-17β-glucuronide a fast time-dependent efflux with similar efficiencies from SER and RER but no clear protein-mediated uptake was shown, indicating an asymmetric transport system for this substrate. Estrone-3-sulfate uptake was time-dependent and higher in SER than in RER vesicles. Inhibition of steroidsulfatase mediated estrone-3-sulfate hydrolysis decreased estrone-3-sulfate uptake but had no effect on taurocholate or estradiol-17β-glucuronide transport.Based on inhibition studies and transport characteristics, three different transport mechanisms are suggested to be involved in the transport of taurocholate, estrone-3-sulfate and estradiol-17β-glucuronide across the ER membrane. The hepatic drug elimination process occurs in hepatocytes and is classified into four phases including drug uptake (phase 0), phase 1 and 2 biotransformation and excretion of metabolites (phase 3) Both CYPs Accordingly, the co-substrate for UGT mediated glucuronidation, the uridine diphosphate glucuronic acid (UDPGA) has to be translocated from its site of synthesis in the cytoplasm, to its site of action within the ER lumen. After UGT mediated glucuronidation, the resulting products have to be transported from the ER lumen back into the cytoplasm. Based on their physicochemical properties neither UDPGA nor glucuronides are likely to cross the ER membrane by simple diffusion, indicating the involvement of transport system(s) in the translocation of these compounds across the ER membrane. Evidence for the existence of such transport system(s) is given as UDPGA transport has been reported in rat liver microsomes − rats. Finally, taurocholate transport into RER and SER was investigated to test for potential common transport pathways with phase 2 products.Based on the knowledge that STS and UGTs have their catalytic site within the ER lumen, the present study investigated and characterized the transport activity for the UGT model product estradiol-17β-glucuronide (E17βG) as well as the STS model substrate estrone-3-sulfate (E3S) in SER and RER vesicles isolated from rat liver. Transport of sulfated and glucuronidated model compounds across SER and RER membranes were functionally compared. Furthermore, a potential impact of a chronic cytoplasmic accumulation of glucuronides on the activity of these transport systems was investigated using ER fractions isolated from wild type and from the TR22.13H(N)]-estradiol-17β-glucuronide , -estrone-3-sulfate , [3H(G)]-taurocholic acid  as well as the liquid scintillation fluid Filter Count were purchased from Perkin-Elmer . Unlabelled substrates including E3S, E17βG, and TC as well as valinomycin, puromycin, and ATP were purchased from Sigma Aldrich . Compounds used for inhibition studies, including UDP-glucuronic acid (UDPGA), Stx64, and vanadate were purchased from Sigma Aldrich (Buchs Switzerland). Glucose-6-phosphate was purchased from Roche  and bovine serum albumin was purchased from Interchim . All chemicals used were of the highest purity available. HPTLC plates and solvents used for HPTLC analysis were purchased from Merck .[6,7-2.2− rats (180–200 g), were held in the animal facilities of the University Hospital of Zurich. The animals received humane care in accordance with local and federal guidelines. Animal experiments were approved by the local government's animal care committee.Wistar rats, obtained from Charles River , Sprague Dawley rats obtained from Harlan , and TR2.32.3.1Basolateral and canalicular membrane vesicles were isolated from Sprague Dawley rats as described by Meier and colleagues 2.3.2− rats (150-200 g) by density gradient centrifugation following procedures as described previously  with standard procedures. Membrane fractions were probed for Cyp3a2 , Sec61α  or PDI  by either incubation at 4 °C overnight (Sec61α) or at room temperature for 2 h . Either HRP-conjugated goat anti rabbit IgG  or sheep anti mouse IgG  was used as secondary antibody. All antibodies were diluted in TBS-T  containing 5% milk powder. UptiLight™  was used as chemiluminescent substrate for protein detection. Western blot analyses for Cyp3a2 and Sec61α were performed for each SER and RER isolation used in this study.Primary antibodies against Oatp1a1 2.52.5.12, 5 mM MgCl2 and 20 mM Hepes/Tris pH 7.4) and solution was rapidly filtered through a 0.45 μm nitrocellulose acetate filter . For TC transport studies, filters were presoaked with 3 ml of 1 mM TC solution. Filters were dissolved in Filter Count  and radioactivity was measured by liquid scintillation counting in a Betacounter .Uptake of radioactive labelled substrates was measured by the rapid filtration method 2.5.1.12, 20 mM Hepes/Tris, pH 7.4 (Kout > Kin resuspension buffer) and substrate containing incubation buffer (Kout > Kin incubation buffer) consisted of 50 mM sucrose, 100 mM Kgluconate, 0.1 mM Ca(gluconate)2, 2.5 mM Mg(gluconate)2, 20 mM Hepes/Tris pH 7.4. Contrary, an inside negative membrane potential was generated by using 50 mM sucrose, 100 mM Kgluconate, 0.1 mM Ca(gluconate)2, 20 mM Hepes/Tris pH 7.4 as resuspension buffer (Kout < Kin resuspension buffer) and 250 mM sucrose, 0.1 mM Ca(gluconate)2, 2.5 mM Mg(gluconate)2, 20 mM Hepes/Tris, pH 7.4 as incubation buffer (Kout < Kin incubation buffer).To test for the role of the membrane potential on transport activities, vesicles were pretreated with valinomycin , added from a stock solution (10 mg/ml in DMSO), for 30 min at room temperature followed by 30 min incubation on ice prior to transport studies. Subsequently a membrane potential was generated by either an inward or outward directed potassium gradient. To generate an inward positive membrane potential, vesicles were resuspended in 250 mM sucrose, 0.1 mM Ca(gluconate)2.5.1.23, 20 mM Hepes/Tris pH 7.4. The substrate containing incubation buffer either consisted of 50 mM sucrose, 100 mM KNO3, 12.5 mM Mg(NO3)2, 10 mM Hepes/Tris, pH 7.4 alone or with 6.25 mM ATP. Uptake was performed as described in 2.5.1.For ATP dependent uptake studies, vesicles were resuspended in 50 mM sucrose, 100 mM KNO2.5.1.3out > Kin resuspension buffer as described in 2.5.1.1. or 0.25 M sucrose, 0.75 M KCl, 1 mM puromycin, 50 mM Tris/HCl pH 7.4 for 1 h at room temperature. After centrifugation  SER and ribosome-free RER vesicles were resuspended in Kout > Kin resuspension buffer. Protein of SER and RER vesicles was determined using bicinchoninic acid  and bovine serum albumin as standard and uptake of TC was measured at an inward positive membrane potential as described in section 2.5.1 and 2.5.1.1.Attached ribosomes of RER vesicles were removed using puromycin and high KCl concentration as described previously 2.5.1.42, 20 mM Hepes/Tris, pH 7.4. The substrate containing incubation buffer consisted of 50 mM sucrose, 0.1 mM Ca(gluconate)2, 2.5 mM Mg(gluconate)2, 20 mM Hepes/Tris pH 7.4 and either 100 mM NaCl or KCl. Uptake studies were performed as described in section 2.5.1.For sodium dependent TC transport, vesicles were resuspended in 250 mM sucrose, 0.1 mM Ca(gluconate)2.5.1.5Experiments for generating osmolarity plots were performed at an inward positive membrane potential at an incubation time point of 10 min (see section 2.5.1.1.) out > Kin resuspension buffer (see 2.5.1.1.) and the osmolarity outside of the vesicles was increased by adding increasing sucrose concentrations, ranging from 50 mM to 800 mM, to the Kout > Kin incubation buffer (see 2.5.1.1.). Uptake was performed as described in section 2.5.1.Vesicles were resuspended in KUptake (pmol/mg protein) was plotted against the reciprocal osmolarity of the incubation buffer and linear regression analyses were performed using the GraphPad prism software . The amount of substrate binding to vesicles is given at the intercept of the extrapolated linear regression with the y-axis (uptake), whereas uptake rates observed at identical intra- and extravesicular osmolarities corresponding to 250 mM sucrose was taken as 100%.2.5.2For efflux studies, 100 μg vesicles were preincubated with the given concentration of radiolabelled substrate in resuspension buffer in parallel with valinomycin treatment (see section 2.5.1.1). Efflux was initiated by a 60- fold dilution with incubation buffer. After the desired time of incubation at 37 °C, efflux was stopped by the addition of ice-cold stop solution and vesicle associated radioactivity was measured as described in 2.5.1 Membrane potentials during efflux studies were generated as described before (section 2.5.1.1).2.6As described previously 2.7Data are given as means ± SD. The number of independent experiments is given in the figure and table legends.Where indicated, statistical significance was calculated using unpaired t-test and the GraphPad prism software . A p-value <0.05 was considered as statistical significant.33.1To test for successful separation of SER and RER, expression of proteins known to be present in different ER compartments , was analyzed by Western blotting.− rats showed decreased Cyp3a2 expression compared to Wistar rats, which is in accordance to previous data While PDI is reported to be equally distributed between SER and RER To assess potential cross-contamination of isolated vesicles with plasma membranes, protein expression of basolateral and canalicular membrane proteins, including Ntcp, Oatp1a1 and Mrp2, was also tested by Western blotting. In comparison to basolateral or canalicular membrane fractions of Male Sprague Dawley rats, used as positive controls, expression of all three proteins was only faint. Therefore, contamination of RER and SER fractions with plasma membrane is only marginal .3.23.2.1− rat liver (Uptake of taurocholate (TC) into SER and RER vesicles was time-dependent and electrogenic A and 2B,at liver A.− rats showed a 2.5 fold higher TC uptake at this time point of incubation into SER compared to RER vesicles  transport activity for TC was shown in SER and RER fractions compared to basolateral membrane fractions, supporting minimal expression of these proteins observed by Western blotting  in SER and RER vesicles isolated from Wistar and TResicles, A remainiesicles, A. Highercubation B. Conseq− rats a 2.5 fold increase of E3S uptake compared to the RER fraction was observed  analyses were performed to investigate the chemical intactness of the substrate at the end of the uptake studies.HPTLC analysis showed time-dependent E3S hydrolysis in SER A as wellThe effect of STS inhibition on E3S uptake was therefore also studied in SER and RER vesicles either pretreated or not pretreated with Stx64. Similar to the time-dependent hydrolysis, E3S uptake decreased with time in Stx64 treated vesicles E reachin3.5To compare functional characteristics of TC, E3S and E17βG transport in more detail, inhibition studies were performed. Furthermore, a possible involvement of already identified ER transporters, including the glucose-6-phosphate transporter 3.5.1As shown in As Stx64 was shown to inhibit E3S uptake, its effect on TC uptake was also studied, but no effect could be observed. Likewise, neither UDPGA nor G6P (with or without vanadate treatment) affected TC uptake in SER and RER vesicles.3.5.2E3S uptake was also significantly inhibited in vanadate treated vesicles to 54-57% of control in SER. In RER the inhibitory effect was even more pronounced and E3S uptake was almost completely inhibited. The irreversible STS inhibitor Stx64 decreased E3S transport to 80% and 76% in SER and RER fractions respectively, but did not reach statistical significance. No inhibitory effect was observed for UDPGA and G6P.3.5.3For E17βG efflux, trans-inhibition studies were performed at an incubation time of 5 sec. As shown in Neither TC, nor one of the other tested compounds, including UDPGA, Stx64 or G6P (with or without vanadate) affected E17βG efflux.4The extensive network of the ER accounts for more than 50% of the total membrane surface and about 15% of the total volume in hepatocytes − rats, which lack of the efflux pump Mrp2.Therefore we investigated the transport of the STS model substrate E3S and the typical UGT generated E17βG across the membrane of the smooth (SER) and the rough (RER) ER subcompartment isolated from rat liver. Successful separation of SER and RER vesicles isolated as well as only marginal contamination of SER and RER with plasma membrane was shown by Western blot analysis. Electrogenic TC uptake, shown in the present study, is in accordance to previous data + diffusion potential has previously been reported in total microsomes, in ER enriched microsomes Xenopus laevis oocytes showed bidirectional TC transport + diffusion membrane potential in Xenopus laevis oocytes + gradient. Besides Oatps, the major part of TC uptake in hepatocytes is mediated by the Na+- taurocholate co-transporting polypeptide (Ntcp) in a sodium-dependent manner Electrogenic TC uptake with higher uptake rates at an intravesicular positive KUGT mediated glucuronidation takes place within the ER lumen After glucuronidation and translocation of formed glucuronides from the ER lumen back into the cytoplasm, glucuronides have to be excreted out of hepatocytes. At the canalicular membrane, the ATP binding cassette transporter Mrp2 is responsible for the ATP dependent export of glucuronides into the bile 4, showing a bidirectional time-dependent but unsaturable transport in total microsomal vesicles isolated from rat liver Compared to TC and E17βG, no data are available studying the transport of E3S across the ER membrane. Only one study investigated the transport of NaSOIn conclusion, our study showed clear differences in the transport characteristics of TC, E3S, and E17βG across SER and RER membrane vesicles. Furthermore, the two STS inhibitors Stx64 and vanadate caused inhibition of E3S uptake but did not affect TC uptake or E17βG efflux. Therefore, we conclude that three different transport systems (as illustrated in